A method for simultaneously detecting six components in Qingkailing preparation

Abstract

The present invention discloses a method for simultaneously detecting six components of a Qingkailing preparation, the method comprising the following steps: preparing a test solution and a reference solution; adding the test solution and the reference solution to a thin layer plate respectively; using ethyl acetate, acetone, formic acid, and water in a volume ratio of (22-28):(12-18):(1-5):(2-7) as a developing agent to fully react with the thin layer plate obtained in the previous step; and coloring the thin layer plate after the reaction under 250-260 nm ultraviolet light. The method can simultaneously qualitatively detect six effective components of traditional Chinese medicine, namely, geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A, and isochlorogenic acid C. The operation is simple and fast, the color development results are clear and easy to interpret, and the method has good specificity and reproducibility, thereby achieving quality control of compound traditional Chinese medicine preparations, standardizing drug production, improving drug safety, and improving production process stability and controllability. The method has broad application prospects in the identification and quality control of components of compound traditional Chinese medicine preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of identification of traditional Chinese medicine compound preparations, and in particular to a method for simultaneously detecting six components of a Qingkailing preparation. Background Art

[0002] The traditional Chinese medicine (TCM) gardenia is the dried, mature fruit of the Rubiaceae plant Gardenia jasminoides Ellis. It is cold in nature, bitter in flavor, and enters the heart, lung, and triple burner meridians. It has the effects of purging heat and relieving restlessness, clearing heat and promoting urination, and cooling the blood and detoxifying. It can be taken internally to treat fever, restlessness, jaundice, red urine, bloody stranguria, painful and painful eye swelling, and fire-induced sores. It can also be used externally to treat sprains and contusions. The TCM plant gardenia contains over 40 bioactive substances, primarily iridoid glycosides and volatile oils. The active ingredients are primarily iridoid glycosides, and geniposide is the most abundant iridoid glycoside in the plant.

[0003] Honeysuckle is the dried buds or early blooming flowers of Lonicera japonica Thunb., a plant of the Caprifoliaceae family. Pharmacological studies have shown that honeysuckle possesses multiple pharmacological effects, including antibacterial, antiviral, antitumor, immune-enhancing, anti-fertility, antioxidant, and hepatoprotective and choleretic properties. Caffeoylquinic acids are an important class of active ingredients in honeysuckle, primarily present in the form of chlorogenic acid (5-O-caffeoylquinic acid), isochlorogenic acid A, and isochlorogenic acid C.

[0004] Baicalin is a flavonoid extracted and isolated from the dried root of Scutellaria baicalensis (Geochelone baicalensis), a dicotyledonous plant of the Labiatae family. It is a pale yellow powder at room temperature and has a bitter taste. It exhibits significant biological activity, including antibacterial, diuretic, anti-inflammatory, cholesterol-lowering, antithrombotic, asthma-relieving, heat-purging and detoxifying, hemostatic, tocolytic, anti-allergic, and antispasmodic properties. It is also a specific inhibitor of mammalian liver salivary enzymes, which can regulate certain diseases and has strong anti-cancer properties.

[0005] In existing traditional Chinese medicine compositions containing baicalin, gardenia, and honeysuckle, identification is often performed on a single ingredient or medicinal material. Currently, due to the complex composition of traditional Chinese medicine compound preparations, conventional thin-layer chromatography (TLC) identification methods, i.e., a TLC method for each medicinal material or active ingredient, are required. This results in a cumbersome, complex, time-consuming, inefficient, and poorly accurate search results.

[0006] Although the prior art discloses the theoretical basis for simultaneously determining multiple components using thin-layer chromatography, as well as the detection of active ingredients in traditional Chinese medicines, there is currently no method that can simultaneously and rapidly identify multiple active ingredients in baicalin, gardenia, and honeysuckle for different detection objects and traditional Chinese medicine compositions with more complex components. Summary of the Invention

[0007] In order to solve the problem of lack of a method for rapidly and simultaneously identifying multiple active ingredients of traditional Chinese medicines in the prior art, the present invention provides a method for simultaneously detecting multiple ingredients of Qingkailing preparation.

[0008] The first object of the present invention is to provide a developing solvent for thin layer chromatography.

[0009] The second object of the present invention is to provide the use of the above-mentioned developing agent in detecting and / or analyzing the components of Qingkailing preparations.

[0010] The third object of the present invention is to provide a method for simultaneously detecting the six components of Qingkailing preparation.

[0011] The fourth object of the present invention is to provide the application of the above method in component analysis and / or quality control of Qingkailing preparations.

[0012] In order to achieve the above object, the present invention is implemented through the following scheme:

[0013] Thin-layer chromatography (TLC), also known as thin-layer chromatography (TLC), is a type of chromatography. The basic principle of TLC is to separate the components of a mixture by repeatedly adsorbing and distributing them based on their different adsorption properties, solubility properties, or other affinity properties. The key to successfully separating multicomponent samples using TLC lies in the choice of developing solvent, which is adjusted accordingly based on the polarity of the components being analyzed.

[0014] The present invention aims to simultaneously detect six chemical components from three traditional Chinese medicinal materials in the Qingkailing preparation: baicalin, gardenia, and honeysuckle. Gardenia is classified as an iridoid glycoside, while baicalin and luteolin belong to the flavonoid class. Chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C belong to the phenylpropanoid class, all of which are highly polar compounds. To achieve the present invention's detection objectives, the six chemical components must be separated using the same developing solvent system, making the proper preparation of the developing solvent crucial.

[0015] The invention provides a developing agent for thin-layer chromatography. The developing agent comprises ethyl acetate, acetone, formic acid and water in a volume ratio of (22-28): (12-18): (1-5): (2-7).

[0016] Preferably, the volume ratio of ethyl acetate, acetone, formic acid and water is (25-28): (12-15): (1.5-3): (4.5-6).

[0017] More preferably, the volume ratio of ethyl acetate, acetone, formic acid and water is 25:15:3:4.5 or 28:12:1.5:6.

[0018] Further preferably, the volume ratio of ethyl acetate, acetone, formic acid and water is 25:15:3:4.5.

[0019] Ethyl acetate is a weak polar solvent, while acetone, formic acid and water are all strong polar solvents. The four different solvents constitute the developing agent and play a synergistic role. Changes in the proportion of a certain solvent will affect the overall developing agent's separation effect on the components to be tested.

[0020] The use of the above-mentioned developing agent in detecting and / or analyzing the components of the Qingkailing preparation should also be within the scope of protection of the present invention.

