A thin-layer chromatographic method for identifying Xiao Xianxiong Decoction

Abstract

This invention belongs to the field of pharmaceutical analysis, specifically relating to a thin-layer chromatography (TLC) method for identifying Xiao Xianxiong Decoction. The TLC method includes developing the sample using n-butanol-glacial acetic acid-water as the developing solvent. This invention, through specific TLC conditions, enables efficient separation of berberine hydrochloride, a characteristic component of Coptis chinensis, and alanine, valine, and leucine, key amino acids in Trichosanthes kirilowii and Pinellia ternata, in a single TLC development process. Furthermore, the use of a dual-colorimetric method ensures clear color development of these three medicinal components without any interference between them, exhibiting good specificity. This method is suitable for examining the composition of the raw materials in Xiao Xianxiong Decoction, accurately identifying the formula, and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a thin-layer chromatography method for identifying Xiao Xianxiong Decoction. Background Technology

[0002] Xiao Xianxiong Decoction is the 12th prescription in the "Catalogue of Ancient Classic Prescriptions (Second Batch) - Han Medicine" published by the State Administration of Traditional Chinese Medicine in 2023. It originates from the "Differentiation of Pulse and Symptoms of Taiyang Disease and Treatment" section of the *Shanghan Lun* (Treatise on Cold Damage) by Zhang Zhongjing, a physician of the Han Dynasty. Composed of Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis, it has the effects of clearing heat and resolving phlegm, opening the chest and dispersing nodules. In ancient times, it was often used to treat minor chest obstruction caused by internal invasion of pathogenic heat and phlegm-heat accumulation in the epigastrium. The *Shanghan Lun* states: "Minor chest obstruction is located in the epigastrium, painful to the touch, with a floating and slippery pulse; Xiao Xianxiong Decoction is the main treatment." It can "eliminate phlegm-heat accumulation in the heart, abdomen, chest, and diaphragm." Besides treating chest pain, it can also "eliminate phlegm, lower lung qi, invigorate the stomach and spleen, stop vomiting, and remove phlegm from the chest." In modern times, the therapeutic scope of Xiao Xianxiong Decoction has expanded, and it is widely used in the treatment of systemic diseases such as respiratory, digestive, metabolic, and cardiovascular diseases.

[0003] Thin-layer chromatography (TLC) is a commonly used separation and analysis method. By developing extracts of medicinal materials or traditional Chinese medicine preparations on a thin-layer plate and comparing them with the chromatograms of reference standards or reference medicinal materials, the authenticity, purity, and efficacy of the medicinal materials or traditional Chinese medicine preparations can be identified. Currently, there are no reported methods for identifying Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis in Xiao Xianxiong Decoction. Previous experiments attempted to apply the currently reported TLC methods for Trichosanthes kirilowii, Pinellia ternata, or Coptis chinensis to the TLC identification of Xiao Xianxiong Decoction, but it is difficult to simultaneously identify all three medicinal materials in Xiao Xianxiong Decoction with a single TLC development. Therefore, there is an urgent need for a TLC identification method for Xiao Xianxiong Decoction that can quickly and accurately identify Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis in Xiao Xianxiong Decoction with a single TLC development, in order to effectively control the quality of Xiao Xianxiong Decoction. Summary of the Invention

[0004] To address the above problems, this invention provides a thin-layer chromatography method for identifying Xiao Xianxiong Decoction, comprising the following steps:

[0005] a. Preparation of test solution: Take the sample to be tested, extract it with methanol solution containing hydrochloric acid, filter it, and take the filtrate as the test solution;

[0006] b. Preparation of reference solutions: Take amino acid reference standards and dissolve them in 70% methanol solution to prepare reference solution ①; take berberine hydrochloride reference standards and dissolve them in methanol solution containing hydrochloric acid to prepare reference solution ②.

[0007] c. Thin-layer chromatography determination: Take the test solution and reference solutions ① and ②, spot them separately on the same silica gel G plate, develop with n-butanol-glacial acetic acid-water, remove, air dry, examine under ultraviolet light, spray with ninhydrin solution, heat until the spots are clearly visible, and examine under sunlight.

[0008] Further, in step a, the mass-to-volume ratio of the sample to be tested to the methanol solution containing hydrochloric acid is 0.5~5g:10~20ml.

