Thin-layer chromatography detection method, quality control and evaluation of pogonatherum crinitum

Abstract

The invention discloses a thin-layer chromatography detection method, quality control and evaluation of pogonatherum crinitum. The thin-layer chromatography detection method comprises the following steps: providing a pogonatherum crinitum test solution; respectively providing reference substance solutions of hyperoside, quercetin, astragalus smicus glycoside and kaempferol; and dripping the test solution and the reference solution on the same thin layer chromatography plate, developing by using a mixed solution of trichloromethane, methanol and formic acid in a volume ratio of (8-12): (1-2.2): (1-2.2) as a developing solvent, taking out, airing, developing, and inspecting. According to the method, rapid separation and identification of hyperoside, quercetin, astragalin and kaempferol in the pogonatherum crinitum are achieved, thin-layer chromatography spots are clear, the accuracy and comprehensiveness of pogonatherum crinitum quality control are improved, and establishment and overall evaluation of pogonatherum crinitum medicinal material quality standards are facilitated.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine detection technology, and in particular to thin-layer chromatography detection methods, quality control and evaluation of Cuscuta chinensis. Background Technology

[0002] Dodder (Cuscuta chinensis), also known as Aifetimon, is the dried aerial part of the plant *Cuscuta chinensis* Lam., belonging to the Convolvulaceae family. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a superior-grade herb. Modern medical research shows that dodder contains various active ingredients, including flavonoids, sterols, polysaccharides, lignans, alkaloids, and terpenes, possessing pharmacological activities such as antioxidant, anti-inflammatory, immunomodulatory, hepatoprotective, anticancer, neuroprotective, and lipid-lowering effects. Therefore, dodder has high medicinal value in both traditional and modern medicine. However, the active ingredients and medicinal mechanisms of dodder are still unclear, necessitating the isolation and identification of its active components and the establishment of comprehensive quality standards for dodder.

[0003] Modern analytical techniques commonly employ high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for the separation and identification of active ingredients in traditional Chinese medicinal materials. While HPLC offers high precision, its high instrument cost and complex operation make it unsuitable for primary care laboratories or rapid screening. TLC, on the other hand, is simple to operate, low-cost, and fast, and it provides a direct visual representation of the separated components' spots and morphology, facilitating preliminary qualitative identification. It is well-suited as a key preliminary technique for the separation and identification of active ingredients. Currently, there are few TLC detection methods for Cuscuta chinensis, primarily focusing on the identification of quercetin and hyperoside, which cannot meet the accuracy and comprehensiveness requirements for quality control of Cuscuta chinensis. Summary of the Invention

[0004] Therefore, it is necessary to provide a thin-layer chromatography method that can comprehensively detect multiple active ingredients in Cuscuta chinensis, thereby improving the accuracy and comprehensiveness of quality control of Cuscuta chinensis and facilitating the establishment and overall evaluation of quality standards for Cuscuta chinensis.

[0005] In a first aspect, this application provides a thin-layer chromatography method for the detection of dodder, comprising the following steps:

[0006] Provide a sample solution of dodder.

[0007] Provide hyperoside reference solution, quercetin reference solution, astragaloside reference solution and kaempferol reference solution;

[0008] The dodder test solution, hyperoside reference solution, quercetin reference solution, astragaloside reference solution and kaempferol reference solution were spotted on the same thin-layer chromatography plate, developed with a developing solvent, removed, dried and then subjected to color development and inspection.

[0009] The developing agent is a mixed solution of chloroform, methanol and formic acid; the volume ratio of chloroform, methanol and formic acid is (8-12):(1-2.2):(1-2.2).

[0010] In some embodiments, the volume ratio of the trichloromethane, the methanol, and the formic acid is (10.5-11.5):(1-2.2):(1-2.2).

[0011] Optionally, the volume ratio of the trichloromethane, the methanol and the formic acid is (10.5-11.5):(1.8-2.2):(1.8-2.2).