[0021] A method for simultaneously detecting six components of a Qingkailing preparation comprises the following steps:

[0022] S1. The ethyl acetate extract of the test sample was removed from the solvent to obtain a precipitate, which was dissolved in acetonitrile to obtain a test solution; methanol solutions of geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A, and isochlorogenic acid C were used as control solutions;

[0023] S2. Add the test solution and reference solution to the thin layer plate;

[0024] S3. The above-mentioned developing agent is fully reacted with the thin layer plate obtained in step S2;

[0025] S4. Develop the thin layer plate obtained in step S3 under 250-260 nm ultraviolet light.

[0026] In step S1, ethyl acetate is used as a solvent. According to the principle of like dissolves like, the effective ingredients in the traditional Chinese medicine compound preparation with complex components can be extracted to a maximum extent, thereby obtaining a better separation effect and facilitating the accuracy and precision of later detection and judgment. Acetonitrile is added to the filter residue. The polarity of acetonitrile is greater than that of ethyl acetate. The use of acetonitrile can effectively ensure that the components extracted from ethyl acetate are dissolved to a large extent, and solves the problem that some components are difficult to dissolve in methanol.

[0027] Preferably, the Qingkailing preparation is one of oral liquid, granules, injection, dry powder, capsule, tablet or paste.

[0028] More preferably, the Qingkailing preparation is an oral liquid or granules.

[0029] Preferably, in step S1, the sample to be tested is Qingkailing oral liquid, and the preparation method of the ethyl acetate extract of the sample to be tested is: fully mix Qingkailing oral liquid, dilute hydrochloric acid and ethyl acetate in a volume ratio of (10-20): (0.5-1): (20-60) and extract.

[0030] More preferably, in step S1, the Qingkailing oral liquid contains baicalin ≥3.5 mg / mL and geniposide ≥0.2 mg / mL.

[0031] More preferably, in step S1, the volume ratio of the Qingkailing oral liquid, dilute hydrochloric acid and ethyl acetate is 40:1:40.

[0032] More preferably, in step S1, the mass concentration of the dilute hydrochloric acid is 9.5% to 10.5% g / mL.

[0033] Preferably, in step S1, the sample to be tested is Qingkailing granules, and the preparation method of the ethyl acetate extract of the sample is: Qingkailing granules, water and ethyl acetate are fully mixed and extracted in a mass volume ratio of (7-11) g: (35-45) mL: (35-45) mL.

[0034] More preferably, in step S1, each 3 g of the Qingkailing granules contains 18-22 mg / mL of baicalin and ≥1 mg of geniposide.

[0035] More preferably, in step S1, the mass volume ratio of the Qingkailing granules, water and ethyl acetate is 9g:40mL:40mL.

[0036] Preferably, in step S1, the method for removing the solvent is heating evaporation.

[0037] Preferably, in step S1, the ethyl acetate extract of the sample to be tested is extracted by shaking with a separatory funnel.

[0038] More preferably, in step S1, the separation funnel is shaken and extracted for three times.

[0039] Preferably, in step S1, the volume concentration of the methanol solution is 65-75%.

[0040] More preferably, in step S1, the volume concentration of the methanol solution is 70%.

[0041] Preferably, in step S1, the working concentration of the reference substance is 0.5-2.5 mg / mL.

[0042] More preferably, in step S1, the working concentration of the reference substance is 1-2 mg / mL.

[0043] Further preferably, in step S1, the working concentration of the geniposide reference solution is 1 mg / mL, the working concentration of the chlorogenic acid reference solution is 1 mg / mL, the working concentration of the baicalin reference solution is 1 mg / mL, the working concentration of the luteolin reference solution is 2 mg / mL, the working concentration of the isochlorogenic acid A reference solution is 2 mg / mL, and the working concentration of the isochlorogenic acid C reference solution is 2 mg / mL.

[0044] Preferably, in step S2, a control medicinal material solution is also added to the thin layer plate; the control medicinal material solution includes a honeysuckle control medicinal material solution and a gardenia control medicinal material solution.

[0045] The control medicinal material solution is used to determine the sources of different spots in different test samples. If the spots of the test sample have corresponding spots in the same position in the honeysuckle control medicinal material solution, it indicates that the test sample contains the components of the honeysuckle medicinal material; if the spots of the test sample have corresponding spots in the same position in the gardenia control medicinal material solution, it indicates that the test sample contains the components of the gardenia medicinal material.

[0046] More preferably, in step S2, the preparation method of the honeysuckle control medicinal material solution includes the following steps: ultrasonic extraction with methanol and honeysuckle control medicinal materials, filtering, and concentrating; mixing the concentrate with dilute hydrochloric acid and ethyl acetate, and shaking and extracting with a separatory funnel; evaporation; dissolving the evaporated precipitate with methanol to obtain the honeysuckle control medicinal material solution.

[0047] More preferably, in step S2, the preparation method of the honeysuckle control medicinal material solution includes the following steps: according to the mass volume ratio of honeysuckle control medicinal material: methanol 1 = 0.512g: 30mL, the honeysuckle control medicinal material and methanol 1 are fully mixed, ultrasonic extraction is carried out for 30min using an ultrasonic cleaner 250W, 40KHz, filtering, collecting the filtrate, heating the filtrate in a water bath and concentrating it to obtain a concentrated solution, the volume of which is one third of the volume of methanol 1; according to the mass volume ratio of honeysuckle control medicinal material: dilute hydrochloric acid: ethyl acetate = 0.512g: 0.5mL: 20mL, the dilute hydrochloric acid, ethyl acetate and the concentrated solution are fully mixed, and extracted with ethyl acetate twice to obtain a honeysuckle control medicinal material extract; the honeysuckle control medicinal material extract is heated until the solvent is completely evaporated, and the solid matter remaining after evaporation is collected; the solid matter is dissolved with methanol 2 equal to the volume of the concentrate to obtain a honeysuckle control medicinal material solution.

[0048] More preferably, in step S2, the method for preparing the Gardenia jasminoides control medicinal material solution comprises the following steps: ultrasonically extracting the Gardenia jasminoides control medicinal material with methanol, and filtering to obtain the Gardenia jasminoides control medicinal material solution.

[0049] More preferably, in step S2, the method for preparing the gardenia control medicinal material solution comprises the following steps: thoroughly mixing the gardenia control medicinal material and 50% methanol by volume according to the mass volume ratio of gardenia control medicinal material: 50% methanol by volume concentration = 1 g: 10 mL, performing ultrasonic extraction for 40 minutes using an ultrasonic cleaner at 250W and 40KHz, and filtering to obtain the gardenia control medicinal material solution.

