A quality control method for a compound traditional Chinese medicine preparation containing rhubarb

Abstract

This invention belongs to the field of quality control technology, specifically relating to a quality control method for a compound traditional Chinese medicine preparation containing rhubarb. The quality control method of this invention uses thin-layer chromatography (TLC) to identify rhubarb, Viola yedoensis, Kochia scoparia, Cnidium monnieri, Coptis chinensis, or Dictamnus dasycarpus in a rhubarb antipruritic lotion. The method uses a developing solvent with a volume ratio of toluene, acetone, ethyl acetate, and formic acid of 8-10:1-3:1-2:0.1-2 to separate and extract the components to be analyzed from numerous effective drug components. While ensuring identification accuracy, this method allows for the identification of 4-6 traditional Chinese medicine components using a single TLC plate, exhibiting high stability and repeatability. This invention offers the technical advantages of requiring less sampling, simplifying quality testing, and greatly improving detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology, specifically relating to a quality control method for a compound traditional Chinese medicine preparation containing rhubarb. Background Technology

[0002] Quality control of traditional Chinese medicine (TCM) compound preparations is a core aspect of the modernization and standardization of TCM, and its accuracy and reliability directly affect the safety and efficacy of clinical medication. Thin-layer chromatography (TLC), a classic technique in the field of TCM identification, is widely used for the qualitative identification of TCM materials and their preparations due to its advantages of high separation efficiency, simple operation, low cost, and ability to achieve both separation and analysis. This method achieves component separation by utilizing the differences in adsorption-desorption between the stationary phase and the mobile phase in the sample, and combines this with colorimetric or ultraviolet visualization for characteristic identification, significantly improving the sensitivity and specificity of identification work. It has become an important technical means for the quality control of TCM.

[0003] Rhubarb Antipruritic Lotion, a typical compound traditional Chinese medicine preparation containing rhubarb, is composed of fourteen herbs including rhubarb, phellodendron bark, violet, coptis, scutellaria, dandelion, sophora flavescens, kochia fruit, dictamnus root bark, cnidium fruit, raspberry, motherwort, agrimony, and achyranthes root. It has the effects of clearing heat and drying dampness, resolving blood stasis and tonifying deficiency, and killing parasites and relieving itching. Clinically, it is mainly used to treat vulvar pruritus caused by damp-heat accumulation. Because this preparation is composed of multiple herbs and has a complex chemical composition (including anthraquinones, alkaloids, flavonoids, volatile oils, and other active ingredients), its quality control requires consideration of the identification of characteristic components of different herbs to ensure the stability and consistency of the preparation's components.

[0004] However, existing technologies still have significant shortcomings in the quality control of compound Chinese medicines containing rhubarb. On the one hand, traditional identification methods are mostly aimed at single medicinal materials (such as identifying only the anthraquinone components in rhubarb), lacking a simultaneous identification system for multiple medicinal materials such as Kochia scoparia, Cnidium monnieri, Viola yedoensis, Coptis chinensis, and Dictamnus dasycarpus, making it difficult to comprehensively reflect the overall quality of the compound. On the other hand, due to the significant differences in the polarity, volatility, and other physicochemical properties of the effective components of different medicinal materials (e.g., berberine in Coptis chinensis is a polar alkaloid, osthol in Cnidium monnieri is a fat-soluble component, and the flavonoids in Viola yedoensis are of moderate polarity), a single developing system and colorimetric conditions are insufficient to achieve efficient separation and specific identification of multiple components, thus limiting the specificity and applicability of the identification methods. In addition, the components of various medicinal materials in compound preparations may interfere with each other. How to eliminate negative interference and improve identification sensitivity by optimizing the composition of the developing solvent and colorimetric conditions remains a technical problem that urgently needs to be solved in this field.

[0005] Therefore, developing a quality control method based on thin-layer chromatography that can simultaneously identify multiple medicinal materials such as rhubarb, Kochia scoparia, Cnidium monnieri, Viola yedoensis, Coptis chinensis, and Dictamnus dasycarpus in rhubarb antipruritic lotion would not only fill the existing technological gap but also provide technical reference for improving the quality standards of similar compound Chinese medicines, and has important practical significance for promoting the standardization and internationalization of traditional Chinese medicine. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a quality control method for compound traditional Chinese medicine preparations containing rhubarb.

[0007] The technical solution of this invention is as follows:

[0008] On the one hand, the present invention provides a quality control method for a compound traditional Chinese medicine preparation containing rhubarb, wherein the compound traditional Chinese medicine preparation containing rhubarb is a rhubarb antipruritic lotion; the quality control method includes using thin-layer chromatography to identify any one or more of rhubarb, Viola yedoensis, Kochia scoparia, Cnidium monnieri, Coptis chinensis or Dictamnus dasycarpus in the rhubarb antipruritic lotion.

[0009] The quality control method includes: taking the reference solution and the test solution, spotting them separately on the same silica gel G thin-layer plate, developing them with the developing solvent, removing them, drying them, and inspecting them. The same component is located at the corresponding position in the chromatogram of the test solution and the chromatogram of the reference solution, and spots of the same color appear respectively.

