Thin-layer identification method for trichosanthes kirilowii maxim and burdock soup

Abstract

The invention discloses a thin-layer identification method of trichosanthes kirilowii maxim and burdock soup, and belongs to the technical field of pharmaceutical analysis. The thin-layer identification method for the trichosanthes kirilowii maxim and burdock soup, which takes the reference extract as the reference substance solution, is used for detecting adulteration or feeding conditions of multiple medicinal materials in the trichosanthes kirilowii maxim and burdock soup, the method is simple, the cost is low, the result is accurate, the identification process of taking a traditional single standard substance reference solution as the reference substance solution can be simplified, and the identification efficiency is improved. The medication safety of the trichosanthes kirilowii and burdock soup can be effectively ensured, and a foundation is laid for establishing the quality standard of the trichosanthes kirilowii and burdock soup.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and in particular to a thin-layer identification method for Gualou Niubang Decoction. Background Art

[0002] Thin-layer chromatography is a rapid, simple, efficient, economical and widely used chromatographic analysis method for quickly separating and qualitatively analyzing a small amount of substances, and plays an important role in qualitative identification, semi-quantitative and quantitative analysis. Gualou Niubang Decoction is composed of 12 herbs, namely tangerine peel, burdock fruit, gardenia, honeysuckle flower, liquorice root, snakegourd seed, scutellaria root, trichosanthes root, forsythia fruit, Chinese honey locust spine, bupleurum root, and green tangerine peel. It has the effects of soothing the liver and relieving depression, detoxifying and dissipating carbuncles, and is mainly used for treating breast abscess, breast gangrene and other diseases. Modern Chinese medicine clinics mainly use it to treat acute mastitis and other diseases, and have significant curative effects on the treatment of its early inflammatory stage.

[0003] Gualou Niubang Decoction has a long application history. However, as a traditional Chinese medicine decoction prepared by decocting, it contains many water-soluble components, and the impurities interfere with each other seriously, making the identification technology difficult. The Identification Method is not described in Part I of the Chinese Pharmacopoeia 2020 Edition, resulting in imperfect quality control and quality evaluation methods, and being unable to effectively detect problems such as drug adulteration, shortage of ingredients, and leakage of ingredients. In order to ensure the safety of medication, under the current trend of the modernization of traditional Chinese medicine, it is urgent to establish a complete thin-layer chromatography detection method for Gualou Niubang Decoction. Summary of the Invention

[0004] The purpose of the present invention is to provide a thin-layer identification method for Gualou Niubang Decoction to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides an application of a reference extract of Gualou Niubang Decoction in the thin-layer identification of Gualou Niubang Decoction or Trichosanthes kirilowii Maxim. seeds in Gualou Niubang Decoction. The preparation method of the reference extract of Gualou Niubang Decoction is as follows: Load resin SEPABEADS SP825L into a column, and rinse with 95% ethanol and water for 6 column volumes in sequence, shake well until the water level is flush with the resin surface, take the freeze-dried powder of Gualou Niubang Decoction, ultrasonically treat it after adding water, load the sample, rinse with water for 2 column volumes, let it stand for adsorption overnight, rinse with water for 1 column volume the next day, rinse with 80% ethanol for 3 column volumes, collect the eluate, and freeze-dry to obtain the reference extract.

[0007] A thin-layer identification method for Gualou Niubang Decoction includes the following steps:

[0008] (1) Preparation of the test solution: Weigh the freeze-dried powder of Gualou Niubang Decoction, add methanol, ultrasonically treat it, filter, and take the filtrate as the test solution;

[0009] (2) Preparation of the reference substance solution;

[0010] The reference substance solution is the reference extract solution of Trichosanthis and Arctii Decoction or the reference solution of the reference standard;

[0011] (3) Spot the test solution and the reference substance solution on the same silica gel GF 254 thin layer plate, using ethyl acetate - formic acid - glacial acetic acid - water with a volume ratio of 15:1:1:2 as the developing agent, develop, take out, dry in air, and examine under ultraviolet light.

[0012] Furthermore, the preparation method of the test solution is: weigh 0.25 g of the freeze - dried powder of Trichosanthis and Arctii Decoction, add 15 mL of methanol, ultrasonically treat at 350 W and 53 kHz for 30 min, filter, and take the filtrate as the test solution.

[0013] Furthermore, the preparation method of the reference extract solution of Trichosanthis and Arctii Decoction is: add water to the formula of Trichosanthis and Arctii Decoction for decoction and then freeze - dry to obtain the freeze - dried powder of the combined decoction of Trichosanthis and Arctii Decoction. Take SEPABEADS SP825L resin to fill the column, and rinse with 95% ethanol and water for 6 column volumes (1200 mL) in sequence, shake well until the water level is flush with the resin surface. Take 3 g of the freeze - dried powder of the combined decoction of Trichosanthis and Arctii Decoction, add 20 mL of water and ultrasonically treat for 20 minutes, load the sample, rinse with water for 2 column volumes (400 mL), let it stand for adsorption overnight, rinse with water for 1 column volume the next day, rinse with 80% ethanol for 3 column volumes, collect the eluate, and freeze - dry to obtain the reference extract; take 0.25 g of the reference extract, add 15 mL of methanol, ultrasonically treat at 350 W and 53 kHz for 30 min, filter, and take the filtrate as the reference extract solution;

[0014] The reference standard solution includes the reference standard solution of geniposide, hesperidin, arctiin, baicalin, oroxylin A - 7 - O - β - D - glucuronide, and wogonoside with methanol as the solvent.

[0015] Furthermore, the spotting volume of the test solution is 7 μL, and the spotting volume of the reference substance solution is 3 μL; the wavelength of the ultraviolet light is 254 nm.

[0016] The present invention provides a thin - layer identification method for Trichosanthes kirilowii Maxim. in Trichosanthis and Arctii Decoction, including the following steps:

[0017] (1) Preparation of the test solution: weigh the freeze - dried powder of Trichosanthis and Arctii Decoction, add petroleum ether, ultrasonically treat, filter, and take the filtrate as the test solution; weigh the freeze - dried powder of the aqueous decoction of Trichosanthes kirilowii Maxim. alone, add petroleum ether, ultrasonically treat, filter, and the filtrate is used as the solution of Trichosanthes kirilowii Maxim. alone;

[0018] (2) Preparation of the control extract solution: Weigh the control extract of Gualou Niubang Decoction, add petroleum ether, perform ultrasonic treatment, filter, and take the filtrate as the control extract solution;

[0019] (3) Spot the test solution and the control extract solution on the same silica gel GF 254 thin layer plate, use cyclohexane - ethyl acetate with a volume ratio of 5:1 as the developing agent, develop, take out, dry in air, and examine under ultraviolet light.