[0050] Preferably, in step S2, a negative control solution is also added to the thin layer plate; the negative control solution includes a honeysuckle medicinal material negative control solution and a gardenia medicinal material negative control solution.

[0051] The negative control solution is used to indicate whether there is interference in the thin layer chromatography detection system. The honeysuckle medicinal material negative control solution is a Qingkailing preparation lacking honeysuckle medicinal material. The honeysuckle medicinal material negative control solution should lack spots derived from honeysuckle medicinal material. However, if there are corresponding spots in the test results, it means that the spots at this position are not a single component, there is interference in the thin layer chromatography detection system, and the test results are unreliable. The Gardenia medicinal material negative control solution is a Qingkailing preparation lacking Gardenia medicinal material, and it also has the same effect.

[0052] More preferably, in step S2, the method for preparing the honeysuckle medicinal material negative control solution comprises the following steps:

[0053] S21. Mix the isatis root, gardenia, and water in a mass-volume ratio of isatis root: gardenia: water 1 = 20 g: 2.5 g: 50 mL, decoct twice for 1 hour each time, combine the decoctions, filter, and collect filtrate 1; heat filtrate 1 at 50°C, concentrate to a relative density of 1.15-1.20, and cool to obtain concentrate 1; thoroughly mix concentrate 1 with ethanol 1 in a mass-volume ratio of isatis root: ethanol 1 = 20 g: 50 mL, let stand, filter, and recover ethanol 1 to obtain filtrate 2; thoroughly mix filtrate 2 with water 2 in a mass-volume ratio of isatis root: water 2 = 20 g: 50 mL, and let stand to obtain an isatis root-gardenia extract;

[0054] S22. Mix buffalo horn powder: mother of pearl: 98% sulfuric acid in a mass volume ratio of 2.5 g:5 g:10 g, and fully hydrolyze to obtain hydrolyzate 1; fully mix hydrolyzate 1 with water 3 in a mass volume ratio of isatis root: water 3 = 2.5 g:50 mL, and filter to obtain filtrate 3; heat filtrate 3 at 50° C., concentrate to a relative density of 1.05-1.10, and cool to obtain concentrate 2; fully mix concentrate 2 with ethanol 2 in a mass volume ratio of buffalo horn powder: ethanol 2 = 2.5 g:100 mL, let stand, filter, and recover ethanol 2 to obtain filtrate 4; fully mix filtrate 4 with water 4 in a mass volume ratio of buffalo horn powder: water 4 = 2.5 g:100 mL, and let stand to obtain buffalo horn-mother of pearl hydrolyzate;

[0055] S23. According to the mass volume ratio of bile acid: hyodeoxycholic acid: ethanol 3 = 3.25g: 3.75g: 50mL, mixed and fully dissolved to obtain bile acid - hyodeoxycholic acid ethanol solution;

[0056] S24. Combine the above-mentioned Radix Isatidis-Gardeniae extract and the water buffalo horn-mother of pearl hydrolyzed liquid, mix them evenly, and add them to the bile acid-hyodeoxycholic acid ethanol solution. Add ethanol 4 to the bile acid-hyodeoxycholic acid ethanol solution according to the mass volume ratio of bile acid: ethanol 4 = 3.25 g: 50 mL, mix thoroughly, let stand, filter, and recover ethanol 4 to obtain filtrate 5; according to the mass volume ratio of bile acid: water 5 = 3.25 g: 100 mL, mix the filtrate 5 and water 5 thoroughly, let stand; add baicalin in an amount twice that of water buffalo horn powder, adjust the pH value to 8.2-9.4 to dissolve the baicalin; according to the mass volume ratio of Radix Isatidis: honeysuckle medicinal material negative medicinal material solution = 20 g: 1000 mL, add water 6 to make up the remaining volume; adjust the pH value to 7.2-7.5 with sodium hydroxide, stir evenly, let stand, filter, and the obtained filtrate is the honeysuckle medicinal material negative medicinal material solution.

[0057] More preferably, in step S2, the preparation method of the gardenia medicinal material negative control solution is similar to the preparation method of the honeysuckle medicinal material negative control solution, with the only difference being that in step S21, gardenia is replaced by honeysuckle, and the isatis root, honeysuckle and water are mixed according to a mass volume ratio of isatis root: honeysuckle: water 7 = 20 g: 6 g: 50 mL.

[0058] Preferably, in step S2, the volume ratio of the test solution to the reference solution is test solution: gardeniaside reference solution: chlorogenic acid reference solution: baicalin reference solution: luteolin reference solution: isochlorogenic acid A reference solution: isochlorogenic acid C reference solution = 0.8-1.2: 0.8-1.2: 1.8-2.2: 1.8-2.2: 0.8-1.2: 0.8-1.2: 0.8-1.2: 0.8-1.2.

[0059] More preferably, in step S2, the volume ratio of the test solution to the reference solution is test solution: gardeniaside reference solution: chlorogenic acid reference solution: baicalin reference solution: luteolin reference solution: isochlorogenic acid A reference solution: isochlorogenic acid C reference solution = 1:1:2:2:1:1:1.

[0060] More preferably, in step S2, the volume ratio of the test solution to the honeysuckle control medicinal material solution and the gardenia control medicinal material solution is test solution: honeysuckle control medicinal material solution: gardenia control medicinal material solution = 0.8-1.2:0.8-1.2:0.8-1.2.

[0061] Further preferably, in step S2, the volume ratio of the test solution to the honeysuckle control medicinal material solution and the gardenia control medicinal material solution is test solution: honeysuckle control medicinal material solution: gardenia control medicinal material solution = 1:1:1.

[0062] More preferably, in step S2, the volume ratio of the test solution to the honeysuckle medicinal material negative control solution and the gardenia medicinal material negative control solution is test solution: honeysuckle medicinal material negative control solution: gardenia medicinal material negative control solution = 0.8-1.2:0.8-1.2:0.8-1.2.

[0063] Further preferably, in step S2, the volume ratio of the test solution to the honeysuckle medicinal material negative control solution and the gardenia medicinal material negative control solution is test solution: honeysuckle medicinal material negative control solution: gardenia medicinal material negative control solution = 1:1:1.

[0064] Preferably, in step S2, the test solution, reference solution, control medicinal material solution and negative control solution are placed on the same horizontal line 150 to 200 mm away from the bottom edge of the thin layer plate to form a plurality of circular dot-shaped spots with a diameter of 1 to 5 mm. The distance between the spots at both ends and the side edge of the adjacent thin layer plate is 5 to 10 mm, and the spots are spaced equidistantly from each other.