[0009] Furthermore, the sample to be tested is a Xiaoxianxiong Decoction preparation; the Xiaoxianxiong Decoction preparation is a modern Chinese medicine preparation using Trichosanthes kirilowii, Pinellia ternata and Coptis chinensis as raw materials; the modern Chinese medicine preparation includes Xiaoxianxiong Decoction decoction, Xiaoxianxiong Decoction granules, Xiaoxianxiong Decoction powder, Xiaoxianxiong Decoction pills, and Xiaoxianxiong Decoction material reference.

[0010] Furthermore, the mass ratio of Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis is 70~90:30~40:12~14.

[0011] Furthermore, the mass ratio of Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis is 80:34.5:13.8.

[0012] Furthermore, the volume ratio of methanol to hydrochloric acid in the methanol solution containing hydrochloric acid is 100:1.

[0013] Further, the amino acid reference standard described in step b is dissolved in 70% methanol.

[0014] Further, in step b, each 1 ml of the reference solution ① contains 0.5-1.5 mg of each amino acid, and each 1 ml of the reference solution ② contains 0.1-1 mg of berberine hydrochloride; the amino acids are alanine, valine, and leucine.

[0015] Further, the volume ratio of n-butanol-glacial acetic acid-water in step c is 9:1:2.

[0016] Furthermore, the heating temperature in step c is 105°C.

[0017] Furthermore, the wavelength of the ultraviolet lamp in step c is 365nm.

[0018] Further, in step c, the volume of the test solution taken is 5 μL, and the volume of the reference solution taken is 1 μL each.

[0019] Furthermore, the chromatogram of the test solution, when viewed under ultraviolet light, shows spots of the same color at the corresponding positions as the berberine hydrochloride reference standard; when viewed under sunlight, it shows spots of the same color at the corresponding positions as the mixed reference standards of alanine, valine, and leucine.

[0020] This invention discloses a thin-layer chromatography (TLC) method for identifying Xiao Xianxiong Decoction. Under specific TLC conditions, the characteristic component berberine hydrochloride from Coptis chinensis, and the key amino acids alanine, valine, and leucine from Trichosanthes kirilowii and Pinellia ternata can be efficiently separated in a single TLC development process. Furthermore, a dual-colorimetric method ensures clear color development of these three medicinal materials without any interference between them, exhibiting excellent specificity. This method is precisely applicable to the examination of the composition of Xiao Xianxiong Decoction raw materials, effectively achieving accurate identification of the decoction, and possesses significant application value and broad application prospects.

[0021] The Xiaoxianxiong Decoction described in this invention comes from Zhang Zhongjing's "Treatise on Febrile Diseases" from the Han Dynasty. It is one of the hundred classic ancient prescriptions. Its formula consists of one liang of Coptis chinensis, half a sheng of Pinellia ternata, and one large Trichosanthes kirilowii fruit. The above three ingredients are boiled in six sheng of water. First, the Trichosanthes kirilowii fruit is boiled to obtain three sheng of decoction, and the dregs are removed. Then, the other ingredients are added and boiled to obtain two sheng of decoction, and the dregs are removed. The decoction is divided into three warm doses.

[0022] The material standard for Xiao Xianxiong Decoction used in this invention is based on the preparation method of Xiao Xianxiong Decoction recorded in the ancient medical book *Shanghan Lun*. Except for the molding process, the other preparation methods are basically consistent with those recorded in *Shanghan Lun*. The specific preparation method is as follows:

[0023] According to the formula of Xiao Xianxiong Decoction, weigh out the raw materials: 80.00g of Trichosanthes kirilowii, 34.50g of Pinellia ternata, and 13.80g of Coptis chinensis. Add 1200ml of water and soak for 30 minutes. First, boil the Trichosanthes kirilowii slices until 600ml remains. Remove the dregs. Then add the Coptis chinensis slices and Pinellia ternata slices and continue to boil until 400ml remains. Remove the dregs. This gives the material basis of Xiao Xianxiong Decoction.

