A method for identifying the medicinal materials of gardenia and licorice in a traditional Chinese medicine composition containing gypsum and gardenia, and application thereof

Abstract

The application discloses a thin-layer identification method for medicinal materials gardenia and licorice in a traditional Chinese medicine composition containing gypsum and gardenia, which comprises the following steps: (1) pretreating a decoction of the traditional Chinese medicine composition containing gypsum and gardenia to obtain a test sample solution, pretreating a control medicinal material to obtain a control medicinal material solution, pretreating a control substance to obtain a control substance solution, and / or pretreating a decoction of a traditional Chinese medicine composition lacking gardenia or licorice to obtain a negative sample solution; and (2) performing thin-layer chromatography identification analysis on the test sample solution, the control medicinal material solution, the control substance solution, a blank solution and / or the negative sample solution. The above thin-layer identification method provides a theoretical basis for quality control and research on the pharmacodynamic material basis of the traditional Chinese medicine composition containing gypsum and gardenia through detection of medicinal materials gardenia and licorice in the traditional Chinese medicine composition containing gypsum and gardenia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a thin-layer chromatography method for identifying gardenia and licorice in a traditional Chinese medicine composition containing gypsum and gardenia, and its application. Background Technology

[0002] Thin-layer chromatography (TLC) is an analytical technique widely used in chemistry, pharmacy, food science, and other fields. It offers numerous advantages, including simple and rapid operation, low cost, high sensitivity, wide applicability, and intuitive visualization of results. TLC is widely used in the identification of traditional Chinese medicinal materials and prepared Chinese medicines. This technique can effectively determine the source and quality of herbal extracts, ensuring the safety and efficacy of the medicines.

[0003] The prior art does not disclose a thin-layer chromatography method for identifying the medicinal materials gardenia and licorice in a traditional Chinese medicine composition containing gypsum and gardenia, as claimed in this invention, nor does it disclose its application. Summary of the Invention

[0004] Based on this, the present invention provides a thin-layer chromatography method for identifying the medicinal materials gardenia and licorice in a traditional Chinese medicine composition containing gypsum and gardenia. The thin-layer chromatography method includes the following steps: (1) Pre-treating the decoction of a traditional Chinese medicine composition containing gypsum and gardenia to obtain a test solution, pre-treating the reference medicinal material to obtain a reference medicinal material solution, pre-treating the reference standard to obtain a reference standard solution, and / or pre-treating the decoction of a traditional Chinese medicine composition lacking gardenia or licorice to obtain a negative sample solution; and (2) Perform thin-layer chromatography identification analysis on the test solution, the reference medicinal material solution, the reference standard solution, the blank solution and / or the negative sample solution; In the thin-layer chromatography identification analysis step, a developing solvent is used for development, wherein the developing solvent is ethyl acetate-acetone-formic acid, and the volume ratio of ethyl acetate, acetone and formic acid in the developing solvent is (2~10):(4~6):(0.5~2).

[0005] Furthermore, the traditional Chinese medicine composition containing gypsum and gardenia includes gypsum, gardenia, patchouli leaves, saposhnikovia root, and licorice.

[0006] Further, the preparation method of the test solution includes: (a) taking an appropriate amount of the decoction of the traditional Chinese medicine composition containing gypsum and gardenia, concentrating it under reduced pressure to dryness to obtain a first residue; (b) adding a first solvent to the residue for extraction, filtering, evaporating the filtrate to dryness to obtain a second residue; and (c) adding a second solvent to the second residue to dissolve it to obtain the test solution.

[0007] Furthermore, the first solvent is ethyl acetate.

[0008] Furthermore, the mass / volume (g / ml) ratio of the decoction containing gypsum and gardenia to the first solvent is 0.5 to 2, for example, about 1.

[0009] Furthermore, the decoction of the traditional Chinese medicine composition containing gypsum and gardenia has a mass of 80-120 g, for example, about 100 g.

[0010] Furthermore, the volume of the first solvent is 80-120 ml, for example, about 100 ml.

[0011] Furthermore, the extraction is performed using ultrasound.

[0012] Furthermore, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0013] Furthermore, the second solvent is methanol.

[0014] Furthermore, the mass / volume (g / ml) ratio of the decoction containing gypsum and gardenia to the second solvent is 80 to 120, for example, about 100.

[0015] Furthermore, the volume of the second solvent is 0.8 to 1.2 ml, for example, about 1 ml.

[0016] Furthermore, the reference medicinal materials include gardenia reference medicinal materials and / or licorice reference medicinal materials.

[0017] Furthermore, the preparation method of the reference medicinal material solution includes: weighing an appropriate amount of gardenia reference medicinal material and / or licorice reference medicinal material, adding a first solvent for extraction, filtering, evaporating the filtrate to dryness, and obtaining residue; adding a second solvent to the residue to dissolve it, thereby obtaining the reference medicinal material solution.

[0018] Furthermore, the first solvent is ethyl acetate.

[0019] Furthermore, the second solvent is methanol.

[0020] Furthermore, the mass / volume (g / ml) ratio of the gardenia reference material or licorice reference material to the first solvent is 0.01 to 0.1, for example, about 0.02.

[0021] Furthermore, the mass / volume (g / ml) ratio of the gardenia reference material or licorice reference material to the second solvent is 0.8 to 1.2, for example, about 1.

[0022] Furthermore, the mass of the gardenia reference material was 0.8–1.2 g, for example, about 1 g.

[0023] Furthermore, the mass of the licorice reference material is 0.8~1.2 g, for example, about 1 g.

[0024] Furthermore, the extraction is performed using ultrasound.

[0025] Furthermore, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0026] Furthermore, the reference standard includes geniposide reference standard and / or glycyrrhizin reference standard.

[0027] Furthermore, the preparation method of the reference solution is as follows: weigh appropriate amounts of geniposide reference standard and / or glycyrrhizin reference standard, and add methanol to prepare geniposide reference standard solution and / or glycyrrhizin reference standard solution respectively.

[0028] Furthermore, the concentration of geniposide in the geniposide reference solution is 1 to 10 mg / ml, for example, about 4 mg / ml.

[0029] Furthermore, the concentration of glycyrrhizin in the glycyrrhizin reference solution is 0.5~2 mg / ml, for example, about 1 mg / ml.

[0030] Furthermore, the blank solution is methanol.

[0031] Furthermore, the preparation method of the decoction of the traditional Chinese medicine composition containing gypsum and gardenia includes: (A) weighing appropriate amounts of gypsum, gardenia, patchouli leaves, saposhnikovia root and licorice, crushing them into coarse particles, mixing them evenly, adding honey wine to moisten them, stir-frying them, cooling them, pulverizing them, and sieving them to obtain the first traditional Chinese medicine composition; and (B) weighing an appropriate amount of the first traditional Chinese medicine composition, adding a solvent, boiling it over high heat, then simmering it over low heat to obtain a concentrated liquid, filtering it while it is hot to obtain the traditional Chinese medicine compound decoction.

[0032] Furthermore, the concentration of this mead is 10-40%.

[0033] Furthermore, the ratio of honey to wine in the mead is approximately 1:1.

[0034] Furthermore, the humidification period is 1 to 4 hours.

[0035] Furthermore, the frying temperature is between 50°C and 80°C.

[0036] Furthermore, the stir-frying time is 30 to 90 minutes.

[0037] Furthermore, this windproof is a baked windproof.

[0038] Furthermore, this sieve is a No. 4 sieve.