[0065] Preferably, in steps S2 to S4, the thin layer plate is a silica gel GF254 thin layer plate.

[0066] Preferably, in step S4, the thin layer plate obtained in step S3 is developed under 245 nm ultraviolet light.

[0067] Silica gel GF254 thin layer plate is selected. Specific fluorescent agent is added to the silica gel GF254 thin layer plate, which emits green fluorescence at a wavelength of 254nm. Most compounds with aromatic rings or conjugated systems will have ultraviolet absorption at a wavelength of 254nm, thereby masking the green fluorescence and forming dark spots; while baicalin, chlorogenic acid, gardenia glycoside, luteolin, isochlorogenic acid A and isochlorogenic acid C can form more obvious dark spots at a wavelength of 254nm. If other thin layer plates such as silica gel G plate are used instead, the effect is not good and cannot be well distinguished.

[0068] The application of the above method in the component analysis and / or quality control of Qingkailing preparations should also be within the scope of protection of the present invention.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The present invention requires only a single sample preparation method. Using a single silica gel GF254 thin-layer plate and one inspection condition, the method can detect six Chinese herbal ingredients, namely, geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A, and isochlorogenic acid C, in three Chinese herbal flavors, namely, Gardenia jasminoides, Scutellaria baicalensis, and Honeysuckle, without the use of a color developer. This effectively saves solvent and labor costs, shortens detection time by approximately 70%, and can be used for the detection of other products containing the six ingredients. Furthermore, it overcomes the interference of other complex components in Chinese herbal medicines and ensures detection accuracy.

[0071] The present invention successfully achieves simultaneous qualitative detection of six traditional Chinese medicine active ingredients in the Qingkailing preparation: geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A, and isochlorogenic acid C, by preparing test solution and reference solution, and performing thin-layer chromatography detection, as well as various specific control conditions. The detection method provided by the present invention is simple and rapid to operate, with clear color development results, good separation, effective elimination of negative interference, clear inspection, easy interpretation, good specificity and reproducibility, and can effectively achieve quality control of compound traditional Chinese medicine preparations, achieve drug production standardization, improve drug safety, and improve production process stability and controllability. It has broad application prospects in the identification and quality control of compound traditional Chinese medicine preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The test results of Example 1; 1 is the baicalin reference substance; 2 is the chlorogenic acid reference substance; 3 is the gardenia glycoside reference substance; 4 is the luteolin reference substance; 5 is the isochlorogenic acid A reference substance; 6 is the isochlorogenic acid C reference substance; 7 is the honeysuckle reference medicinal material; 8 is the gardenia jasminoides reference medicinal material; 9 to 11 are the test points of Qingkailing oral liquid, corresponding to batch numbers: 1220701, 1220702 and 1220703; 12 is the gardenia jasminoides medicinal material negative control; 13 is the honeysuckle medicinal material negative control.

[0073] Figure 2 The test results of Example 2; 1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is gardenia glycoside reference substance; 4 is luteolin reference substance; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle reference medicinal material; 8 is gardenia jasminoides reference medicinal material; 9 to 11 are Qingkailing granule test sample points, corresponding to batch numbers 5220801, 5220802 and 5220803; 12 is gardenia jasminoides medicinal material negative control; 13 is honeysuckle medicinal material negative control.

[0074] Figure 3The test results of Example 3; 1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is gardenia glycoside reference substance; 4 is luteolin reference substance; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle reference medicinal material; 8 is gardenia jasminoides reference medicinal material; 9 to 11 are Qingkailing granule test sample points, corresponding to batch numbers 5220801, 5220802 and 5220803; 12 is gardenia jasminoides medicinal material negative control; 13 is honeysuckle medicinal material negative control.

[0075] Figure 4 The test results of Comparative Example 1; 1 is the baicalin reference substance; 2 is the chlorogenic acid reference substance; 3 is the gardenia glycoside reference substance; 4 is the luteolin reference substance; 5 is the isochlorogenic acid A reference substance; 6 is the isochlorogenic acid C reference substance; 7 is the honeysuckle reference medicinal material; 8 is the gardenia jasminoides reference medicinal material; 9 to 11 are the test points of Qingkailing oral liquid, corresponding to batch numbers 1220701, 1220702 and 1220703; 12 is the gardenia jasminoides medicinal material negative control; 13 is the honeysuckle medicinal material negative control.

[0076] Figure 5 The test results of Comparative Example 2; A is the 254nm ultraviolet light color development result; B is the sunlight color development result; 1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is gardenia glycoside reference substance; 4 is luteolin reference substance; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle reference medicinal material; 8 is gardenia jasminoides reference medicinal material; 9 is Qingkailing oral liquid test sample batch number 1220701; 10 is gardenia jasminoides medicinal material negative control; 11 is honeysuckle medicinal material negative control.

[0077] Figure 6 The test results of Comparative Example 3; A is the 254nm ultraviolet light color development result; B is the sunlight color development result; 1 is baicalin reference; 2 is chlorogenic acid reference; 3 is gardenia glycoside reference; 4 is luteolin reference; 5 is isochlorogenic acid A reference; 6 is isochlorogenic acid C reference; 7 is honeysuckle reference medicinal material; 8 is gardenia jasminoides reference medicinal material; 9 is Qingkailing oral liquid test sample batch number 1220701; 10 is gardenia jasminoides medicinal material negative control; 11 is honeysuckle medicinal material negative control.

[0078] Figure 7 These are the test results of Comparative Example 4; 1 is the baicalin reference substance; 2 is the chlorogenic acid reference substance; 3 is the gardenia glycoside reference substance; 4 is the luteolin reference substance; 5 is the isochlorogenic acid A reference substance; 6 is the isochlorogenic acid C reference substance; 7 is the honeysuckle reference medicinal material; 8 is the gardenia jasminoides reference medicinal material; 9 is the Qingkailing oral liquid test sample batch number 1220701.

[0079] Figure 8The test results of Comparative Example 5; 1 is the baicalin reference substance; 2 is the chlorogenic acid reference substance; 3 is the gardenia glycoside reference substance; 4 is the luteolin reference substance; 5 is the isochlorogenic acid A reference substance; 6 is the isochlorogenic acid C reference substance; 7 is the honeysuckle reference medicinal material; 8 is the gardenia jasminoides reference medicinal material; 9 is the Qingkailing oral liquid test sample batch number 1220701. DETAILED DESCRIPTION

[0080] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0081] In the following embodiments, the instruments and consumables involved include: an ultrasonic cleaner (KQ-250DE model, 250W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.) and a silica gel GF254 thin layer plate (Qingdao Ocean Chemical Plant Branch, Tianjin Silida Technology Co., Ltd., Qingdao Yumin Chemical Plant).