[0024] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0025] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0026] Figure 1 Thin-layer chromatography (TLC) of toluene-ethyl formate-formic acid (10:10:1)-sunlight (1 is a negative solution for Pinellia ternata lacking Qingbanxia; 2 is a negative solution for Coptis chinensis lacking Huanglian; 3 is a reference sample solution for Xiaoxianxiong Decoction; 4 is a negative solution for Trichosanthes kirilowii lacking Gualou; 5 is a reference solution for berberine hydrochloride; 6 is a mixed reference solution for alanine, valine, and leucine).

[0027] Figure 2 Thin-layer chromatography (TLC) of the development system—n-butanol-anhydrous ethanol-glacial acetic acid-water (8:2:2:1)-sunlight (1 is the reference sample solution of Xiaoxianxiong Decoction; 2 is the negative solution lacking Trichosanthes kirilowii; 3 is the negative solution lacking Pinellia ternata; 4 is the negative solution lacking Coptis chinensis; 5 is the mixed reference solution of alanine, valine, and leucine; 6 is the reference solution of berberine hydrochloride).

[0028] Figure 3 Thin-layer chromatography (TLC) at 365 nm using a development system of n-butanol-glacial acetic acid-water (9:1:2) (1 is berberine hydrochloride reference solution; 2 is a mixed reference solution of alanine, valine, and leucine; 3 is the Xiaoxianxiong decoction reference sample solution; 4 is a negative solution lacking Trichosanthes kirilowii; 5 is a negative solution lacking Pinellia ternata; 6 is a negative solution lacking Coptis chinensis).

[0029] Figure 4 Thin-layer chromatography (TLC) of development system - n-butanol - glacial acetic acid - water (9:1:2) - sunlight (1 is berberine hydrochloride reference solution; 2 is a mixed reference solution of alanine, valine, and leucine; 3 is Xiaoxianxiong decoction reference sample solution; 4 is a negative solution lacking Trichosanthes kirilowii; 5 is a negative solution lacking Pinellia ternata; 6 is a negative solution lacking Coptis chinensis).

[0030] Figure 5 Thin-layer chromatography (TLC) at -365 nm was used to investigate sample volume (1-3 were mixed reference solutions of alanine, valine, and leucine, 1 μl, 5 μl, and 8 μl, respectively; 4-6 were reference solutions of Xiaoxianxiong Decoction, 1 μl, 5 μl, and 8 μl, respectively; 7-9 were reference solutions of berberine hydrochloride, 1 μl, 5 μl, and 8 μl, respectively).

[0031] Figure 6 Sample volume determination - Thin-layer chromatography (TLC) of sunlight (1-3 are mixed reference solutions of alanine, valine, and leucine, 1 μl, 5 μl, and 8 μl, respectively; 4-6 are reference solutions of Xiaoxianxiong Decoction, 1 μl, 5 μl, and 8 μl; 7-9 are reference solutions of berberine hydrochloride, 1 μl, 5 μl, and 8 μl).

[0032] Figure 7 Specificity assessment - 365nm thin-layer chromatography (1 is blank; 2 is berberine hydrochloride reference solution; 3 is a mixed reference solution of alanine, valine, and leucine; 4 is a negative solution lacking Trichosanthes kirilowii; 5 is a reference sample solution of Xiaoxianxiong Decoction; 6 is a negative solution lacking Pinellia ternata; 7 is a negative solution lacking Coptis chinensis; 8 is a reference sample solution of Trichosanthes kirilowii; 9 is a reference sample solution of Pinellia ternata; 10 is a reference sample solution of Coptis chinensis).

[0033] Figure 8Specificity assessment - Thin-layer chromatography (1 is blank; 2 is berberine hydrochloride reference solution; 3 is a mixed reference solution of alanine, valine, and leucine; 4 is Xiaoxianxiong decoction reference solution; 5 is negative solution for Trichosanthes kirilowii (Gualou) lacking; 6 is negative solution for Pinellia ternata lacking; 7 is negative solution for Coptis chinensis lacking; 8 is reference solution for Trichosanthes kirilowii (Gualou); 9 is reference solution for Pinellia ternata; 10 is reference solution for Coptis chinensis).