[0010] Specifically, the developing solvent is composed of toluene, acetone, ethyl acetate and formic acid, and the volume ratio of toluene, acetone, ethyl acetate and formic acid is 8-10:1-3:1-2:0.1-2.

[0011] Preferably, the volume ratio of toluene, acetone, ethyl acetate and formic acid is 8-9 or 9-10: 1-2 or 2-3: 1-1.5 or 1.5-2: 0.1-0.5, 0.5-1.0, 1.0-1.5 or 1.5-2.0.

[0012] More preferably, the volume ratio of toluene, acetone, ethyl acetate and formic acid is 8-10:1-2:1-2:2.

[0013] More preferably, the volume ratio of toluene, acetone, ethyl acetate and formic acid is 8:1:1:2.

[0014] Specifically, the preparation method of the test solution includes: taking 5-15 mL of rhubarb antipruritic lotion, extracting it 2-3 times with 10-30 mL of water-saturated organic solvent, combining the extracted organic solvents, washing with 5-15 mL of ammonia solution, allowing it to stand and separate into layers, discarding the ammonia solution, then adding 5-15 mL of water to wash, allowing it to stand and separate into layers, and discarding the water wash solution; evaporating the washed organic solvent to dryness, adding methanol to the residue after evaporation to dissolve it, and obtaining the test solution.

[0015] Preferably, the organic solvent includes any one or more of diethyl ether, ethyl acetate, n-butanol, isopropanol, and dichloromethane.

[0016] More preferably, the organic solvent is n-butanol and / or ethyl acetate.

[0017] More preferably, the organic solvent is n-butanol.

[0018] More preferably, the preparation method of the test solution includes: taking 10 mL of rhubarb antipruritic lotion, extracting it three times with 20 mL of water-saturated n-butanol, combining the n-butanol obtained from the three extractions, adding 10 mL of ammonia test solution to wash, allowing it to stand and separate into layers, discarding the ammonia test solution, adding 10 mL of water to wash again, allowing it to stand and separate into layers, discarding the water wash solution; evaporating the washed n-butanol to dryness, adding 1 mL of methanol to the residue after evaporation to dissolve, and obtaining the test solution.

[0019] Specifically, the reference solutions include rhubarb reference medicinal material solution, violet reference medicinal material solution, kochia scoparia reference medicinal material solution, cnidium monnieri reference medicinal material solution, coptis chinensis reference medicinal material solution, or dictamnus dasycarpus reference medicinal material solution.

[0020] Preferably, the preparation method of the rhubarb reference herb solution includes: adding water to the rhubarb reference herb, sonicating it, adding ethyl acetate, shaking, centrifuging, and using the supernatant as the rhubarb reference herb solution.

[0021] More preferably, the preparation method of the rhubarb reference herb solution includes: taking 0.1g of rhubarb reference herb and placing it in a centrifuge tube, adding 5mL of water and sonicating for 30 minutes, adding 2mL of ethyl acetate and shaking, centrifuging, and using the supernatant as the rhubarb reference herb solution.

[0022] Preferably, the preparation method of the Viola yedoensis reference herb solution includes: adding methanol to the Viola yedoensis reference herb, ultrasonicating, filtering, evaporating the filtrate to dryness, dissolving in water, filtering again, evaporating the filtrate to dryness, adding methanol to dissolve, and thus obtaining the Viola yedoensis reference herb solution.

[0023] More preferably, the preparation method of the Viola yedoensis reference herb solution includes: taking 0.4g of Viola yedoensis reference herb, adding 20mL of methanol and sonicating for 30 minutes, filtering, evaporating the filtrate to dryness, adding 10mL of water to dissolve the residue, filtering, evaporating the filtrate to dryness, adding 2mL of methanol to dissolve the residue, and using this as the Viola yedoensis reference herb solution.

[0024] Preferably, the preparation method of the Kochia scoparia reference medicinal material solution includes: adding Kochia scoparia reference medicinal material to methanol, ultrasonicating, filtering, and using the filtrate as the Kochia scoparia reference medicinal material solution.

[0025] More preferably, the preparation method of the Kochia scoparia reference medicinal material solution includes: taking 0.2g of Kochia scoparia reference medicinal material, adding 2mL of methanol, sonicating for 30 minutes, filtering, and taking the filtrate as the Kochia scoparia reference medicinal material solution.

[0026] Preferably, the method for preparing the Cnidium monnieri reference medicinal material solution includes: adding methanol to the Cnidium monnieri reference medicinal material, ultrasonicating, filtering, and using the filtrate as the Cnidium monnieri reference medicinal material solution.

[0027] More preferably, the preparation method of the Cnidium monnieri reference medicinal material solution includes: taking 0.15g of Cnidium monnieri reference medicinal material, adding 5mL of methanol, sonicating for 30 minutes, filtering, and taking the filtrate as the Cnidium monnieri reference medicinal material solution.