[0082] In the following examples, the organic solvents involved, such as methanol, ethyl acetate, chloroform, acetone and formic acid, were all of analytical grade.

[0083] In the following examples, the reference substances involved include: baicalin (China Food and Drug Inspection Institute, batch number: 110715-202122); chlorogenic acid (China Food and Drug Inspection Institute, batch number: 110753-202119); gardeniaside (China Food and Drug Inspection Institute, batch number: 110749-201919); luteolin (China Food and Drug Inspection Institute, batch number: 111720-202111); isochlorogenic acid A (ChemFaces, batch number: CFN99118); isochlorogenic acid C (NATURE STANDARD, batch number: RS06601020).

[0084] In the following examples, the control medicinal materials involved include: honeysuckle control medicinal material (China Food and Drug Inspection Institute, batch number: 121060-201608); Gardenia control medicinal material (China Food and Drug Inspection Institute, batch number: 120986-201610).

[0085] In the following examples, the dilute hydrochloric acid used was prepared according to the 2020 edition of the Pharmacopoeia, and its mass concentration was 9.5% to 10.5% (g / mL).

[0086] Example 1 A method for simultaneously detecting six components of Qingkailing oral liquid

[0087] 1. Preparation of test solution

[0088] (1) Take 20 mL of the sample to be tested (the sample to be tested contains baicalin ≥3.5 mg / mL and geniposide ≥0.2 mg / mL), add 0.5 mL of dilute hydrochloric acid and 20 mL of ethyl acetate to it, shake and extract with a separatory funnel, and collect the organic phase extract; add 20 mL of ethyl acetate again, repeat the extraction twice, collect the organic phase, and obtain the sample extract.

[0089] (2) Place the sample extract in a container and heat it until the solvent in the sample extract is completely evaporated. Collect the solid matter remaining after evaporation and record it as solid matter 1.

[0090] (3) Dissolve the solid 1 in 2 mL of acetonitrile to obtain the test solution.

[0091] 2. Preparation of reference solution

[0092] Take 10.82 mg of gardeniaside, 10.61 mg of chlorogenic acid, 10.17 mg of baicalin, 20.81 mg of luteolin, 20.12 mg of isochlorogenic acid A, and 20.34 mg of isochlorogenic acid C, and dissolve them in 70% methanol solution to prepare 1 mg / mL gardeniaside reference solution, 1 mg / mL chlorogenic acid reference solution, 1 mg / mL baicalin reference solution, 2 mg / mL luteolin reference solution, 2 mg / mL isochlorogenic acid A reference solution, and 2 mg / mL isochlorogenic acid C reference solution.

[0093] 3. Preparation of control medicinal material solution

[0094] (1) Take 0.512 g of honeysuckle control medicinal material, add 30 mL of methanol, use an ultrasonic cleaner at 250 W and 40 kHz to extract for 30 min, filter, and collect the filtrate, which is recorded as filtrate 1.

[0095] The filtrate 1 was heated in a water bath until it was concentrated to 10 mL, 0.5 mL of dilute hydrochloric acid and 20 mL of ethyl acetate were added thereto, and the mixture was shaken with a separatory funnel to extract the mixture, and the organic phase extract was collected; 20 mL of ethyl acetate was added again, and the extraction was repeated twice, and the organic phase was collected to obtain a honeysuckle control medicinal material extract.

[0096] The honeysuckle control medicinal material extract was placed in a container and heated until the solvent was completely evaporated, and the solid remaining after evaporation was collected and recorded as solid 2; solid 2 was dissolved in 2 mL of methanol to obtain the honeysuckle control medicinal material solution.

[0097] (2) Take 1 g of Gardenia jasminoides reference medicinal material, add 10 mL of 50% methanol by volume, use an ultrasonic cleaner at 250 W and 40 kHz for 40 min, filter, and record the resulting filtrate as filtrate 2, which is the Gardenia jasminoides reference medicinal material solution.

[0098] 4. Preparation of negative control solution

[0099] (1) Negative control solution of honeysuckle (i.e., Qingkailing preparation without honeysuckle)

[0100] Weigh 20 g of Radix Isatidis and 2.5 g of Gardenia, add 50 mL of water, and boil twice for 1 hour each time. Combine the decoctions, filter, and collect the filtrate; heat the filtrate at 50°C, concentrate to a relative density of 1.15 to 1.20, and cool; add 50 mL of ethanol, let stand, filter, recover the ethanol, add 50 mL of water, and let stand to obtain the Radix Isatidis-Gardenia extract.

[0101] Weigh 2.5 g of buffalo horn powder and 5 g of mother-of-pearl, add 10 mL of 98% sulfuric acid, fully hydrolyze, add 50 mL of water, and filter; adjust the pH of the filtrate to 4 with 15% calcium hydroxide solution, filter, and collect the filtrate; heat the filtrate at 50° C., concentrate to a relative density of 1.05 to 1.10, and cool; add 100 mL of ethanol, let stand, filter, recover the ethanol, add 100 mL of water, and let stand to obtain a buffalo horn-mother-of-pearl hydrolyzate.

[0102] Weigh 3.25 g of cholic acid and 3.75 g of hyodeoxycholic acid, add 50 mL of ethanol, and fully dissolve to obtain cholic acid-hyodeoxycholic acid ethanol solution.

[0103] Combine the above-mentioned Radix Isatidis-Gardeniae extract and the buffalo horn-mother of pearl hydrolyzed solution, mix well, add to the cholic acid-hyodeoxycholic acid ethanol solution, add 50 mL of ethanol, and let it stand; filter, recover ethanol from the filtrate, add 100 mL of water, and let it stand; add 5 g of baicalin, adjust the pH value to 8.2-9.4 to dissolve the baicalin; add water to make up the volume to 1000 mL; adjust the pH value to 7.2-7.5 with sodium hydroxide, stir well, let it stand, and filter. The resulting filtrate is the honeysuckle medicinal material negative medicinal material solution.