[0039] Furthermore, the solvent is water.

[0040] Furthermore, the mass ratio of the prescription dose to the mass / volume of the solvent is 0.02~0.03, in g / mL.

[0041] Furthermore, the simmering time is 30-60 minutes.

[0042] Furthermore, the volume ratio of the concentrate to the solvent is 0.4 to 0.6.

[0043] Furthermore, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is (10~30):(1~10):(20~40):(100~200):(100~200).

[0044] Furthermore, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is (15~25):(2~6):(25~35):(140~180):(110~140).

[0045] Furthermore, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is approximately 20.65: approximately 4.13: approximately 28.91: approximately 165.20: approximately 123.90.

[0046] Furthermore, the preparation method of the negative sample solution includes weighing an appropriate amount of the decoction of the traditional Chinese medicine composition lacking gardenia or licorice, and the subsequent steps are the same as the preparation method of the test sample solution.

[0047] Furthermore, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (4~6):(4.5~5.5):(0.8~1.2).

[0048] Furthermore, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is approximately 5: approximately 5: approximately 1.

[0049] Furthermore, the thin-layer plate is a silicone G thin-layer plate.

[0050] Furthermore, the silicone G thin film is a Merck silicone G thin film, or a Qingdao Ocean silicone G thin film, or a Yinlong silicone G thin film.

[0051] Furthermore, the inspection conditions for the thin-layer chromatography identification analysis steps are as follows: remove the developed thin-layer plate, air dry it, spray it with sulfuric acid ethanol solution, heat it until the spots are clear, and examine it under sunlight.

[0052] Furthermore, the concentration of the sulfuric acid ethanol solution is 8% to 12%, for example, about 10%.

[0053] Furthermore, the heating temperature is approximately 105°C.

[0054] Furthermore, the sample volume of the test solution is 3 to 9 μl, for example, about 5 μl.

[0055] Furthermore, when the reference herb is Gardenia jasminoides, the amount of the reference herb solution applied is 1.5~2.5 μl, for example, about 2 μl.

[0056] Furthermore, when the reference herb is licorice, the amount of the reference herb solution applied is 1.5~2.5 μl, for example, about 2 μl.

[0057] Furthermore, when the reference standard is geniposide reference standard, the sample volume of the geniposide reference standard solution is 1.5~2.5 μl, for example, about 2 μl.

[0058] Furthermore, when the reference standard is glycyrrhizin, the sample volume of the glycyrrhizin solution is 3-9 μl, for example, about 5 μl.

[0059] Furthermore, the amount of the negative sample solution spotted was approximately 15 μl.

[0060] Furthermore, the amount of the blank solution spotted was approximately 10 μl.

[0061] Furthermore, the temperature for this thin-layer identification is 4~30℃.

[0062] Furthermore, the relative humidity for this thin-layer identification is 42% to 72%.

[0063] Furthermore, the criteria for thin-layer identification include one selected from the following: (1) Check whether the thin-layer plate shows characteristic spots of the same color as the reference medicinal material and reference standard at the corresponding positions under sunlight; if the thin-layer plate has such characteristic spots under sunlight, then the tested Chinese medicine composition contains gardenia and licorice; or (2) Check whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions of the reference medicinal material and the reference standard under sunlight, and whether the negative sample and / or blank solution interfere; if the thin-layer plate has the characteristic spots under sunlight and the negative sample and / or blank solution does not interfere, then the detected Chinese medicine composition contains gardenia and licorice.

[0064] According to another aspect of the present invention, the use of the above-described thin-layer identification method in identifying traditional Chinese medicine compositions containing gypsum and gardenia is provided.

[0065] According to another aspect of the present invention, a kit is provided for identifying a traditional Chinese medicine composition containing gypsum and gardenia, the kit comprising a thin-layer plate and a developing solvent, the developing solvent being ethyl acetate-acetone-formic acid, and the thin-layer plate being a silica gel G thin-layer plate.

[0066] Furthermore, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (2~10):(4~6):(0.5~2).

[0067] Furthermore, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (4~6):(4.5~5.5):(0.8~1.2).

[0068] Furthermore, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is approximately 5: approximately 5: approximately 1.

[0069] Furthermore, the silicone G thin film is a Merck silicone G thin film, or a Qingdao Ocean silicone G thin film, or a Yinlong silicone G thin film.

[0070] The beneficial effects of this invention are: The thin-layer chromatography identification method of the present invention provides a theoretical basis for the quality control and pharmacodynamic material basis research of traditional Chinese medicine compound prescriptions containing gypsum and gardenia by detecting the medicinal materials gardenia and licorice in traditional Chinese medicine compositions containing gypsum and gardenia. Attached Figure Description

[0071] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0072] Figure 1 The thin-layer identification chromatogram of ethyl acetate-acetone-formic acid-water (5:5:1:1) as the developing solvent in the thin-layer method (1). Wherein, 1 is 5 μl of XHKL2023112708-L; S is 2 μl of geniposide reference standard.

[0073] Figure 2 The thin-layer identification chromatogram of ethyl acetate-acetone-formic acid-water (5:5:1:1) as the developing solvent in the thin-layer method (2). Wherein, 1 is 5 μl of XHKL2023112708-L; S is 2 μl of geniposide reference standard.

[0074] Figure 3The thin-layer identification chromatogram of ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent in the thin-layer method (3) is shown. Among them, 1 is XHKL2023112708-L 5μl; S1 is geniposide reference standard 2μl; S2 is glycyrrhizin reference standard 5μl.

[0075] Figure 4 The thin-layer identification chromatogram using ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent in the thin-layer method (4) is shown below. In this chromatogram, 1 is 5 μl of XHKL2023112708-L; 2 is 2 μl of Gardenia reference material solution; 3 is 2 μl of Glycyrrhiza reference material solution; 4 is 15 μl of Gardenia negative standard solution; 5 is 15 μl of Glycyrrhiza negative standard solution; S1 is 2 μl of geniposide reference solution; S2 is 5 μl of glycyrrhizin reference solution; and 6 is 10 μl of blank solution (methanol).

[0076] Figure 5 This is a specific thin-layer chromatography chromatogram for the reference sample of the traditional Chinese medicine composition containing Poria cocos and Cinnamomum cassia of the present invention. Wherein, 1-3 are 5 μl of XHKL2023120507 / 08 / 09-L; 4 is 2 μl of Gardenia jasminoides reference material solution; 5 is 2 μl of Glycyrrhiza uralensis reference material solution; 6 is 15 μl of Gardenia jasminoides negative control solution; 7 is 15 μl of Glycyrrhiza uralensis negative control solution; S1 is 2 μl of geniposide reference solution; S2 is 5 μl of glycyrrhizin reference solution; 8 is 10 μl of blank solution (methanol). Detailed Implementation

[0077] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0078] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0079] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0080] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0081] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0082] As described in the background section, the prior art does not disclose a thin-layer chromatography method for identifying the medicinal materials cinnamon twig, atractylodes macrocephala, and licorice in a traditional Chinese medicine composition containing poria cocos and cinnamon twig, as claimed in this invention, nor its application. To address the above problem, this invention provides a thin-layer chromatography method for identifying the medicinal materials gardenia and licorice in a traditional Chinese medicine composition containing gypsum and gardenia, the method comprising the following steps: (1) Pre-treating the decoction of a traditional Chinese medicine composition containing gypsum and gardenia to obtain a test solution, pre-treating the reference medicinal material to obtain a reference medicinal material solution, pre-treating the reference standard to obtain a reference standard solution, and / or pre-treating the decoction of a traditional Chinese medicine composition lacking gardenia or licorice to obtain a negative sample solution; and (2) Perform thin-layer chromatography identification analysis on the test solution, the reference medicinal material solution, the reference standard solution, the blank solution and / or the negative sample solution; In the thin-layer chromatography identification analysis step, a developing solvent is used for development, wherein the developing solvent is ethyl acetate-acetone-formic acid, and the volume ratio of ethyl acetate, acetone and formic acid in the developing solvent is (2~10):(4~6):(0.5~2).