[0020] Furthermore, the preparation method of the test solution is as follows: Weigh 0.1 g of the freeze - dried powder of the combined decoction of Gualou Niubang Decoction, add 0.8 mL of petroleum ether, perform ultrasonic treatment at 350 W and 53 kHz for 10 min, filter, and take the filtrate as the test solution.

[0021] Furthermore, the preparation method of the control extract solution is as follows: Take the formula of Gualou Niubang Decoction, decoct it with water and then freeze - dry to obtain the freeze - dried powder of the combined decoction of Gualou Niubang Decoction. Load the resin SEPABEADS SP825L into a column, rinse it successively with 95% ethanol and water for 6 column volumes (1200 mL), shake well until the water level is flush with the resin surface. Take 3 g of the freeze - dried powder of the combined decoction of Gualou Niubang Decoction, add 20 mL of water, then perform ultrasonic treatment for 20 minutes, load the sample, rinse with water for 2 column volumes (400 mL), let it stand for adsorption overnight, rinse with water for 1 column volume the next day, rinse with 80% ethanol for 3 column volumes, collect the eluate, and freeze - dry to obtain the control extract;

[0022] Take 0.1 g of the control extract of Gualou Niubang Decoction, add 0.8 mL of petroleum ether, perform ultrasonic treatment at 350 W and 53 kHz for 10 min, filter, and take the filtrate as the control extract solution.

[0023] Furthermore, the spotting volume of the test solution is 3 μL, and the spotting volume of the control extract solution is 3 μL; the wavelength of the ultraviolet light is 254 nm and 365 nm.

[0024] The present invention also provides the application of the above - mentioned thin - layer identification method in the quality detection or quality evaluation of Gualou Niubang Decoction.

[0025] The present invention discloses the following technical effects:

[0026] The present invention provides a method for qualitatively detecting the drug components in the traditional decoction Gualou Niubang Decoction by using thin - layer chromatography technology. The present invention prepares a control extract of Gualou Niubang Decoction. The preparation method of this control extract is simple, the application is convenient, the spot position is determined, it can be used for the thin - layer identification of multiple components in Gualou Niubang Decoction, with strong specificity, good durability, and no interference from negative controls.

[0027] The present invention establishes a thin-layer chromatography identification method for Trichosanthis and Arctii Decoction using a control extract as a reference solution, which is used to detect the adulteration or ingredient addition of multiple medicinal materials in Trichosanthis and Arctii Decoction. The method is simple, low-cost, and accurate in results. It can simplify the identification process of using a traditional single reference standard solution as a reference solution, effectively ensure the medication safety of Trichosanthis and Arctii Decoction, and lay a foundation for the establishment of the quality standard of Trichosanthis and Arctii Decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0029] Figure 1 TLC diagrams of different dissolution solvents for Example 1; among them, the left is ultraviolet color development at 254 nm; the right is ultraviolet color development at 365 nm; the bands A - E are test samples prepared with absolute ethanol, 95% ethanol, 80% ethanol, methanol, and 80% methanol in sequence; the bands 1 - 7 are wogonoside, oroxylin A, liquiritin, baicalin, arctiin, geniposide, and hesperidin in sequence;

[0030] Figure 2 TLC diagrams of different color development methods for Example 1; among them, the left is ultraviolet color development at 254 nm; the right is ultraviolet color development at 365 nm; the bands A - B are test samples prepared with pure methanol and 80% methanol in sequence; the band C is the reference extract; the bands 1 - 7 are wogonoside, oroxylin A, liquiritin, baicalin, arctiin, geniposide, and hesperidin in sequence;

[0031] Figure 3 TLC diagrams of different developing agent systems for Example 1; among them, (1) is ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2); (2) is ethyl acetate - formic acid - glacial acetic acid - water (10:1:1:2); (3) is ethyl acetate - formic acid - water (15:2:2); (4) is toluene - ethyl acetate - methanol - formic acid (10:3:1:2); (5) is dichloromethane - methanol - water (20:4:0.5); the bands A - B are test samples prepared with pure methanol and 80% methanol in sequence; the band C is the reference extract; the bands 1 - 7 are wogonoside, oroxylin A, liquiritin, baicalin, arctiin, geniposide, and hesperidin in sequence;

[0032] Figure 4TLC chromatogram for the finger identification of Trichosanthis and Arctii Decoction in Example 1; among them, (1) is UV visualization at 254 nm; (2) is UV visualization at 365 nm; band A is the control extract; band B is the test solution; bands 1-6 are baicalin, wogonoside, oroxylin A-7-O-β-D-glucuronide, arctiin, geniposide, and hesperidin in sequence;

[0033] Figure 5 TLC chromatogram for different sample application volumes in Example 1; among them, bands 1-3 are the control extract with sample application volumes of 2 μL, 3 μL, and 5 μL in sequence; bands 4-10 are the test samples with sample application volumes of 3 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, and 10 μL in sequence;

[0034] Figure 6 TLC chromatogram for different temperature and humidity conditions in Example 1; among them, (1) temperature is 25 °C and humidity is 80%; (2) temperature is 25 °C and humidity is 50%; (3) temperature is 25 °C and humidity is 30%; (4) temperature is 35 °C and humidity is 50%; (5) temperature is 25 °C and humidity is 50%; (6) temperature is 15 °C and humidity is 50%; band A is the control extract; band B is the test solution;

[0035] Figure 7 GF of different manufacturers in Example 1 254 TLC chromatogram of TLC plates; among them, (1) is Yantai Yinlong; (2) is Merck, Germany; (3) is MN, Germany; band A is the test sample; band B is the control extract;

[0036] Figure 8 TLC chromatogram for the specificity investigation in Example 1; among them, band A is the test sample; band B is the control extract; band 1 is the single herb of Gardenia jasminoides; band 2 is the negative sample of Gardenia jasminoides; band 3 is the single herb of Glycyrrhiza uralensis; band 4 is the negative sample of Glycyrrhiza uralensis; band 5 is the single herb of Citrus reticulata Blanco; band 6 is the negative sample of Citrus reticulata Blanco; band 7 is the single herb of Citrus reticulata Blanco var. austera; band 8 is the negative sample of Citrus reticulata Blanco var. austera; band 9 is the single herb of Arctium lappa; band 10 is the negative sample of Arctium lappa; band 11 is Scutellaria baicalensis; band 12 is the negative sample of Scutellaria baicalensis; band 13 is the single herb of Bupleurum chinense; band 14 is the negative sample of Bupleurum chinense; band 15 is the single herb of Lonicera japonica; band 16 is the negative sample of Lonicera japonica; band 17 is the single herb of Gleditsia sinensis Lam.; band 18 is the negative sample of Gleditsia sinensis Lam.; band 19 is the single herb of Trichosanthes kirilowii Maxim.; band 20 is the negative sample of Trichosanthes kirilowii Maxim.; band 21 is the single herb of Trichosanthes kirilowii Maxim. seeds; band 22 is the negative sample of Trichosanthes kirilowii Maxim. seeds; band 23 is the single herb of Forsythia suspense; band 24 is the negative sample of Forsythia suspense;