[0028] Preferably, the preparation method of the Coptis chinensis reference herb solution includes: adding methanol to the Coptis chinensis reference herb, ultrasonicating it, filtering it, and using the filtrate as the Coptis chinensis reference herb solution.

[0029] More preferably, the preparation method of the Coptis chinensis reference herb solution includes: taking 0.03g of Coptis chinensis reference herb, adding 5 mL of methanol, sonicating for 30 minutes, filtering, and taking the filtrate as the Coptis chinensis reference herb solution.

[0030] Preferably, the preparation method of the Dictamnus dasycarpus reference medicinal material solution includes: adding methanol to the Dictamnus dasycarpus reference medicinal material, ultrasonicating, filtering, and concentrating the filtrate to obtain the Dictamnus dasycarpus reference medicinal material solution.

[0031] More preferably, the preparation method of the Dictamnus dasycarpus reference medicinal material solution includes: taking 0.5g of Dictamnus dasycarpus reference medicinal material, adding 20mL of methanol, sonicating for 30 minutes, filtering, and concentrating the filtrate to 1mL as the Dictamnus dasycarpus reference medicinal material solution.

[0032] Specifically, the sample volume of the test solution is 3-9 μL.

[0033] Preferably, the sample volume of the test solution is 3-4 μL, 4-5 μL, 5-6 μL, 6-7 μL, 7-8 μL or 8-9 μL.

[0034] More preferably, the sample volume of the test solution is 3 μL, 6 μL, or 9 μL.

[0035] More preferably, the sample volume of the test solution is 6 μL.

[0036] Specifically, the sample volume of the reference solution is 1-5 μL.

[0037] Preferably, the sample volume of the reference solution is 1-2 μL, 2-3 μL, 3-4 μL, or 4-5 μL.

[0038] More preferably, the sample volume of the reference solution is 1 μL, 3 μL, or 5 μL.

[0039] More preferably, the sample volume of the reference solution is 3 μL.

[0040] Preferably, the inspection method includes: inspection using a 365nm ultraviolet lamp; or inspection by spraying with a 10% sulfuric acid ethanol solution followed by heating to develop color; or inspection by spraying with a 10% sulfuric acid ethanol solution, heating to develop color, and then inspection using a 365nm ultraviolet lamp.

[0041] Specifically, the quality control method is carried out under conditions of relative humidity ≤50%.

[0042] Preferably, the quality control method is carried out under conditions of relative humidity of 20-50%.

[0043] More preferably, the quality control method is carried out under relative humidity conditions of 20-25%, 25%-30%, 30-35%, 35%-40%, 40-45%, or 45%-50%.

[0044] The beneficial effects of this invention are as follows:

[0045] This invention provides a quality control method for compound traditional Chinese medicine preparations containing rhubarb. Thin-layer chromatography (TLC) is used to isolate and extract the target components from a rhubarb antipruritic lotion composed of fourteen ingredients, including rhubarb, Kochia scoparia, Cnidium monnieri, Viola yedoensis, Coptis chinensis, and Dictamnus dasycarpus. While ensuring identification accuracy, this method allows for the identification of 4-6 different traditional Chinese medicine components using a single TLC plate, exhibiting high stability and repeatability. This invention offers the technical advantages of requiring less sampling, simplifying quality testing, and significantly improving detection efficiency. Attached Figure Description

[0046] Figure 1 Thin-layer chromatography (365 nm) of the test sample under different development systems.

[0047] Figure 2The images show thin-layer chromatography (365 nm) with different ratios of developing solvent. In developing solvents A to H, 1 represents rhubarb reference solution, 2 represents Viola yedoensis reference solution, 3 represents Cnidium monnieri reference solution, 4 represents the test solution, 5 represents Dictamnus dasycarpus reference solution, 6 represents Kochia scoparia reference solution, and 7 represents Coptis chinensis reference solution. In developing solvent I, 1 represents Viola yedoensis reference solution, 2 represents rhubarb reference solution, 3 represents Kochia scoparia reference solution, 4 represents test solution 1, 5 represents test solution 2, 6 represents test solution 3, 7 represents Dictamnus dasycarpus reference solution, 8 represents Cnidium monnieri reference solution, and 9 represents Coptis chinensis reference solution.

[0048] Figure 3 These are thin-layer chromatography (TLC) spectra with different ratios of developing solvent (sulfuric acid-sunlight). In the developing solvents A to H, 1 represents rhubarb reference solution, 2 represents Viola yedoensis reference solution, 3 represents Cnidium monnieri reference solution, 4 represents the test solution, 5 represents Dictamnus dasycarpus reference solution, 6 represents Kochia scoparia reference solution, and 7 represents Coptis chinensis reference solution. In developing solvent I, 1 represents Viola yedoensis reference solution, 2 represents rhubarb reference solution, 3 represents Kochia scoparia reference solution, 4 represents test solution 1, 5 represents test solution 2, 6 represents test solution 3, 7 represents Dictamnus dasycarpus reference solution, 8 represents Cnidium monnieri reference solution, and 9 represents Coptis chinensis reference solution.

