Thin-layer identification method for traditional Chinese medicine compound preparation containing angelica sinensis and pericarpium citri reticulatae and application of thin-layer identification method

Abstract

The invention discloses a thin-layer identification method of a traditional Chinese medicine compound preparation containing angelica sinensis and pericarpium citri reticulatae, which comprises the following steps: (1) pretreating the traditional Chinese medicine compound preparation containing angelica sinensis and pericarpium citri reticulatae to obtain a test solution, and pretreating the traditional Chinese medicine compound preparation without pericarpium citri reticulatae or angelica sinensis to obtain a negative sample solution, pretreating a reference substance to obtain a reference substance solution; and (2) performing thin-layer chromatography identification analysis on the test solution, the negative sample solution and the reference solution. According to the thin-layer identification method disclosed by the invention, the Chinese angelica and the dried orange peel in the Chinese herbal compound containing the Chinese angelica and the dried orange peel are detected, so that a theoretical basis is provided for quality control and research on pharmacodynamic material basis of the Chinese herbal compound containing the Chinese angelica and the dried orange peel.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a thin-layer chromatography method for identifying traditional Chinese medicine compound preparations containing angelica and tangerine peel, and its application. Background Technology

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

[0003] The thin-layer chromatography method for identifying a traditional Chinese medicine compound preparation containing angelica and tangerine peel, as claimed in this invention, and its application, are not disclosed in the prior art. Summary of the Invention

[0004] Based on this, the present invention provides a thin-layer chromatography (TLC) identification method for a traditional Chinese medicine compound preparation containing Angelica sinensis and Citrus reticulata peel, the TLC identification method comprising the following steps:

[0005] (1) A traditional Chinese medicine compound preparation containing Angelica sinensis and Citrus reticulata peel is pretreated to obtain a test solution; a traditional Chinese medicine compound preparation lacking Citrus reticulata peel or Angelica sinensis is pretreated to obtain a negative sample solution; and a reference standard is pretreated to obtain a reference standard solution; and

[0006] (2) Thin-layer chromatography was performed to identify the test solution, the negative sample solution, and the reference solution;

[0007] In the thin-layer chromatography identification analysis step, a developing solvent is used for development. The developing solvent includes a first developing solvent and a second developing solvent. The first developing solvent is cyclohexane-ethyl acetate, and the second developing solvent is a lower layer solution of chloroform-methanol-water. The volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.5~2):(0.5~2), and the volume ratio of chloroform, methanol and water in the second developing solvent is (5~20):(1~6):(0.5~2).

[0008] Furthermore, this traditional Chinese medicine compound preparation containing angelica and tangerine peel includes angelica, rehmannia, tangerine peel, pinellia, poria, ginger, and licorice.

[0009] Furthermore, the mass ratio of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is (350~500): (750~900): (250~400): (350~500): (350~500): (200~350): (150~300).

[0010] Furthermore, the mass ratio of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is approximately 414.44: 828.89: 311.11: 414.44: 414.44: 277.78: 207.22g.

[0011] Furthermore, the mass ratio of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is (1~3):(3~5):(1~2):(1~3):(1~3):(1~2):(0.5~1.5).

[0012] Furthermore, the mass ratio of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is approximately 2:4:1.5:2:2:1.34:1.

[0013] Furthermore, this Pinellia ternata is Qing Pinellia ternata.

[0014] Furthermore, this licorice is stir-fried licorice.

[0015] Furthermore, the dosage form of this traditional Chinese medicine compound preparation containing angelica and tangerine peel is granules.

[0016] Furthermore, the preparation method of the granules includes the following steps: A suitable amount of fresh ginger is cut into thick slices, and aromatic water is extracted by steam distillation with a suitable amount of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, and Glycyrrhiza uralensis. The aqueous solution is then encapsulated with hydroxypropyl beta-cyclodextrin, and the encapsulation solution is concentrated under reduced pressure to a relative density of 1.10–1.20 (approximately 60°C), dried, and set aside. The dregs are added to 4–8 times their volume of water, decocted for 30–60 minutes, filtered, and the filtrate is concentrated under reduced pressure to a clear extract with a relative density of 1.20–1.30 (approximately 60°C), dried, and the encapsulation solution and a suitable amount of maltodextrin are added. Granulation is then performed to produce approximately 1000g of granules, thus obtaining the granules.

[0017] Furthermore, the preparation method of the granules includes the following steps: A suitable amount of fresh ginger is cut into thick slices, and aromatic water is extracted by steam distillation with a suitable amount of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, and Glycyrrhiza uralensis. The aqueous solution is then encapsulated with hydroxypropyl beta-cyclodextrin, and the encapsulated solution is concentrated under reduced pressure to a relative density of 1.10–1.20 (approximately 60°C), dried, and the encapsulated solution is set aside. The dregs are added to approximately 6 times their volume of water, decocted for approximately 45 minutes, filtered, and the filtrate is concentrated under reduced pressure to a clear extract with a relative density of 1.20–1.30 (approximately 60°C). The extract is dried, and the encapsulated solution and a suitable amount of maltodextrin are added. Granulation is then performed to produce approximately 1000g of granules, thus obtaining the granules.

[0018] Furthermore, the negative sample solution is a negative sample solution lacking dried tangerine peel and / or a negative sample solution lacking angelica.

[0019] Furthermore, the reference standard is hesperidin reference standard, norihesperidin reference standard and / or ligustrolactone I reference standard.

[0020] Furthermore, the preparation method of the test sample solution includes: (1) weighing an appropriate amount of Chinese herbal compound preparation granules containing Angelica sinensis and Citrus reticulata, grinding them, dissolving them in water, extracting them by shaking with a first solvent, collecting and merging the organic layers, evaporating them to dryness, and obtaining the residue; and (2) adding a second solvent to the residue to dissolve it, thereby obtaining the test sample solution.

[0021] Furthermore, the first solvent is ethyl acetate.

[0022] Furthermore, the mass / volume (g / ml) ratio of the compound Chinese medicine preparation granules containing angelica and tangerine peel to the water is 0.1 to 0.5, for example, about 0.12.

[0023] Furthermore, the volume ratio of the water to the first solvent is 0.5 to 2, for example, about 1.

[0024] Furthermore, the shaking extraction is performed 2 to 4 times, for example, 3 times.

[0025] Furthermore, the mass of the compound Chinese medicine preparation granules containing angelica and tangerine peel is 1-5 g, for example, about 3.6 g.

[0026] Furthermore, the volume of the water is 20-40 ml, for example, about 30 ml.

[0027] Furthermore, the volume of the ethyl acetate is 20 to 40 ml, for example, about 30 ml.

[0028] Furthermore, the second solvent is an alcohol, such as methanol.

[0029] Furthermore, the mass / volume (g / ml) ratio of the compound Chinese medicine preparation granules containing angelica and tangerine peel to the second solvent is 3 to 4, for example, about 3.6.

[0030] Furthermore, the volume of the methanol is 0.5 to 2 ml, for example, about 1 ml.

[0031] Furthermore, the preparation method of the reference solution includes: taking an appropriate amount of hesperidin, norihesperidin or ligustrazine I reference standard, adding methanol to obtain hesperidin reference solution, norihesperidin reference solution or ligustrazine I reference solution respectively.

[0032] Furthermore, the concentration of hesperidin in the hesperidin reference solution is 1 to 3 mg / ml, for example, about 2 mg / ml.

[0033] Furthermore, the concentration of norepinephrine in the norepinephrine reference solution is 1-3 mg / ml, for example, about 2 mg / ml.

[0034] Furthermore, the concentration of ligustilide I in the ligustilide I reference solution is 0.1~1 mg / ml, for example, about 0.5 mg / ml.