[0037] Figure 9 TLC chromatogram of 15 batches of Trichosanthis and Arctii Decoction samples in Example 1; among them, band A is the control extract; bands 1-15 are the test samples of 15 batches of Trichosanthis and Arctii Decoction;

[0038] Figure 10 TLC diagrams of different dissolution solvents in Example 2; among them, the left is the ultraviolet color development at 254 nm; the right is the ultraviolet color development at 365 nm; the bands 1-5 are the reference substance solutions prepared with petroleum ether, ethanol, methanol, ethyl acetate, and 95% ethanol in sequence;

[0039] Figure 11 TLC diagrams of different developing agent systems in Example 2 (ultraviolet color development at 254 nm); among them, (1) ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2); (2) cyclohexane - ethyl acetate (5:1); (3) petroleum ether - ethyl acetate (5:1); (4) toluene - ethyl acetate (5:1); the bands 1-4 are the test samples of Gualou Niubang Decoction, the control medicinal material of Trichosanthes kirilowii Maxim seeds, the negative sample without Trichosanthes kirilowii Maxim seeds, and the reference extract respectively;

[0040] Figure 12 TLC diagrams of different developing agent systems in Example 2 (ultraviolet color development at 365 nm); among them, (1) ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2); (2) cyclohexane - ethyl acetate (5:1); (3) petroleum ether - ethyl acetate (5:1); (4) toluene - ethyl acetate (5:1); the bands 1-4 are the test samples of Gualou Niubang Decoction, the control medicinal material of Trichosanthes kirilowii Maxim seeds, the negative sample without Trichosanthes kirilowii Maxim seeds, and the reference extract respectively;

[0041] Figure 13 TLC diagrams of different temperature and humidity conditions in Example 2; among them, (1) temperature 25°C, humidity 80%; (2) temperature 25°C, humidity 50%; (3) temperature 25°C, humidity 30%; (4) temperature 35°C, humidity 50%; (5) temperature 25°C, humidity 50%; (6) temperature 15°C, humidity 50%; the bands 1-3 are the control medicinal material of Trichosanthes kirilowii Maxim seeds, the test samples of Gualou Niubang Decoction, and the reference extract respectively;

[0042] Figure 14 GF of different manufacturers in Example 2 254 TLC diagrams of thin layer plates; among them, (1) is Yantai Yinlong; (2) is Merck of Germany; (3) is MN of Germany; the bands 1-3 are the single herb of Trichosanthes kirilowii Maxim seeds, the test samples of Gualou Niubang Decoction, and the reference extract respectively;

[0043] Figure 15 TCL diagrams of Trichosanthes kirilowii Maxim seeds in 15 batches of Gualou Niubang Decoction samples in Example 2; among them, the bands 1-15 are the test samples of 15 batches of Gualou Niubang Decoction; the band 16 is the reference extract. Detailed implementation methods

[0044] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0048] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0049] Example 1

[0050] 1. Instruments and Materials

[0051] FW-100 High-Speed Pulverizer (Tianjin Test Instrument Co., Ltd.); SK7210HP Ultrasonic Cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); ZF-90 Multifunctional Dark-Box Ultraviolet Transilluminator (Shanghai Baoshan Gucun Electro-Optical Instrument Factory); Drug Stability Test Chamber (Model IMT-150L, Shiduokai Instrument and Equipment Co., Ltd.). Glass spotting capillary (Specification: inner diameter 0.3 mm, length 100 mm; Manufacturer: Beijing Ruizekang Technology Co., Ltd.). Thin-layer chromatography double-tank developing chamber (Model: P-1, 100*100 mm, 200*100 mm; Manufacturer: Shanghai Xinyi Instrument Factory Co., Ltd.); Silver Dragon silica gel GF 254Glass plates (specification: 200*100mm; batch number: 20241127; manufacturer: Yantai Chemical Industry Research Institute); German MN-HPTLC-20UV254 glass plates (specification: 200*100mm; manufacturer: Macherey-Nagel GmbH, Germany); German Merck GF 254 Aluminum plates (specification: 200*100mm; manufacturer: Merck KGaA, Germany); Analytical glass columns (inner diameter 4.9 cm, column length 50 cm); Imported resin S: SEPABEADS SP825L (Mitsubishi), purchased from Beijing Greenherbs Technology Development Co., Ltd.; Domestic resin H: HPD-600, domestic resin A: ADS-17; All purchased from Cangzhou Baen Adsorbent Materials Technology Co., Ltd.

[0052] Geniposide (110749-202320, 98.1%), Hesperidin (110721-202220, 97.2%), Arctiin (110819-202414, 96.6%), Baicalin (110715-202223, 97.2%), Liquiritin (111610-202209, 95.2%) were all purchased from the National Institutes for Food and Drug Control; Oroxylin A-7-O-β-D-glucuronide (23111396, 98%), Wogonoside (23080601, 98%) were all purchased from Beijing Beite Renkang Biomedical Technology Co., Ltd.; Single herbs and whole formula samples of Gualou Niubang Decoction; Methanol, ethyl acetate, formic acid, glacial acetic acid and petroleum ether (60-90°C), etc. were all of analytical grade; Wahaha purified water.

[0053] 2. Establishment of thin-layer identification method

[0054] 2.1 Preparation of test solution

[0055] Take 3.73 g of dried tangerine peel, 3.73 g of great burdock achene, 3.73 g of gardenia fruit, 3.73 g of honeysuckle flower, 3.73 g of liquorice root, 3.73 g of snakegourd seed, 3.73 g of baical skullcap root, 3.73 g of trichosanthes root, 3.73 g of forsythia fruit, 3.73 g of Chinese honeylocust spine, 1.85 g of bupleurum root and 1.85 g of green tangerine peel, decoct with 400 mL of water until 160 mL remains, and freeze-dry to obtain the combined decoction freeze-dried powder of Gualou Niubang Decoction.

[0056] Take 0.25 g of the combined decoction freeze-dried powder of Gualou Niubang Decoction, add 15 mL of methanol, ultrasonicate (350 W, 53 kHz) for 30 min, filter, and use the filtrate as the test solution.