[0034] Figure 9 Thin-layer chromatography study—365nm—Merck's thin-layer chromatography spectrum (1 is a mixed standard solution of alanine, valine, and leucine, 1μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5μl; 3 is a standard solution of berberine hydrochloride, 1μl);

[0035] Figure 10 Thin-layer chromatography (TLC) studies – sunlight – Merck TLC (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0036] Figure 11 Thin-layer chromatography (TLC) analysis—365nm—TLC spectrum of Qingdao Ocean (1 is a mixed standard solution of alanine, valine, and leucine, 1μl; 2 is a standard solution of Xiaoxianxiong decoction, 5μl; 3 is a standard solution of berberine hydrochloride, 1μl).

[0037] Figure 12 Thin-layer chromatography (TLC) studies—Sunlight—Qingdao Ocean (1: A mixed standard solution of alanine, valine, and leucine, 1 μl; 2: Xiaoxianxiong decoction standard sample solution, 5 μl; 3: Berberine hydrochloride standard solution, 1 μl).

[0038] Figure 13 Thin-layer chromatography (TLC) at 8℃–365nm (1 is a mixed standard solution of alanine, valine, and leucine, 1μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5μl; 3 is a standard solution of berberine hydrochloride, 1μl).

[0039] Figure 14 Thin-layer chromatography at 8℃ under sunlight (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0040] Figure 15 Thin-layer chromatography (TLC) at 35℃–365nm (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0041] Figure 16Thin-layer chromatography (TLC) at 35℃–365nm (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0042] Figure 17 Thin-layer chromatography (TLC) spectra at 35% humidity – 365 nm (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0043] Figure 18 Thin-layer chromatography (TLC) at 35% humidity under sunlight (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0044] Figure 19 Thin-layer chromatography (TLC) at 72% humidity – 365 nm (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0045] Figure 20 Thin-layer chromatography (TLC) at 72% humidity under sunlight (1 is a mixed standard solution of alanine, valine, and leucine, 1 μl; 2 is a standard sample solution of Xiaoxianxiong decoction, 5 μl; 3 is a standard solution of berberine hydrochloride, 1 μl).

[0046] Figure 21 Multiple batches of validation-365nm thin-layer chromatography (1 is berberine hydrochloride reference solution, 1μl; 2 is a mixed reference solution of alanine, valine, and leucine, 1μl; 3-5 are Xiaoxianxiong decoction reference sample solutions).

[0047] Figure 22 Multiple batches of validation - thin-layer chromatography (1 is berberine hydrochloride reference solution, 1 μl; 2 is a mixed reference solution of alanine, valine, and leucine, 1 μl; 3-5 are Xiaoxianxiong decoction reference sample solutions). Detailed Implementation

[0048] The raw materials, equipment, and reagents used in the specific embodiments of this invention are all known products and were obtained by purchasing commercially available products. The preparation method of the Xiaoxianxiong Decoction reference sample is as follows: weigh the raw materials according to the Xiaoxianxiong Decoction formula, add 1200ml of water, soak for 30 minutes, first boil the Trichosanthes kirilowii (Trichosanthes tangutorum) slices to 600ml, remove the dregs, then add the Coptis chinensis slices and Pinellia ternata slices, continue to boil to 400ml, remove the dregs, and the sample is obtained.

[0049] Preparation method of Trichosanthes kirilowii (Gualou) reference sample: Weigh 80.00g of Trichosanthes kirilowii (Gualou), add 1200ml of water, soak for 30 minutes, boil down to 600ml, remove the dregs, and the sample is obtained.

[0050] Preparation method of Pinellia ternata reference sample: Weigh 34.50g of Pinellia ternata slices, add 600ml of hot water, soak for 30 minutes, boil down to 400ml, remove the dregs, and the sample is obtained.

[0051] Preparation method of Coptis chinensis reference sample: Weigh 13.80g of Coptis chinensis slices, add 600ml of hot water, soak for 30 minutes, boil down to 400ml, remove the dregs, and the sample is obtained.

[0052] Preparation method of reference sample for Trichosanthes kirilowii (Gualou): Weigh 34.50g of Pinellia ternata slices and 13.80g of Coptis chinensis slices, add 600ml of hot water, soak for 30 minutes, boil down to 400ml, and remove the dregs.