[0035] Furthermore, the preparation method of the negative sample solution includes: weighing appropriate amounts of traditional Chinese medicine compound preparation granules lacking tangerine peel or angelica, and the subsequent steps are the same as the preparation method of the test sample solution.

[0036] Furthermore, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.8~1.2):(0.8~1.2).

[0037] Furthermore, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is approximately 1:approximately 1.

[0038] Furthermore, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (8~16):(2~5):(0.6~1.4).

[0039] Furthermore, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (10~14):(3~4):(0.8~1.2).

[0040] Furthermore, the volume ratio of the trichloromethane, the methanol, and the water in the second developing agent is approximately 12: approximately 3.5: approximately 1.

[0041] Furthermore, the thin-layer plate is made of silicone GF. 254 Thin-layer plate.

[0042] Furthermore, the silicone GF254 Thin-layer plates are made of Merck GF silicone rubber from Germany. 254 Thin-layer plate or Yantai Jiangyou silicone GF 254 Thin-layer plate or Qingdao Marine GF silicone 254 Thin-layer plate.

[0043] Furthermore, the inspection conditions for tangerine peel in the thin-layer chromatography identification analysis steps are as follows: place the thin-layer plate in a developing tank, develop it using the first developing solvent, remove the developed thin-layer plate, air dry it, and inspect it under ultraviolet light.

[0044] Furthermore, the inspection conditions for Angelica sinensis in the thin-layer chromatography identification analysis steps are as follows: place the thin-layer plate back into the developing tank, develop it using a second developing solvent, remove the developed thin-layer plate, air dry it, and inspect it under ultraviolet light.

[0045] Furthermore, the detection wavelength of this ultraviolet lamp is 254 nm.

[0046] Furthermore, in the inspection conditions of dried tangerine peel, the developing tank was saturated with ammonia vapor for about 20 minutes.

[0047] Furthermore, in the inspection conditions of Angelica sinensis, the developing tank was saturated with ammonia vapor for about 10 minutes.

[0048] Furthermore, the sample volume of the test solution is 3~12 μl, for example 5~10 μl.

[0049] Furthermore, the spotting volume of the hesperidin reference solution is 0.1 to 2 μl, for example 0.5 to 1.5 μl, for example about 1 μl.

[0050] Furthermore, the amount of the hesperidin reference solution applied is 0.1~2 μl, for example 0.5~1.5 μl, for example about 1 μl.

[0051] Furthermore, the amount of the sample of the ligustrolactone I reference solution is 3-7 μl, for example 4-6 μl, for example about 5 μl.

[0052] Furthermore, the amount of the negative sample solution applied is 5 to 15 μl, for example, about 10 μl.

[0053] Furthermore, the temperature for this thin-layer identification is 2 to 30°C, for example, about 8°C or about 24°C.

[0054] Furthermore, the relative humidity for this thin-layer identification is 38% to 95%, for example, about 42% or about 88%.

[0055] Furthermore, the criteria for thin-layer chromatography identification include: after the first development using the first developing solvent, checking whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions as the hesperidin and tangeretin reference standards; after the second development using the second developing solvent, checking whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions as the ligustrolactone I reference standard; and whether there is interference from negative samples; if the thin-layer plate has the characteristic spots after both the first and second developments and the negative samples do not interfere, then the detected traditional Chinese medicine compound preparation containing angelica and tangerine peel contains angelica and tangerine peel medicinal materials.

[0056] According to another aspect of the present invention, the above-described thin-layer identification method is provided for use in identifying traditional Chinese medicine compound preparations containing angelica and tangerine peel.

[0057] According to another aspect of the present invention, a kit is provided for identifying traditional Chinese medicine compound preparations containing Angelica sinensis and Citrus reticulata peel. The kit includes a thin-layer chromatography plate and a developing solvent, the developing solvent comprising a first developing solvent and a second developing solvent, wherein the first developing solvent is cyclohexane-ethyl acetate, the second developing solvent is a lower layer solution of chloroform-methanol-water, and the thin-layer plate is silica gel GF. 254 Thin-layer plate.

[0058] Furthermore, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.5~2):(0.5~2).

[0059] Furthermore, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.8~1.2):(0.8~1.2).

[0060] Furthermore, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is approximately 1:approximately 1.

[0061] Furthermore, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (5~20):(1~6):(0.5~2).

[0062] Furthermore, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (8~16):(2~5):(0.6~1.4).

[0063] Furthermore, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (10~14):(3~4):(0.8~1.2).

[0064] Furthermore, the volume ratio of the trichloromethane, the methanol, and the water in the second developing agent is approximately 12: approximately 3.5: approximately 1.

[0065] The beneficial effects of this invention are:

[0066] The thin-layer chromatography identification method of the present invention provides a theoretical basis for the quality control and pharmacodynamic material basis research of traditional Chinese medicine compound formulas containing angelica and tangerine peel by detecting angelica and tangerine peel. Attached Figure Description

[0067] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0068] Figure 1 This is a schematic diagram showing the results of the thin-layer chromatography (TLC) development conditions. S1 represents hesperidin, S2 represents norihesperidin, and S3 represents ligustrolactone I; 1 is a negative control solution lacking tangerine peel, 2 is a test solution of a traditional Chinese medicine compound containing angelica and tangerine peel, and 3 is a negative control solution lacking angelica; A represents the first development, and B represents the second development.

[0069] Figure 2 Thin-layer chromatograms (first development) were used to investigate the sample quantity. The samples included: S1, hesperidin (1 μl); S2, hesperidin (3 μl); S3, hesperidin (5 μl); S4, tangeretin (1 μl); S5, tangeretin (3 μl); S6, tangeretin (5 μl); S7, ligustrazine I (3 μl); S8, ligustrazine I (5 μl); S9, ligustrazine I (7 μl); 1, negative control solution lacking tangerine peel (5 μl); 2, test solution of traditional Chinese medicine compound containing angelica and tangerine peel (5 μl); 3, test solution of traditional Chinese medicine compound containing angelica and tangerine peel (10 μl); 4, test solution of traditional Chinese medicine compound containing angelica and tangerine peel (15 μl); 5, negative control solution lacking angelica (5 μl).

[0070] Figure 3 Thin-layer chromatograms (second development) were used to examine the sample quantity. The samples included: S1, hesperidin (1 μl); S2, hesperidin (3 μl); S3, hesperidin (5 μl); S4, tangeretin (1 μl); S5, tangeretin (3 μl); S6, tangeretin (5 μl); S7, ligustrazine I (3 μl); S8, ligustrazine I (5 μl); S9, ligustrazine I (7 μl); 1, negative control solution lacking tangerine peel (5 μl); 2, test solution of traditional Chinese medicine compound containing angelica and tangerine peel (5 μl); 3, test solution of traditional Chinese medicine compound containing angelica and tangerine peel (10 μl); 4, test solution of traditional Chinese medicine compound containing angelica and tangerine peel (15 μl); 5, negative control solution lacking angelica (5 μl).

[0071] Figure 4 Thin-layer chromatograms for temperature investigation (first development). S1: hesperidin; S2: norihesperidin; S3: ligustrolactone I; 1: negative control solution lacking tangerine peel; 2: test solution of traditional Chinese medicine compound containing angelica and tangerine peel; 3: negative control solution lacking angelica.

[0072] Figure 5 The thin-layer chromatogram for temperature investigation (second development) shows the following: S1, hesperidin; S2, norihesperidin; S3, ligustrolactone I; 1, negative control solution lacking tangerine peel; 2, test solution of traditional Chinese medicine compound containing angelica and tangerine peel; 3, negative control solution lacking angelica.