[0057] 2.2 Preparation of reference extract

[0058] Take 200 mL of SEPABEADS SP825L resin and pack it into a column. Rinse the column with 95% ethanol for 6 column volumes (1200 mL), then rinse it with water for 6 column volumes. Shake well and wait for the resin to settle naturally until the volume remains unchanged. When the water level is flush with the resin surface, take 3 g of the co-boiled freeze-dried powder of Trichosanthis Fruit and Arctii Fruit Decoction, add 20 mL of water, and ultrasonicate for 20 min until completely dissolved. Load the sample, rinse with water for 2 column volumes (400 mL), let it stand for adsorption overnight. The next day, rinse with water for 1 column volume, then rinse with 80% ethanol for 3 column volumes. Collect the eluate, freeze-dry it to prepare the reference extract.

[0059] Take 0.25 g of the freeze-dried powder of the reference extract, add 15 mL of methanol, ultrasonicate (350 W, 53 kHz) for 30 min, filter, and the filtrate is the reference extract solution.

[0060] 2.3 Preparation of the standard control solution

[0061] Precisely weigh 0.53 mg of geniposide, 1.04 mg of hesperidin, 1.03 mg of arctiin, 0.51 mg of baicalin, 0.52 mg of oroxylin A-7-O-β-D-glucuronide, 0.50 mg of wogonoside, and 0.66 mg of liquiritin. Dissolve them separately in a 5 mL volumetric flask with methanol and ultrasonicate until completely dissolved. Pass through a 0.22 μm microporous membrane to obtain standard control solutions with concentrations of 106 μg / mL, 208 μg / mL, 206 μg / mL, 102 μg / mL, 104 μg / mL, 100 μg / mL, and 132 μg / mL respectively.

[0062] 2.4 Preparation of single-herb and negative sample solutions

[0063] Take 41 g of Gleditsiae Spina, Trichosanthis Semen, Trichosanthis Radix, Forsythiae Fructus, Bupleuri Radix, Gardeniae Fructus, Arctii Fructus, Scutellariae Radix, Lonicerae Japonicae Flos, Glycyrrhizae Radix et Rhizoma, Citri Reticulatae Viride Pericarpium, and Citri Reticulatae Pericarpium respectively. Decoct with 400 mL of water until the volume is reduced to 160 mL, and then freeze-dry to obtain the freeze-dried powder of the single-herb decoction.

[0064] According to the preparation method of the co-boiled freeze-dried powder of Trichosanthis Fruit and Arctii Fruit Decoction in "2.1 Preparation of the test solution", prepare the negative freeze-dried powder without the medicinal herb.

[0065] Precisely weigh an appropriate amount of the freeze-dried powder of the single-herb decoction of Gleditsiae Spina, Trichosanthis Semen, Trichosanthis Radix, Forsythiae Fructus, Bupleuri Radix, Gardeniae Fructus, Arctii Fructus, Scutellariae Radix, Lonicerae Japonicae Flos, Glycyrrhizae Radix et Rhizoma, Citri Reticulatae Viride Pericarpium, and Citri Reticulatae Pericarpium respectively. Dissolve them separately in 5 mL of methanol, ultrasonicate until completely dissolved, and pass through a 0.22 μm microporous membrane to obtain single-herb solutions with a concentration of 10 μg / mL.

[0066] Accurately weigh appropriate amount of negative freeze-dried powder sample lacking Gleditsia sinensis spines, negative freeze-dried powder sample lacking Trichosanthes kirilowii seeds, negative freeze-dried powder sample lacking Trichosanthes kirilowii seeds, negative freeze-dried powder sample lacking Forsythia suspensa, negative freeze-dried powder sample lacking Bupleurum chinense, negative freeze-dried powder sample lacking Gardenia jasminoides, negative freeze-dried powder sample lacking Arctium lappa, negative freeze-dried powder sample lacking Scutellaria baicalensis, negative freeze-dried powder sample lacking Honeysuckle, negative freeze-dried powder sample lacking Licorice, negative freeze-dried powder sample lacking Citrus aurantium, and negative freeze-dried powder sample lacking Tangerine peel, and dissolve them respectively with 15 mL of methanol, ultrasonicate until completely dissolved, pass through a 0.22 microporous filter membrane to obtain a negative sample solution with a concentration of 16.7 μg / mL.

[0067] 2.5 Thin layer identification operation

[0068] 5 μL of the test solution, reference solution and control extract solution were taken and spotted on the same silica gel GF 254 The thin layer plate was developed using dichloromethane-methanol-water (40:8:1) as the developing agent, taken out, dried, and examined under ultraviolet light (254 nm).

[0069] 3. Optimization of dissolution extraction

[0070] The extraction effects of the following five different solvents on the components of the sample of Gualou Niubang Decoction were compared: ethanol; 95% ethanol; 80% ethanol; methanol; 80% methanol. Figure 1 As shown. When examined at 254nm and 365nm, it can be seen that methanol and 80% methanol are clearly visible in the thin layer spots, and there is no obvious difference between the two; on the same thin layer plate, the separation effect of components extracted with methanol is better than that extracted with ethanol, and the spots are also more clearly visible. Therefore, methanol is selected as the final extraction solvent.

[0071] 4. Comparison of color development methods

[0072] Compare the following two color development methods: 254nm UV color development and 365nm UV color development. The results are as follows Figure 2 As shown in the figure, 254nm color development is clearer and more sensitive, so 254nm ultraviolet color development is selected.

[0073] 5. Expand system selection

[0074] The following five development systems were compared: (1) ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2); (2) ethyl acetate-formic acid-glacial acetic acid-water (10:1:1:2); (3) ethyl acetate-formic acid-water (15:2:2); (4) toluene-ethyl acetate-methanol-formic acid (10:3:1:2); (5) dichloromethane-methanol-water (20:4:0.5).

[0075] The results are as follows Figure 3As shown. Clear spots can be obtained with all 5 developing agent systems. Among them, the Rf values of (4) and (5) are on the low side, the separation effect of the test solution is poor, the spots are not clear, the spots of the reference solution cannot correspond one by one with the test solution, and there is an obvious trailing phenomenon; compared with (1) and (2)(3), the Rf values are reasonably distributed (0.35 - 0.60), each spot is clear, the shape and size are appropriate, the trailing phenomenon is not obvious, and clear spots exist at the corresponding positions of most of the reference substances and the test solution, which can characteristically identify the components in Trichosanthis and Arctii Decoction. Therefore, the developing agent was finally determined to be ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2). f value is on the low side, the separation effect of the test solution is poor, the spots are not clear, the spots of the reference solution cannot correspond one by one with the test solution, and there is an obvious trailing phenomenon; compared with (1) and (2)(3), the R f f values are reasonably distributed (0.35 - 0.60), each spot is clear, the shape and size are appropriate, the trailing phenomenon is not obvious, and clear spots exist at the corresponding positions of most of the reference substances and the test solution, which can characteristically identify the components in Trichosanthis and Arctii Decoction. Therefore, the developing agent was finally determined to be ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2).