[0012] In some embodiments, the method for preparing the dodder test solution includes:

[0013] The dodder extract was prepared by reflux extraction of the dodder sample in ethanol solution, solid-liquid separation, drying of the filtrate;

[0014] The dodder extract was dissolved in a solvent to prepare the dodder test solution.

[0015] In some embodiments, the mass-to-volume ratio of the dodder sample to the ethanol solution is 1 g: (10-50) mL; and / or, the mass fraction of the ethanol solution is 50%-70%; and / or, the reflux extraction is performed 2-3 times; and / or, the reflux extraction time is 1-3 hours; and / or, the drying temperature is 55°C-65°C; and / or, the solvent includes one or more of ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide, acetone, and water.

[0016] Optionally, the solvent includes ethanol, wherein the mass fraction of the ethanol is 50%-70%.

[0017] In some embodiments, the dodder sample includes one or more of the dodder herb and preparations containing dodder;

[0018] Optionally, the dodder herb material includes one or more parts of the dodder plant, such as seeds, roots, stems, leaves, and flowers; more preferably, the dodder herb material includes the whole dodder plant.

[0019] Optionally, the dosage forms of preparations containing dodder include decoction pieces, granules, tablets, decoctions, pills, powders, ointments, tablets, granules, lozenges, capsules, liquids, or tinctures.

[0020] In some embodiments, the preparation method of the hyperoside reference solution, the quercetin reference solution, the astragaloside reference solution, and the kaempferol reference solution includes: preparing the hyperoside reference standard, quercetin reference standard, astragaloside reference standard, and kaempferol reference standard by independently mixing each with methanol;

[0021] And / or, the concentrations of the hyperoside reference solution, the quercetin reference solution, the astragaloside reference solution, and the kaempferol reference solution are each independently 0.02 mg / mL to 2 mg / mL;

[0022] And / or, the concentration of dodder extract in the dodder test solution is 5 mg / mL to 500 mg / mL.

[0023] In some embodiments, the thin-layer chromatography plate includes a silica gel thin-layer plate;

[0024] Optionally, the silicone sheet may include a silicone G sheet, a silicone H sheet, or a GF254 sheet.

[0025] In some embodiments, the colorimetric agent used in the colorimetric treatment includes aluminum trichloride solution;

[0026] Optionally, the aluminum trichloride solution includes an ethanol solution of aluminum trichloride;

[0027] Optionally, the mass fraction of the aluminum trichloride solution is 0.1%-6%.

[0028] In some embodiments, the light source used for inspection includes an ultraviolet light source;

[0029] Optionally, the wavelength of the ultraviolet light source is 365nm.

[0030] Compared with traditional technologies, the beneficial effects of the technical solution in this application include:

[0031] This application provides a thin-layer chromatography (TLC) method for the detection of dodder. The method uses a TLC plate to separate dodder samples, employing a mixed solution of chloroform, methanol, and formic acid in a volume ratio of (8-12):(1-2.2):(1-2.2) as the developing solvent. This allows for the rapid separation and identification of hyperoside, quercetin, astragaloside, and kaempferol in the dodder sample. The TLC spots in this method are clear, improving the accuracy and comprehensiveness of dodder quality control and facilitating the establishment and overall evaluation of dodder medicinal material quality standards. Attached Figure Description

[0032] Figures 1-8 The following are the thin-layer chromatography detection results obtained using developing solvents 1-8 in Example 1 of this application, with the specific ratio of chloroform-methanol-formic acid as the developing solvent: Figure 1 The ratio is 11:2:2. Figure 2 The ratio is 10:2:2. Figure 3 The ratio is 8:2:2. Figure 4 The ratio is 5:2:2. Figure 5 The ratio is 11:1:1. Figure 6 The ratio is 11:1:2. Figure 7 The ratio is 11:4:1. Figure 8 The ratio is 11:1:4.