[0073] Figure 6 Thin-layer chromatogram for humidity investigation (first development). S1: hesperidin; S2: norihesperidin; S3: ligustrolactone I; 1: negative control solution lacking tangerine peel; 2: test solution of traditional Chinese medicine compound containing angelica and tangerine peel; 3: negative control solution lacking angelica.

[0074] Figure 7 Thin-layer chromatogram for humidity investigation (second development). S1: hesperidin; S2: norihesperidin; S3: ligustrolactone I; 1: negative control solution lacking tangerine peel; 2: test solution of traditional Chinese medicine compound containing angelica and tangerine peel; 3: negative control solution lacking angelica.

[0075] Figure 8 Thin-layer chromatograms (first development) were examined for different manufacturers' thin-layer plates. The following components were tested: S1, hesperidin; S2, norihesperidin; S3, ligustrolactone I; 1, negative control solution lacking tangerine peel; 2, test solution of a traditional Chinese medicine compound containing angelica and tangerine peel; 3, negative control solution lacking angelica.

[0076] Figure 9 Thin-layer chromatograms (second development) were examined for different manufacturers' thin-layer plates. Among them, S1 was hesperidin, S2 was norihesperidin, and S3 was ligustrolactone I; 1. Negative control solution lacking tangerine peel, 2. Test solution of traditional Chinese medicine compound containing angelica and tangerine peel, and 3. Negative control solution lacking angelica.

[0077] Figure 10 Schematic diagram to verify the experimental results. S1, hesperidin (1 μl); S2, norihesperidin (1 μl); S3, ligustrolactone I (5 μl); 1, JSLJJBXY241201-P (5 μl); 2, JSLJJBXY241201-P (5 μl); 3, JSLJJBXY241203-P (5 μl); A represents the first development, B represents the second development. Detailed Implementation

[0078] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0079] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0080] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0081] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0082] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0083] As described in the background section, the prior art does not disclose a thin-layer chromatography (TLC) identification method for a traditional Chinese medicine compound preparation containing Angelica sinensis and Citrus reticulata peel, as claimed in this invention, nor its application. To address the above problem, this invention provides a TLC identification method for a traditional Chinese medicine compound preparation containing Angelica sinensis and Citrus reticulata peel, comprising the following steps:

[0084] (1) A traditional Chinese medicine compound preparation containing Angelica sinensis and Citrus reticulata peel is pretreated to obtain a test solution; a traditional Chinese medicine compound preparation lacking Citrus reticulata peel or Angelica sinensis is pretreated to obtain a negative sample solution; and a reference standard is pretreated to obtain a reference standard solution; and

[0085] (2) Thin-layer chromatography was performed to identify the test solution, the negative sample solution, and the reference solution;

[0086] In the thin-layer chromatography identification analysis step, a developing solvent is used for development. The developing solvent includes a first developing solvent and a second developing solvent. The first developing solvent is cyclohexane-ethyl acetate, and the second developing solvent is a lower layer solution of chloroform-methanol-water. The volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.5~2):(0.5~2), and the volume ratio of chloroform, methanol and water in the second developing solvent is (5~20):(1~6):(0.5~2).

[0087] In this invention, when volume ratio, number of times, mass, ratio, volume, concentration, spotting amount, temperature, or other values ​​or parameters are expressed as a range, preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value and any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range "0.5~2" is disclosed, the described range should be interpreted as including ranges "0.5~2", "0.5~1.7", "0.5~1.4", "0.5~1.1", "0.5~0.8", "0.8~2", "0.8~1.7", "0.8~1.4", "0.8~1.1", "1.1~2", "1.1~1.7", "1.1~1.4", "1.4~2", "1.4~1.7", "1.7~2", etc. When a range of values ​​is described in this document, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0088] In a preferred embodiment, the traditional Chinese medicine compound preparation containing angelica and tangerine peel includes angelica, rehmannia, tangerine peel, pinellia, poria, ginger, and licorice.

[0089] In a preferred embodiment, the mass ratio of the Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is (350~500):(750~900):(250~400):(350~500):(350~500):(200~350):(150~300).

[0090] In a preferred embodiment, the mass ratio of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is about 414.44: about 828.89: about 311.11: about 414.44: about 414.44: about 277.78: about 207.22g.

[0091] In a preferred embodiment, the mass ratio of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is (1~3):(3~5):(1~2):(1~3):(1~3):(1~2):(0.5~1.5).

[0092] In a preferred embodiment, the mass ratio of the angelica, the rehmannia, the tangerine peel, the pinellia, the poria, the ginger, and the licorice is about 2: about 4: about 1.5: about 2: about 2: about 1.34: about 1.

[0093] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 414.44" includes ±5% of 414.44, or from 393.718 to 435.162; "about 828.89" includes ±5% of 828.89, or from 787.4455 to 870.3345; "about 311.11" includes ±5% of 311.11, or from 295.5545 to 326.6655; "about 277.78" includes ±5% of 277.78, or from 263.891 to 291.669; "About 207.22" includes 207.22 ± 5%, or from 196.859 to 217.581; "About 2" includes 2 ± 5%, or from 1.9 to 2.1; "About 4" includes 4 ± 5%, or from 3.8 to 4.2; "About 1.5" includes 1.5 ± 5%, or from 1.425 to 1.575; "About 1.34" includes 1.34 ± 5%, or from 1.273 to 1.407; "About 1" includes 1 ± 5%, or from 0.95 to 1.05.

[0094] In a preferred embodiment, the Pinellia ternata is processed Pinellia ternata.

[0095] In a preferred embodiment, the licorice is stir-fried licorice.

[0096] In a preferred embodiment, the preparation method of the granules includes the following steps: A suitable amount of fresh ginger is cut into thick slices, and aromatic water is extracted by steam distillation with a suitable amount of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, and Glycyrrhiza uralensis. The extract is then infused with hydroxypropyl beta-cyclodextrin, and the infusion solution is concentrated under reduced pressure to a relative density of 1.10–1.20 (approximately 60°C), dried, and the infusion compound is set aside. The dregs are added to 4–8 times their volume of water, decocted for 30–60 minutes, filtered, and the filtrate is concentrated under reduced pressure to a clear extract with a relative density of 1.20–1.30 (approximately 60°C). The extract is dried, and the infusion compound and a suitable amount of maltodextrin are added. Granulation is then performed to produce approximately 1000g of granules, thus obtaining the granules.

[0097] In a preferred embodiment, the preparation method of the granules includes the following steps: A suitable amount of fresh ginger is cut into thick slices, and aromatic water is extracted by steam distillation with a suitable amount of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, and Glycyrrhiza uralensis. The extract is then infused with hydroxypropyl beta-cyclodextrin, and the infusion solution is concentrated under reduced pressure to a relative density of 1.10–1.20 (approximately 60°C), dried, and the infusion compound is set aside. The dregs are added to approximately 6 times their volume of water, decocted for approximately 45 minutes, filtered, and the filtrate is concentrated under reduced pressure to a clear extract with a relative density of 1.20–1.30 (approximately 60°C). The extract is dried, and the infusion compound and a suitable amount of maltodextrin are added. Granulation is then performed to produce approximately 1000g of granules, thus obtaining the granules.

[0098] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.12" includes ±5% of 0.12, or from 0.114 to 0.126.

[0099] In a preferred embodiment, the preparation method of the granules includes the following steps: 828.89g of Rehmannia glutinosa (processed), 414.44g of Angelica sinensis, 414.44g of Pinellia ternata (processed), 311.11g of Citrus reticulata peel, 414.44g of Poria cocos, 277.78g of fresh ginger, and 207.22g of stir-fried Glycyrrhiza uralensis. The above seven herbs are combined with thickly sliced ​​fresh ginger and the aromatic water extracted by steam distillation with the other six herbs. The extract is then infused with hydroxypropyl beta-cyclodextrin, and the infusion solution is concentrated under reduced pressure to a relative density of 1.10–1.20 (60°C). The solution is dried to obtain the infusion compound, which is then set aside. The dregs are decocted with 6 times the amount of water for 45 minutes, filtered, and the filtrate is concentrated under reduced pressure to a clear extract with a relative density of 1.20–1.30 (60°C). The extract is dried, and the infusion compound and an appropriate amount of maltodextrin are added. Granulation is then performed to produce 1000g of granules, thus obtaining the granules.

