A thin layer identification method for a patrinia formula granule and application thereof

Abstract

The application discloses a kind of baijiaocao formula granules thin layer identification method and application thereof, the thin layer identification method includes: test sample powder is dissolved after water is added and is extracted with ethyl acetate, evaporates dry, residue is dissolved with methanol to obtain test sample solution;Take baijiaocao control medicinal material, heat reflux, filter, concentrate, add methanol, ultrasonic, filter, evaporate dry, dissolve with water, extract with ethyl acetate, evaporate dry, residue is dissolved with methanol to obtain control medicinal material solution;Sample is applied to the same thin layer plate, with cyclohexane-ethyl acetate-methanol-formic acid as developing agent, after color development, view;When the same color spot is shown in the corresponding position of test sample chromatogram with control medicinal material chromatogram, then it indicates that test sample is baijiaocao formula granules.The application method can detect multiple components in sample, greatly improve the specificity of method;Can distinguish between yellow flower and white flower baijiaocao;Baijiaocao can be easily distinguished from counterfeit, with higher practical value.

Description

Technical Field

[0001] This invention relates to a thin-layer chromatography method for identifying Patrinia scabiosifolia formulation granules and its application, belonging to the field of quality control of Patrinia scabiosifolia formulation granules. Background Technology

[0002] Patrinia scabiosaefolia Fisch. or Patrinia villosa Juss., both belonging to the Valerianaceae family, are dried whole herbs. They are harvested before flowering in summer, sun-dried until semi-dry, bundled, and then air-dried. Patrinia scabiosaefolia is mainly produced in Jiangsu, Zhejiang, Anhui, and Hubei provinces, and is known for its heat-clearing, detoxifying, blood-activating, and pus-draining properties. Studies have found that Patrinia scabiosaefolia contains various active ingredients, including terpenes, flavonoids, lignans, and volatile oils.

[0003] Traditional Chinese medicine (TCM) granules represent a breakthrough in the application of traditional Chinese medicine decoction pieces. They are typically made from decoction pieces through standardized extraction (usually water extraction), concentration, drying, and granulation. Their properties, flavors, meridian tropism, and efficacy remain largely consistent with the original decoction pieces, while offering advantages such as no need for decoction, ease of dispensing, storage, and convenient administration, leading to their widespread adoption. However, the 2020 edition of the Chinese Pharmacopoeia does not include quality standards for *Patrinia scabiosifolia* (Herba Patriniae) or its granules under Part I.

[0004] In existing technologies, most literature focuses on thin-layer chromatography (TLC) identification of *Patrinia scabiosifolia* herbal extracts, rather than water extracts such as formulation granules. Literature such as "An Improved Quality Detection Method for Longqing Capsules," "Research on Quality Standards of Yiren Liumiao Granules," and "Identification Method of *Patrinia scabiosifolia* in Compound Traditional Chinese Medicine Preparations" focuses on TLC identification of *Patrinia scabiosifolia* components in compound granule preparations. However, since traditional Chinese medicine formulation granules undergo water extraction, their lipid-soluble components are relatively few, making TLC identification methods for herbal extracts potentially unsuitable. Furthermore, traditional Chinese medicine formulation granules are single-ingredient formulas, and their composition differs significantly from that of compound granules; methods for compound granules may not be applicable to TLC identification of formulation granules. Moreover, the TLC chromatographic spots for *Patrinia scabiosifolia* in the literature are few, indicating weak specificity and unsatisfactory identification results. Therefore, establishing a rapid, simple, and low-cost TLC identification method for *Patrinia scabiosifolia* formulation granules is an important research topic. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a rapid, simple and low-cost thin-layer chromatography method for identifying Patrinia scabiosifolia granules and its application.

[0006] Technical Solution: To solve the above-mentioned technical problems, this invention provides a thin-layer chromatography method for identifying Patrinia scabiosifolia formula granules, comprising the following steps:

[0007] (1) Preparation of test solution: Dissolve the test powder in water and extract with ethyl acetate by shaking. Evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution.

[0008] (2) Preparation of reference herb solution: Take Patrinia scabiosifolia reference herb, add water and heat under reflux, filter, concentrate the filtrate to near dryness, add methanol to the residue, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, evaporate the ethyl acetate to dryness, dissolve the residue in methanol to obtain the reference herb solution.