[0033] Figures 9-16 The following are the thin-layer chromatography detection results obtained using developing solvents 9-16 in Example 1 of this application, with the specific developing solvents being: Figure 9 The ratio of methanol-glacial acetic acid-water is 6:1:4. Figure 10 The ratio of ethyl acetate - butanone - formic acid - water is 10:6:1:2. Figure 11 The ratio of dichloromethane-methanol-glacial acetic acid is 7:3:0.5. Figure 12 The ratio of chloroform to methanol is 10:1. Figure 13 The ratio of chloroform to methanol is 11:2. Figure 14 The ratio of chloroform-methanol-acetic acid is 11:2:2. Figure 15 The ratio of chloroform-methanol-acetic acid is 10:4:1. Figure 16 The ratio of chloroform-methanol-water is 80:20:1.

[0034] Figures 17-22 The following are the thin-layer chromatography detection results obtained using colorimetric reagents 1-6 in Example 2 of this application, respectively. The specific colorimetric reagents are: Figure 17 An ethanol solution of 1.0 wt% aluminum trichloride. Figure 18 An ethanol solution of 0.1 wt% aluminum trichloride Figure 19 An ethanol solution of 0.5 wt% aluminum trichloride Figure 20 It is a 2.0 wt% aluminum trichloride ethanol solution. Figure 21 A 4.0 wt% aluminum trichloride ethanol solution Figure 22 It is a 6.0 wt% aluminum trichloride ethanol solution.

[0035] Figures 23-26 The following are the thin-layer chromatography detection results of thin-layer plates 1-4 in Example 3 of this application, and the specific thin-layer plates are as follows: Figure 23 For silicone G thin film, Figure 24 For polyamide thin-layer plates, Figure 25 For silicone H thin film, Figure 25 It is a GF254 thin-layer plate.

[0036] Figures 27-29 The following are the thin-layer chromatography detection results of different test solutions 1-3 in Example 4 of this application, respectively. The specific test solutions are as follows: Figure 27 The test solution was prepared using 60% ethanol as the extraction solvent. Figure 28 2. The test solution was prepared using 60% methanol as the extraction solvent. Figure 29 The test solution 3 was prepared using water as the extraction solvent.

[0037] The markings in the attached figure are as follows: A is hyperoside standard, B is quercetin standard, C is astragaloside standard, D is kaempferol standard, E-1 is test batch 1, E-2 is test batch 2, and E-3 is test batch 3. Detailed Implementation

[0038] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0040] As used herein, "optional," "optional," and "optional" refer to either "with" or "without" parallel options. If multiple "optional" entries appear in a technical solution, each "optional" entry is independent unless otherwise specified and there are no contradictions or mutual constraints. The term "and / or" as used herein includes any and all combinations of one or more related listed items. Unless otherwise specified, "multiple," "multiple," etc., as used herein refer to a quantity greater than 2 or equal to 2; for example, "one or more" indicates one, two, or more than two. In open-ended technical features or solutions described herein using words such as "containing," "including," and "comprising," unless otherwise specified, additional members beyond the listed members are not excluded. This can be considered as providing both a closed-ended feature or solution consisting of the listed members and an open-ended feature or solution that includes additional members beyond the listed members.

[0041] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0042] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0043] In research on dodder medicinal materials, the mature seeds are often used as the main medicinal component. For example, the dodder seed listed in the 2020 edition of the Chinese Pharmacopoeia (Part I) is the dried, mature seed of the dodder plant. Similarly, in classic Uyghur medicine prescriptions, the whole dodder plant (including stems and leaves) is frequently used. However, quality control measures for dodder medicinal materials are not currently included in the Chinese Pharmacopoeia. Therefore, research on the quality control of dodder medicinal materials is needed to provide insights for establishing quality standards for dodder medicinal materials.

[0044] Existing research techniques indicate that hyperoside and quercetin are among the main active components of Cuscuta chinensis. However, relying solely on hyperoside and quercetin is insufficient for comprehensive quality control and evaluation of Cuscuta chinensis. Therefore, this application aims to provide a thin-layer chromatography method capable of simultaneously detecting multiple active components in Cuscuta chinensis, thereby improving the accuracy and comprehensiveness of quality control and evaluation of Cuscuta chinensis.

[0045] In a first aspect, this application provides a thin-layer chromatography method for the detection of dodder, comprising the following steps:

[0046] S10. Provide a sample solution of dodder.