[0083] In this invention, when boiling range, volume ratio, ratio, volume, number of times, time, mass, voltage, mass ratio, sample quantity, temperature, relative humidity, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “2~10” is disclosed, the described range should be interpreted as including ranges “2~10”, “2~8”, “2~6”, “2~4”, “4~10”, “4~8”, “4~6”, “6~10”, “6~8”, “8~10”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0084] In a preferred embodiment, the traditional Chinese medicine composition comprising gypsum and gardenia includes gypsum, gardenia, patchouli leaves, saposhnikovia root, and licorice.

[0085] In a preferred embodiment, the method for preparing the test solution includes: (a) taking an appropriate amount of a decoction of a traditional Chinese medicine composition containing gypsum and gardenia, concentrating it under reduced pressure to dryness to obtain a first residue; (b) adding a first solvent to the residue for extraction, filtering, evaporating the filtrate to dryness to obtain a second residue; and (c) adding a second solvent to the second residue to dissolve it to obtain the test solution.

[0086] In a preferred embodiment, the first solvent is ethyl acetate.

[0087] In a preferred embodiment, the mass / volume (g / ml) ratio of the decoction containing gypsum and gardenia to the first solvent is 0.5 to 2, for example, about 1.

[0088] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0089] In a preferred embodiment, the decoction of the traditional Chinese medicine composition containing gypsum and gardenia has a mass of 80-120g, for example, about 100g.

[0090] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0091] In a preferred embodiment, the volume of the first solvent is 80-120 ml, for example, about 100 ml.

[0092] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0093] In a preferred embodiment, the extraction is ultrasonic extraction.

[0094] In a preferred embodiment, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0095] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0096] In a preferred embodiment, the second solvent is methanol.

[0097] In a preferred embodiment, the mass / volume (g / ml) ratio of the decoction containing gypsum and gardenia to the second solvent is 80 to 120, for example, about 100.

[0098] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0099] In a preferred embodiment, the volume of the second solvent is 0.8 to 1.2 ml, for example, about 1 ml.

[0100] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0101] In a preferred embodiment, the reference medicinal materials include gardenia reference medicinal materials and / or licorice reference medicinal materials.

[0102] In a preferred embodiment, the method for preparing the reference herb solution includes: weighing an appropriate amount of gardenia reference herb and / or licorice reference herb, adding a first solvent for extraction, filtering, evaporating the filtrate to dryness, and obtaining a residue; adding a second solvent to the residue to dissolve it, thereby obtaining the reference herb solution.

[0103] In a preferred embodiment, the first solvent is ethyl acetate.

[0104] In a preferred embodiment, the second solvent is methanol.

[0105] In a preferred embodiment, the mass / volume (g / ml) ratio of the gardenia reference material or licorice reference material to the first solvent is 0.01 to 0.1, for example, about 0.02.

[0106] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.02" includes ±5% of 0.02, or from 0.019 to 0.021.

[0107] In a preferred embodiment, the mass / volume (g / ml) ratio of the gardenia reference material or licorice reference material to the second solvent is 0.8 to 1.2, for example, about 1.

[0108] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0109] In a preferred embodiment, the mass of the gardenia reference material is 0.8 to 1.2 g, for example, about 1 g.

[0110] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0111] In a preferred embodiment, the licorice reference material has a mass of 0.8 to 1.2 g, for example, about 1 g.

[0112] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0113] In a preferred embodiment, the extraction is ultrasonic extraction.

[0114] In a preferred embodiment, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0115] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0116] In a preferred embodiment, the reference standard includes geniposide reference standard and / or glycyrrhizin reference standard.

[0117] In a preferred embodiment, the preparation method of the reference solution is as follows: weigh appropriate amounts of geniposide reference standard and / or glycyrrhizin reference standard, and add methanol to prepare geniposide reference standard solution and / or glycyrrhizin reference standard solution respectively.

[0118] In a preferred embodiment, the concentration of geniposide in the geniposide reference solution is 1 to 10 mg / ml, for example, about 4 mg / ml.

[0119] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0120] In a preferred embodiment, the concentration of glycyrrhizin in the glycyrrhizin reference solution is 0.5 to 2 mg / ml, for example, about 1 mg / ml.

[0121] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0122] In a preferred embodiment, the blank solution is methanol.

[0123] In a preferred embodiment, the method for preparing the decoction of the traditional Chinese medicine composition containing gypsum and gardenia includes: (A) weighing appropriate amounts of gypsum, gardenia, patchouli leaves, saposhnikovia root and licorice, crushing them into coarse particles, mixing them evenly, adding honey wine to moisten them, stir-frying them, cooling them, pulverizing them, and sieving them to obtain the first traditional Chinese medicine composition; and (B) weighing an appropriate amount of the first traditional Chinese medicine composition, adding a solvent, boiling it over high heat, then simmering it over low heat to obtain a concentrated liquid, filtering it while it is hot to obtain the traditional Chinese medicine compound decoction.

[0124] In a preferred embodiment, the concentration of the mead is 10-40%.

[0125] In a preferred embodiment, the ratio of honey to wine in the mead is approximately 1:approximately 1.

[0126] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0127] In a preferred embodiment, the soaking time is 1 to 4 hours.

[0128] In a preferred embodiment, the frying temperature is 50°C to 80°C.

[0129] In a preferred embodiment, the frying time is 30 to 90 minutes.

[0130] In a preferred embodiment, the windproof is a baked windproof.

[0131] In a preferred embodiment, the sieve is a No. 4 sieve.

[0132] In a preferred embodiment, the solvent is water.

[0133] In a preferred embodiment, the mass ratio of the prescription dose to the mass / volume ratio of the solvent is 0.02 to 0.03, in g / mL.

[0134] In a preferred embodiment, the simmering time is 30-60 minutes.

[0135] In a preferred embodiment, the volume ratio of the concentrate to the solvent is 0.4 to 0.6.

[0136] In a preferred embodiment, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is (10~30):(1~10):(20~40):(100~200):(100~200).

[0137] In a preferred embodiment, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is (15~25):(2~6):(25~35):(140~180):(110~140).

[0138] In a preferred embodiment, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is about 20.65: about 4.13: about 28.91: about 165.20: about 123.90.

[0139] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20.65" includes ±5% of 20.65, or from 19.6175 to 21.6825; "about 4.13" includes ±5% of 4.13, or from 3.9235 to 4.3365; "about 28.91" includes ±5% of 28.91, or from 27.4645 to 30.3555; "about 165.20" includes ±5% of 165.20, or from 156.94 to 173.46; "about 123.90" includes ±5% of 123.90, or from 117.705 to 130.095.

[0140] In a preferred embodiment, the method for preparing the negative sample solution includes weighing appropriate amounts of the decoction of the traditional Chinese medicine composition lacking gardenia or licorice, and the subsequent steps are the same as those for preparing the test sample solution.