[0009] (3) Spot the reference medicinal material solution and the test solution on the same thin layer plate, develop them with cyclohexane-ethyl acetate-methanol-formic acid as the developing solvent, remove them, air dry them, spray them with sulfuric acid ethanol solution, heat them until the spots are clearly visible, examine them, and compare the thin layer chromatograms for identification.

[0010] (4) When the test sample chromatogram shows spots of the same color at the corresponding positions as the reference medicinal material chromatogram, it indicates that the test sample is a formula granule of Patrinia scabiosifolia.

[0011] Among them, Patrinia scabiosifolia includes white-flowered Patrinia scabiosifolia or yellow-flowered Patrinia scabiosifolia.

[0012] The temperature of the unfolding process in step (3) is 4 to 40°C.

[0013] In step (3), the humidity of the unfolded part is 18-88%.

[0014] This invention also provides the application of the thin-layer identification method in the identification of the authenticity of Patrinia scabiosifolia formula granules.

[0015] This invention also provides a method for distinguishing between *Patrinia scabiosifolia* formula granules, *Illicium verum* formula granules, and *Euphorbia hirta* formula granules, comprising the following steps:

[0016] (1) Preparation of test solution: Dissolve the test powder in water and extract with ethyl acetate by shaking. Evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution.

[0017] (2) Preparation of reference herb solution: Take Patrinia scabiosifolia reference herb, add water and heat under reflux, filter, concentrate the filtrate to near dryness, add methanol to the residue, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, evaporate the ethyl acetate to dryness, dissolve the residue in methanol to obtain the reference herb solution.

[0018] (3) Spot the reference medicinal material solution and the test solution on the same thin layer plate, develop them with cyclohexane-ethyl acetate-methanol-formic acid as the developing solvent, remove them, air dry them, spray them with sulfuric acid ethanol solution, heat them until the spots are clearly visible, examine them, and compare the thin layer chromatograms for identification.

[0019] (4) When the test sample chromatogram shows spots of the same color at the corresponding positions as the reference medicinal material chromatogram, it indicates that the test sample is a formula granule of Patrinia scabiosifolia.

[0020] The temperature of the unfolding process in step (3) is 4 to 40°C.

[0021] In step (3), the humidity of the unfolded part is 18-88%.

[0022] This invention also provides a method for distinguishing between granules of *Patrinia scabra* and *Patrinia glutinosa*, comprising the following steps:

[0023] (1) Preparation of test solution: Dissolve the test powder in water and extract with ethyl acetate by shaking. Evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution.

[0024] (2) Preparation of reference herb solution: Take Patrinia scabiosifolia reference herb, add water and heat under reflux, filter, concentrate the filtrate to near dryness, add methanol to the residue, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, evaporate the ethyl acetate to dryness, dissolve the residue in methanol to obtain the reference herb solution.

[0025] (3) Spot the reference medicinal material solution and the test solution on the same thin layer plate, develop them with cyclohexane-ethyl acetate-methanol-formic acid as the developing solvent, remove them, air dry them, spray them with sulfuric acid ethanol solution, heat them until the spots are clearly visible, examine them, and compare the thin layer chromatograms for identification.

[0026] (4) When the chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material, and there are no yellow-brown spots but blue spots at an Rf value of about 0.2, it indicates that the test sample is a formula granule of Patrinia scabiosifolia; when the chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material, and there are yellow-brown spots at an Rf value of about 0.2 but no blue spots, it indicates that the test sample is a formula granule of Patrinia scabiosifolia.

[0027] The temperature of the unfolding process in step (3) is 4 to 40°C.

[0028] In step (3), the humidity of the unfolded part is 18-88%.

[0029] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. It solves the problem that most existing literature focuses on the identification of medicinal materials rather than the identification of granules; 2. It solves the problem that some literature, although targeting granules for identification, has few spots and the identification effect is not ideal; 3. Using reference medicinal materials as reference substances, the sample is treated with a simple method and examined under ultraviolet light (365nm). The number of spots is large and the separation is good, which can detect multiple components in the sample, greatly improving the specificity of the method; 4. This method can distinguish between the two sources of Patrinia scabiosifolia, Patrinia scabiosifolia var. scabiosifolia and Patrinia scabiosifolia var. scabiosifolia; 5. In addition, it can easily distinguish Patrinia scabiosifolia from adulterants such as Astragalus membranaceus and Astragalus membranaceus, which has high practical value. Attached Figure Description

[0030] Figure 1 TLC spectra of the test sample solution and the control medicinal material solution under different preparation conditions;