[0047] S20. Provide hyperoside reference solution, quercetin reference solution, astragaloside reference solution and kaempferol reference solution.

[0048] S30. Spot the Cuscuta chinensis test solution, hyperoside reference solution, quercetin reference solution, astragaloside reference solution and kaempferol reference solution onto the same thin-layer chromatography plate, develop with the developing solvent, remove, air dry, and then perform color development and inspection.

[0049] S40, the developing agent is a mixed solution of chloroform, methanol and formic acid; the volume ratio of chloroform, methanol and formic acid is (8-12):(1-2.2):(1-2.2).

[0050] This application uses a mixed solution of chloroform, methanol, and formic acid in a volume ratio of (8-12):(1-2.2):(1-2.2) as the developing solvent, which significantly improves the separation effect, accuracy, and sensitivity of the active ingredients of Cuscuta chinensis. It can rapidly separate and identify hyperoside, quercetin, astragaloside, and kaempferol in Cuscuta chinensis simultaneously, and the obtained thin-layer chromatographic spots have clear morphology, thereby improving the accuracy and comprehensiveness of quality control and evaluation of Cuscuta chinensis.

[0051] In some embodiments, in step S40, the volume ratio of the trichloromethane, the methanol, and the formic acid includes, but is not limited to, 8:1:1, 8:2:2, 8:2.2:2.2, 10:1:1, 10:1:2, 10:2:1, 11:1:1, 11:1:2, 12:2:2, 12:2.2:2.2, or any of the foregoing ranges and values ​​within those ranges.

[0052] In some embodiments, the volume ratio of the trichloromethane, the methanol, and the formic acid is (10.5-11.5):(1-2.2):(1-2.2), including but not limited to 11:1:1, 11:1:2, 11:1:2.2, 11:2:1, 11:2:2, 11:2:2.2, 11:2.2:1, 11:2.2:2, 11:2.2:2.2, or any of the foregoing ranges and values ​​within those ranges.

[0053] In some embodiments, the volume ratio of the trichloromethane, the methanol, and the formic acid is (10.5-11.5):(1.8-2.2):(1.8-2.2), including but not limited to 11:1.8:1.8, 11:1.8:2, 11:1.8:2.2, 11:2:2, 11:2:2.2, 11:2.2:1.8, 11:2.2:2, 11:2.2:2.2, or any of the foregoing ranges and values ​​within those ranges.

[0054] In some embodiments, the method for preparing the dodder test solution includes:

[0055] S11. The dodder sample was extracted by reflux in ethanol solution, solid-liquid separation was performed, the filtrate was dried, and dodder extract was prepared.

[0056] S12. Dissolve the dodder extract in a solvent to prepare the dodder test solution.

[0057] In some embodiments, the mass-to-volume ratio of the dodder sample to the ethanol solution is 1g:(10-50)mL, including but not limited to 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL, 1g:30mL, 1g:35mL, 1g:40mL, 1g:45mL, 1g:50mL, or any of the foregoing ranges and values ​​within those ranges.

[0058] In some embodiments, the ethanol solution has a mass fraction of 50%-70%, including but not limited to 50%, 55%, 60%, 65%, 70%, or any combination thereof and values ​​within that range.

[0059] In some embodiments, the reflux extraction is performed 2-3 times, more preferably 2 times.

[0060] In some embodiments, the reflux extraction time is 1-3 hours, including but not limited to 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any range formed by both of the foregoing and values ​​within that range. It is understood that the reflux extraction time refers to the time of one reflux. For example, the dodder sample is refluxed twice in an ethanol solution, with each reflux extraction lasting 1-3 hours.

[0061] In some embodiments, the temperature for drying the filtrate is 55°C-65°C, including but not limited to 55°C, 58°C, 60°C, 62°C, 65°C, or any combination thereof and values ​​within that range.

[0062] In some embodiments, the solvent includes one or more of ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide, acetone, and water. Exemplarily, the solvent includes ethanol, wherein the ethanol mass fraction is 50%-70%, including but not limited to 50%, 55%, 60%, 65%, 70%, or any combination thereof and values ​​within that range.