[0104] (2) Gardenia jasminoides negative control solution (i.e., Qingkailing preparation without Gardenia jasminoides)

[0105] Weigh 20 g of Radix Isatidis and 6 g of Flos Lonicerae, add 50 mL of water, and boil twice for 1 hour each time. Combine the decoctions, filter, and collect the filtrate; heat the filtrate at 50°C, concentrate to a relative density of 1.15 to 1.20, and cool; add 50 mL of ethanol, let stand, filter, recover the ethanol, add 50 mL of water, and let stand to obtain the Radix Isatidis-Flos Lonicerae extract.

[0106] Weigh 2.5 g of buffalo horn powder and 5 g of mother-of-pearl, add 10 mL of 98% sulfuric acid, fully hydrolyze, add 50 mL of water, and filter; adjust the pH of the filtrate to 4 with 15% calcium hydroxide solution, filter, and collect the filtrate; heat the filtrate at 50° C., concentrate to a relative density of 1.05 to 1.10, and cool; add 100 mL of ethanol, let stand, filter, recover the ethanol, add 100 mL of water, and let stand to obtain a buffalo horn-mother-of-pearl hydrolyzate.

[0107] Weigh 3.25 g of cholic acid and 3.75 g of hyodeoxycholic acid, add 50 mL of ethanol, and fully dissolve to obtain cholic acid-hyodeoxycholic acid ethanol solution.

[0108] Combine the above-mentioned Radix Isatidis-Lonicera Japonica extract and the buffalo horn-mother of pearl hydrolyzed solution, mix well, add to the cholic acid-hyodeoxycholic acid ethanol solution, add 50 mL of ethanol, and let it stand; filter, recover ethanol from the filtrate, add 100 mL of water, and let it stand; add 5 g of baicalin, adjust the pH value to 8.2-9.4 to dissolve the baicalin; add water to make up the volume to 1000 mL; adjust the pH value to 7.2-7.5 with sodium hydroxide, stir well, let it stand, and filter. The resulting filtrate is the Gardenia jasminoides negative medicinal material solution.

[0109] 5. Thin layer chromatography detection

[0110] (1) Sample

[0111] Using a quantitative capillary tube, dispense 10 μL of the test solution, 10 μL of the 1 mg / mL geniposide reference solution, 20 μL of the 1 mg / mL chlorogenic acid reference solution, 20 μL of the 1 mg / mL baicalin reference solution, 10 μL of the 2 mg / mL luteolin reference solution, 10 μL of the 2 mg / mL isochlorogenic acid A reference solution, 10 μL of the 2 mg / mL isochlorogenic acid C reference solution, 10 μL of the honeysuckle control solution, 10 μL of the gardenia control solution, 10 μL of the honeysuckle negative control solution, and 10 μL of the gardenia negative control solution. Spot the sample onto the same horizontal line 150–200 mm from the bottom edge of a silica gel GF254 thin-layer plate, forming multiple circular spots with a diameter of 1–5 mm. The spots at both ends should be 5–10 mm away from the adjacent edge of the plate, and each spot should be equally spaced. This creates a spot plate.

[0112] (2) Expand

[0113] A developing solvent was prepared according to the volume ratio of ethyl acetate:acetone:formic acid:water=25:15:3:4.5.

[0114] Add the developing agent to the chromatography tank and let it stand for 30 minutes for pre-saturation. Then put the spot plate into the chromatography tank and develop it until the spot extends to 15 cm from the bottom edge of the spot plate.

[0115] Allow the sample to dry until all the developer has evaporated.

[0116] (3) Inspection

[0117] Place the dried spot plate under 254nm ultraviolet light for color development and inspection.

[0118] Example 2 A method for simultaneously detecting six components of Qingkailing oral liquid

[0119] The preparation of the test solution, the preparation of the reference solution, the preparation of the control medicinal material solution, the preparation of the negative control solution and the thin layer chromatography detection were the same as in Example 1, except that in the "inspection" step of the thin layer chromatography detection, the developing solvent was prepared according to the volume ratio of ethyl acetate: acetone: formic acid: water = 28:12:1.5:6.

[0120] Example 3 A method for simultaneously detecting six components of Qingkailing granules

[0121] 1. Preparation of test solution

[0122] (1) Take 3 bags of Qingkailing granules (3 g of Qingkailing granules per bag, containing 18-22 mg / mL of baicalin and ≥1 mg of gardeniaside) as the test sample, grind them into powder, add 40 mL of hot water, shake them thoroughly to dissolve, let them cool, and shake them with a 40 mL ethyl acetate separatory funnel for extraction. Extract them twice in total, combine the extracts, and use them as the sample extract solution.

[0123] (2) Place the sample extract in a container and heat it in a water bath until the solvent in the sample extract is completely evaporated. Collect the solid matter remaining after evaporation and record it as solid matter 1.

[0124] (3) Dissolve the solid 1 in 2 mL of acetonitrile to obtain the test solution.

[0125] 2. The preparation of reference solution, reference medicinal material solution, negative control solution and thin layer chromatography detection were the same as in Example 1.

[0126] Comparative Example 1: A method for detecting the ingredients of Qingkailing preparation

[0127] The preparation of the test solution, the preparation of the reference solution, the preparation of the control medicinal material solution, the preparation of the negative control solution and the thin layer chromatography detection were the same as in Example 1, with the only difference being that in the "development" step of the thin layer chromatography detection, the dried spot plate was placed under 365 nm ultraviolet light for color development.

[0128] Comparative Example 2: A method for detecting the ingredients of Qingkailing preparations

[0129] 1. The preparation of the test solution, the preparation of the reference solution, the preparation of the control medicinal material solution, and the preparation of the negative control solution are the same as those in Example 1.

[0130] 2. Thin layer chromatography detection

[0131] (1) Sample

[0132] Using a quantitative capillary tube, dispense 10 μL of the test solution, 10 μL of the 1 mg / mL geniposide reference solution, 20 μL of the 1 mg / mL chlorogenic acid reference solution, 20 μL of the 1 mg / mL baicalin reference solution, 10 μL of the 2 mg / mL luteolin reference solution, 10 μL of the 2 mg / mL isochlorogenic acid A reference solution, 10 μL of the 2 mg / mL isochlorogenic acid C reference solution, 10 μL of the honeysuckle reference solution, and 10 μL of the gardenia reference solution. Spot the sample onto the same horizontal line 150–200 mm from the bottom edge of a silica gel GF254 thin-layer plate, forming multiple circular spots with a diameter of 1–5 mm. The spots at both ends should be 5–10 mm away from the adjacent edge of the plate, and each spot should be equally spaced. This creates a spot plate.