[0031] Figure 2 TLC chromatograms of the test solution and the reference herb solution at different spotting amounts;

[0032] Figure 3 For the specificity of the samples;

[0033] Figure 4 TLC spectra of the test solution and the control herbal solution at different temperatures;

[0034] Figure 5 TLC spectra of Patrinia scabiosifolia formulation granules under different humidity conditions;

[0035] Figure 6 TLC spectra of Patrinia scabiosifolia formulation granules from different manufacturers on thin-layer plates;

[0036] Figure 7 Thin-layer chromatograms for different batches of Patrinia scabiosifolia formulation granules;

[0037] Figure 8 Thin-layer chromatograms for identification of Patrinia scabiosifolia (yellow-flowered Patrinia scabiosifolia), Patrinia scabiosifolia (white-flowered Patrinia scabiosifolia), Astragalus membranaceus, and Astragalus membranaceus. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0039] 1. Instruments and reagents

[0040] Instruments: CAMAG TLC VISUALIZER automatic thin-layer imaging system, METTLERTOLEDO balance (0.0001 g), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), silica gel G thin-layer plates (Qingdao Ocean Chemical Co., Ltd., Qingdao Kangyexin Pharmaceutical Silica Gel Desiccant Co., Ltd., Yantai Xincheng Silica Gel Material Co., Ltd.).

[0041] Reagents: Methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), cyclohexane (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), formic acid (Sinopharm Chemical Reagent Co., Ltd.), and sulfuric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.) were all of analytical grade, and water was also used; Patrinia scabiosaefolia reference material (batch number: 121271-201201) was purchased from the National Institutes for Food and Drug Control. The following herbal formula granules are provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.: Patrinia scabiosifolia (batch numbers: 18041129, 18041139, 18041149), Patrinia scabiosifolia (yellow-flowered Patrinia scabiosifolia) (11803650, 11803651, 11803652), Patrinia scabiosifolia (white-flowered Patrinia scabiosifolia) (1806028, 1806029, 1806030), Astragalus membranaceus (2407001, 2407025, 2407026), and Astragalus membranaceus (2206047, 2206050, 2206052).

[0042] 2. Experimental Methods

[0043] 2.1 Thin-layer identification conditions

[0044] Using silica gel G thin-layer plates, cyclohexane-ethyl acetate-methanol-formic acid (6:4:0.5:0.1) was used as the developing solvent. After development, the plates were removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under ultraviolet light (365nm).

[0045] 2.2 Solution Preparation

[0046] 2.2.1 Preparation of the test solution

[0047] Method 1: Take 1g of Patrinia scabiosifolia formula granules, grind them into a fine powder, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0048] Method 2: Take 1g of Patrinia scabiosifolia formula granules, grind them into a fine powder, add 20ml of water to dissolve them, extract them twice with ethyl acetate, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0049] Method 3: Take 1g of Patrinia scabiosifolia formula granules, grind them into a fine powder, add 25ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, extract twice with ethyl acetate, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0050] 2.2.2 Preparation of the control herbal solution

[0051] Method 1: Take 2g of Patrinia scabiosifolia reference material, add 50ml of water, heat under reflux for 60 minutes, cool, filter, concentrate the filtrate to about 20ml, extract twice with ethyl acetate, 20ml each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1ml of methanol, and use as the reference material solution.

[0052] Method 2: Take 2g of Patrinia scabiosifolia reference material, add 50ml of water, heat under reflux for 60 minutes, cool, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to the residue to dissolve, extract twice with ethyl acetate (20ml each time), combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use as the reference material solution.

[0053] 2.2.3 Preparation of negative control solution

[0054] Take 1g of negative sample lacking Patrinia scabiosifolia and prepare a negative control solution with the test sample "Method 2".

[0055] Example 1: Investigation of the preparation methods of the test solution and the control medicinal material solution

[0056] The test solutions and reference solutions prepared using the above-mentioned methods for preparing the three test solutions and two reference solutions of Patrinia scabiosifolia granules (batch number: 18041139) and Patrinia scabiosifolia reference material (batch number: 121271-201201) were spotted separately onto the same silica gel G thin-layer plate. The plates were developed under the above-mentioned thin-layer chromatographic conditions, removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under ultraviolet light (365nm). The development temperature was 26.5℃ and the humidity was 37%. Figure 1 In the table: 1: Test sample, Method 1; 2: Test sample, Method 2; 3: Test sample, Method 3; S1: *Patrinia scabiosifolia* reference material (Method 1); S2: *Patrinia scabiosifolia* reference material (Method 2). Results are as follows: Figure 1 As shown, the test solutions prepared by methods two and three have more spots and better separation. Considering that method two is simpler to process and the reagents used have lower toxicity, method two was chosen as the sample processing method for this thin-layer identification. The Patrinia scabiosifolia control material (method two) corresponds better with the granular spots, so method two was chosen as the control material processing method.