[0100] In a preferred embodiment, the dosage form of the traditional Chinese medicine compound preparation containing angelica and tangerine peel is granules.

[0101] In a preferred embodiment, the negative sample solution is a negative sample solution lacking dried tangerine peel and / or a negative sample solution lacking angelica.

[0102] In a preferred embodiment, the reference standard is hesperidin reference standard, norihesperidin reference standard and / or ligustrazine I reference standard.

[0103] In a preferred embodiment, the preparation method of the test sample solution includes: (1) weighing an appropriate amount of Chinese herbal compound preparation granules containing Angelica sinensis and Citrus reticulata, grinding them, dissolving them in water, extracting them by shaking with a first solvent, collecting and merging the organic layers, evaporating them to dryness, and obtaining the residue; and (2) adding a second solvent to the residue to dissolve it, thereby obtaining the test sample solution.

[0104] In a preferred embodiment, the first solvent is ethyl acetate.

[0105] In a preferred embodiment, the mass / volume (g / ml) ratio of the compound Chinese medicine preparation granules containing angelica and tangerine peel to the water is 0.1 to 0.5, for example, about 0.12.

[0106] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.12" includes ±5% of 0.12, or from 0.114 to 0.126.

[0107] In a preferred embodiment, the volume ratio of the water to the first solvent is 0.5 to 2, for example, about 1.

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

[0109] In a preferred embodiment, the shaking extraction is performed 2 to 4 times, for example, 3 times.

[0110] In a preferred embodiment, the mass of the compound Chinese medicine preparation granules containing angelica and tangerine peel is 1-5g, for example, about 3.6g.

[0111] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.6" includes ±5% of 3.6, or from 3.42 to 3.78.

[0112] In a preferred embodiment, the volume of the water is 20 to 40 ml, for example, about 30 ml.

[0113] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0114] In a preferred embodiment, the volume of the ethyl acetate is 20 to 40 ml, for example, about 30 ml.

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

[0116] In a preferred embodiment, the second solvent is an alcohol, such as methanol.

[0117] In a preferred embodiment, the mass / volume (g / ml) ratio of the compound Chinese medicine preparation granules containing angelica and tangerine peel to the second solvent is 3 to 4, for example, about 3.6.

[0118] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.6" includes ±5% of 3.6, or from 3.42 to 3.78.

[0119] In a preferred embodiment, the volume of methanol is 0.5 to 2 ml, for example, about 1 ml.

[0120] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0121] In a preferred embodiment, the method for preparing the reference solution includes: taking an appropriate amount of hesperidin, norihesperidin, or ligustrazine I reference standard, adding methanol, and obtaining hesperidin reference solution, norihesperidin reference solution, or ligustrazine I reference solution, respectively.

[0122] In a preferred embodiment, the concentration of hesperidin in the hesperidin reference solution is 1 to 3 mg / ml, for example, about 2 mg / ml.

[0123] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0124] In a preferred embodiment, the concentration of norepinephrine in the norepinephrine reference solution is 1-3 mg / ml, for example, about 2 mg / ml.

[0125] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0126] In a preferred embodiment, the concentration of ligustilide I in the ligustilide I reference solution is 0.1~1 mg / ml, for example, about 0.5 mg / ml.

[0127] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0128] In a preferred embodiment, the preparation method of the negative sample solution includes: weighing appropriate amounts of traditional Chinese medicine compound preparation granules lacking tangerine peel or angelica, and the subsequent steps are the same as the preparation method of the test sample solution.

[0129] In a preferred embodiment, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.8~1.2):(0.8~1.2).

[0130] In a preferred embodiment, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is about 1:about 1.

[0131] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0132] In a preferred embodiment, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is (8~16):(2~5):(0.6~1.4).

[0133] In a preferred embodiment, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is (10~14):(3~4):(0.8~1.2).

[0134] In a preferred embodiment, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is about 12: about 3.5: about 1.

[0135] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6; "about 3.5" includes ±5% of 3.5, or from 3.325 to 3.675; and "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0136] In a preferred embodiment, the thin-layer plate is silicone GF. 254 Thin-layer plate.

[0137] In a preferred embodiment, the silicone GF 254 Thin-layer plates are made of Merck GF silicone rubber from Germany. 254 Thin-layer plate or Yantai Jiangyou silicone GF 254 Thin-layer plate or Qingdao Marine GF silicone 254 Thin-layer plate.

[0138] In a preferred embodiment, the inspection conditions for tangerine peel in the thin-layer chromatography identification analysis step are as follows: the thin-layer plate is placed in a developing tank, developed using a first developing solvent, and the developed thin-layer plate is removed, dried, and inspected under ultraviolet light.

[0139] In a preferred embodiment, the inspection conditions for Angelica sinensis in the thin-layer chromatography identification analysis step are as follows: the thin-layer plate is placed back into the developing tank, developed using a second developing solvent, and the developed thin-layer plate is removed, dried, and inspected under ultraviolet light.

[0140] In a preferred embodiment, the detection wavelength of the ultraviolet lamp is 254 nm.

[0141] In a preferred embodiment, under the inspection conditions of dried tangerine peel, the developing tank is a developing tank saturated with ammonia vapor for about 20 minutes.

[0142] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0143] In a preferred embodiment, under the inspection conditions of Angelica sinensis, the developing cylinder is a developing cylinder saturated with ammonia vapor for about 10 minutes.

[0144] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0145] In a preferred embodiment, the sample volume of the test solution is 3-12 μl, for example 5-10 μl.

[0146] In a preferred embodiment, the amount of hesperidin reference solution applied is 0.1 to 2 μl, for example 0.5 to 1.5 μl, for example about 1 μl.

[0147] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0148] In a preferred embodiment, the amount of the noriheptacorlin reference solution applied is 0.1-2 μl, for example 0.5-1.5 μl, for example about 1 μl.

[0149] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0150] In a preferred embodiment, the amount of the ligustilide I reference solution is 3-7 μl, for example 4-6 μl, for example about 5 μl.

[0151] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0152] In a preferred embodiment, the amount of the negative sample solution applied is 5 to 15 μl, for example, about 10 μl.

[0153] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0154] In a preferred embodiment, the temperature for thin-layer identification is 2 to 30°C, for example, about 8°C or about 24°C.

[0155] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4; "about 24" includes ±5% of 24, or from 22.8 to 25.2.

[0156] In a preferred embodiment, the relative humidity for thin-layer identification is 38% to 95%, for example, about 42% or about 88%.

[0157] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 42%" includes 42% ± 5%, or from 39.9% to 44.1%; "about 88%" includes 88% ± 5%, or from 83.6% to 92.4%.

[0158] In a preferred embodiment, the criteria for thin-layer chromatography identification include: after a first development using the first developing solvent, checking whether the thin-layer plate displays characteristic spots of the same color at the corresponding positions as the hesperidin and tangeretin reference standards; after a second development using the second developing solvent, checking whether the thin-layer plate displays characteristic spots of the same color at the corresponding positions as the ligustrolactone I reference standard; and whether there is interference from negative samples; if the thin-layer plate has the characteristic spots after both the first and second developments and the negative samples do not interfere, then the detected traditional Chinese medicine compound preparation containing angelica and tangerine peel contains angelica and tangerine peel medicinal materials.