[0141] In a preferred embodiment, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (4~6):(4.5~5.5):(0.8~1.2).

[0142] In a preferred embodiment, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is about 5: about 5: about 1.

[0143] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25; "about 1" includes 1 ± 5%, or from 0.95 to 1.05.

[0144] In a preferred embodiment, the thin film is a silicone G thin film.

[0145] In a preferred embodiment, the silicone G thin film is a Merck silicone G thin film, or a Qingdao Ocean silicone G thin film, or a Yinlong silicone G thin film.

[0146] In a preferred embodiment, the inspection conditions for the thin-layer chromatography identification analysis step are as follows: the developed thin-layer plate is removed, dried, sprayed with sulfuric acid ethanol solution, heated until the spots are clear, and then inspected under sunlight.

[0147] In a preferred embodiment, the concentration of the sulfuric acid ethanol solution is 8% to 12%, for example, about 10%.

[0148] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10%" includes 10% ± 5%, or from 9.5% to 10.5%.

[0149] In a preferred embodiment, the heating temperature is approximately 105°C.

[0150] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 105" includes ±5% of 105, or from 99.75 to 110.25.

[0151] In a preferred embodiment, the sample volume of the test solution is 3 to 9 μl, for example, about 5 μl.

[0152] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0153] In a preferred embodiment, when the reference herb is gardenia, the amount of the reference herb solution is 1.5~2.5 μl, for example, about 2 μl.

[0154] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0155] In a preferred embodiment, when the reference herb is licorice, the amount of the reference herb solution applied is 1.5 to 2.5 μl, for example, about 2 μl.

[0156] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0157] In a preferred embodiment, when the reference standard is geniposide reference standard, the amount of the geniposide reference standard solution spotted is 1.5~2.5 μl, for example, about 2 μl.

[0158] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0159] In a preferred embodiment, when the reference standard is glycyrrhizin, the amount of the solution of the glycyrrhizin reference standard is 3 to 9 μl, for example, about 5 μl.

[0160] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0161] In a preferred embodiment, the amount of the negative sample solution spotted is about 15 μl.

[0162] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0163] In a preferred embodiment, the amount of blank solution spotted is about 10 μl.

[0164] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0165] In a preferred embodiment, the temperature for thin-layer identification is 4~30°C.

[0166] In a preferred embodiment, the relative humidity for thin-layer identification is 42% to 72%.

[0167] In a preferred embodiment, the criteria for thin-layer identification include one selected from the following: (1) Check whether the thin-layer plate shows characteristic spots of the same color as the reference medicinal material and reference standard at the corresponding positions under sunlight; if the thin-layer plate has such characteristic spots under sunlight, then the tested Chinese medicine composition contains gardenia and licorice; or (2) Check whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions of the reference medicinal material and the reference standard under sunlight, and whether the negative sample and / or blank solution interfere; if the thin-layer plate has the characteristic spots under sunlight and the negative sample and / or blank solution does not interfere, then the detected Chinese medicine composition contains gardenia and licorice.

[0168] According to another aspect of the present invention, the use of the above-described thin-layer identification method in identifying traditional Chinese medicine compositions containing gypsum and gardenia is provided.

[0169] According to another aspect of the present invention, a kit is provided for identifying a traditional Chinese medicine composition containing gypsum and gardenia, the kit comprising a thin-layer plate and a developing solvent, the developing solvent being ethyl acetate-acetone-formic acid, and the thin-layer plate being a silica gel G thin-layer plate.

[0170] In a preferred embodiment, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (2~10):(4~6):(0.5~2).

[0171] In a preferred embodiment, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (4~6):(4.5~5.5):(0.8~1.2).

[0172] In a preferred embodiment, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is about 5: about 5: about 1.

[0173] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25; "about 1" includes 1 ± 5%, or from 0.95 to 1.05.

[0174] In a preferred embodiment, the silicone G thin film is a Merck silicone G thin film, or a Qingdao Ocean silicone G thin film, or a Yinlong silicone G thin film.

[0175] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0176] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0177] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0178] Example 1. Experimental instruments, reagents and reagents 1.1 Experimental Apparatus Electronic balance (1%) Model: XY1000-2C, Manufacturer: Changzhou Lucky Electronic Equipment Co., Ltd. Rotary evaporator model: R-1001LN / VN, manufacturer: Zhengzhou Changcheng Industry & Trade Co., Ltd. CNC ultrasonic cleaner model: KQ-500DB, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd. Water bath model: DZKW-S-6, manufacturer: Beijing Yongguangming Automatic Thin-Layer Sampling Instrument Model: ATS4 Manufacturer: CAMAG Constant Temperature Drying Oven Model: GZX-9140MBE, Manufacturer: Shanghai Boxun Experimental Equipment Co., Ltd. Four-in-one UV analyzer, model: WFH-203C, manufacturer: Shanghai Yixin Scientific Instruments Co., Ltd. Pharmaceutical Refrigerated Storage Cabinet Model: FL-1200D Manufacturer: Boxing County Dingchi Electric Appliance Co., Ltd. Silicone G precast panel (batch number: HX17906326, model: 100×200mm, manufacturer: Merck, Germany); Silicone G precast panel (batch number: 20221020, model: 100×200mm, manufacturer: Yinlong New Materials Co., Ltd.); Silicone G precast panels (batch numbers: 20231201, 20230902, model: 100×200mm, manufacturer: Qingdao Haiyang Chemical Co., Ltd.); 1.2 Reagents Ethanol, ethyl acetate, acetone, n-butanol, chloroform, glacial acetic acid, vanillin, and phosphomolybdic acid were of analytical grade; formic acid and methanol were of chromatographic grade; sulfuric acid was of analytical grade; and water was purified water.

[0179] 1.3 Drug Testing Gardenin reference standard (batch number: 110744-202320, content: 98.1%, China National Institutes for Food and Drug Control), glycyrrhizin reference standard (batch number: 111610-202209, content: 95.2%, China National Institutes for Food and Drug Control), gardenia reference medicinal material (batch number: 120986-202111, China National Institutes for Food and Drug Control), glycyrrhiza reference medicinal material (batch number: 120904-202021, China National Institutes for Food and Drug Control). (Institute of Testing and Inspection), XHKL reference sample decoction (XHKL2023112708-L, XHKL2023120507-L, XHKL2023120508-L, XHKL2023120509-L), XHKL reference sample Gardenia negative decoction (batch number: XHKL2023120301-L), XHKL reference sample Glycyrrhiza negative decoction (batch number: XHKL2023112701-L); Gypsum (batch number: ZB08923031601; origin: Fengyang County, Chuzhou City, Anhui Province); Gardenia (batch number: ZC07123032002; origin: Fuding City, Ningde City, Fujian Province); Patchouli leaf (batch number: ZB18223072601; origin: Bobai County, Yulin City, Guangxi Zhuang Autonomous Region); Saposhnikovia divaricata (batch number: ZB03923031708; origin: Zhalute Banner, Tongliao City, Inner Mongolia); Licorice (batch number: ZA00423081408; origin: Hangjin Banner, Ordos City, Inner Mongolia Autonomous Region). The preparation method of this traditional Chinese medicine compound decoction includes the following steps: Take appropriate amounts of each medicinal herb slices, crush them into coarse particles, weigh out 20.65g of gypsum, 4.13g of gardenia, 28.91g of patchouli leaves, 165.20g of saposhnikovia root (baked), and 123.90g of licorice root, mix them well, add honey wine to moisten, stir-fry, let cool, pulverize, and pass through a No. 4 sieve; take one dose of the prescription, add water, cover, heat over high heat to boiling, then simmer over low heat to obtain a concentrated liquid, filter while hot to obtain the traditional Chinese medicine compound decoction.