[0057] Example 2: Investigation of different sample sizes

[0058] Patrinia scabiosifolia granules (batch number: 18041139) and Patrinia scabiosifolia reference material (batch number: 121271-201201) were prepared into test solution and reference solution according to the preparation methods for the test solution and reference material solution as determined above. The samples were spotted separately on the same silica gel G thin-layer plate under the thin-layer chromatographic conditions specified in the experimental method. After development, the plates were removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under ultraviolet light (365nm). The development temperature was 26.5℃, and the humidity was 37%. The results are as follows: Figure 2 As shown, Figure 2 In the table: 1: 1 μl of test sample; 2: 2 μl of test sample; 3: 5 μl of test sample; 4: 5 μl of reference medicinal material; 5: 10 μl of reference medicinal material; 6: 15 μl of reference medicinal material. Figure 2 It can be seen that when the sample volume of the test solution is 2 μl and the sample volume of the reference medicinal material solution is 10 μl, the spots in the chromatogram of the test solution and the corresponding positions in the chromatogram of the reference medicinal material are clearly corresponding and there is no other interference. Therefore, the sample volume of the test solution is selected as 2 μl and the sample volume of the reference medicinal material solution is 10 μl.

[0059] Example 3 Specificity test of the sample

[0060] Patrinia scabiosifolia granules (batch number: 18041139) and Patrinia scabiosifolia reference material (batch number: 121271-201201) were prepared into test solution, reference material solution, and negative control solution according to the preparation methods for the test solution and reference material solution determined above. The samples were spotted separately on the same silica gel G thin-layer plate under the thin-layer chromatographic conditions specified in the experimental method. After development, the plates were removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under ultraviolet light (365nm). The development temperature was 26.5℃, and the humidity was 37%. Figure 3 1–3: *Patrinia scabiosifolia* formula granules (18041139); 4: negative control; S: *Patrinia scabiosifolia* control material. (From...) Figure 3 It is evident that the chromatograms of the Patrinia scabiosifolia formula granules and the reference medicinal material show fluorescent spots of the same color at the corresponding positions, and the negative control shows no interference, indicating that the thin-layer identification method has good specificity.

[0061] Example 4: Investigation at different temperatures

[0062] Prepare test solutions and reference solutions using the prescribed methods for preparing the test solution and reference solution of *Patrinia scabiosifolia* granules (batch number: 18041139) and reference herb (batch number: 121271-201201) as described above. Spot the samples on the same silica gel G thin-layer plate under the thin-layer chromatographic conditions specified in the experimental method. Develop the plates at 40°C and 4°C, respectively. Remove the plates, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly visible. Examine the plates under ultraviolet light (365nm). The developing humidity was 37%. Figure 4 1-3: *Patrinia scabiosifolia* formula granules (18041139); S: *Patrinia scabiosifolia* reference material. From Figure 4 As can be seen, under different temperature conditions, the test sample chromatogram and the reference herb chromatogram of Patrinia scabiosifolia granules showed fluorescent spots of the same color at corresponding positions, and the separation effect was good in both cases. The experimental results indicate that temperature has no effect on the thin-layer identification of Patrinia scabiosifolia granules, suggesting that this thin-layer identification method is robust to different temperatures.

[0063] Example 5: Investigation of different humidity levels

[0064] Prepare test solutions and reference solutions using the prescribed methods for preparing the test solution and reference solution of *Patrinia scabiosifolia* granules (batch number: 18041139) and reference herb (batch number: 121271-201201) as described above. Spot the samples on the same silica gel G thin-layer plate under the thin-layer chromatographic conditions specified in the experimental method. Develop the plates under different humidity conditions (18% and 88%). Remove the plates, air dry them, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine the plates under ultraviolet light (365nm). The development temperature was 26.5℃. Figure 5 1-3: *Patrinia scabiosifolia* formula granules (18041139); S: *Patrinia scabiosifolia* reference material. From Figure 5 As can be seen, under different humidity conditions, the test sample chromatogram and the reference herb chromatogram of Patrinia scabiosifolia granules showed fluorescent spots of the same color at corresponding positions, and the separation effect was good in both cases. The experimental results indicate that humidity has no effect on the thin-layer identification of Patrinia scabiosifolia granules, demonstrating that this thin-layer identification method is robust to different humidity levels.