[0159] According to another aspect of the present invention, the above-described thin-layer identification method is provided for use in identifying traditional Chinese medicine compound preparations containing angelica and tangerine peel.

[0160] According to another aspect of the present invention, a kit is provided for identifying traditional Chinese medicine compound preparations containing Angelica sinensis and Citrus reticulata peel. The kit includes a thin-layer chromatography plate and a developing solvent, the developing solvent comprising a first developing solvent and a second developing solvent, wherein the first developing solvent is cyclohexane-ethyl acetate, the second developing solvent is a lower layer solution of chloroform-methanol-water, and the thin-layer plate is silica gel GF. 254 Thin-layer plate.

[0161] In a preferred embodiment, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.5~2):(0.5~2).

[0162] In a preferred embodiment, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.8~1.2):(0.8~1.2).

[0163] In a preferred embodiment, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is about 1:about 1.

[0164] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0165] In a preferred embodiment, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is (5~20):(1~6):(0.5~2).

[0166] In a preferred embodiment, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is (8~16):(2~5):(0.6~1.4).

[0167] In a preferred embodiment, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is (10~14):(3~4):(0.8~1.2).

[0168] In a preferred embodiment, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is about 12: about 3.5: about 1.

[0169] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6; "about 3.5" includes ±5% of 3.5, or from 3.325 to 3.675; and "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0170] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0171] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0172] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0173] Example

[0174] 1. Instruments and reagents

[0175] Instrument: 1% electronic balance: JY2002 Shanghai Puchun Metrology Instrument Co., Ltd.;

[0176] Thin-layer plate: Qingdao Marine Silicone GF 254 Thin-layer board, Yantai Huayang silicone GF 254 Thin-layer plate, Merck GF silicone rubber from Germany 254 Thin-layer plate;

[0177] Digital display constant water bath: DZKW-S-6 Beijing Yongguangming Medical Instrument Co., Ltd.;

[0178] Thin-layer imager: Visualizer 2 CAMAG;

[0179] Medicine Cooling Cabinet: FL-1200D, Shandong Sanjiu Electric Appliance Co., Ltd.

[0180] Reagents: Ethyl acetate, cyclohexane, chloroform, methanol, and concentrated ammonia solution were all of analytical grade; sulfuric acid was of analytical grade; and water was purified water.

[0181] Test drugs: hesperidin (batch number: 112054-202102, content calculated at 99.7%, China National Institutes for Food and Drug Control), norihesperidin (batch number: 112055-202102, content calculated at 99.7%, China National Institutes for Food and Drug Control), and ligustrolactone I (batch number: 112071-202101, content calculated at 99.2%, China National Institutes for Food and Drug Control);

[0182] Samples: Traditional Chinese medicine compound preparation granules containing angelica and tangerine peel, batch numbers: JSLJJBX2024040901-P, JSLJJBXY241201-P, JSLJJBXY241202-P and JSLJJBXY241203-P;

[0183] The negative control batch number for a traditional Chinese medicine compound preparation containing angelica and tangerine peel but lacking tangerine peel is: JSLJJBX2024042203-L;

[0184] The batch number of the negative control of the traditional Chinese medicine compound preparation containing angelica and tangerine peel is missing angelica: JSLJJBX2024042204-L.

[0185] 2. Investigation on thin-layer chromatographic identification method of granular tangerine peel and angelica root

[0186] 2.1 Investigation of Thin-Layer Development Conditions

[0187] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 10 µl each of the above test solution, negative control solution lacking tangerine peel, and negative control solution lacking angelica sinensis, 1 µl each of hesperidin reference solution and norihesperidin reference solution, and 5 µl of ligustrazine lactone I reference solution were spotted separately onto the same silica gel G thin-layer plate to form bands. Petroleum ether (60-90℃)-chloroform-ethyl acetate (2:1:3) was used as the developing solvent, and the development distance was about 8 cm. The plate was removed, dried, sprayed with vanillin-sulfuric acid test solution, and heated at 105℃ until the spots were clearly visible. The results are as follows.

[0188] The results showed that the negative control solutions lacking tangerine peel and angelica root had the same color spots at the corresponding positions on the chromatograms of the test samples, indicating that the chromatographic conditions were not suitable for the identification of tangerine peel and angelica root.

[0189] The thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition) was used. 10 µl each of the above test solution, the negative control solution lacking tangerine peel, and the negative control solution lacking angelica root, 1 µl each of the hesperidin reference solution and the norihesperidin reference solution, and 5 µl of the ligustrazine lactone I reference solution were spotted onto the same silica gel GF254 thin-layer plate to form bands. Cyclohexane-ethyl acetate (8:1) was used as the developing solvent. The development distance was approximately 8 cm. The plate was removed, dried, and examined under ultraviolet light (254 nm). The results are as follows.

[0190] The results showed that under ultraviolet light (254 nm), the test sample chromatogram showed spots of the same color at the corresponding positions as the chromatograms of hesperidin, norihesperidin, and ligustrazine I reference standards. However, the negative samples showed interference, and the Rf values ​​were lower. The presence of ligustrazine I reference standard at the origin indicated that the developing solvent polarity was too low. The developing solvent system was adjusted, and the number of development cycles was increased.

[0191] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition), 10 µl each of the above test solution, negative control solution lacking tangerine peel, and negative control solution lacking angelica sinensis, 1 µl each of hesperidin reference solution and norihesperidin reference solution, and 5 µl of ligustrazine lactone I reference solution were spotted separately onto the same silica gel GF254 thin-layer plate to form bands. Cyclohexane-ethyl acetate (8:1) was used as the developing solvent, and the plate was developed to a distance of approximately 8 cm. The plate was removed, dried, and examined under ultraviolet light (254 nm) (tangerine peel); then, the lower layer of chloroform-methanol-water (12:5:1) was used as the developing solvent, and the plate was developed, dried, and examined under ultraviolet light (254 nm) (angelica sinensis). The results are as follows.

[0192] The results showed that after the first development, when examined under ultraviolet light (254 nm), the test sample chromatogram showed spots of the same color at the corresponding positions as those of the hesperidin reference standard, norihesperidin reference standard, and ligustrazine lactone I reference standard. However, the negative results showed interference, and the Rf value was low.

[0193] After the second development, the sample was examined under ultraviolet light (254 nm). The chromatogram of the test sample showed spots of the same color at the corresponding positions as the chromatogram of the reference standard, ligustrazine I. However, the negative test showed interference, and the Rf value was higher. Therefore, the ratio of the developing solvent was adjusted.

[0194] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition), 10 µl each of the above test solution, negative control solution lacking tangerine peel, and negative control solution lacking angelica sinensis, 1 µl each of hesperidin reference solution and norihesperidin reference solution, and 5 µl of ligustrazine lactone I reference solution were spotted separately onto the same silica gel GF254 thin-layer plate to form bands. The plate was developed using cyclohexane-ethyl acetate (1:1) as the developing solvent, with a development distance of approximately 8 cm. The plate was removed, dried, and examined under ultraviolet light (254 nm) (tangerine peel); then, the lower layer of chloroform-methanol-water (12:3.5:1) was used as the developing solvent, developed, removed, dried, and examined under ultraviolet light (254 nm) (angelica sinensis). The results are as follows.

[0195] The results showed that after the first development, when examined under ultraviolet light (254 nm), the test sample chromatogram showed spots of the same color at the corresponding positions as those of the hesperidin reference standard, norihesperidin reference standard, and ligustrazine lactone I reference standard, but the negative results showed interference.

[0196] After the second development, the sample was examined under a UV lamp (254nm). In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference standard Ligusticum lactone I, but there was interference from the negative test.