[0180] 2. Development of thin-layer methods 2.1 Development of thin-layer methods (1) Take 100g of the decoction of this product, concentrate it to dryness under reduced pressure, add 10ml of 50% methanol, sonicate for 40 minutes, filter, and take the filtrate as the test solution; take geniposide reference standard, add ethanol to prepare a solution containing 4mg per ml, as the reference solution; perform thin-layer chromatography (General Chapter 0502 of Chinese Pharmacopoeia 2020), take 5μl of the above test solution and 2μl of the reference solution, respectively, and spot them on the same silica gel G plate, use ethyl acetate-acetone-formic acid-water (5:5:1:1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight.

[0181] Results Analysis: Figure 1 As can be seen, no spots of the same color as the reference substance appeared at the corresponding positions in the chromatogram of the test sample, and the spots were not clearly separated. Therefore, this method is not suitable for the thin-layer chromatography identification of gardenia. Further optimization of the method is needed.

[0182] 2.2 Development of Thin-Layer Methods (2) Take 100g of the decoction of this product, concentrate it to dryness under reduced pressure, add 100ml of ethyl acetate to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to prepare the test solution; then take geniposide reference standard and add methanol to prepare a solution containing 4mg per ml to prepare the reference solution; perform thin-layer chromatography (General Chapter 0502 of Chinese Pharmacopoeia 2020), take 5μl of the above test solution and 2μL of the reference solution respectively, spot them on the same silica gel G plate, use ethyl acetate-acetone-formic acid-water (5:5:1:1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight.

[0183] Results Analysis: Figure 2 It can be seen that in the chromatogram of the test sample, no spots of the same color as the reference standard appear at the corresponding positions, and the spots are not clearly separated. Therefore, this method is not suitable for the thin-layer identification of gardenia. Further optimization of the method is needed. The yellow spots are suspected to be glycyrrhizin; therefore, a glycyrrhizin reference standard can be added to the next method.

[0184] 2.3 Development of thin-layer methods (3) Take 100g of the decoction of this product, concentrate it to dryness under reduced pressure, add 100ml of ethyl acetate to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0185] Prepare a reference solution containing 4 mg of geniposide per ml by dissolving it in methanol. Prepare a reference solution containing 1 mg of glycyrrhizin per ml by dissolving it in methanol. Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020). Apply 5 μl of the above test solutions, 2 μl of the geniposide reference solution, and 5 μl of the glycyrrhizin reference solution to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clear. Examine the plate under sunlight.

[0186] Results Analysis: Figure 3 It can be seen that by adjusting the developing solvent and reducing its polarity, spots of the same color as the reference substance appear at the corresponding positions in the chromatogram of the test sample, and the spots are clearly separated. In the next method, negative samples of licorice and gardenia can be added to confirm whether the negative sample of this method is interfered with.

[0187] 2.4 Development of thin-layer methods (4) Preparation of the reference sample test solution: Take 100g of the decoction of this product, concentrate it to dryness under reduced pressure, add 100mL of ethyl acetate to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution.

[0188] Preparation of Gardenia negative solution as reference sample: Take 100g of Gardenia negative decoction as reference sample, concentrate to dryness under reduced pressure, add 100ml of ethyl acetate to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and obtain Gardenia negative solution.

[0189] Preparation of licorice negative solution for reference sample: Take 100g of licorice negative decoction for reference sample, concentrate it to dryness under reduced pressure, add 100ml of ethyl acetate to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and obtain the licorice negative solution.

[0190] Preparation of Gardenia reference material solution: Take 1g of Gardenia reference material, add 50mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to obtain Gardenia reference material solution.

[0191] Preparation of licorice reference material solution: Take 1g of licorice reference material, add 50ml of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and obtain the licorice reference material solution.

[0192] Gardenoside reference solution: Take an appropriate amount of gardenoside reference standard, add methanol to prepare a reference solution containing 4 mg per 1 mL.

[0193] Glycyrrhizin reference solution: Take an appropriate amount of glycyrrhizin reference standard, add methanol to prepare a reference solution containing 1 mg per 1 ml.

[0194] Perform the thin-layer chromatography test (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition). Apply 5 μl of the test solution, 2 μl of the gardenia reference solution, 2 μl of the licorice reference solution, 15 μl of the gardenia negative standard solution, 15 μl of the licorice negative standard solution, 2 μl of the geniposide reference solution, 5 μl of the glycyrrhizin reference solution, and 10 μl of the blank solution (methanol) to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clear. Examine the plate under sunlight.

[0195] Results Analysis: Figure 4 It can be seen that in the chromatogram of the test sample, spots of the same color as the reference sample appear at the corresponding positions, and the spots are clearly separated with no interference from the negative. Therefore, this method is suitable for the thin-layer identification of gardenia and licorice in this product.

[0196] 2.5 Investigation of the composition of the developing solvent According to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020), 5 μl of the test solution, 2 μl of gardenia reference material solution, 2 μl of licorice reference material solution, 15 μl of gardenia negative standard solution, 15 μl of licorice negative standard solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, and 10 μl of blank solution (methanol) were spotted onto the same silica gel G thin-layer plate. The developing solvents were ethyl acetate-acetone-formic acid (5:5:1), n-butanol-chloroform-methanol-water (2:2:0.5:0.1), and n-butanol-glacial acetic acid-water (7:1:1), respectively. After development, the plate was removed, dried, and then sprayed with 10% sulfuric acid ethanol solution. The plate was heated at 105℃ until the spots were clear and examined under sunlight.

[0197] The results showed that, under the three developing solvents, the ethyl acetate-acetone-formic acid (5:5:1) solvent produced the most suitable Rf value and produced spots of the same color at the corresponding positions of the reference sample and the control medicinal material and the control standard at the corresponding positions in the chromatogram of the reference sample. The chromatographic spots were well separated and there was no interference from the negative. The other two developing solvents had poor chromatographic spot separation. Therefore, ethyl acetate-acetone-formic acid (5:5:1) was selected as the developing solvent.

[0198] 2.6 Investigation of the developing solvent ratio Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 5 μl of the test solution, 2 μl of the gardenia reference solution, 2 μl of the licorice reference solution, 15 μl of the gardenia negative standard solution, 15 μl of the licorice negative standard solution, 2 μl of the geniposide reference solution, 5 μl of the glycyrrhizin reference solution, and 10 μl of the blank solution (methanol) to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-formic acid (10:5:1), ethyl acetate-acetone-formic acid (5:5:1), and ethyl acetate-acetone-formic acid (2:5:1) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol, and heat at 105℃ until the spots are clear. Examine the plate under sunlight.

[0199] The results showed that, under the three ratios of developing solvent, the Rf value of the ethyl acetate-acetone-formic acid (5:5:1) developing solvent was moderate at the corresponding positions of the reference medicinal material and the reference standard, and the spots showed the same color. The chromatographic spots were well separated and there was no interference from the negative. The other two developing solvents had poor chromatographic spot separation. Therefore, ethyl acetate-acetone-formic acid (5:5:1) was selected as the developing solvent.