[0065] Example 6: Investigation of Thin-Layer Laminates from Different Manufacturers

[0066] Patrinia scabiosifolia granules (batch number: 18041139) and Patrinia scabiosifolia reference material (batch number: 121271-201201) were prepared into test solutions and reference solutions according to the methods specified above. These solutions were spotted onto silica gel G thin-layer plates from different manufacturers. The plates were developed under the thin-layer chromatographic conditions described in the experimental method at 26.5℃ and 37% humidity, respectively. The plates were then removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The results were then examined under ultraviolet light (365nm). The results are as follows: Figure 6 As shown in Figure 6, 1-3 represent *Patrinia scabiosifolia* formula granules (18041139); S represents *Patrinia scabiosifolia* reference material. Figure 6 shows that under different manufacturers' thin-layer plate conditions, the corresponding positions of the *Patrinia scabiosifolia* formula granules and the reference material chromatograms show spots of the same color, and the separation effect is good in both cases. The experimental results indicate that the manufacturer of the thin-layer plate has no significant impact on the thin-layer identification of *Patrinia scabiosifolia* formula granules, suggesting that this thin-layer identification method is durable for different manufacturers' thin-layer plates.

[0067] Based on the above research results, the thin-layer chromatography method for identifying Patrinia scabiosifolia formula granules is determined as follows:

[0068] Take 1g of *Patrinia scabiosifolia* granules, grind them finely, add 20ml of water to dissolve them, and extract twice with ethyl acetate, 20ml each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 2g of *Patrinia scabiosifolia* reference material, add 50ml of water, heat under reflux for 60 minutes, cool, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, and prepare the reference material solution using the same method. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 2 μl of the test solution and 10 μl of the reference herb solution separately to the same silica gel G thin-layer plate. Develop the plate using cyclohexane-ethyl acetate-methanol-formic acid (6:4:0.5:0.1) as the developing solvent. Remove the plate, air-dry it, spray with 10% sulfuric acid in ethanol, and heat at 105℃ until the spots are clearly visible. Examine the plate 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 reference herb.

[0069] Example 7: Thin-layer chromatography identification of different batches of Patrinia scabiosifolia formulation granules

[0070] Different batches of *Patrinia scabiosaefolia* formula granules were used to prepare test solutions and control solutions according to the above-described method. These solutions were spotted onto the same silica gel G thin-layer plate, developed, removed, and air-dried. The plates were then sprayed with 10% sulfuric acid ethanol solution and heated at 105℃ until the spots were clearly visible. The plates were then examined under ultraviolet light (365nm). The results are as follows: Figure 7As shown in the figure, 1: *Patrinia scabiosifolia* formula granules (18041129); 2: *Patrinia scabiosifolia* formula granules (18041139); 3: *Patrinia scabiosifolia* formula granules (18041149); S: *Patrinia scabiosifolia* control material. (From...) Figure 7 As can be seen, the chromatograms of the Patrinia scabiosifolia formula granules and the reference herb show fluorescent spots of the same color at the corresponding positions.

[0071] Example 8: Comparison of Yellow Flower Patrinia, White Flower Patrinia and Counterfeits