[0053] Preparation method of reference sample for Coptis chinensis deficiency: Weigh 80.00g of Trichosanthes kirilowii and 34.50g of Pinellia ternata slices, add 1200ml of water, soak for 30 minutes, first boil Trichosanthes kirilowii slices to 600ml, remove the dregs, then add Pinellia ternata slices, continue to boil to 400ml, remove the dregs.

[0054] Preparation method of reference sample of Pinellia ternata without clear water: Weigh 80.00g of Trichosanthes kirilowii and 13.80g of Coptis chinensis slices, add 1200ml of water, soak for 30 minutes, first boil Trichosanthes kirilowii slices to 600ml, remove the dregs, then add Coptis chinensis slices, continue to boil to 400ml, remove the dregs.

[0055] Example 1: Thin-layer chromatographic detection of Xiao Xianxiong Decoction

[0056] a. Preparation of test solution: Weigh 1.0g of the Xiaoxianxiong Decoction preparation to be tested, add 15ml of methanol-hydrochloric acid solution with a volume ratio of 100:1, sonicate for 10 minutes, filter, and use as the test solution.

[0057] b. Preparation of reference solution: Take alanine reference standard, valine reference standard and leucine reference standard, add 70% methanol to prepare a mixed solution containing 1 mg of each reference standard per 1 ml, as reference solution ①;

[0058] Take berberine hydrochloride reference standard and add methanol hydrochloric acid solution with a volume ratio of 100:1 to prepare a solution containing 0.5 mg of reference standard per 1 ml, as reference solution ②;

[0059] c. Take 5 μl of the test solution and 1 μl each of the reference solution ① and reference solution ②, and spot them separately on the same silica gel G thin layer plate. Develop with n-butanol-glacial acetic acid-water (9:1:2) as the developing solvent. Remove the plate, air dry it, examine it under ultraviolet light (365nm), spray it with ninhydrin solution, heat it until the spots are clearly visible, and examine it under sunlight.

[0060] d. Identification

[0061] In the chromatogram of the test solution examined under ultraviolet light, spots of the same color should appear at the corresponding positions as the berberine hydrochloride reference standard; in the chromatogram of the test solution examined under sunlight, spots of the same color should appear at the corresponding positions as the mixed reference standards of alanine, valine, and leucine. The test sample is Xiaoxianxiong Decoction.

[0062] The following experimental examples illustrate the beneficial effects of the present invention:

[0063] Experimental Example 1: Study on Thin-Layer Chromatography Method for Xiaoxianxiong Decoction

[0064] 1. Instruments and reagents

[0065] 1.1 Instruments

[0066] Thin-layer imaging system: CAMAG TLC Visualizer; Silicone G thin-layer plate: Qingdao Ocean Chemical Plant, batch number: 20240504; Merck, Germany, batch number: HX32019153).

[0067] 1.2 Reagents

[0068] Methanol, hydrochloric acid, n-butanol, glacial acetic acid, 95% ethanol, ninhydrin, and water were all of analytical grade.

[0069] 1.3 Drug Test

[0070] Berberine hydrochloride (batch number: 110887-202305, purity 99.6%), alanine (batch number: 140680-202206, purity 99.6%), valine (batch number: 110887-202305, purity 99.6%), and leucine (batch number: 110887-202305, purity 99.6%) were all purchased from the National Institutes for Food and Drug Control; Trichosanthes kirilowii (batch number: GL(GL)-BT-01) and Pinellia ternata (batch number: The following samples were provided by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.: QBX-BT-01, Coptis chinensis reference sample (batch number: HL(HL)-BT-01), Trichosanthes kirilowii (batch number: YX-BT-01(GL(GL))), Pinellia ternata (batch number: YX-BT-01(QBX)), Coptis chinensis (batch number: YX-BT-01(HL(HL)); and Xiaoxianxiong Decoction reference samples (batch numbers: BT-01, BT-02, BT-03).

[0071] 2.3 Establishment of Thin-Layer Method

[0072] 2.3.1 Preparation of the test solution

[0073] Take 1.0g of Xiaoxianxiong Decoction as a reference sample, add 15ml of methanol:hydrochloric acid (100:1), sonicate for 10 minutes, filter, and use as the test solution.

[0074] 2.3.2 Preparation of reference solution

[0075] Prepare a mixed solution containing 1 mg of each of alanine, valine, and leucine reference standards per ml by adding 70% methanol.