[0076] 6. Identification by thin - layer chromatography

[0077] Spot the reference solutions of 6 reference substances including geniposide, hesperidin, arctiin, baicalin, oroxylin A - 7 - O - β - D - glucuronide, and wogonoside, the reference extract, and the test solution on the same silica gel GF 254 thin - layer plate and compare the separation of the spots. The results are as Figure 4 shown. Under ultraviolet light at 254 nm, it can be clearly identified that Trichosanthis and Arctii Decoction contains baicalin, wogonoside, oroxylin A - 7 - O - β - D - glucuronide, geniposide, arctiin, and hesperidin; and the spots separated from the reference extract and the test solution correspond one by one, and the spots of the reference extract are clearer. Under ultraviolet light at 365 nm, baicalin, wogonoside, oroxylin A - 7 - O - β - D - glucuronide, and hesperidin can also be clearly identified. This indicates that the reference extract can be used as a reference chromatogram for Trichosanthis and Arctii Decoction to comprehensively identify and evaluate the quality of Trichosanthis and Arctii Decoction.

[0078] 7. Determination of the spotting volume

[0079] Compare different spotting volumes: the spotting volumes of the reference extract solution are 2, 3, and 5 μL; the spotting volumes of the test solution are 3, 5, 6, 7, 8, 9, and 10 μL. The results are as Figure 5 shown. When the spotting volume of the reference extract is 3 μL and the spotting volume of the test solution is 6 - 7 μL, the spots are clearly visible.

[0080] 8. Durability investigation

[0081] 8.1 Investigation under different temperature and humidity

[0082] A comparative investigation was carried out on different temperatures and humidities. The conditions are as follows: (1) temperature 25 °C, humidity 80%; (2) temperature 25 °C, humidity 50%; (3) temperature 25 °C, humidity 30%; (4) temperature 35 °C, humidity 50%; (5) temperature 25 °C, humidity 50%; (6) temperature 15 °C, humidity 50%. The results are asFigure 6 As shown, at different temperatures and humidities, the thin-layer chromatography spots are relatively clear, indicating that this method has good durability.

[0083] 8.2 GF from different manufacturers 254 Investigation of thin-layer plates

[0084] Comparison of GF from different manufacturers 254 Thin-layer plates: Yantai Yinlong, Merck of Germany, and MN of Germany. The results are as Figure 7 shown. The Rf values of the GF thin-layer plates of Yantai Yinlong, Merck of Germany, and MN of Germany are basically the same, and the chromatography spots are relatively clear. Generally speaking, the GF thin-layer plates of the three manufacturers can all meet the requirements of TLC identification, indicating that the durability of this method is good. 254 value of the thin-layer plate f is basically the same, and the chromatography spots are relatively clear. Generally speaking, the GF thin-layer plates of the three manufacturers can all meet the requirements of TLC identification, indicating that the durability of this method is good. 254 thin-layer plates can all meet the requirements of TLC identification, indicating that the durability of this method is good.

[0085] 9. Specificity investigation

[0086] 12 single herbs contained in Gualou Niubang Decoction and their respective negative sample solutions were spotted (7 μL) on silica gel GF thin-layer plates, developed with ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2) as the developing agent, developed, taken out and dried, and inspected under ultraviolet light. The results are as 254 shown. Figure 8 shown.

[0087] For Gardenia jasminoides Ellis medicinal materials, dark blue spots can be seen at positions with an Rf value of approximately 0.28 when inspected at ultraviolet 254 nm and 365 nm, and there are no corresponding spots on the thin-layer plate for the Gardenia jasminoides Ellis negative sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that Gualou Niubang Decoction contains Gardenia jasminoides Ellis medicinal materials. f value of approximately 0.28, and there are no corresponding spots on the thin-layer plate for the Gardenia jasminoides Ellis negative sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that Gualou Niubang Decoction contains Gardenia jasminoides Ellis medicinal materials.

[0088] For Glycyrrhiza uralensis Fisch. medicinal materials, dark blue spots can be seen at positions with an Rf value of approximately 0.17 - 0.47 when inspected at ultraviolet 254 nm and 365 nm, and there are no corresponding spots for the Glycyrrhiza uralensis Fisch. negative sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that Gualou Niubang Decoction contains Glycyrrhiza uralensis Fisch. medicinal materials. f value of approximately 0.17 - 0.47, and there are no corresponding spots for the Glycyrrhiza uralensis Fisch. negative sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that Gualou Niubang Decoction contains Glycyrrhiza uralensis Fisch. medicinal materials.

[0089] For Citrus reticulata Blanco medicinal materials, no dark spots are shown at ultraviolet 254 nm, and dark blue fluorescence can be seen at a position with an Rf value of approximately 0.38 when inspected at 365 nm, and there are no corresponding spots on the thin-layer plate for the Citrus reticulata Blanco negative sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that Gualou Niubang Decoction contains Citrus reticulata Blanco medicinal materials. f value of approximately 0.38, and there are no corresponding spots on the thin-layer plate for the Citrus reticulata Blanco negative sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that Gualou Niubang Decoction contains Citrus reticulata Blanco medicinal materials.

[0090] The green tangerine peel medicinal material does not show dark spots under ultraviolet light at 254 nm, and under inspection at 365 nm, a dark blue fluorescence can be seen at the position where the R value is approximately 0.93. There are no corresponding spots on the thin-layer plate for the negative sample solution of green tangerine peel, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains green tangerine peel medicinal material. f At the position where the R value is about 0.93, there is a dark blue fluorescence. There are no corresponding spots on the thin-layer plate for the negative sample solution of green tangerine peel, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains green tangerine peel medicinal material.

[0091] For the Arctii Fructus medicinal material, under inspection at ultraviolet 254 nm and 365 nm, dark blue spots can be seen at the positions where the R value is approximately 0.33 - 0.80. These correspond to the spots of the negative sample solution of Arctii Fructus in ultraviolet 365 nm. The polarity of arctiin in Arctii Fructus is similar to that of oroxylin A - 7 - O - β - D - glucuronide, and its interference may come from the Scutellariae Radix medicinal material. f For the Arctii Fructus medicinal material, under inspection at ultraviolet 254 nm and 365 nm, dark blue spots can be seen at the positions where the R value is approximately 0.33 - 0.80. These correspond to the spots of the negative sample solution of Arctii Fructus in ultraviolet 365 nm. The polarity of arctiin in Arctii Fructus is similar to that of oroxylin A - 7 - O - β - D - glucuronide, and its interference may come from the Scutellariae Radix medicinal material.