[0133] (2) Expand

[0134] A developing solvent was prepared according to the volume ratio of ethyl acetate:acetone:formic acid:water=2:10:1:0.5.

[0135] Add the developing agent to the chromatography tank and let it stand for 30 minutes for pre-saturation. Then put the spot plate into the chromatography tank and develop it until the spot extends to 15 cm from the bottom edge of the spot plate.

[0136] Allow the sample to dry until all the developer has evaporated.

[0137] (3) Color development

[0138] Take 57 mL of 98% sulfuric acid and dilute it to 1000 mL with ethanol to obtain a 10% sulfuric acid ethanol solution as a color developer.

[0139] Spray the developer onto the thin layer plate, dry the developer, and heat in an oven at 105°C for 3 min until the spots are clear.

[0140] (4) Inspection

[0141] Place the dried spot sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

[0142] Comparative Example 3: A method for detecting the ingredients of Qingkailing preparations

[0143] 1. The preparation of the test solution, the preparation of the reference solution, the preparation of the control medicinal material solution, and the preparation of the negative control solution are the same as those in Example 1.

[0144] 2. Thin layer chromatography detection

[0145] (1) Sample

[0146] Using a quantitative capillary tube, dispense 10 μL of the test solution, 10 μL of the 1 mg / mL geniposide reference solution, 20 μL of the 1 mg / mL chlorogenic acid reference solution, 20 μL of the 1 mg / mL baicalin reference solution, 10 μL of the 2 mg / mL luteolin reference solution, 10 μL of the 2 mg / mL isochlorogenic acid A reference solution, 10 μL of the 2 mg / mL isochlorogenic acid C reference solution, 10 μL of the honeysuckle reference solution, and 10 μL of the gardenia reference solution. Spot the sample onto the same horizontal line 150–200 mm from the bottom edge of a silica gel GF254 thin-layer plate, forming multiple circular spots with a diameter of 1–5 mm. The spots at both ends should be 5–10 mm away from the adjacent edge of the plate, and each spot should be equally spaced. This creates a spot plate.

[0147] (2) Expand

[0148] A developing solvent was prepared according to the volume ratio of ethyl acetate:acetone:formic acid:water=5:5:1:1.

[0149] Add the developing agent to the chromatography tank and let it stand for 30 minutes for pre-saturation. Then put the spot plate into the chromatography tank and develop it until the spot extends to 15 cm from the bottom edge of the spot plate.

[0150] Allow the sample to dry until all the developer has evaporated.

[0151] (3) Color development

[0152] Take 57 mL of 98% sulfuric acid and dilute it to 1000 mL with ethanol to obtain a 10% sulfuric acid ethanol solution as a color developer.

[0153] Spray the developer onto the thin layer plate, dry the developer, and heat in an oven at 105°C for 3 min until the spots are clear.

[0154] (4) Inspection

[0155] Place the dried spot sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

[0156] Comparative Example 4: A method for detecting the ingredients of Qingkailing preparations

[0157] 1. The preparation of the test solution, the preparation of the reference solution and the preparation of the reference medicinal material solution are the same as those in Example 1.

[0158] 2. Thin layer chromatography detection

[0159] (1) Sample

[0160] Using a quantitative capillary tube, dispense 10 μL of the test solution, 10 μL of the 1 mg / mL geniposide reference solution, 20 μL of the 1 mg / mL chlorogenic acid reference solution, 20 μL of the 1 mg / mL baicalin reference solution, 10 μL of the 2 mg / mL luteolin reference solution, 10 μL of the 2 mg / mL isochlorogenic acid A reference solution, 10 μL of the 2 mg / mL isochlorogenic acid C reference solution, 10 μL of the honeysuckle control solution, 10 μL of the gardenia control solution, 10 μL of the honeysuckle negative control solution, and 10 μL of the gardenia negative control solution. Spot the sample onto the same horizontal line 150–200 mm from the bottom edge of a silica gel GF254 thin-layer plate, forming multiple circular spots with a diameter of 1–5 mm. The spots at both ends should be 5–10 mm away from the adjacent edge of the plate, and each spot should be equally spaced. This creates a spot plate.

[0161] (2) Expand

[0162] A developing solvent was prepared according to the volume ratio of ethyl acetate:formic acid:water=8:3:2.

[0163] Add the developing agent to the chromatography tank and let it stand for 30 minutes for pre-saturation. Then put the spot plate into the chromatography tank and develop it until the spot extends to 15 cm from the bottom edge of the spot plate.

[0164] Allow the sample to dry until all the developer has evaporated.

[0165] (3) Color development

[0166] Take 57 mL of 98% sulfuric acid and dilute it to 1000 mL with ethanol to obtain a 10% sulfuric acid ethanol solution as a color developer.

[0167] Spray the developer onto the thin layer plate, dry the developer, and heat in an oven at 105°C for 3 min until the spots are clear.

[0168] (4) Inspection

[0169] Place the dried spot sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

[0170] Comparative Example 5: A method for detecting the ingredients of Qingkailing preparations

[0171] 1. The preparation of the test solution, the preparation of the reference solution and the preparation of the reference medicinal material solution are the same as those in Example 1.

[0172] 2. Thin layer chromatography detection

[0173] (1) Sample

[0174] Using a quantitative capillary tube, dispense 10 μL of the test solution, 10 μL of the 1 mg / mL geniposide reference solution, 20 μL of the 1 mg / mL chlorogenic acid reference solution, 20 μL of the 1 mg / mL baicalin reference solution, 10 μL of the 2 mg / mL luteolin reference solution, 10 μL of the 2 mg / mL isochlorogenic acid A reference solution, 10 μL of the 2 mg / mL isochlorogenic acid C reference solution, 10 μL of the honeysuckle control solution, 10 μL of the gardenia control solution, 10 μL of the honeysuckle negative control solution, and 10 μL of the gardenia negative control solution. Spot the sample onto the same horizontal line 150–200 mm from the bottom edge of a silica gel GF254 thin-layer plate, forming multiple circular spots with a diameter of 1–5 mm. The spots at both ends should be 5–10 mm away from the adjacent edge of the plate, and each spot should be equally spaced. This creates a spot plate.

[0175] (2) Expand

[0176] A developing solvent was prepared according to the volume ratio of chloroform: ethyl acetate: methanol: water = 15:40:22:10.

[0177] Add the developing agent to the chromatography tank and let it stand for 30 minutes for pre-saturation. Then put the spot plate into the chromatography tank and develop it until the spot extends to 15 cm from the bottom edge of the spot plate.