[0049] Figure 4 These are thin-layer chromatography (Thin-layer chromatography) spectra with different ratios of developing solvent (sulfuric acid - 365 nm). In developing solvents A to H, 1 represents rhubarb reference solution, 2 represents Viola yedoensis reference solution, 3 represents Cnidium monnieri reference solution, 4 represents the test solution, 5 represents Dictamnus dasycarpus reference solution, 6 represents Kochia scoparia reference solution, and 7 represents Coptis chinensis reference solution. In developing solvent I, 1 represents Viola yedoensis reference solution, 2 represents rhubarb reference solution, 3 represents Kochia scoparia reference solution, 4 represents test solution 1, 5 represents test solution 2, 6 represents test solution 3, 7 represents Dictamnus dasycarpus reference solution, 8 represents Cnidium monnieri reference solution, and 9 represents Coptis chinensis reference solution.

[0050] Figure 5The results are for specificity testing; A in the figure represents the results under ultraviolet light (365nm); B represents the results under sulfuric acid-sunlight; C represents the results under sulfuric acid-365nm; 1 represents the negative test solution of Viola yedoensis; 2 represents the control solution of Viola yedoensis; 3 represents the negative test solution of Rheum palmatum; 4 represents the control solution of Rheum palmatum; 5 represents the negative test solution of Kochia scoparia; 6 represents the control solution of Kochia scoparia; 7 represents test solution 1; 8 represents test solution 2; 9 represents test solution 3; 10 represents the control solution of Dictamnus dasycarpus; 11 represents the negative test solution of Dictamnus dasycarpus; 12 represents the control solution of Cnidium monnieri; 13 represents the negative test solution of Cnidium monnieri; 14 represents the control solution of Coptis chinensis; 15 represents the negative test solution of Coptis chinensis.

[0051] Figure 6 The figures show the results of the influence of sample volume; A in the figure represents the results under ultraviolet light (365nm); B represents the results under sulfuric acid-sunlight; C represents the results under sulfuric acid-365nm; 1 in the figure represents a 1μL sample of Viola yedoensis control herb solution; 2 represents a 3μL sample of Viola yedoensis control herb solution; 3 represents a 5μL sample of Viola yedoensis control herb solution; 4 represents a 1μL sample of Rheum palmatum control herb solution; 5 represents a 3μL sample of Rheum palmatum control herb solution; 6 represents a 5μL sample of Rheum palmatum control herb solution; 7 represents a 1μL sample of Kochia scoparia control herb solution; 8 represents a 3μL sample of Kochia scoparia control herb solution; 9 represents a 5μL sample of Kochia scoparia control herb solution. 10 represents a 3 μL sample of the test solution; 11 represents a 6 μL sample of the test solution; 12 represents a 9 μL sample of the test solution; 13 represents a 1 μL sample of the *Dictamnus dasycarpus* root bark reference solution; 14 represents a 3 μL sample of the *Dictamnus dasycarpus* root bark reference solution; 15 represents a 5 μL sample of the *Dictamnus dasycarpus* root bark reference solution; 16 represents a 1 μL sample of the *Cnidium monnieri* fruit reference solution; 17 represents a 3 μL sample of the *Cnidium monnieri* fruit reference solution; 18 represents a 5 μL sample of the *Cnidium monnieri* fruit reference solution; 19 represents a 1 μL sample of the *Coptis chinensis* fruit reference solution; 20 represents a 3 μL sample of the *Coptis chinensis* fruit reference solution; 21 represents a 5 μL sample of the *Coptis chinensis* fruit reference solution.

[0052] Figure 7 The results of the temperature effect investigation are shown in the figure; A in the figure is the result under ultraviolet light (365nm); B is the result under sulfuric acid-sunlight; C is the result under sulfuric acid-365nm; 1 in the figure represents the Viola yedoensis reference medicinal material solution; 2 represents the Rheum palmatum reference medicinal material solution; 3 represents the Kochia scoparia reference medicinal material solution; 4 represents test solution 1; 5 represents test solution 2; 6 represents test solution 3; 7 represents the Dictamnus dasycarpus reference medicinal material solution; 8 represents the Cnidium monnieri reference medicinal material solution; 9 represents the Coptis chinensis reference medicinal material solution.

[0053] Figure 8 The results of the investigation on the influence of relative humidity are shown in the figure. A in the figure represents the results under ultraviolet light (365nm); B represents the results under sulfuric acid-sunlight; C represents the results under sulfuric acid-365nm; 1 in the figure represents the Viola yedoensis reference medicinal material solution; 2 represents the Rheum palmatum reference medicinal material solution; 3 represents the Kochia scoparia reference medicinal material solution; 4 represents test solution 1; 5 represents test solution 2; 6 represents test solution 3; 7 represents the Dictamnus dasycarpus reference medicinal material solution; 8 represents the Cnidium monnieri reference medicinal material solution; 9 represents the Coptis chinensis reference medicinal material solution.