[0200] 2.7 Investigation of the colorimetric reagent According to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020), 5 μl of the test solution, 2 μl of gardenia reference material solution, 2 μl of licorice reference material solution, 15 μl of gardenia negative standard solution, 15 μl of licorice negative standard solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, and 10 μl of blank solution (methanol) were spotted onto the same silica gel G thin-layer plate. The plates were developed using ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. The plates were then sprayed with 10% sulfuric acid ethanol solution, 5% phosphomolybdic acid solution, and 2% vanillin sulfuric acid-ethanol (4:1) mixed solution. The plates were heated at 105℃ until the spots were clearly visible and examined under sunlight.

[0201] The results showed that no spots were observed in the licorice reference material and licorice reference standard under 5% phosphomolybdic acid solution. However, spots of the same color as the reference material and the reference standard were observed under 2% vanillin-sulfuric acid-ethanol (4:1) mixed solution and 10% sulfuric acid-ethanol solution. The chromatographic spots were well separated and there was no interference from negative samples. According to the pharmacopoeia, 10% sulfuric acid-ethanol solution is commonly used as the colorimetric reagent for gardenia. Therefore, 10% sulfuric acid-ethanol solution was selected as the colorimetric reagent.

[0202] 2.8 Examination of Inspection Methods The thin-layer chromatography method (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) was followed. 5 μl of the test solution, 2 μl of gardenia reference material solution, 2 μl of licorice reference material solution, 15 μl of gardenia negative control solution, 15 μl of licorice negative control solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, and 10 μl of blank solution (methanol) were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. The plate was removed, air-dried, and examined under sunlight, 254 nm UV lamp, and 365 nm UV lamp. Then, 10% sulfuric acid ethanol solution was sprayed onto the plate, and the plate was heated at 105℃ until the spots were clearly visible. The plates were then examined under sunlight, 254 nm UV lamp, and 365 nm UV lamp.

[0203] The results showed that, without the color-developing agent, under the three viewing methods, the chromatogram of the test sample did not show fluorescent spots of the same color at the corresponding positions as the reference medicinal material and the reference standard, and the chromatographic resolution was poor. After the color-developing agent was sprayed, at 254 nm and 365 nm, the chromatogram of the test sample did not show fluorescent spots of the same color at the corresponding positions as the reference medicinal material and the reference standard, and the chromatographic resolution was poor. However, under sunlight, the chromatogram of the test sample showed spots of the same color at the corresponding positions as the reference medicinal material and the reference standard, the chromatographic spot resolution was good, and there was no interference from negative samples. Therefore, the examination under sunlight was chosen.

[0204] 2.9 Investigation of sample quantity Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) using 2 μl, 5 μl, and 10 μl of the test solution; 1 μl, 2 μl, and 3 μl of the gardenia reference material solution; 1 μl, 2 μl, and 3 μl of the licorice reference material solution; 1 μl, 2 μl, and 3 μl of the geniposide reference solution; 2 μl, 5 μl, and 10 μl of the glycyrrhizin reference solution; 15 μl of the gardenia negative control solution, 15 μl of the licorice negative control solution, and 10 μl of the blank solution. Develop the plate with ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under sunlight.

[0205] The results showed that the chromatogram of the test sample showed spots of the same color at the corresponding positions as the reference medicinal material and the reference standard. The chromatographic spots were well separated, and there was no negative interference. Based on the separation and clarity of the spots, the spotting volume of the test sample solution was determined to be 5 μl, the spotting volume of gardenia reference medicinal material was 2 μl, the spotting volume of licorice reference medicinal material was 2 μl, the spotting volume of geniposide reference standard solution was 2 μl, and the spotting volume of glycyrrhizin reference standard solution was 5 μl.

[0206] 2.10 Method Determination Based on the type and ratio of developing solvent, different colorimetric reagents, inspection methods, and different sample amounts, the following preliminary method for thin-layer chromatography identification of gardenia and licorice was determined: Take 100g of the decoction of this product, concentrate it to dryness under reduced pressure, add 100ml of ethyl acetate to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to prepare the test solution. Take 1g of gardenia reference material, add 50ml of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to prepare the gardenia reference material solution. Take 1g of licorice reference material, add 50mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to prepare the licorice reference material solution. Take an appropriate amount of geniposide reference standard, add methanol to prepare a reference solution containing 4mg per ml; then take an appropriate amount of glycyrrhizin reference standard, add methanol to prepare a reference solution containing 1mg per ml. Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 5 μl of the test solution, 2 μl of the reference herb solution, 2 μl of the geniposide reference solution, 5 μl of the glycyrrhizin reference solution, and 10 μl of the blank solution (methanol) to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. Remove the plate, air dry, and then spray with 10% sulfuric acid in ethanol. Heat at 105℃ until the spots are clear and examine under sunlight. In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatograms of the corresponding reference herb and reference standard.

[0207] 3. Methodological Validation 3.1 Specificity Examination According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), take 2 μl of licorice reference material solution, 2 μl of gardenia reference material solution, 15 μl of gardenia negative standard solution, 15 μl of licorice negative standard solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, 5 μl of test solution, and 10 μl of blank solution, and spot them on the same silica gel G thin-layer plate. Develop with ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. After development, remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine it under sunlight.

[0208] Results Analysis: Figure 5 It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as the reference medicinal material and the reference standard. The chromatographic spots are well separated, and there is no interference from negative samples. Therefore, the specificity is good.

[0209] 3.2 Durability Test 3.2.1 Investigation at different temperatures Experimental conditions and control methods: Temperature range: 10–30℃; relative humidity: 65%; prepare the solution by mixing 34 ml of sulfuric acid with 100 ml of water. Add the solution to one side of a 100×100 mm double-chamber chromatography tank (add 12 ml of the prepared sulfuric acid solution). Place the spotted thin-layer plate into the other side of the tank, seal, and let stand for 20 minutes. Add developing solvent, develop, remove, and air dry. Repeat the process, adding developing solvent again, developing, removing, and air drying. Spray with 10% sulfuric acid ethanol solution and heat at 105℃ until the spots are clearly visible. Examine under sunlight.

[0210] Low-temperature (4–10℃) investigation: Experimental conditions and control methods: The temperature was controlled within the range of 4–10℃ using a refrigerator temperature setting of 4℃. The relative humidity in the developing tank was controlled at 65%. 34 ml of sulfuric acid was mixed with 100 ml of water to prepare the solution. This solution was added to one side of a dual-tank chromatography vessel (100×100 mm tank, with 12 ml of the prepared sulfuric acid solution added). The spotted thin-layer plate was placed in the other side tank, sealed, and left to stand for 20 minutes. Developing solvent was added, and the plate was developed. It was then removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plate was then examined under sunlight.

[0211] According to the thin-layer chromatography method in the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0502, 2 μl of licorice reference material solution, 2 μl of gardenia reference material solution, 15 μl of gardenia negative standard solution, 15 μl of licorice negative standard solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, 5 μl of test solution, and 10 μl of blank solution were spotted onto the same silica gel G thin-layer plate. The plates were developed at room temperature and low temperature (6.5℃) using the developing solvent determined under the section on developing solvent testing. After development, the plates were removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under sunlight.

[0212] The results showed that, under normal and low temperature conditions, the test sample chromatogram showed spots of the same color at the corresponding positions as the reference medicinal material and reference standard chromatograms. The chromatographic spot separation effect was good, and there was no negative interference. The experimental results showed that temperature had no significant effect on thin-layer identification, and the thin-layer identification method had good temperature resistance.