[0076] Take berberine hydrochloride reference standard and add methanol:hydrochloric acid (100:1) to prepare a solution containing 0.5 mg per ml, which is used as the reference solution.

[0077] 2.3.3 Determination Method

[0078] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 5 μl of the test solution and 1 μl of the reference solution separately to the same silica gel G thin-layer plate. Develop the plate using n-butanol-glacial acetic acid-water (9:1:2) as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the berberine hydrochloride reference standard. Then spray with ninhydrin solution and heat at 105℃ until the spots are clearly visible. Examine the plate under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the mixed reference standards of alanine, valine, and leucine.

[0079] 2.4 Examination of Different Deployment Systems

[0080] Based on the experimental conditions outlined above, the developing solvent was adjusted, and the effects of three different developing systems—toluene-ethyl formate-formic acid (10:10:1), n-butanol-anhydrous ethanol-glacial acetic acid-water (8:2:2:1), and n-butanol-glacial acetic acid-water (9:1:2)—on the detection results were investigated. The results are shown below. Figures 1-4 .

[0081] The results showed that when toluene-ethyl formate-formic acid (10:10:1) was used as the developing solvent, berberine hydrochloride, alanine, valine, and leucine did not show color on the thin-layer chromatography (TLC) pattern after ninhydrin development. When n-butanol-anhydrous ethanol-glacial acetic acid-water (8:2:2:1) was used as the developing solvent, alanine, valine, and leucine all showed color on the TLC pattern of the amino acid reference standard after ninhydrin development, but the spots were light in color, and the amino acid reference standard was similar to that provided by Xiaoxianxiong Decoction. Differences in the corresponding positions of the samples affected the judgment of qualitative results. When using n-butanol-glacial acetic acid-water (9:1:2) as the developing solvent, under ultraviolet light, the Xiaoxianxiong Decoction sample and berberine hydrochloride reference standard showed fluorescent spots of the same color at the same positions on the thin-layer chromatography spectrum. After ninhydrin development, the Xiaoxianxiong Decoction sample and alanine, valine, and leucine reference standards showed fluorescent spots of the same color at the same positions, which could clearly distinguish Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis. Therefore, n-butanol-glacial acetic acid-water (9:1:2) was ultimately determined as the developing solvent for thin-layer chromatography identification of Xiaoxianxiong Decoction.

[0082] 2.5 Methodological Examination

[0083] 2.5.1 Sampling quantity investigation

[0084] Under the proposed experimental conditions, 1 μl, 5 μl, and 8 μl of berberine hydrochloride reference solution, alanine, valine, and leucine mixed reference solution, and Xiaoxianxiong decoction standard sample solution were respectively spotted onto the same silica gel G thin-layer plate. The results are shown below. Figures 5-6 .

[0085] The results showed that under ultraviolet light (365 nm), the mixed reference solution of alanine, valine, and leucine showed no fluorescent spots, while the fluorescent spots of the berberine hydrochloride reference solution and the test solution were clearly visible. Under sunlight, the mixed reference solution of alanine, valine, and leucine showed purplish-red spots, while the berberine hydrochloride reference solution showed no purplish-red spots. When the sample volume was 1-8 μl, the spots were clear and the separation was good, and the sample volume was determined to be 1-8 μl.

[0086] 2.5.2 Specificity Examination

[0087] Negative solutions, berberine hydrochloride reference solutions, mixed reference solutions of alanine, valine, and leucine, and Xiaoxianxiong decoction reference sample solutions were prepared according to the above preparation methods. Thin-layer chromatography identification experiments were performed, and the results are shown in the figure. Figures 7-8 .

[0088] As shown in the figure, under ultraviolet light (365 nm), the mixed reference solution of alanine, valine, and leucine showed no fluorescent spots in thin-layer chromatography, while the reference solution of berberine hydrochloride showed fluorescent spots. The negative sample solutions lacking Trichosanthes kirilowii, Pinellia ternata, Xiaoxianxiong Decoction, and Coptis chinensis showed clear fluorescent spots at the corresponding positions of berberine hydrochloride in thin-layer chromatography. The negative sample solutions lacking Coptis chinensis, Pinellia ternata, and Trichosanthes kirilowii showed no fluorescent spots at the corresponding positions of berberine hydrochloride in thin-layer chromatography.