[0054] Figure 9 The results of the influence investigation on the shelf manufacturer are shown in the figure; A in the figure is the result under ultraviolet light (365nm); B is the result under sulfuric acid-sunlight; C is the result under sulfuric acid-365nm; 1 in the figure represents the Viola yedoensis reference herb solution; 2 represents the Rheum palmatum reference herb solution; 3 represents the Kochia scoparia reference herb solution; 4 represents test solution 1; 5 represents test solution 2; 6 represents test solution 3; 7 represents the Dictamnus dasycarpus reference herb solution; 8 represents the Cnidium monnieri reference herb solution; 9 represents the Coptis chinensis reference herb solution. Detailed Implementation

[0055] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are all commercially available.

[0056] The reagents used in this invention, including ethyl acetate, methanol, petroleum ether, formic acid, and sulfuric acid, were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0057] The thin-layer plates used in this invention are: pre-formed Guiyou G thin-layer plate (HX73416553 1.05553.0001, 20×20cm, Merck, Germany); pre-formed silicone G thin-layer plate (Yantai Chemical Industry Research Institute, 20190307, ​​100×100mm); and pre-formed Guiyou G (Qingdao Marine Chemical Plant, 20240117, 100×100mm).

[0058] The reference standards used in this invention are: Kochia scoparia (National Institutes for Food and Drug Control 121148-201804); Rheum palmatum (National Institutes for Food and Drug Control 121249-202105); Viola yedoensis (National Institutes for Food and Drug Control 121429-202106); Coptis chinensis (National Institutes for Food and Drug Control 121752-201801); Dictamnus dasycarpus (National Institutes for Food and Drug Control 120978-202006); and Cnidium monnieri (National Institutes for Food and Drug Control 121030-201808).

[0059] The preparation method of the rhubarb antipruritic lotion used in this invention is as follows:

[0060] Weigh out 12g rhubarb, 12g dandelion, 12g raspberry, 15g coptis, 15g scutellaria, 15g phellodendron, 15g violet, 15g achyranthes, 25g kochia, 25g dictamnus root bark, 25g cnidium, 25g motherwort, 25g agrimony, and 25g sophora flavescens. Add water and extract for 1.5 hours. Filter and collect the filtrate. Repeat this step three times, combine the three filtrates, concentrate to 1000mL, filter, and bottle to obtain rhubarb antipruritic lotion.

[0061] Example 1: Products of the test sample and the control sample

[0062] 1. Preparation of the test solution

[0063] Preparation method of test sample: Take 10 mL of rhubarb antipruritic lotion, extract it 3 times with 20 mL of water-saturated n-butanol each time, combine the n-butanol solutions, add 10 mL of ammonia test solution to wash, discard the ammonia test solution, wash with 10 mL of water, discard the water wash solution, take the n-butanol solution and evaporate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test sample solution.

[0064] 2. Preparation of reference solution

[0065] (1) Coptis chinensis reference material solution: Take 0.03g of Coptis chinensis reference material, add 5 mL of methanol and sonicate for 30 minutes, filter, and take the filtrate as Coptis chinensis reference material solution.

[0066] (2) Rhubarb reference material solution: Take 0.1g of rhubarb reference material and place it in a centrifuge tube. Add 5mL of water and sonicate for 30 minutes. Add 2mL of ethyl acetate, shake, centrifuge, and use the supernatant as the rhubarb reference material solution.

[0067] (3) Kochia scoparia reference medicinal material solution: Take 0.2g of Kochia scoparia reference medicinal material, add 2mL of methanol and sonicate for 30 minutes, filter, and take the filtrate as Kochia scoparia reference medicinal material solution.

[0068] (4) Dictamnus dasycarpus reference material solution: Take 0.5g of Dictamnus dasycarpus reference material, add 20mL of methanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 1mL as Dictamnus dasycarpus reference material solution.

[0069] (5) Cnidium monnieri reference medicinal material solution: Take 0.15g of Cnidium monnieri reference medicinal material, add 5mL of methanol and sonicate for 30 minutes, filter, and take the filtrate as Cnidium monnieri reference medicinal material solution.

[0070] (6) Viola yedoensis reference material solution: Take 0.4g of Viola yedoensis reference material, add 20mL of methanol and sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 10mL of water to dissolve the residue, filter, evaporate the filtrate to dryness, add 2mL of methanol to dissolve the residue, and use it as Viola yedoensis reference material solution.

[0071] 3. Preparation of odorless negative test solution

[0072] (1) Coptis chinensis negative test solution: Take 10 mL of the washing solution lacking Coptis chinensis and prepare the Coptis chinensis negative test solution according to the test solution method.

[0073] (2) Rhubarb negative test solution: Take 10 mL of the washing solution lacking rhubarb and prepare the Coptis chinensis negative test solution according to the test solution method.

[0074] (3) Kochia scoparia negative test solution: Take 10 mL of the washing solution without Kochia scoparia and prepare the Coptis chinensis negative test solution according to the test solution method.

[0075] (4) White Dictamnus Cortex Negative Test Solution: Take 10 mL of the washing solution lacking White Dictamnus Cortex and prepare the Coptis chinensis negative test solution according to the test solution method.