[0063] In some embodiments, the concentration of dodder extract in the dodder test solution is 5 mg / mL to 500 mg / mL, including but not limited to 5 mg / mL, 10 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, or any combination thereof and values ​​within such ranges.

[0064] In some embodiments, the preparation method of the hyperoside reference solution, the quercetin reference solution, the astragaloside reference solution, and the kaempferol reference solution includes: preparing each of the hyperoside reference standard, quercetin reference standard, astragaloside reference standard, and kaempferol reference standard by independently mixing them with methanol.

[0065] In some embodiments, the concentrations of the hyperoside reference solution, the quercetin reference solution, the astragaloside reference solution, and the kaempferol reference solution are each independently 0.02 mg / mL to 2 mg / mL, including but not limited to 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or any range formed by both of the foregoing and values ​​within that range.

[0066] In some embodiments, the dodder sample includes one or more of the dodder herb and preparations containing dodder. As a non-limiting example, the dodder herb includes one or more parts of the dodder plant, such as seeds, roots, stems, leaves, and flowers. Furthermore, in this application, using the whole dodder plant better demonstrates the technical advantages of the thin-layer chromatography technique. It is understood that the whole dodder plant (scientific name: *Cuscuta chinensis* Lam.) is an annual parasitic herb belonging to the genus *Cuscuta* in the family Convolvulaceae, and its dried aerial parts or fresh product can be used medicinally. Dodder lacks leaves and roots; its stems are slender and thread-like, yellow or orange-yellow in color, and it obtains nutrients by attaching to host plants such as those in the families Leguminosae and Asteraceae through haustoria.

[0067] As a non-limiting example, dosage forms of preparations containing dodder include slices, granules, tablets, decoctions, pills, powders, ointments, tablets, granules, lozenges, capsules, liquids, or tinctures.

[0068] In some embodiments, the thin-layer chromatography plate comprises a silica gel thin-layer plate. As a non-limiting example, the silica gel thin-layer plate includes a silica gel G thin-layer plate, a silica gel H thin-layer plate, or a GF254 thin-layer plate. Furthermore, the inclusion of a silica gel G thin-layer plate allows for better separation of multiple active ingredients in Cuscuta chinensis.

[0069] In some embodiments, the colorimetric agent used in the colorimetric treatment includes an aluminum trichloride solution. As a non-limiting example, the aluminum trichloride solution includes an ethanol solution of aluminum trichloride. Further, the mass fraction of the aluminum trichloride solution is 0.1%-6%, including but not limited to 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, or any range formed by both of the foregoing and values ​​within that range.

[0070] In some embodiments, the light source used for inspection includes an ultraviolet light source; further, the wavelength of the ultraviolet light source is 365 nm.

[0071] It should be noted that experimental methods in the following embodiments of this application, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products, or can be prepared by those skilled in the art using known methods.

[0072] The dodder herb, batches TSC-YP-240903, TSC-YP-250315, and TSC-YP-250721, were all purchased from Xinjiang Xinlvbao Pharmaceutical Co., Ltd.

[0073] Hyperoside reference standard (batch number 111521-202310), quercetin reference standard (batch number 100081-201610), and kaempferol reference standard (batch number 110861-202214) were all purchased from the National Institutes for Food and Drug Control.

[0074] The astragaloside reference standard (batch number WP25011008) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0075] Pure water, ethanol, chloroform, methanol, glacial acetic acid, ethyl acetate, butanone, dichloromethane, and aluminum trichloride were all of analytical grade.

[0076] Silicone G thin-layer plate was purchased from Qingdao Haiyang Chemical Co., Ltd., silicone H thin-layer plate was purchased from Qingdao Haiyang Chemical Co., Ltd., GF254 thin-layer plate was purchased from Qingdao Haiyang Chemical Co., Ltd., and polyamide thin-layer plate was purchased from Taizhou Luqiao Sijia Biochemical Plastics Factory.