[0092] For the Scutellariae Radix medicinal material, under inspection at ultraviolet 254 nm and 365 nm, dark blue spots can be seen at the positions where the R value is approximately 0.20 - 0.43. There are no corresponding spots on the thin-layer plate for the negative sample solution of Scutellariae Radix, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains Scutellariae Radix medicinal material. f For the Scutellariae Radix medicinal material, under inspection at ultraviolet 254 nm and 365 nm, dark blue spots can be seen at the positions where the R value is approximately 0.20 - 0.43. There are no corresponding spots on the thin-layer plate for the negative sample solution of Scutellariae Radix, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains Scutellariae Radix medicinal material.

[0093] For the Bupleuri Radix medicinal material, there are no relatively clear spots under inspection at ultraviolet 254 nm and 365 nm.

[0094] For the Lonicerae Japonicae Flos medicinal material, under inspection at ultraviolet 254 nm and 365 nm, dark blue spots can be seen at the positions where the R value is approximately 0.24 - 0.80. There are no corresponding spots on the thin-layer plate for the negative sample solution of Lonicerae Japonicae Flos, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains Lonicerae Japonicae Flos medicinal material. f For the Lonicerae Japonicae Flos medicinal material, under inspection at ultraviolet 254 nm and 365 nm, dark blue spots can be seen at the positions where the R value is approximately 0.24 - 0.80. There are no corresponding spots on the thin-layer plate for the negative sample solution of Lonicerae Japonicae Flos, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains Lonicerae Japonicae Flos medicinal material.

[0095] For the Gleditsiae Spina medicinal material, it does not show dark spots under ultraviolet light at 254 nm, and under inspection at 365 nm, a dark blue fluorescence can be seen at the positions where the R value is approximately 0.28 - 0.88. There are no corresponding spots on the thin-layer plate for the negative sample solution of Gleditsiae Spina, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains Gleditsiae Spina medicinal material. f For the Gleditsiae Spina medicinal material, it does not show dark spots under ultraviolet light at 254 nm, and under inspection at 365 nm, a dark blue fluorescence can be seen at the positions where the R value is approximately 0.28 - 0.88. There are no corresponding spots on the thin-layer plate for the negative sample solution of Gleditsiae Spina, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains Gleditsiae Spina medicinal material.

[0096] For the Trichosanthis Radix medicinal material, it does not show dark spots under ultraviolet light at 254 nm, and under inspection at 365 nm, a dark blue fluorescence can be seen at the positions where the R value is approximately 0.28 - 0.88. There are no corresponding spots on the thin-layer plate for the negative sample solution of Trichosanthis Radix, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthis and Arctii Decoction contains Trichosanthis Radix medicinal material. fThere is dark blue fluorescence at the position with a value of approximately 0.16 - 0.32, and there are no corresponding spots on the thin layer plate for the negative Trichosanthes kirilowii Maxim. sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthes and Arctium Decoction contains Trichosanthes kirilowii Maxim. medicinal material.

[0097] There are no relatively clear spots for the Fructus Trichosanthis medicinal material under ultraviolet inspection at 254 nm and 365 nm.

[0098] For the Forsythia suspensa (Thunb.) Vahl. medicinal material, dark blue spots can be seen at the position with an Rf value of approximately 0.12 - 0.44 under ultraviolet inspection at 254 nm and 365 nm. f There are no corresponding spots on the thin layer plate for the negative Forsythia suspensa (Thunb.) Vahl. sample solution, indicating that the negative sample has no interference. By comparing the spot positions of the test solution, reference extract, and reference medicinal material, it is determined that the Trichosanthes and Arctium Decoction contains Forsythia suspensa (Thunb.) Vahl. medicinal material.

[0099] 11. Thin layer identification of samples

[0100] The finally determined thin layer identification method for the Trichosanthes and Arctium Decoction is as follows: Take 0.25 g of the freeze-dried powder of the Trichosanthes and Arctium Decoction, add 15 mL of methanol, ultrasonically treat (350 W, 53 kHz) for 30 minutes, filter, and take the filtrate as the test solution. Additionally, take 0.25 g of the reference extract of the Trichosanthes and Arctium Decoction and prepare a reference solution in the same manner. Pipette 7 μL of the test solution and 3 μL of the reference extract solution, and spot them on the same silica gel GF 254 thin layer plate. Use ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2) as the developing solvent, develop, take out, dry in air, and examine under an ultraviolet lamp (254 nm). If the spots at the corresponding positions in the test solution chromatogram and the reference extract chromatogram show the same color, it is judged that the Trichosanthes and Arctium Decoction is a qualified product.

[0101] Or, take 0.25 g of the freeze-dried powder of the Trichosanthes and Arctium Decoction, add 15 mL of methanol, ultrasonically treat (350 W, 53 kHz) for 30 minutes, filter, and take the filtrate as the test solution. Additionally, take reference substances of geniposide, hesperidin, arctiin, baicalin, oroxylin A - 7 - O - β - D - glucuronide, and wogonoside, dissolve them in 5 mL of methanol to prepare a standard reference solution. Pipette 7 μL of the test solution and 3 μL of the standard reference solution, and spot them on the same silica gel GF 254 thin layer plate. Use ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2) as the developing solvent, develop, take out, dry in air, and examine under an ultraviolet lamp (254 nm). If the spots at the corresponding positions in the test solution chromatogram and the standard reference solution chromatogram show the same color, it is judged that the Trichosanthes and Arctium Decoction contains Gardenia jasminoides Ellis, Citrus reticulata Blanco, Citrus reticulata Blanco var. austera Swingle, Arctium lappa L., and Scutellaria baicalensis Georgi.

[0102] Using the above determined TLC conditions and methods, 15 batches of Gualou Niubang decoction samples were subjected to TLC identification. Figure 9 As shown, the chromatogram of the sample of the Gualou Niubo Decoction shows the same spots as the chromatogram of the control extract at the corresponding positions. Therefore, the thin layer chromatography identification method established in the present invention can be used for the identification of Gualou Niubo Decoction.

[0103] Example 2 Establishment of TLC Identification Method for Fructus Trichosanthis in Fructus Trichosanthis and Burdock Decoction

[0104] 1. Sample solution preparation

[0105] 1.1 Preparation of test solution

[0106] Take 0.1 g of freeze-dried powder of Trichosanthes and Burdock Decoction (same as in Example 1), add 0.8 mL of petroleum ether, ultrasonicate (350 W, 53 kHz) for 10 min, filter, and use the filtrate as the test solution of the decoction.