[0076] (5) Cnidium monnieri negative test solution: Take 10 mL of the washing solution lacking Cnidium monnieri and prepare Coptis chinensis negative test solution according to the test solution method.

[0077] (6) Viola yedoensis negative test solution: Take 10 mL of the washing solution lacking Viola yedoensis and prepare Coptis chinensis negative test solution according to the test solution method.

[0078] Example 2 Selection of developing solvent system

[0079] Prepare different developing agents according to the developing system in Table 1.

[0080] Table 1

[0081]

[0082] According to the thin-layer chromatography method (General Chapter 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, 6 μL of the test solution was applied to a silica gel G thin-layer plate. Different developing solvents were used (see Table 1). After development, the plate was removed, dried, and examined under ultraviolet light (365 nm). The results are shown in the table below. Figure 1 .

[0083] Figure 1 The results of developing the sample under different developing systems showed significant differences. In developing system 1, the spots were mainly concentrated at the origin. In developing system 2, the number of spots increased. In developing system 3, the spots were mainly yellow (Coptis chinensis). In developing systems 4 and 5, the number of spots increased significantly, with the yellow (Coptis chinensis) spots in developing system 5 showing the largest increase. f The value is higher than that of development system 4, making it more suitable for the identification of Coptis chinensis. Therefore, development system 5, i.e., toluene:acetone:ethyl acetate:formic acid with a volume ratio of 8:1:1:2, was chosen.

[0084] Example 3: Determination of developing solvent ratio and colorimetric method

[0085] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, 3 μL of the test solution and each reference solution were spotted onto a silica gel G thin-layer plate. Different developing solvents were used (see Table 2). The plate was then removed, air-dried, and examined under ultraviolet light (365 nm). The results are shown in the table below. Figure 2 Spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly visible, and observe the results as follows: Figure 3 Then examine under ultraviolet light (365nm), the results are shown below. Figure 4 .

[0086] Table 2

[0087]

[0088] Figure 2 For thin-layer chromatography observation at 365 nm UV, rhubarb showed spots corresponding to the test sample under different developing solvent ratios. f The value is relatively high; Viola yedoensis showed spots corresponding to the test sample under different developing solvent ratios, R f The values ​​differ, but all are suitable; the main spot of the Coptis chinensis control herb remained at the origin under developing solvents A-D, while the R value under developing solvent E was... f The values ​​were low, and the spots were poorly separated with developing solvent F. Spots corresponding to the sample were observed with developing solvents G, H, and I. Therefore, under UV 365 nm, the ratio of developing solvent G, H, and I can be used to observe rhubarb, violet, and coptis.

[0089] Figure 3For thin-layer chromatography with sulfuric acid development, only under developing solvent I conditions will the medicinal material and the test sample show corresponding spots. Therefore, developing solvent I is used, and after sulfuric acid development on thin-layer plates, the root bark of Dictamnus dasycarpus and the fruit of Kochia scoparia can be observed.

[0090] Figure 4 The results are from observation under UV 365nm after sulfuric acid development on thin-layer plates. Cnidium monnieri showed spots corresponding to the test sample under different developing solvent ratios. R f The values ​​are suitable; the results of Coptis chinensis at 365 nm are the same as those obtained directly, and observations are possible under developing solvent G, developing solvent H, and developing solvent I conditions; Viola yedoensis shows spots corresponding to the test sample under different developing solvent ratios, R f The initial value was suitable, but subsequent studies found that negative results caused interference. Therefore, Viola yedoensis is best observed directly at 365 nm. Thus, the ratio of developing solvent G, developing solvent H, and developing solvent I can be used to observe Cnidium monnieri and Coptis chinensis at 365 nm using sulfuric acid on a thin-layer plate.

[0091] In summary:

[0092] Using developing solvent A-developing solvent I, i.e., toluene:acetone:ethyl acetate:formic acid in a volume ratio of 8-10:1-3:1-2:0.1-2, a single thin-layer plate can be used to identify four medicinal materials: rhubarb, violet, kochia, and cnidium.

[0093] Using developing solvent G-developing solvent I, i.e., toluene:acetone:ethyl acetate:formic acid in a volume ratio of 8-10:1-2:1-2:2, a single thin-layer plate can be used to identify five medicinal materials: rhubarb, violet, kochia, dictamnus dasycarpus, and cnidium monnieri.

[0094] Using solvent I, i.e., toluene:acetone:ethyl acetate:formic acid in a volume ratio of 8:1:1:2, a single thin-layer plate can be used to identify six medicinal materials: rhubarb, violet, coptis, kochia, dictamnus dasycarpus, and cnidium monnieri.

[0095] Example 4: Specificity of the developing solvent

[0096] Test solution, odorless negative test solution, and reference solution; perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 6 μL each of the test solution and odorless negative test solution, and 3 μL each of the reference herb solution to the same silica gel G thin-layer plate, forming bands. Use toluene:acetone:ethyl acetate:formic acid (volume ratio 8:1:1:2) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (365 nm). Results are shown in the figure. Figure 5 A; then spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly visible and examine. The results are shown in [the original text]. Figure 5B in the image; then examine under ultraviolet light (365nm), the results are shown in [the image]. Figure 5 C in the middle.