[0213] 3.2.2 Investigation of different humidity levels High humidity (72% relative humidity) experimental condition control method: This experiment investigated the effect of 72% relative humidity on thin-layer chromatography identification. 27.5 ml of sulfuric acid was mixed with 100 ml of water and added to one side of a dual-chamber chromatography tank (100×100 mm tank, with 12 ml of the prepared sulfuric acid solution). The spotted thin-layer plate was placed in the other side of the tank, sealed, and left for 20 minutes. Developing solvent was added, the plate was developed, removed, and dried. A specific colorimetric reagent was sprayed on top, and the plate was heated at 105℃ until the spots were clearly visible. The plate was then examined using a specific inspection method.

[0214] Low humidity (42% relative humidity) experimental condition control method: This experiment investigated the effect of 42% relative humidity on thin-layer chromatography identification. 57 ml of sulfuric acid was mixed with 100 ml of water to prepare the solution. This solution was added to one side of a dual-chamber chromatography tank (100×100 mm tank, with 12 ml of the prepared sulfuric acid solution added). The spotted thin-layer plate was placed in the other side of the tank, sealed, and left for 20 minutes. Developing solvent was added, the plate was developed, removed, and dried. A specific colorimetric reagent was sprayed onto the plate, and it was heated at 105℃ until the spots were clearly visible. The plate was then examined using a specific inspection method.

[0215] According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), 2 μl of licorice reference material solution, 2 μl of gardenia reference material solution, 15 μl of gardenia negative standard solution, 15 μl of licorice negative standard solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, 5 μl of test solution, and 10 μl of blank solution were spotted onto the same silica gel G thin-layer plate. The plates were developed with ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent at 42% and 72% humidity, respectively. The plates were removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under sunlight.

[0216] The results showed that under both high and low humidity conditions, the test sample chromatogram showed spots of the same color at the corresponding positions as the reference medicinal material and reference standard chromatograms. The chromatographic spot separation effect was good, and there was no interference from negative samples. The experimental results indicate that humidity has no significant effect on thin-layer identification, and this thin-layer identification method has good durability against humidity.

[0217] 3.2.3 Durability Test of Thin-Layer Laminates from Different Manufacturers According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), 2 μl of licorice reference material solution, 2 μl of gardenia reference material solution, 15 μl of gardenia negative control solution, 15 μl of licorice negative control solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, 5 μl of test solution, and 10 μl of blank solution were spotted onto the same silica gel G thin-layer plate from different manufacturers (Merck silica gel G thin-layer plate, Qingdao Ocean silica gel G thin-layer plate, and Yinlong silica gel G thin-layer plate). The plates were developed with ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. After development, the plates were removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under sunlight.

[0218] The results showed that when using thin-layer silica gel plates from different manufacturers, the test sample chromatogram showed spots of the same color at the corresponding positions as the reference medicinal material and reference standard chromatograms. The chromatographic spot separation effect was good, and there was no negative interference. The experimental results indicate that thin-layer silica gel plates from different manufacturers have no significant impact on thin-layer identification, and this thin-layer identification method has good durability with thin-layer silica gel plates from different manufacturers.

[0219] 3.2.4 Durability assessment for different personnel According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), three personnel, A, B, and C, prepared the test solution respectively. They applied 2 μl of licorice reference material solution, 2 μl of gardenia reference material solution, 15 μl of gardenia negative standard solution, 15 μl of licorice negative standard solution, 2 μl of geniposide reference solution, 5 μl of glycyrrhizin reference solution, 5 μl of test solution, and 10 μl of blank solution to the same silica gel G thin-layer plate. The plate was developed with ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. The plates were then removed, dried, sprayed with 10% sulfuric acid ethanol solution, and examined under sunlight.

[0220] The results showed that, under different experimental operations by different personnel, the chromatograms of the test sample showed spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard. The chromatographic spot separation effect was good, and there was no negative interference. The experimental results indicate that different experimental personnel have no significant impact on thin-layer identification, and the robustness of this thin-layer identification method is good.

[0221] 4. Verification Summary In summary, this method demonstrates good specificity and robustness, and can be used for thin-layer chromatography identification of the herbal flavor of gardenia and licorice in reference samples of traditional Chinese medicine compositions containing gypsum and gardenia. The method is as follows: Take 100g of the decoction of this product, concentrate it to dryness under reduced pressure, add 100mL of ethyl acetate to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution. Take 1g of gardenia reference material, add 50mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the gardenia reference material solution. Take 1g of licorice reference material, add 50mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the licorice reference material solution. Take an appropriate amount of geniposide reference standard, add methanol to prepare a reference solution containing 4mg per 1mL; then take an appropriate amount of glycyrrhizin reference standard, add methanol to prepare a reference solution containing 1mg per 1mL. According to the thin-layer chromatography method (General Rule 0502), 5 μl of the test solution, 2 μl of the reference herb solution, 2 μl of the geniposide reference solution, 5 μl of the glycyrrhizin reference solution, and 10 μl of the blank solution (methanol) were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-acetone-formic acid (5:5:1) as the developing solvent. After development, the plate was removed, air-dried, and then sprayed with 10% sulfuric acid ethanol solution. The plate was heated at 105℃ until the spots were clear and examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatograms of the corresponding reference herb and reference standards. The results are shown in Table 1.

[0222] Table 1 Summary of the validation results of the thin-layer chromatography method for identifying Gardenia and Licorice

[0223] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A thin-layer chromatography method for identifying the medicinal materials gardenia and licorice in a traditional Chinese medicine composition containing gypsum and gardenia, characterized in that, The thin-layer identification method includes the following steps: (1) The decoction of the traditional Chinese medicine composition containing gypsum and gardenia is pretreated to obtain the test solution, the reference medicinal material is pretreated to obtain the reference medicinal material solution, the reference standard is pretreated to obtain the reference standard solution, and / or the decoction of the traditional Chinese medicine composition lacking gardenia or licorice is pretreated to obtain the negative sample solution. as well as (2) Thin-layer chromatography identification analysis was performed on the test solution, the reference medicinal material solution, the reference standard solution, the blank solution and / or the negative sample solution; In the thin-layer chromatography identification analysis step, a developing solvent is used for development, wherein the developing solvent is ethyl acetate-acetone-formic acid, and the volume ratio of ethyl acetate, acetone and formic acid in the developing solvent is (2~10):(4~6):(0.5~2).

2. The thin-layer identification method according to claim 1, characterized in that, The traditional Chinese medicine composition containing gypsum and gardenia includes gypsum, gardenia, patchouli leaf, saposhnikovia root, and licorice. Preferably, the method for preparing the test solution includes: (a) taking an appropriate amount of a decoction of a traditional Chinese medicine composition containing gypsum and gardenia, concentrating it under reduced pressure to dryness to obtain a first residue; (b) adding a first solvent to the residue for extraction, filtering, evaporating the filtrate to dryness to obtain a second residue; and (c) adding a second solvent to the second residue to dissolve it to obtain the test solution. More preferably, the first solvent is ethyl acetate; Preferably, the mass / volume (g / ml) ratio of the decoction containing gypsum and gardenia to the first solvent is 0.5 to 2, for example, about 1; Preferably, the decoction of the traditional Chinese medicine composition containing gypsum and gardenia has a mass of 80-120 g, for example, about 100 g; Preferably, the volume of the first solvent is 80-120 ml, for example, about 100 ml; Preferably, the extraction is ultrasonic extraction; More preferably, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes; More preferably, the second solvent is methanol; More preferably, the mass / volume (g / ml) ratio of the decoction of the traditional Chinese medicine composition containing gypsum and gardenia to the second solvent is 80 to 120, for example, about 100; More preferably, the volume of the second solvent is 0.8 to 1.2 ml, for example, about 1 ml.