[0089] Under sunlight, the mixed reference solution of alanine, valine, and leucine showed purplish-red spots in thin-layer chromatography. The reference solution of berberine hydrochloride and the reference solution of Coptis chinensis showed no purplish-red spots, but yellow spots. The negative control solutions lacking Trichosanthes kirilowii and Pinellia ternata showed purple and yellow spots at the corresponding positions of the amino acid references in the thin-layer chromatography, indicating that Coptis chinensis in the two negative control solutions affected the qualitative analysis of amino acids. The negative control solutions lacking Coptis chinensis and the reference solution of Xiaoxianxiong Decoction showed clear purplish-red spots at the corresponding positions of the amino acid references, indicating that Coptis chinensis in the reference solution of Xiaoxianxiong Decoction did not interfere with the qualitative analysis of amino acids in the thin-layer chromatography.

[0090] Under ultraviolet light, the presence of fluorescence at the corresponding position of berberine hydrochloride can distinguish negative samples lacking Coptis chinensis, standard sample solutions of Pinellia ternata, and standard samples of Trichosanthes kirilowii from the standard sample of Xiao Xianxiong Decoction. Under sunlight, the presence of clear purplish-red spots at the corresponding positions of amino acids can further distinguish negative samples lacking Trichosanthes kirilowii, negative samples lacking Pinellia ternata, and standard samples of Coptis chinensis from the standard sample of Xiao Xianxiong Decoction. Ultimately, this method effectively achieves accurate identification of Xiao Xianxiong Decoction, demonstrating good specificity.

[0091] 2.5.3 Durability Assessment

[0092] 2.5.3.1 Comparison of different thin-layer plates

[0093] Precast panels (silicone G-plate) from the Qingdao Marine Chemical Plant branch and Merck (silicone G-plate) from Germany were tested according to the proposed test methods. (See attached text.) Figure 9-12 .

[0094] The results showed that under ultraviolet light (365 nm), the mixed reference solution of alanine, valine, and leucine showed no fluorescent spots in the thin-layer chromatography, while the fluorescent spots of the berberine hydrochloride reference solution and the test solution were clearly visible. Under sunlight, the mixed reference solution of alanine, valine, and leucine showed purplish-red spots, while the berberine hydrochloride reference solution showed no purplish-red spots. The Xiaoxianxiong decoction reference sample solution showed clear purplish-red spots at the corresponding positions of the amino acid references. Both thin-layer chromatography plates met the identification requirements.

[0095] 2.5.3.2 Comparison of different temperatures

[0096] Prepare the test solution and reference solution according to the prescribed method and spot them. Take the spotted thin-layer plates and develop them at low temperature (8℃) and high temperature (35℃), respectively. See Figures 13-16 .

[0097] The results showed that under ultraviolet light (365 nm), the mixed reference solution of alanine, valine, and leucine showed no fluorescent spots in thin-layer chromatography, while the fluorescent spots of the berberine hydrochloride reference solution and the test solution were clearly visible. Under sunlight, the mixed reference solution of alanine, valine, and leucine showed purplish-red spots, while the berberine hydrochloride reference solution showed no purplish-red spots. The Xiaoxianxiong decoction reference sample solution showed clear purplish-red spots at the corresponding positions of the amino acid references. This method showed good adaptability to different temperatures.

[0098] 2.5.3.3 Comparison of different humidity levels

[0099] Prepare the test solution and reference solution according to the prescribed method and spot them. Take the spotted thin-layer plates and develop them in humidity environments of 35% RH and 72% RH, respectively. See Figures 17-20 .

[0100] The results showed that under ultraviolet light (365 nm), the mixed reference solution of alanine, valine, and leucine showed no fluorescent spots in thin-layer chromatography, while the fluorescent spots of the berberine hydrochloride reference solution and the test solution were clearly visible. Under sunlight, the mixed reference solution of alanine, valine, and leucine showed purplish-red spots, while the berberine hydrochloride reference solution showed no purplish-red spots. The Xiaoxianxiong decoction reference sample solution showed clear purplish-red spots at the corresponding positions of the amino acid references. This method showed good adaptability to different humidity levels.