[0097] Figure 5 The results showed that the test sample chromatogram had spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the separation was good. In the negative control chromatogram, there were no spots at the corresponding positions as the reference medicinal material chromatogram. The results showed that the negative control had no interference and good specificity.

[0098] Example 5: Effect of Sample Amount

[0099] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, different volumes of the test solution and the reference solution were spotted separately onto the same silica gel G thin-layer plate. The developing solvent was toluene:acetone:ethyl acetate:formic acid in a volume ratio of 8:1:1:2. After development, the plate was removed, dried, and examined under ultraviolet light (365 nm). The results are shown in the figure. Figure 6 A; then spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly visible and examine. The results are shown in [the original text]. Figure 6 B in the image; then examine under ultraviolet light (365nm), the results are shown in [the image]. Figure 6 C in the middle.

[0100] Figure 6 The letter 'A' indicates that when the sample volume of the test solution is 3-9 μL, corresponding spots will appear at the corresponding positions as in the reference herbs Viola yedoensis, rhubarb, and Coptis chinensis, with 6 μL being preferred. For Viola yedoensis reference herb solution, 1-5 μL produces clear spots, with 3 μL being preferred; for rhubarb reference herb solution, 1-5 μL produces clear spots, with 3 μL being preferred; for Coptis chinensis reference herb solution, 1-5 μL produces clear spots, with 3 μL being preferred.

[0101] Figure 6 -B indicates that when the sample volume of the test solution is 3-9 μL, corresponding spots will appear at the corresponding positions as in the reference herbs Kochia scoparia and Dictamnus dasycarpus, with 6 μL being preferred. When the Kochia scoparia reference herb solution is 1-5 μL, the spots are clear, with 3 μL being preferred; when the Dictamnus dasycarpus reference herb solution is 1-5 μL, the spots are clear, with 3 μL being preferred.

[0102] Figure 6 -C indicates that when the sample volume of the test solution is 3-9 μL, there will be corresponding spots at the corresponding positions as the reference herbs Cnidium monnieri and Coptis chinensis, preferably 6 μL. When the sample volume of the Cnidium monnieri reference herb solution is 1-5 μL, the spots are clear, preferably 3 μL; when the sample volume of the Coptis chinensis reference herb solution is 1-5 μL, the spots are clear, preferably 3 μL.

[0103] Example 6: Effect of Temperature

[0104] The experiment was conducted at three temperatures: 30℃ (constant temperature incubator), 25℃ (room temperature), and 8℃ (refrigerator). Following the thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, different volumes of the test solution and reference solution were spotted separately onto the same silica gel G thin-layer plate. The developing solvent was toluene:acetone:ethyl acetate:formic acid at a volume ratio of 8:1:1:2. After development, the plate was removed, dried, and examined under ultraviolet light (365nm). The results are shown in the figure. Figure 7 A; then spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly visible and examine. The results are shown in [the original text]. Figure 7 B in the image; then examine under ultraviolet light (365nm), the results are shown in [the image]. Figure 7 C in the middle.

[0105] from Figure 7 It can be seen that under the above-mentioned different temperature conditions, the chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material, and the separation is good. f The value did not change significantly, indicating that the temperature range of 8-30℃ has no significant impact on the use of the developing agent in thin-layer chromatography for identification, and there is no need to control the ambient temperature.

[0106] Example 7: The Influence of Relative Humidity Environment

[0107] Three relative humidity levels were used in the experiment: 20% (indoors), 50% (achieved by placing 43.4% sulfuric acid solution in a developing tank and sealing for 2 hours), and 80% (achieved by placing 26.2% sulfuric acid solution in a developing tank and sealing for 2 hours). The thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia was followed. Different volumes of the test solution and reference solution were spotted onto the same silica gel G thin-layer plate. The developing solvent was toluene:acetone:ethyl acetate:formic acid at a volume ratio of 8:1:1:2. After development, the plate was removed, dried, and examined under ultraviolet light (365 nm). The results are shown in the figure. Figure 8 A; then spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly visible and examine. The results are shown in [the original text]. Figure 8 B in the image; then examine under ultraviolet light (365nm), the results are shown in [the image]. Figure 8 C in the middle.

[0108] from Figure 8 It can be seen that at relative humidity of 50% and below, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference herb chromatogram, and the separation is good. However, at 80% relative humidity, the Rf values ​​of the spots of Kochia scoparia and Coptis chinensis decrease and the separation becomes worse. This indicates that the relative humidity in the range of 20-50% has little impact on the development solvent of this invention for thin-layer identification, and the relative humidity of the environment should be controlled to be below 50%.