[0077] Example 1

[0078] Preparation of test solution: Take 5 g of each of three batches of Cuscuta chinensis herb, add 100 mL of 60 wt% ethanol solution, heat and reflux to extract twice, 1.5 h each time, filter, combine the two filtrates, evaporate the filtrate to dryness at 60℃, take the evaporated solid and add 2 mL of 60 wt% ethanol solution to dissolve, to prepare a test solution with a concentration of 100 mg / mL.

[0079] Preparation of reference solutions: Take hyperoside, quercetin, astragaloside, and kaempferol reference standards and prepare reference solutions containing 100 μg per mL with methanol.

[0080] The thin-layer chromatography method (Chinese Pharmacopoeia 2010 Edition, Part I, Appendix VI B) was followed. 2 μL each of the above-mentioned test solution, hyperoside reference solution, quercetin reference solution, astragaloside reference solution, and kaempferol reference solution were spotted onto the same silica gel G thin-layer plate. The plate was developed using the 16 groups of developing solvents shown in Table 1. The plate was then removed, dried, and sprayed with 1 wt% aluminum trichloride ethanol solution for color development. The results were examined at 365 nm. The results are shown in the appendix. Figure 1 To be continued Figure 16 As shown.

[0081] Table 1: Composition of the developing solvent

[0082]

[0083] See appendix Figure 1 To be continued Figure 16 The thin-layer chromatography results show that the developing solvents 1-3 and 5-6 can successfully separate hyperoside, quercetin, astragaloside and kaempferol in the test sample. The spots in the chromatographic plate have good separation, good morphology, clear edges, no obvious tailing phenomenon, and appropriate specific migration value (Rf value).

[0084] Example 2

[0085] Preparation of test solution: Take 5 g of each of three batches of Cuscuta chinensis herb, add 100 mL of 60 wt% ethanol solution to each batch, heat and reflux to extract twice, 1.5 h each time, filter, combine the two filtrates, evaporate the filtrate to dryness at 60℃, take the evaporated solids and add 2 mL of 60 wt% ethanol solution to dissolve, to prepare a test solution with a concentration of 100 mg / mL.

[0086] Preparation of reference solutions: Take hyperoside, quercetin, astragaloside, and kaempferol reference standards and prepare reference solutions containing 100 μg per mL with methanol.

[0087] The thin-layer chromatography method (Chinese Pharmacopoeia 2010 Edition, Part I, Appendix VI B) was used. 2 μL each of the above-mentioned test solution, hyperoside reference solution, quercetin reference solution, astragaloside reference solution, and kaempferol reference solution were spotted onto the same silica gel G thin-layer plate. The plate was developed using chloroform-methanol-formic acid (11:2:2, v / v). After development, the plate was removed, dried, and sprayed with the six colorimetric reagents shown in Table 2. The results were examined at 365 nm. The results are shown in the appendix. Figure 17 To be continued Figure 22 As shown.

[0088] Table 2: Composition of the colorimetric reagent

[0089]

[0090] See appendix Figure 17 To be continued Figure 22 The thin-layer chromatography results show that the colorimetric reagents in groups 1-6 of Table 2 can all develop color in the active substances in the test sample, and the colorimetric effect is good, and the relevant components can be clearly identified.

[0091] Example 3

[0092] Preparation of test solution: Take 5 g of each of three batches of Cuscuta chinensis herb, add 100 mL of 60 wt% ethanol solution to each batch, heat and reflux to extract twice, 1.5 h each time, filter, combine the two filtrates, evaporate the filtrate to dryness at 60℃, take the evaporated solids and add 2 mL of 60 wt% ethanol solution to dissolve, to prepare a test solution with a concentration of 100 mg / mL.

[0093] Preparation of reference solutions: Take hyperoside, quercetin, astragaloside, and kaempferol reference standards and prepare reference solutions containing 100 μg per mL with methanol.