[0197] Based on the above separation results, it is considered to improve the separation in both development processes by adding concentrated ammonia solution.

[0198] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 10 µl each of the test solution, the negative control solution lacking tangerine peel, and the negative control solution lacking angelica root; 1 µl each of the hesperidin reference solution and the norihesperidin reference solution; and 5 µl of the ligustrazine lactone I reference solution. Spot each solution onto the same silica gel GF plate. 254On the thin-layer plate, cyclohexane-ethyl acetate (1:1) was used as the developing solvent. The plate was placed in a developing tank saturated with ammonia vapor for 20 minutes, developed, removed, dried, and examined under ultraviolet light (254nm) to detect the aroma of dried tangerine peel. Then, the lower layer solution of chloroform-methanol-water (12:3.5:1) was used as the developing solvent. The plate was placed in a developing tank saturated with ammonia vapor for 10 minutes, developed, removed, dried, and examined under ultraviolet light (254nm) to detect the aroma of angelica.

[0199] The results are as follows Figure 1 As shown, after the first development, under ultraviolet light (254 nm), the chromatogram of the test sample showed spots of the same color at the corresponding positions as the chromatograms of the reference standards of hesperidin and norihesperidin, with no interference from negative sources, indicating good spot separation. After the second development, under ultraviolet light (254 nm), the chromatogram of the test sample showed spots of the same color at the corresponding positions as the reference chromatogram of ligustrazine I, with no interference from negative sources, indicating good spot separation. Therefore, this method is suitable for the thin-layer chromatographic identification of tangerine peel and angelica in compound preparations of traditional Chinese medicine containing angelica and tangerine peel. Therefore, the next step is to investigate the sample spotting volume.

[0200] 2.2. Sample Quantity Investigation

[0201] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) using 5 μl, 10 μl, and 15 μl of the above-mentioned test solution; 10 μl each of the negative control solutions lacking tangerine peel and angelica sinensis; 1 μl, 3 μl, and 5 μl each of the hesperidin and norihesperidin reference solutions; and 3 μl, 5 μl, and 7 μl of ligustrazine lactone I reference solution. Spot these solutions onto the same silica gel GF plate. 254 On a thin-layer plate, the strips were formed. Using cyclohexane-ethyl acetate (1:1) as the developing solvent, the plate was placed in a developing tank saturated with ammonia vapor for 20 minutes. The developing distance was about 8 cm. The plate was then removed, dried, and examined under a UV lamp (254 nm) (dried tangerine peel). Then, using the lower layer solution of chloroform-methanol-water (12:3.5:1) as the developing solvent, the plate was placed in a developing tank saturated with ammonia vapor for 10 minutes. The plate was then removed, dried, and examined under a UV lamp (254 nm) (angelica sinensis).

[0202] The results are as follows Figure 2 and Figure 3As shown, after the first development, under ultraviolet light (254 nm), the chromatogram of the test sample showed spots of the same color at the corresponding positions as those of the hesperidin and limonene reference standards, with no interference from negative light. When the spotting volume of the hesperidin and limonene reference solutions was 1 μl, the spots were clear and of moderate size. When the spotting volume was 3 μl and 5 μl, the spots showed obvious diffusion and were larger. Therefore, the spotting volume of the hesperidin and limonene reference solutions was determined to be 1 μl. After the second development, under ultraviolet light (254 nm), the chromatogram of the test sample showed spots of the same color at the corresponding positions as those of the chromatogram of ligustrolactone I reference standard, with no interference from negative light. When the spotting volume of the ligustrazine I reference solution was 3 μl, 5 μl, and 7 μl, the spots were clear and of moderate size with no obvious diffusion. Therefore, the spotting volume of the ligustrazine I reference solution was determined to be 5 μl. Comparing the spotting volumes of the test solution, when the spotting volume was 5–10 μl, the spots were clear and the separation was good. When the spotting volume increased to 15 μl, the test sample spots were larger and the spot separation was poor. Therefore, a spotting volume of 5–10 μl for the test solution was deemed appropriate. In summary, the spotting volume of the test solution was determined to be 5–10 μl, the spotting volume of the hesperidin and norihesperidin reference solutions was 1 μl, and the spotting volume of the ligustrazine I reference solution was 5 μl.

[0203] 2.3. Investigation at different temperatures

[0204] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) using 5 µl of the above test solution, 10 µl each of the negative control solutions lacking tangerine peel and angelica sinensis, 1 µl each of the hesperidin and norihesperidin reference solutions, and 5 µl of ligustrazine lactone I reference solution, and spot them separately onto the same silica gel GF plate. 254 On thin-layer plates, cyclohexane-ethyl acetate (1:1) was used as the developing solvent. The plates were placed in a developing tank saturated with ammonia vapor for 20 minutes and developed at room temperature (10~30℃) and low temperature (2~10℃), respectively. The plates were then removed, dried, and examined for the medicinal aroma of dried tangerine peel under ultraviolet light (254nm). The lower layer solution of chloroform-methanol-water (12:3.5:1) was used as the developing solvent. The plates were placed in a developing tank saturated with ammonia vapor for 10 minutes and developed at room temperature (10~30℃) and low temperature (2~10℃), respectively. The plates were then removed, dried, and examined for the medicinal aroma of angelica root under ultraviolet light (254nm).

[0205] The results are as follows Figure 4 and Figure 5As shown, after the first development, under ultraviolet light (254 nm) and examination, spots of the same color appeared at the corresponding positions in the chromatograms of hesperidin and norihesperidin reference standards under both room temperature and low temperature conditions, with no interference from negative samples. After the second development, under ultraviolet light (254 nm) and examination, spots of the same color appeared at the corresponding positions in the chromatograms of ligustrolactone I reference standards under both room temperature and low temperature conditions, with no interference from negative samples. Therefore, temperature has no significant effect on this thin-layer chromatography identification, and this thin-layer chromatography identification method has good temperature robustness.

[0206] 2.4 Investigation of different humidity levels

[0207] High humidity (88% relative humidity) experimental condition control method: This experiment investigated the effect of 88% relative humidity on thin-layer chromatography identification. 10.8 ml of sulfuric acid was measured and 100 ml of water was added, and the mixture was stirred well to prepare the solution. This solution was then added to one side of a dual-chamber chromatography tank (200*100 mm chromatography tank, with 12 ml of the prepared sulfuric acid solution added).

[0208] Low humidity (42% relative humidity) experimental condition control method: This experiment investigated the effect of 42% relative humidity on thin-layer chromatography identification. 57 ml of sulfuric acid was measured and 100 ml of water was added, and the mixture was stirred well. The solution was then added to one side of a dual-chamber chromatography tank (200*100 mm chromatography tank, with 12 ml of the prepared sulfuric acid solution added).

[0209] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) using 5 µl of the above test solution, 10 µl each of the negative control solutions lacking tangerine peel and angelica sinensis, 1 µl each of the hesperidin and norihesperidin reference solutions, and 5 µl of ligustrazine lactone I reference solution, and spot them separately onto the same silica gel GF plate. 254 On thin-layer chromatography plates, after saturation for 30 minutes under high humidity (88%) and low humidity (42%) conditions respectively, the plates were developed in a developing tank saturated with ammonia vapor for 20 minutes using cyclohexane-ethyl acetate (1:1) as the developing solvent. The plates were then removed, dried, and examined for the medicinal aroma of dried tangerine peel under ultraviolet light (254 nm). Next, the plates were saturated for 30 minutes under high humidity (88%) and low humidity (42%) conditions respectively, and developed in a developing tank saturated with ammonia vapor for 10 minutes using the lower layer of chloroform-methanol-water (12:3.5:1) as the developing solvent. The plates were then removed, dried, and examined for the medicinal aroma of angelica root under ultraviolet light (254 nm). The results are as follows.