3. The thin-layer identification method according to claim 1, characterized in that, The reference medicinal materials include gardenia reference medicinal materials and / or licorice reference medicinal materials; Preferably, the method for preparing the reference herb solution includes: weighing an appropriate amount of gardenia reference herb and / or licorice reference herb, adding a first solvent for extraction, filtering, evaporating the filtrate to dryness, and obtaining a residue; adding a second solvent to the residue to dissolve it, thereby obtaining the reference herb solution; More preferably, the first solvent is ethyl acetate; More preferably, the second solvent is methanol; Preferably, the mass / volume (g / ml) ratio of the gardenia reference material or licorice reference material to the first solvent is 0.01 to 0.1, for example, about 0.02; Preferably, the mass / volume (g / ml) ratio of the gardenia reference material or licorice reference material to the second solvent is 0.8 to 1.2, for example, about 1; More preferably, the mass of the gardenia reference material is 0.8~1.2 g, for example, about 1 g; More preferably, the mass of the licorice reference material is 0.8~1.2 g, for example, about 1 g; More preferably, the extraction is ultrasonic extraction; Particularly preferably, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes.

4. The thin-layer identification method according to claim 1, characterized in that, The reference standards include geniposide reference standard and / or glycyrrhizin reference standard; Preferably, the method for preparing the reference solution is as follows: weigh appropriate amounts of geniposide reference standard and / or glycyrrhizin reference standard, and add methanol to prepare geniposide reference solution and / or glycyrrhizin reference solution respectively; More preferably, the concentration of geniposide in the geniposide reference solution is 1 to 10 mg / ml, for example, about 4 mg / ml; More preferably, the concentration of glycyrrhizin in the glycyrrhizin reference solution is 0.5~2 mg / ml, for example, about 1 mg / ml; Preferably, the blank solution is methanol.

5. The thin-layer identification method according to claim 1, characterized in that, The method for preparing the decoction of the traditional Chinese medicine composition containing gypsum and gardenia includes: (A) weighing appropriate amounts of gypsum, gardenia, patchouli leaves, saposhnikovia root and licorice, crushing them into coarse particles, mixing them, adding honey wine to moisten them, stir-frying them, cooling them, pulverizing them, and sieving them to obtain the first traditional Chinese medicine composition; and (B) weighing an appropriate amount of the first traditional Chinese medicine composition, adding a solvent, boiling it over high heat, then simmering it over low heat to obtain a concentrated liquid, filtering it while it is hot to obtain the traditional Chinese medicine compound decoction. Preferably, the concentration of the mead is 10-40%; Preferably, the ratio of honey to wine in the mead is approximately 1:approximately 1; Preferably, the soaking time is 1-4 hours; Preferably, the frying temperature is 50°C to 80°C; Preferably, the frying time is 30-90 minutes; Preferably, the windproof is a baked windproof; Preferably, the sieve is a No. 4 sieve; Preferably, the solvent is water; Preferably, the mass ratio of the prescription dose to the mass / volume ratio of the solvent is 0.02~0.03, in g / mL; Preferably, the simmering time is 30-60 minutes; Preferably, the volume ratio of the concentrate to the solvent is 0.4 to 0.6; Preferably, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is (10~30):(1~10):(20~40):(100~200):(100~200). More preferably, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is (15~25):(2~6):(25~35):(140~180):(110~140). Preferably, the mass ratio of the gypsum, the gardenia, the patchouli leaf, the saposhnikovia root, and the licorice is about 20.65: about 4.13: about 28.91: about 165.20: about 123.90; More preferably, the method for preparing the negative sample solution includes weighing appropriate amounts of the decoction of the traditional Chinese medicine composition lacking gardenia or licorice, and the subsequent steps are the same as the method for preparing the test sample solution.

6. The thin-layer identification method according to claim 1, characterized in that, The volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (4~6):(4.5~5.5):(0.8~1.2). Preferably, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is about 5: about 5: about 1.

7. The thin-layer identification method according to claim 1, characterized in that, The thin-layer plate is a silicone G thin-layer plate; Preferably, the silicone G thin film is a Merck silicone G thin film, or a Qingdao Ocean silicone G thin film, or a Yinlong silicone G thin film; More preferably, the inspection conditions for the thin-layer chromatography identification analysis step are as follows: take out the developed thin-layer plate, air dry it, spray it with sulfuric acid ethanol solution, heat it until the spots are clear, and inspect it under sunlight. Preferably, the concentration of the sulfuric acid ethanol solution is 8% to 12%, for example, about 10%; Preferably, the heating temperature is approximately 105°C; More preferably, the sample volume of the test solution is 3 to 9 μl, for example, about 5 μl; More preferably, when the reference herb is gardenia, the amount of the reference herb solution is 1.5~2.5 μl, for example, about 2 μl; More preferably, when the reference herb is licorice, the amount of the reference herb solution applied is 1.5~2.5 μl, for example, about 2 μl; More preferably, when the reference standard is geniposide reference standard, the sample volume of the geniposide reference standard solution is 1.5~2.5 μl, for example, about 2 μl; More preferably, when the reference standard is glycyrrhizin reference standard, the sample volume of the glycyrrhizin reference standard solution is 3~9 μl, for example, about 5 μl; More preferably, the amount of the negative sample solution spotted is about 15 μl; More preferably, the amount of blank solution spotted is about 10 μl; Particularly preferred is that the temperature for thin-layer identification is 4~30℃; Particularly preferred is that the relative humidity for the thin-layer identification is 42% to 72%.

8. The thin-layer identification method according to claim 1, characterized in that, The criteria for thin-layer chromatography identification include one of the following: (1) Check whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions as the control medicinal material and the control standard under sunlight; if the thin-layer plate has the characteristic spots under sunlight, then the detected Chinese medicine composition contains gardenia and licorice; or (2) Check whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions of the control medicinal material and the control standard under sunlight, and whether the negative sample and / or blank solution interfere; if the thin-layer plate has the characteristic spots under sunlight and the negative sample and / or blank solution does not interfere, then the detected Chinese medicine composition contains gardenia medicinal material and licorice medicinal material.

9. The use of the thin-layer identification method according to any one of claims 1 to 8 in identifying traditional Chinese medicine compositions containing gypsum and gardenia.

10. A kit for identifying traditional Chinese medicine compositions containing gypsum and gardenia, characterized in that, The kit includes a thin-layer plate and a developing solvent, wherein the developing solvent is ethyl acetate acetone formic acid and the thin-layer plate is a silica gel G thin-layer plate; Preferably, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (2~10):(4~6):(0.5~2). More preferably, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is (4~6):(4.5~5.5):(0.8~1.2). Preferably, the volume ratio of the ethyl acetate, acetone and formic acid in the developing solvent is about 5: about 5: about 1; More preferably, the silicone G thin film is a Merck silicone G thin film, or a Qingdao Ocean silicone G thin film, or a Yinlong silicone G thin film.

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