[0107] Take 0.1 g of the lyophilized powder of the single herbal decoction of Trichosanthes fruit (same as in Example 1), add 0.8 mL of petroleum ether, ultrasonicate (350 W, 53 kHz) for 10 min, filter, and use the filtrate as the single herbal solution of Trichosanthes fruit.

[0108] 1.2 Preparation of control extract

[0109] Take 0.1 g of the reference extract of Gualou Niubang decoction (same as in Example 1), add 0.8 mL of petroleum ether, ultrasonicate (350 W, 53 kHz) for 10 min, filter, and the filtrate is the reference extract solution.

[0110] 1.3 Preparation of negative sample solution

[0111] Accurately weigh an appropriate amount of the freeze-dried powder sample of the negative water decoction of Trichosanthes kirilowii, dissolve it in 0.8 mL of petroleum ether, and sonicate until completely dissolved to obtain a negative sample solution with a concentration of 125 μg / mL.

[0112] 2. Thin layer identification operation

[0113] 3 μL of the test solution and the control extract solution, and 10 μL of the single herb solution of Trichosanthes fruit were taken and spotted on the same silica gel GF. 254 The thin layer plate was developed using cyclohexane-ethyl acetate (5:1) as the developing solvent, taken out, dried, and examined under ultraviolet light (254nm and 365nm).

[0114] 3. Optimization of sample extraction solvent

[0115] The extraction effects of the following five different solvents were compared: petroleum ether, anhydrous ethanol, methanol, ethyl acetate and 95% ethanol. Figure 10As shown, under the inspection at 254 nm and 365 nm, the Rf values of the thin-layer plates extracted with 5 different solvents are basically the same. However, the color development is clearer after extraction with petroleum ether. Therefore, petroleum ether is selected as the final extraction solvent. f The Rf value is basically the same, but the color development is clearer after extraction with petroleum ether. Therefore, petroleum ether is selected as the final extraction solvent.

[0116] 4. Selection of developing agent system

[0117] Compare the following 4 developing agent systems: (1) ethyl acetate - formic acid - glacial acetic acid - water (15:1:1:2); (2) cyclohexane - ethyl acetate (5:1); (3) petroleum ether - ethyl acetate (5:1); (4) toluene - ethyl acetate (5:1). The results are as Figure 11 and Figure 12 shown. Spots can be shown with all 4 developing agents. In (2), under the inspection at 254 nm of ultraviolet light, the spots are observed to be clear, with appropriate shape and size. The spots of the test sample are consistent with those of the reference extract, and it can be characteristically identified that Trichosanthes and Arctium Decoction contains Trichosanthes kirilowii Maxim. seeds. Under the inspection at 365 nm of ultraviolet light, fluorescence can be observed at the Rf value of 0.62. The Rf values of systems (1) and (4) are on the high side, the spots are not clear enough, and the separation effect is poor. In system (3), under the inspection at 365 nm of ultraviolet light, obvious fluorescence can be observed at the Rf value of 0.53. Both cyclohexane and petroleum ether are of low toxicity, but cyclohexane has lower volatility, reducing the risk of inhalation. Therefore, the final selected developing agent is cyclohexane - ethyl acetate (5:1). f For (2), under the inspection at 254 nm of ultraviolet light, the spots are observed to be clear, with appropriate shape and size. The spots of the test sample are consistent with those of the reference extract, and it can be characteristically identified that Trichosanthes and Arctium Decoction contains Trichosanthes kirilowii Maxim. seeds. Under the inspection at 365 nm of ultraviolet light, fluorescence can be observed at the Rf value of 0.62. The Rf values of systems (1) and (4) are on the high side, the spots are not clear enough, and the separation effect is poor. In system (3), under the inspection at 365 nm of ultraviolet light, obvious fluorescence can be observed at the Rf value of 0.53. Both cyclohexane and petroleum ether are of low toxicity, but cyclohexane has lower volatility, reducing the risk of inhalation. Therefore, the final selected developing agent is cyclohexane - ethyl acetate (5:1). f values are on the high side, the spots are not clear enough, and the separation effect is poor. In system (3), under the inspection at 365 nm of ultraviolet light, obvious fluorescence can be observed at the Rf value of 0.53. Both cyclohexane and petroleum ether are of low toxicity, but cyclohexane has lower volatility, reducing the risk of inhalation. Therefore, the final selected developing agent is cyclohexane - ethyl acetate (5:1). f In system (3), under the inspection at 365 nm of ultraviolet light, obvious fluorescence can be observed at the Rf value of 0.53. Both cyclohexane and petroleum ether are of low toxicity, but cyclohexane has lower volatility, reducing the risk of inhalation. Therefore, the final selected developing agent is cyclohexane - ethyl acetate (5:1).

[0118] 5. Investigation of different temperature and humidity

[0119] Investigate different temperatures and humidities, and the conditions are as follows: (1) temperature 25°C, humidity 80%; (2) temperature 25°C, humidity 50%; (3) temperature 25°C, humidity 30%; (4) temperature 35°C, humidity 50%; (5) temperature 25°C, humidity 50%; (6) temperature 15°C, humidity 50%. As Figure 13 shown, under different temperatures and humidities, the thin-layer chromatography spots are relatively clear, indicating that the method has good durability.

[0120] 6. Investigation of GF thin-layer plates from different manufacturers 254 The experiment investigated the influence of GF thin-layer plates from 3 brands, namely Yantai Yinlong, Merck of Germany, and MN of Germany, on the experimental results. The results are as

[0121] shown. The Rf values of the thin-layer plates from the three manufacturers are basically the same, the spots are clear, the separation effect is good, and all can meet the TLC identification requirements, indicating that the durability of this method is good. 254 The experiment investigated the influence of GF thin-layer plates from 3 brands, namely Yantai Yinlong, Merck of Germany, and MN of Germany, on the experimental results. The results are as Figure 14 shown. The Rf values of the thin-layer plates from the three manufacturers are basically the same, the spots are clear, the separation effect is good, and all can meet the TLC identification requirements, indicating that the durability of this method is good. f The Rf values of the thin-layer plates from the three manufacturers are basically the same, the spots are clear, the separation effect is good, and all can meet the TLC identification requirements, indicating that the durability of this method is good.