[0101] 2.5.4 Verification

[0102] Thin-layer chromatography was performed on three batches of Xiaoxianxiong Decoction reference samples according to the proposed method. The experimental results are shown in [Figure 1]. Figure 21-22 .

[0103] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the berberine hydrochloride reference standard; spray with ninhydrin solution and heat at 105℃ until the spots are clearly visible. Examine under sunlight. In the chromatogram of the test sample, clearly visible spots of the same color appear at the corresponding positions as in the chromatogram of the mixed reference standards of alanine, valine, and leucine.

[0104] 2.6 Determination of identification points between Coptis chinensis, Trichosanthes kirilowii, and Pinellia ternata in the reference samples of Xiao Xian Xiong Tang.

[0105] The results showed that under ultraviolet light (365 nm), the mixed reference solution of alanine, valine, and leucine showed no fluorescent spots, while the berberine hydrochloride reference solution and the Xiao Xian Xiong Tang test solution showed clear fluorescent spots. Under sunlight, the mixed reference solution of alanine, valine, and leucine showed purplish-red spots, while the berberine hydrochloride reference solution showed no purplish-red spots. The Xiao Xian Xiong Tang test sample showed clear purplish-red spots at the corresponding positions of the amino acid references. Thin-layer chromatography, using two colorimetric methods in combination, can distinguish between Coptis chinensis and Trichosanthes kirilowii (Gualou) and Pinellia ternata in the classic formula Xiao Xian Xiong Tang, thus enabling the examination of the raw material composition of Xiao Xian Xiong Tang.

Claims

1. A thin layer chromatography method for identifying Xiaoxianxiong decoction, characterized in that: It comprises the following steps: ​ a. Preparation of test sample solution: take the Xiaxianxiong decoction preparation to be tested, extract with methanol solution containing hydrochloric acid, filter, and take the filtrate as the test sample solution; b. Preparation of control sample solution: take amino acid control sample, dissolve with 70% methanol solution, and take as control sample solution ①; Take hydrochloric acid berberine control sample, dissolve with methanol solution containing hydrochloric acid, and take as control sample solution ②; c. Thin layer chromatography determination: take the test sample solution and control sample solution ① and control sample solution ②, respectively, and point on the same silica gel G plate, develop with n-butanol-glacial acetic acid-water (volume ratio 9:1:2), take out, air dry, and observe under ultraviolet light, then spray with ninhydrin solution, heat until the spots develop clear color, and observe under daylight; The Xiaxianxiong decoction preparation is a modern traditional Chinese medicine preparation using Gualou, Qingbanxia and Huanglian as raw medicinal materials; The amino acid is alanine, valine and leucine; The chromatogram of the test sample solution, when observed under ultraviolet light, shows the same color spots at the positions corresponding to the hydrochloric acid berberine control sample; when observed under daylight, shows the same color spots at the positions corresponding to the mixed control sample of alanine, valine and leucine, and the sample to be tested is Xiaxianxiong decoction.

2. The thin layer chromatography method of claim 1, wherein: The mass-volume ratio of the Xiaxianxiong decoction preparation to be tested to the methanol solution containing hydrochloric acid in step a is 0.5-5g:10-20ml.

3. Thin layer chromatography method according to claim 1 or 2, characterized in that: The modern traditional Chinese medicine preparation includes Xiaxianxiong decoction, Xiaxianxiong decoction granules, Xiaxianxiong decoction powder, Xiaxianxiong decoction pills and Xiaxianxiong decoction substance reference.

4. The thin layer chromatography method according to claim 1 or 2, characterized in that: The volume ratio of methanol to hydrochloric acid in the methanol solution containing hydrochloric acid is 100:

1.

5. The thin layer chromatography method of claim 1, wherein: The control sample solution ① in step b contains 0.5-1.5mg of each amino acid per 1ml, and the control sample solution ② contains 0.1-1mg of hydrochloric acid berberine per 1ml.

6. The thin layer chromatography method of claim 1, wherein: The heating temperature in step c is 105℃, and the wavelength of the ultraviolet lamp is 365nm.

7. The thin layer chromatography method of claim 1, wherein: The suction amount of the test sample solution in step c is 5μL, and the suction amount of the control sample solution is 1μL each.

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