[0210] The results are as follows Figure 6 and Figure 7As shown, after the first development, under ultraviolet light (254 nm), spots of the same color appeared at the corresponding positions in the chromatograms of hesperidin and norimethamine reference standards under both high and low humidity conditions, with no interference from negative samples. After the second development, under ultraviolet light (254 nm), spots of the same color appeared at the corresponding positions in the chromatograms of ligustrolactone I reference standards under both high and low humidity conditions, with no interference from negative samples. Therefore, humidity has no significant effect on this thin-layer chromatography identification method, and this method has good robustness to humidity.

[0211] 2.5. Silicone GF from different manufacturers 254 Thin-layer plate investigation

[0212] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 5 µl of the above test solution, 10 µl each of the negative control solutions lacking tangerine peel and angelica sinensis, 1 µl each of the hesperidin and norihesperidin reference solutions, and 5 µl of ligustrazine lactone I reference solution were spotted onto silica gel GF from different manufacturers. 254 On the thin-layer plate, cyclohexane-ethyl acetate (1:1) was used as the developing solvent. The plate was placed in a developing tank saturated with ammonia vapor for 20 minutes, developed, removed, dried, and examined under ultraviolet light (254nm) to detect the aroma of dried tangerine peel. Then, the lower layer solution of chloroform-methanol-water (12:3.5:1) was used as the developing solvent. The plate was placed in a developing tank saturated with ammonia vapor for 10 minutes, developed, removed, dried, and examined under ultraviolet light (254nm) to detect the aroma of angelica.

[0213] The results are as follows Figure 8 and Figure 9 As shown, after the first unfolding, the images were examined under a UV lamp (254nm). The results showed that silicone GF from different manufacturers... 254 In the chromatogram of the test sample on the thin-layer plate, spots of the same color appeared at the corresponding positions as those of the hesperidin and norepinephrine reference standards, with no negative interference; after the second development, the sample was examined under ultraviolet light (254 nm) on silica gel GF plates from different manufacturers. 254 In the chromatogram of the test sample on the thin-layer plate, spots of the same color appeared at the corresponding positions as in the reference chromatogram of ligustilide I, and there was no interference from negative samples. Therefore, silica gel GF from different manufacturers... 254 Thin-layer plates have no significant impact on this thin-layer chromatographic identification method. This thin-layer chromatographic identification method is compatible with silica gel GF from different manufacturers. 254 Thin-layer boards have good durability.

[0214] 3. Determination of thin-layer chromatographic identification method for tangerine peel and angelica in granules

[0215] The above analytical method validation results show that this thin-layer chromatography identification method has good specificity and is effective against temperature, humidity, and different manufacturers' silica gel GF. 254The thin-layer chromatography (TLC) plate exhibited good durability. Therefore, the final TLC identification method for the tangerine peel and angelica root extracts in compound Chinese medicine preparations containing angelica and tangerine peel was determined as follows: Take 3.6g of the product, grind it finely, add 30ml of water to dissolve it, extract with ethyl acetate three times (30ml each time), combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution. Separately, prepare reference solutions containing 2mg of hesperidin, 2mg of norepinephrine, and 0.5mg of ligustrazine I per ml using methanol. Perform the TLC test according to the Chinese Pharmacopoeia 2020 General Chapter 0502, applying 5-10µl of the above test solution, 1µl each of the hesperidin and norepinephrine reference solutions, and 5µl of the ligustrazine I reference solution to the same silica gel GF plate. 254 On a thin-layer plate, using cyclohexane-ethyl acetate (1:1) as the developing solvent, the plate was developed in a developing tank saturated with ammonia vapor for 20 minutes. After development, the plate was removed, dried, and examined for the aroma of dried tangerine peel under ultraviolet light (254 nm). Then, using the lower layer solution of chloroform-methanol-water (12:3.5:1) as the developing solvent, the plate was developed in a developing tank saturated with ammonia vapor for 10 minutes. After development, the plate was removed, dried, and examined for the aroma of angelica root under ultraviolet light (254 nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0216] 4. Verification Experiment

[0217] Experimental results are as follows Figure 10 As shown. After the first development, under ultraviolet light (254 nm), the chromatograms of the three batches of test samples showed spots of the same color at the corresponding positions as the chromatograms of hesperidin and norihesperidin reference standards. After the second development, under ultraviolet light (254 nm), the chromatograms of the three batches of test samples showed spots of the same color at the corresponding positions as the chromatograms of ligustrazine I reference standard. The spots were clear, and the Rf values ​​were suitable. This indicates that the thin-layer chromatography identification method is stable and feasible.

[0218] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A thin-layer chromatography method for identifying a traditional Chinese medicine compound preparation containing Angelica sinensis and Citrus reticulata peel, characterized in that, The thin-layer identification method includes the following steps: (1) The traditional Chinese medicine compound preparation containing angelica and tangerine peel is pretreated to obtain the test solution, the traditional Chinese medicine compound preparation lacking tangerine peel or angelica is pretreated to obtain the negative sample solution, and the reference standard is pretreated to obtain the reference solution. as well as (2) Thin-layer chromatography identification analysis was performed on the test solution, the negative sample solution and the reference solution; In the thin-layer chromatography identification analysis step, a developing solvent is used for development. The developing solvent includes a first developing solvent and a second developing solvent. The first developing solvent is cyclohexane-ethyl acetate, and the second developing solvent is a lower layer solution of chloroform-methanol-water. The volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.5~2):(0.5~2), and the volume ratio of chloroform, methanol and water in the second developing solvent is (5~20):(1~6):(0.5~2).

2. The thin-layer identification method according to claim 1, characterized in that, The traditional Chinese medicine compound preparation containing angelica and tangerine peel includes angelica, rehmannia, tangerine peel, pinellia, poria, ginger and licorice. Preferably, the mass ratio of the Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale and Glycyrrhiza uralensis is (350~500):(750~900):(250~400):(350~500):(350~500):(200~350):(150~300). Preferably, the mass ratio of the Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale, and Glycyrrhiza uralensis is approximately 414.44: approximately 828.89: approximately 311.11: approximately 414.44: approximately 414.44: approximately 277.78: approximately 207.22 g; Preferably, the mass ratio of the Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale, and Glycyrrhiza uralensis is (1~3):(3~5):(1~2):(1~3):(1~3):(1~2):(0.5~1.5). Preferably, the mass ratio of the Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, Zingiber officinale, and Glycyrrhiza uralensis is approximately 2: approximately 4: approximately 1.5: approximately 2: approximately 2: approximately 1.34: approximately 1; Preferably, the Pinellia ternata is processed Pinellia ternata; Preferably, the licorice is stir-fried licorice; Preferably, the dosage form of the traditional Chinese medicine compound preparation containing angelica and tangerine peel is granules; Preferably, the preparation method of the granules includes the following steps: A suitable amount of ginger is cut into thick slices, and aromatic water is extracted by steam distillation with a suitable amount of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, and Glycyrrhiza uralensis. The aqueous solution is then encapsulated with hydroxypropyl beta-cyclodextrin, and the encapsulation solution is concentrated under reduced pressure to a relative density of 1.10–1.20 (approximately 60°C), dried, and the encapsulation compound is prepared for later use. The dregs are added to 4–8 times their volume of water, decocted for 30–60 minutes, filtered, and the filtrate is concentrated under reduced pressure to a clear extract with a relative density of 1.20–1.30 (approximately 60°C). The extract is dried, and the encapsulation compound and a suitable amount of maltodextrin are added. Granulation is then performed to produce approximately 1000g of granules, thus obtaining the granules. Preferably, the preparation method of the granules includes the following steps: A suitable amount of ginger is cut into thick slices, and aromatic water is extracted by steam distillation with a suitable amount of Angelica sinensis, Rehmannia glutinosa, Citrus reticulata peel, Pinellia ternata, Poria cocos, and Glycyrrhiza uralensis. The extract is then infused with hydroxypropyl beta-cyclodextrin, and the infusion solution is concentrated under reduced pressure to a relative density of 1.10–1.20 (approximately 60°C), dried, and the infusion compound is prepared for later use. The dregs are added to approximately 6 times their volume of water, decocted for approximately 45 minutes, filtered, and the filtrate is concentrated under reduced pressure to a clear extract with a relative density of 1.20–1.30 (approximately 60°C), dried, and the infusion compound and a suitable amount of maltodextrin are added. Granulation is then performed to produce approximately 1000g of granules, thus obtaining the granules. More preferably, the negative sample solution is a negative sample solution lacking dried tangerine peel and / or a negative sample solution lacking angelica root; More preferably, the reference standard is hesperidin reference standard, norihesperidin reference standard and / or ligustrazine I reference standard.