[0122] 7. Thin-layer Identification of Samples

[0123] The finally determined thin-layer identification method for Trichosanthis Semen in Trichosanthis and Arctii Decoction is as follows: Take 0.10 g of the freeze-dried powder of the combined decoction of Trichosanthis and Arctii Decoction, add 0.80 mL of petroleum ether, treat it by ultrasonic wave (350 W, 53 kHz) for 10 minutes, filter, and take the filtrate as the test solution. Additionally, take 0.10 g of the reference extract of Trichosanthis and Arctii Decoction and prepare a reference extract solution in the same manner. Pipette 3 μL of the test solution and 3 μL of the reference extract solution, and spot them on the same silica gel GF 254 thin-layer plate. Use cyclohexane-ethyl acetate (5:1) as the developing solvent, develop, take out, air dry, and examine under an ultraviolet lamp (254 nm and 365 nm). In the chromatogram of the test sample, at the corresponding positions of the chromatograms of Trichosanthis and Arctii Decoction and the reference extract, spots of the same color appear.

[0124] Using the determined thin-layer identification method, thin-layer identification was performed on 15 batches of Trichosanthis and Arctii Decoction samples. The results are as Figure 15 shown. As can be seen from the figure, at the corresponding positions of the chromatograms of Trichosanthis and Arctii Decoction samples and the reference extract, the same spots are shown, indicating that the thin-layer chromatography identification method established in this experiment can be used for the identification of Trichosanthis Semen in Trichosanthis and Arctii Decoction.

[0125] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An application of a reference extract of Gualou Niubang decoction in thin layer identification of Trichosanthes fruit in Gualou Niubang decoction or Gualou Niubang decoction, characterized in that: The preparation method of the control extract of trichosanthes and burdock soup is as follows: taking resin SEPABEADS SP825L to pack a column, washing 6 column volumes with 95% ethanol and water in sequence, shaking until the water surface is flush with the resin surface, taking the freeze-dried powder of trichosanthes and burdock soup, adding water and then ultrasonically treating it, loading the sample, washing it with water for 2 column volumes, standing it for adsorption overnight, washing it with water for 1 column volume the next day, washing it with 80% ethanol for 3 column volumes, collecting the eluate, and freeze-drying it to obtain the control extract.

2. A thin layer chromatography identification method for trichosanthes and burdock soup, characterized in that: The steps include: (1) Preparation of test solution: Weigh the freeze-dried powder of Gualou Niubang decoction, add methanol, ultrasonically treat, filter, and take the filtrate as the test solution; (2) Preparation of reference solution; The reference substance solution is a reference extract solution of Trichosanthes and Arctium Decoction or a standard reference solution; (3) Spot the test solution and the reference solution on the same silica gel GF 254 On the thin layer plate, use ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 15:1:1:2 as the developing agent, develop, take out, dry, and examine under ultraviolet light.

3. The thin layer identification method according to claim 1, characterized in that: The preparation method of the test solution is as follows: weigh 0.25 g of the freeze-dried powder of Trichosanthes and Burdock Decoction, add 15 mL of methanol, perform ultrasonic treatment at 350 W and 53 kHz for 30 min, filter, and take the filtrate as the test solution.

4. The thin layer identification method according to claim 1, characterized in that: The preparation method of the control extract solution of trichosanthes and burdock soup is as follows: take a resin SEPABEADS SP825L column, wash 6 column volumes with 95% ethanol and water in sequence, shake until the water surface is flush with the resin surface, take a freeze-dried powder of trichosanthes and burdock soup, add water and then ultrasonically treat it, load it, wash it with water for 2 column volumes, let it stand for adsorption overnight, wash it with water for 1 column volume the next day, wash it with 80% ethanol for 3 column volumes, collect the eluate, and freeze-dry it to obtain a control extract; take 0.25g of the control extract, add 15mL of methanol, ultrasonically treat it at 350W and 53kHz for 30min, filter it, and take the filtrate as the control extract solution; The standard reference solutions include a gardenia glycoside standard reference solution, a hesperidin standard reference solution, an arctiin standard reference solution, a baicalin standard reference solution, a melaleuca alternifolia A-7-O-β-D-glucuronide standard reference solution and a wogonin standard reference solution, all of which use methanol as a solvent.

5. The thin layer identification method according to claim 1, characterized in that: The spotting volume of the test solution was 7 μL, and the spotting volume of the reference solution was 3 μL; the wavelength of the ultraviolet light was 254 nm.

6. A thin layer chromatography identification method for Trichosanthes kirilowii seeds in Trichosanthes kirilowii and Burdock soup, characterized in that: The steps include: (1) Preparation of test solution: Weigh the lyophilized powder of Gualou Niubang decoction, add petroleum ether, ultrasonically treat, filter, and use the filtrate as the test solution; weigh the lyophilized powder of Gualou Zi single herb decoction, add petroleum ether, ultrasonically treat, filter, and use the filtrate as the Gualou Zi single herb solution; (2) Preparation of control extract solution: weigh the control extract of Gualou Niubang decoction, add petroleum ether, ultrasonically treat, filter, and take the filtrate as the control extract solution; (3) Spot the test solution and the control extract solution on the same silica gel GF 254 On the thin layer plate, use cyclohexane-ethyl acetate with a volume ratio of 5:1 as the developing agent, develop, take out, dry, and examine under ultraviolet light.

7. The thin layer identification method according to claim 6, characterized in that: The preparation method of the test solution is as follows: weigh 0.1 g of the freeze-dried powder of Trichosanthes and Burdock Decoction, add 0.8 mL of petroleum ether, perform ultrasonic treatment at 350 W and 53 kHz for 10 min, filter, and take the filtrate as the test solution.

8. The thin layer identification method according to claim 6, characterized in that: The preparation method of the control extract solution is as follows: taking resin SEPABEADS SP825L to load the column, washing 6 column volumes with 95% ethanol and water in sequence, shaking until the water level is flush with the resin surface, taking the freeze-dried powder of Gualou Niubang decoction, adding water and then ultrasonic treatment, loading the sample, washing with water for 2 column volumes, standing for adsorption overnight, washing with water for 1 column volume the next day, washing with 80% ethanol for 3 column volumes, collecting the eluate, and freeze-drying to obtain the control extract; Take 0.1 g of the control extract of Trichosanthes and Burdock Decoction, add 0.8 mL of petroleum ether, ultrasonically treat at 350 W, 53 kHz for 10 min, filter, and take the filtrate as the control extract solution.

9. The thin layer identification method according to claim 6, characterized in that: The spotting volume of the test solution is 3 μL, and the spotting volume of the control extract solution is 3 μL; the wavelength of the ultraviolet light is 254 nm and 365 nm.

10. Use of the thin layer identification method according to any one of claims 2 to 5 or the thin layer identification method according to any one of claims 6 to 9 in quality detection or quality evaluation of Trichosanthes and Burdock Decoction.

Images