[0178] Allow the sample to dry until all the developer has evaporated.

[0179] (3) Color development

[0180] Take 57 mL of 98% sulfuric acid and dilute it to 1000 mL with ethanol to obtain a 10% sulfuric acid ethanol solution as a color developer.

[0181] Spray the developer onto the thin layer plate, dry the developer, and heat in an oven at 105°C for 3 min until the spots are clear.

[0182] (4) Inspection

[0183] Place the dried spot sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

[0184] Application Examples

[0185] 1. Experimental Methods

[0186] Using the methods of Examples 1 to 3 and Comparative Examples 1 to 5, the contents of gardenia glycoside, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A, and isochlorogenic acid C in Qingkailing oral liquid (batch numbers: 1220701; 1220702; 1220703) and Qingkailing granules (batch numbers: 5220801; 5220802; 5220803) produced by Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd. were detected.

[0187] 2. Experimental Results

[0188] like Figure 1 and Figure 2 As shown, in the chromatogram of the test sample under 254nm ultraviolet light, the chlorogenic acid reference solution, the geniposide reference solution, the baicalin reference solution, the luteolin reference solution, the isochlorogenic acid A reference solution, the isochlorogenic acid C reference solution, the gardenia control medicinal material, and the honeysuckle control medicinal material showed the same spots at the corresponding positions on the chromatogram. The honeysuckle negative medicinal material solution and the gardenia negative medicinal material solution lacked spots of the corresponding medicinal material-derived components at the corresponding positions. This shows that the methods of Examples 1 and 2 can simultaneously detect gardeniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A, and isochlorogenic acid C in Qingkailing oral liquid.

[0189] like Figure 3 As shown, in the chromatogram of the test sample under 254nm ultraviolet light irradiation, the chlorogenic acid reference solution, the gardenia jasminoides reference solution, the baicalin reference solution, the luteolin reference solution, the isochlorogenic acid A reference solution, the isochlorogenic acid C reference solution, the gardenia jasminoides reference medicinal material, and the honeysuckle control medicinal material showed the same spots at the corresponding positions on the chromatogram. The honeysuckle negative medicinal material solution and the gardenia jasminoides negative medicinal material solution lacked spots of the corresponding medicinal material-derived components at the corresponding positions. This indicates that the method of Example 3 can simultaneously detect gardenia jasminoides, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A, and isochlorogenic acid C in Qingkailing Granules.

[0190] like Figure 4 As shown, in the chromatogram of the test sample of Comparative Example 1 under 365nm ultraviolet light, the chlorogenic acid reference solution, luteolin reference solution, isochlorogenic acid A reference solution, isochlorogenic acid C reference solution, and honeysuckle reference medicinal material showed the same spots at the corresponding positions on the chromatogram, while the baicalin reference solution and the geniposide reference solution had no obvious spots. This shows that the method of Comparative Example 1 cannot detect baicalin and geniposide in Qingkailing oral liquid.

[0191] like Figure 5 and Figure 6 As shown, the developing agents of Comparative Examples 2 and 3 were unable to separate the various components in the test sample. Even when 10% sulfuric acid ethanol solution was used as the color developer, no obvious spots of the test sample could be observed at the corresponding positions of the chromatograms in the baicalin reference solution, chlorogenic acid reference solution, geniposide reference solution, honeysuckle reference medicinal material, and gardenia reference medicinal material under ultraviolet light and sunlight.

[0192] like Figure 7 and Figure 8As shown, the developing agent of Comparative Example 4 (chloroform: ethyl acetate: methanol: water = 15:40:22:10) and the developing agent of Comparative Example 5 (ethyl acetate: formic acid: water = 8:3:2) have poor separation effects on the components in the test sample, and there is a tailing phenomenon. Moreover, when the color developer is superimposed, it is still impossible to observe obvious spots of the test sample in the corresponding positions of the chromatogram in the baicalin reference solution, chlorogenic acid reference solution, geniposide reference solution, honeysuckle reference medicinal material, and gardenia reference medicinal material under sunlight or ultraviolet light (fluorescence results are not shown).

[0193] In summary, the methods of Examples 1 to 3 can simultaneously detect gardeniaside, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A and isochlorogenic acid C in Qingkailing oral liquid and Qingkailing granules. The detection can be completed without the use of a color developer. The operation is simple and quick, the color development results are clear, the separation is good, negative interference is effectively eliminated, and the inspection is clear and easy to interpret. It has good specificity and reproducibility, can effectively achieve quality control of the compound preparation, realize drug production standardization, and can better meet the drug safety, production process stability and controllability.

[0194] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for simultaneously detecting six components of Qingkailing preparation, characterized in that: The following steps are involved: S1. The ethyl acetate extract of the test sample was removed from the solvent to obtain a precipitate, which was dissolved in acetonitrile to obtain a test solution; methanol solutions of geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A and isochlorogenic acid C were used as reference solutions; S2. The test solution and the reference solution were added to a thin layer plate, the thin layer plate being a silica gel GF254 thin layer plate; S3. Fully reacting the developing agent with the thin layer plate obtained in step S2, wherein the developing agent comprises ethyl acetate, acetone, formic acid, and water in a volume ratio of (22-28): (12-18): (1-5): (2-7); S4. The thin layer plate obtained after the reaction in step S3 is developed under 250-260 nm ultraviolet light.

2. The method according to claim 1, characterized in that In step S1, the sample to be tested is Qingkailing oral liquid, and the preparation method of the ethyl acetate extract of the sample to be tested is: fully mix Qingkailing oral liquid, dilute hydrochloric acid and ethyl acetate in a volume ratio of (10-20): (0.5-1): (20-60) and extract.

3. The method according to claim 1, characterized in that In step S1, the sample to be tested is Qingkailing granules, and the preparation method of the ethyl acetate extract of the sample to be tested is: Qingkailing granules, water and ethyl acetate are fully mixed and extracted according to a mass volume ratio of (7-11) g: (35-45) mL: (35-45) mL.

4. The method according to claim 1, wherein In step S1, the volume concentration of the methanol solution is 65-75%.

5. The method according to claim 1, wherein In step S1, the working concentration of the reference solution is 0.5 to 2.5 mg / mL.

6. The method according to claim 1, characterized in that In step S2, a control medicinal material solution is also added to the thin layer plate; the control medicinal material solution includes a honeysuckle control medicinal material solution and a gardenia control medicinal material solution.

7. Use of the method according to any one of claims 1 to 6 in component analysis and / or quality control of Qingkailing preparations.

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