3. The thin-layer identification method according to claim 1, characterized in that, The preparation method of the test solution includes: (1) weighing an appropriate amount of Chinese herbal compound preparation granules containing Angelica sinensis and Citrus reticulata, grinding them, dissolving them in water, extracting them by shaking with a first solvent, collecting and merging the organic layers, evaporating them to dryness, and obtaining the residue; and (2) adding a second solvent to the residue to dissolve it, thereby obtaining the test solution. Preferably, the first solvent is ethyl acetate; Preferably, the mass / volume (g / ml) ratio of the compound Chinese medicine preparation granules containing angelica and tangerine peel to the water is 0.1 to 0.5, for example, about 0.12; Preferably, the volume ratio of the water to the first solvent is 0.5 to 2, for example, about 1; Preferably, the shaking extraction is performed 2 to 4 times, for example, 3 times; More preferably, the mass of the compound Chinese medicine preparation granules containing angelica and tangerine peel is 1-5 g, for example, about 3.6 g; More preferably, the volume of the water is 20 to 40 ml, for example, about 30 ml; More preferably, the volume of the ethyl acetate is 20-40 ml, for example, about 30 ml; Preferably, the second solvent is an alcohol, such as methanol; Preferably, the mass / volume (g / ml) ratio of the compound Chinese medicine preparation granules containing angelica and tangerine peel to the second solvent is 3-4, for example, about 3.6; More preferably, the volume of the methanol is 0.5 to 2 ml, for example, about 1 ml.

4. The thin-layer identification method according to claim 1, characterized in that, The method for preparing the reference solution includes: taking an appropriate amount of hesperidin, norihesperidin or ligustrazine I reference standard, adding methanol to obtain hesperidin reference solution, norihesperidin reference solution or ligustrazine I reference solution respectively. Preferably, the concentration of hesperidin in the hesperidin reference solution is 1-3 mg / ml, for example, about 2 mg / ml; Preferably, the concentration of norepinephrine in the norepinephrine reference solution is 1-3 mg / ml, for example, about 2 mg / ml; Preferably, the concentration of ligustilide I in the ligustilide I reference solution is 0.1~1 mg / ml, for example, about 0.5 mg / ml.

5. The thin-layer identification method according to claim 1, characterized in that, The preparation method of the negative sample solution includes: weighing appropriate amounts of traditional Chinese medicine compound preparation granules lacking tangerine peel or angelica, and the subsequent steps are the same as the preparation method of the test sample solution.

6. The thin-layer identification method according to claim 1, characterized in that, The volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.8~1.2):(0.8~1.2). Preferably, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is about 1:about 1; More preferably, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (8~16):(2~5):(0.6~1.4). Preferably, the volume ratio of chloroform, methanol and water in the second developing agent is (10~14):(3~4):(0.8~1.2). More preferably, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is about 12: about 3.5: about 1.

7. The thin-layer identification method according to claim 1, characterized in that, The thin-layer plate is silicone GF. 254 Thin-layer plate; Preferably, the silicone GF 254 Thin-layer plates are made of Merck GF silicone rubber from Germany. 254 Thin-layer plate or Yantai Jiangyou silicone GF 254 Thin-layer plate or Qingdao Marine GF silicone 254 Thin-layer plate; More preferably, the inspection conditions for tangerine peel in the thin-layer chromatography identification analysis step are as follows: place the thin-layer plate in the developing tank, develop it with the first developing solvent, take out the thin-layer plate after development, dry it, and inspect it under ultraviolet light. More preferably, the inspection conditions for Angelica sinensis in the thin-layer chromatography identification analysis step are as follows: place the thin-layer plate back into the developing tank, develop it with a second developing solvent, remove the developed thin-layer plate, dry it, and inspect it under ultraviolet light. Preferably, the detection wavelength of the ultraviolet lamp is 254 nm; Preferably, in the inspection conditions of dried tangerine peel, the developing tank is a developing tank saturated with ammonia vapor for about 20 minutes; Preferably, in the inspection conditions of Angelica sinensis, the developing cylinder is a developing cylinder saturated with ammonia vapor for about 10 minutes; More preferably, the sample volume of the test solution is 3~12 μl, for example 5~10 μl; More preferably, the amount of hesperidin reference solution applied is 0.1 to 2 μl, for example 0.5 to 1.5 μl, for example about 1 μl; More preferably, the amount of the nobiletin reference solution applied is 0.1~2 μl, for example 0.5~1.5 μl, for example about 1 μl; More preferably, the amount of the sample spotting of the ligustrazine I reference solution is 3-7 μl, for example 4-6 μl, for example about 5 μl; Particularly preferably, the amount of the negative sample solution applied is 5-15 μl, for example, about 10 μl; Particularly preferred is that the temperature for thin-layer identification is 2~30°C, for example about 8°C or about 24°C; Particularly preferred is that the relative humidity for the thin-layer identification is 38% to 95%, for example, about 42% or about 88%.

8. The thin-layer identification method according to claim 1, characterized in that, The criteria for thin-layer chromatography identification include: after the first development using the first developing solvent, checking whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions as the hesperidin and tangeretin reference standards; after the second development using the second developing solvent, checking whether the thin-layer plate shows characteristic spots of the same color at the corresponding positions as the ligustrolactone I reference standard; and whether there is interference from negative samples; if the thin-layer plate has the characteristic spots after both the first and second developments and the negative samples do not interfere, then the detected traditional Chinese medicine compound preparation containing angelica and tangerine peel contains angelica and tangerine peel medicinal materials.

9. The use of the thin-layer identification method according to any one of claims 1 to 8 in identifying traditional Chinese medicine compound preparations containing angelica and tangerine peel.

10. A kit for identifying traditional Chinese medicine compound preparations containing angelica and tangerine peel, characterized in that, The kit includes a thin-layer plate and a developing solvent, wherein the developing solvent comprises a first developing solvent and a second developing solvent, wherein the first developing solvent is cyclohexane-ethyl acetate, and the second developing solvent is a lower layer solution of chloroform-methanol-water. The thin-layer plate is silica gel GF. 254 Thin-layer plate; Preferably, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.5~2):(0.5~2). More preferably, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is (0.8~1.2):(0.8~1.2). Preferably, the volume ratio of cyclohexane to ethyl acetate in the first developing solvent is about 1:about 1; More preferably, the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is (5~20):(1~6):(0.5~2); Particularly preferably, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (8~16):(2~5):(0.6~1.4). Particularly preferably, the volume ratio of the trichloromethane, methanol and water in the second developing agent is (10~14):(3~4):(0.8~1.2). Particularly preferred is that the volume ratio of the trichloromethane, the methanol and the water in the second developing agent is about 12: about 3.5: about 1.

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