[0109] Example 8: Impact of Thin-Layer Board Manufacturers

[0110] Three thin-layer chromatography (TLC) plates from three different manufacturers (Merck, Germany; Yantai Chemical Industry Research Institute; and Qingdao Marine Chemical Plant) were selected for the experiments. Following the TLC method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, different volumes of the test solution and reference solution were spotted onto the same silica gel G TLC plate. The developing solvent was toluene:acetone:ethyl acetate:formic acid at a volume ratio of 8:1:1:2. After development, the plates were removed, dried, and examined under ultraviolet light (365 nm). The results are shown in the figure below. Figure 9 A; then spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly visible and examine. The results are shown in [the original text]. Figure 9 B in the image; then examine under ultraviolet light (365nm), the results are shown in [the image]. Figure 9 C in the middle.

[0111] Based on the development results of thin-layer plates from different manufacturers, the chromatograms of the test sample showed spots of the same color at the corresponding positions as those of the reference medicinal material, with good separation and no negative interference. f The values ​​vary slightly. Thin-layer plates from all three manufacturers can be used for identification of rhubarb antipruritic lotion.

[0112] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A quality testing method for a compound traditional Chinese medicine preparation containing rhubarb, characterized in that, The aforementioned rhubarb-containing compound traditional Chinese medicine preparation is a rhubarb antipruritic lotion; the aforementioned quality detection method includes using thin-layer chromatography to identify rhubarb, Viola yedoensis, Kochia scoparia, Cnidium monnieri, Coptis chinensis, and Dictamnus dasycarpus in the rhubarb antipruritic lotion; The quality testing method includes: taking the reference solution and the test solution, spotting them separately on the same silica gel G thin-layer plate, developing them with the developing solvent, removing them, drying them, and inspecting them. The same component is located at the corresponding position in the chromatogram of the test solution and the chromatogram of the reference solution, and spots of the same color appear respectively. The developing solvent is composed of toluene, acetone, ethyl acetate and formic acid; the volume ratio of toluene:acetone:ethyl acetate:formic acid is 8-10:1-3:1-2:0.1-2 to identify four medicinal materials: rhubarb, violet, dictamnus dasycarpus, and cnidium monnieri. The preparation method of the rhubarb antipruritic lotion is as follows: Weigh 12g rhubarb, 12g dandelion, 12g raspberry, 15g coptis, 15g scutellaria, 15g phellodendron, 15g violet, 15g achyranthes, 25g kochia, 25g dictamnus root bark, 25g cnidium, 25g motherwort, 25g agrimony, and 25g sophora flavescens, add water, extract for 1.5h, filter, collect the filtrate, repeat this step three times, combine the three filtrates, concentrate to 1000mL, filter, and fill into bottles to obtain the rhubarb antipruritic lotion; The preparation method of the test solution includes: taking 5-15 mL of rhubarb antipruritic lotion, extracting it 2-3 times with 10-30 mL of water-saturated organic solvent, combining the extracted organic solvents, washing with 5-15 mL of ammonia solution, allowing it to stand and separate into layers, discarding the ammonia solution, then adding 5-15 mL of water to wash, allowing it to stand and separate into layers, discarding the water wash solution; evaporating the washed organic solvent to dryness, dissolving it in methanol in the residue after evaporation to obtain the test solution, wherein the organic solvent is n-butanol; The reference solutions include rhubarb reference medicinal material solution, violet reference medicinal material solution, kochia fruit reference medicinal material solution, cnidium fruit reference medicinal material solution, coptis fruit reference medicinal material solution, or dictamnus root bark reference medicinal material solution: the solvent for the rhubarb reference medicinal material solution is ethyl acetate; the solvent for the violet reference medicinal material solutions, the kochia fruit reference medicinal material solutions, the cnidium fruit reference medicinal material solutions, the coptis fruit reference medicinal material solutions, and the dictamnus root bark reference medicinal material solutions is methanol; The inspection methods include: inspecting rhubarb, violet, and coptis with a 365nm ultraviolet lamp; inspecting dictamnus dasycarpus and kochia scoparia by spraying with a 10% sulfuric acid ethanol solution and heating to develop color; and inspecting cnidium monnieri by spraying with a 10% sulfuric acid ethanol solution, heating to develop color, and then inspecting cnidium monnieri with a 365nm ultraviolet lamp.

2. The quality inspection method according to claim 1, characterized in that, The volume ratio of toluene, acetone, ethyl acetate and formic acid is 8-10:1-2:1-2:2, used to identify five medicinal materials: rhubarb, violet, coptis, dictamnus dasycarpus, and cnidium monnieri.

3. The quality inspection method according to claim 2, characterized in that, The volume ratio of toluene, acetone, ethyl acetate and formic acid is 8:1:1:2, used to identify six medicinal materials: rhubarb, violet, coptis, kochia, dictamnus dasycarpus, and cnidium monnieri.

4. The quality inspection method according to claim 1, characterized in that, The sample volume of the test solution is 3-9 μL, and the sample volume of the control solution is 1-5 μL.

5. The quality inspection method according to claim 1, characterized in that, The sample volume of the test solution is 6 μL, and the sample volume of the control solution is 3 μL.

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