[0094] The thin-layer chromatography method (Chinese Pharmacopoeia 2010 Edition, Part I, Appendix VI B) was followed. 2 μL each of the above-mentioned test solution, hyperoside reference solution, quercetin reference solution, astragaloside reference solution, and kaempferol reference solution were spotted onto the same thin-layer plate (see Table 3 for specific plate types and models). The plate was developed using chloroform-methanol-formic acid (11:2:2, v / v). After development, the plate was removed, dried, and sprayed with 1 wt% aluminum trichloride ethanol solution for color development. The results were examined at 365 nm. The results are shown in the appendix. Figure 23 To be continued Figure 26 As shown.

[0095] Table 3: Types and Models of Thin-Layer Laminates

[0096]

[0097] See appendix Figure 23 To be continued Figure 26 The thin-layer chromatography results show that among the different thin-layer chromatography plates in groups 1-4 of Table 3, silica gel G thin-layer plate, silica gel H thin-layer plate and GF254 thin-layer plate have better separation effect, while the active ingredients in polyamide thin-layer plate have trailing spots and poor morphology, making it difficult to clearly identify the relevant active layer components.

[0098] Example 4

[0099] Preparation of test solution 1: Take 5 g of each of three batches of Cuscuta chinensis herb, add 100 mL of 60 wt% ethanol solution to each batch, heat and reflux to extract twice, 1.5 h each time, filter, combine the two filtrates, evaporate the filtrate to dryness at 60℃, take the evaporated solids and add 2 mL of 60 wt% ethanol solution to dissolve, to prepare a test solution with a concentration of 100 mg / mL.

[0100] Preparation of test solution 2: Take 5 g of each of the three batches of Cuscuta chinensis herb, add 100 mL of 60 wt% methanol solution to each batch, heat and reflux to extract twice, 1.5 h each time, filter, combine the two filtrates, evaporate the filtrate to dryness at 60℃, take the evaporated solid and add 2 mL of 60 wt% ethanol solution to dissolve, to prepare a test solution with a concentration of 100 mg / mL.

[0101] Preparation of test solution 3: Take 5 g of each of the three batches of Cuscuta chinensis herb, add 100 mL of water to each batch, heat and reflux at 90 °C twice for 1.5 h each time, filter, combine the two filtrates, evaporate the filtrate to dryness at 60 °C, add 2 mL of 60 wt% ethanol solution to dissolve the evaporated solids, and prepare a test solution with a concentration of 100 mg / mL.

[0102] Preparation of reference solutions: Take hyperoside, quercetin, astragaloside, and kaempferol reference standards and prepare reference solutions containing 100 μg per mL with methanol.

[0103] Perform thin-layer chromatography (Chinese Pharmacopoeia 2010 Edition, Part I, Appendix VI B). Apply 2 μL each of the above-mentioned test solution, hyperoside reference solution, quercetin reference solution, astragaloside reference solution, and kaempferol reference solution to the same silica gel G thin-layer plate. Develop the plate using chloroform-methanol-formic acid (11:2:2, v / v). Remove the plate, air dry, and spray with 1 wt% aluminum trichloride ethanol solution for color development. Examine the plate at 365 nm. The results are shown in the appendix. Figure 27 To be continued Figure 29 As shown.

[0104] See appendix Figure 27 To be continued Figure 29 Thin-layer chromatography results show that the test samples prepared by extraction with different solvents all exhibited good separation effects in the thin-layer chromatographic plate, among which... Figure 27 and attached Figure 28 The spots are more clearly visible, indicating that alcohol-based extract solvents are more effective than water-based solvents, resulting in higher extraction concentrations.

[0105] This application analyzes the preparation methods of thin-layer chromatography developing agents, colorimetric agents, and test solutions using comprehensive chromatographic detection methods, achieving accurate and comprehensive quality control of Cuscuta chinensis. The technical solution of this application has high sensitivity, is easy to operate, has low cost, and short detection time, which is conducive to the establishment and overall evaluation of Cuscuta chinensis quality standards.

[0106] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A thin-layer chromatographic detection method for Cuscuta chinensis, characterized in that, Includes the following steps: Provide a sample solution of dodder. Provide hyperoside reference solution, quercetin reference solution, astragaloside reference solution and kaempferol reference solution; The dodder test solution, hyperoside reference solution, quercetin reference solution, astragaloside reference solution and kaempferol reference solution were spotted on the same thin-layer chromatography plate, developed with a developing solvent, removed, dried and then subjected to color development and inspection. The developing agent is a mixed solution of chloroform, methanol and formic acid; the volume ratio of chloroform, methanol and formic acid is (8-12):(1-2.2):(1-2.2).

2. The thin-layer chromatography detection method for Cuscuta chinensis according to claim 1, characterized in that, The volume ratio of the trichloromethane, the methanol, and the formic acid is (10.5-11.5):(1-2.2):(1-2.2). Optionally, the volume ratio of the trichloromethane, the methanol and the formic acid is (10.5-11.5):(1.8-2.2):(1.8-2.2).

3. The thin-layer chromatography detection method for Cuscuta chinensis according to claim 1, characterized in that, The preparation method of the dodder test solution includes: The dodder extract was prepared by reflux extraction of the dodder sample in ethanol solution, solid-liquid separation, drying of the filtrate; The dodder extract was dissolved in a solvent to prepare the dodder test solution.

4. The thin-layer chromatography detection method for Cuscuta chinensis according to claim 3, characterized in that, The mass-to-volume ratio of the dodder sample to the ethanol solution is 1 g: (10-50) mL; and / or, the mass fraction of the ethanol solution is 50%-70%; and / or, the reflux extraction is performed 2-3 times; and / or, the reflux extraction time is 1-3 hours; and / or, the drying temperature is 55℃-65℃; and / or, the solvent includes one or more of ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide, acetone, and water. Optionally, the solvent includes ethanol, wherein the mass fraction of the ethanol is 50%-70%.

5. The thin-layer chromatography detection method for Cuscuta chinensis according to claim 3, characterized in that, The dodder sample includes one or more of the following: dodder herb and preparations containing dodder. Optionally, the dodder herb material includes one or more parts of the dodder plant, such as seeds, roots, stems, leaves, and flowers; more preferably, the dodder herb material includes the whole dodder plant. Optionally, the dosage forms of preparations containing dodder include decoction pieces, granules, tablets, decoctions, pills, powders, ointments, tablets, granules, lozenges, capsules, liquids, or tinctures.

6. The thin-layer chromatography detection method for Cuscuta chinensis according to any one of claims 1 to 3, characterized in that, The preparation method of the hyperoside reference solution, the quercetin reference solution, the astragaloside reference solution and the kaempferol reference solution includes: preparing each of the hyperoside reference standard, quercetin reference standard, astragaloside reference standard and kaempferol reference standard by independently mixing them with methanol. And / or, the concentrations of the hyperoside reference solution, the quercetin reference solution, the astragaloside reference solution, and the kaempferol reference solution are each independently 0.02 mg / mL to 2 mg / mL; And / or, the concentration of dodder extract in the dodder test solution is 5 mg / mL to 500 mg / mL.

7. The thin-layer chromatography detection method for Cuscuta chinensis according to any one of claims 1 to 3, characterized in that, The thin-layer chromatography plate includes a silica gel thin-layer plate; Optionally, the silicone sheet may include a silicone G sheet, a silicone H sheet, or a GF254 sheet.

8. The thin-layer chromatography detection method for Cuscuta chinensis according to any one of claims 1 to 3, characterized in that, The colorimetric reagent used in the colorimetric treatment includes aluminum trichloride solution; Optionally, the aluminum trichloride solution includes an ethanol solution of aluminum trichloride; Optionally, the mass fraction of the aluminum trichloride solution is 0.1%-6%.

9. The thin-layer chromatography detection method for Cuscuta chinensis according to any one of claims 1 to 3, characterized in that, The light sources used in the inspection included ultraviolet light sources; Optionally, the wavelength of the ultraviolet light source is 365nm.

10. The application of the thin-layer chromatography detection method of Cuscuta chinensis according to any one of claims 1 to 9 in the quality control and evaluation of Cuscuta chinensis preparations.

Images