[0072] Take 1g each of the following herbal formulas: *Patrinia scabiosifolia* (yellow-flowered), *Patrinia scabiosifolia* (white-flowered), *Ipomoea aquatica*, and *Euphorbia hirta*. Grind them into a fine powder, add 20ml of water to dissolve, and extract twice with 20ml of ethyl acetate each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 2g of *Patrinia scabiosifolia* reference material, add 50ml of water, heat under reflux for 60 minutes, cool, filter, concentrate the filtrate to near dryness, add 25ml of methanol to the residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, and prepare the reference material solution using the same method. The thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was used. Following the method in Example 7, 2 μl of the above-mentioned test solution and 10 μl of the reference herb solution were spotted separately onto the same silica gel G thin-layer plate. Cyclohexane-ethyl acetate-methanol-formic acid (6:4:0.5:0.1) was used as the developing solvent. After development, the plate was removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly visible. The plates were then examined under ultraviolet light (365 nm). The results are as follows: Figure 8 As shown in the figure, 1-3: *Patrinia scabiosifolia* (yellow-flowered *Patrinia scabiosifolia*) formula granules (11803650, 11803651, 11803652); 4-6: *Patrinia scabiosifolia* (white-flowered *Patrinia scabiosifolia*) formula granules (1806028, 1806029, 1806030); 7-9: *Ipomoea quamoclit* formula granules (2407001, 2407025, 2407026); 10-12: *Euphorbia hirta* formula granules (2206047, 2206050, 2206052); S: *Patrinia scabiosifolia* control material. Figure 8 It can be seen that, compared with the granules of *Patrinia scabiosifolia* (yellow-flowered), the granules of *Patrinia scabiosifolia* (white-flowered) lack yellow-brown spots and have increased blue spots at around Rf value 0.2. Therefore, this thin-layer chromatography method can distinguish between the granules of *Patrinia scabiosifolia* (yellow-flowered) and *Patrinia scabiosifolia* (white-flowered). The granules of *Ipomoea quamoclit*, *Atractylodes macrocephala*, and *Patrinia scabiosifolia* show significant differences in the number and position of spots, making them easy to distinguish. Therefore, this thin-layer chromatography method can distinguish between the granules of *Patrinia scabiosifolia*, *Ipomoea quamoclit*, and *Atractylodes macrocephala*.

Claims

1. A thin-layer chromatography method for identifying granules of Patrinia scabiosifolia, characterized in that, Includes the following steps: (1) Preparation of test solution: Dissolve the test powder in water and extract with ethyl acetate by shaking. Evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution. (2) Preparation of reference herb solution: Take Patrinia scabiosifolia reference herb, add water and heat under reflux, filter, concentrate the filtrate to near dryness, add methanol to the residue, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, evaporate the ethyl acetate to dryness, dissolve the residue in methanol to obtain the reference herb solution. (3) Take the reference medicinal material solution and the test solution and spot them on the same silica gel G thin layer plate. Develop the plate with cyclohexane-ethyl acetate-methanol-formic acid in a volume ratio of 6:4:0.5:0.

1. Remove the plate, air dry it, spray it with sulfuric acid ethanol solution, heat it until the spots are clearly visible, examine it, and compare the thin layer chromatograms. (4) If the test sample shows a spot of the same color at the corresponding position in the chromatogram of the reference medicinal material, it indicates that the test sample is a formula granule of Patrinia scabiosifolia.

2. The thin-layer chromatography identification method according to claim 1, characterized in that, The temperature for unfolding in step (3) is 4~40℃.

3. The thin-layer chromatography identification method according to claim 1, characterized in that, The humidity level during the unfolding process in step (3) is 18-88%.

4. The application of the thin-layer chromatography method according to any one of claims 1 to 3 in the identification of the authenticity of Patrinia scabiosifolia formula granules.

5. A method for distinguishing between *Patrinia scabiosifolia* formula granules, *Illicium verum* formula granules, and *Euphorbia hirta* formula granules, characterized in that... Includes the following steps: (1) Preparation of test solution: Dissolve the test powder in water and extract with ethyl acetate by shaking. Evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution. (2) Preparation of reference herb solution: Take Patrinia scabiosifolia reference herb, add water and heat under reflux, filter, concentrate the filtrate to near dryness, add methanol to the residue, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, evaporate the ethyl acetate to dryness, dissolve the residue in methanol to obtain the reference herb solution. (3) Take the reference medicinal material solution and the test solution and spot them on the same silica gel G thin layer plate. Develop the plate with cyclohexane-ethyl acetate-methanol-formic acid in a volume ratio of 6:4:0.5:0.

1. Remove the plate, air dry it, spray it with sulfuric acid ethanol solution, heat it until the spots are clearly visible, examine it, and compare the thin layer chromatograms. (4) If the test sample shows a spot of the same color at the corresponding position in the chromatogram of the reference medicinal material, it indicates that the test sample is a formula granule of Patrinia scabiosifolia.

6. The method according to claim 5, characterized in that, The temperature for unfolding in step (3) is 4~40℃.

7. The method according to claim 5, characterized in that, The humidity level during the unfolding process in step (3) is 18-88%.

8. A method for distinguishing between *Patrinia scabiosifolia* and *Patrinia nigra* granules, characterized in that, Includes the following steps: (1) Preparation of test solution: Dissolve the test powder in water and extract with ethyl acetate by shaking. Evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution. (2) Preparation of reference herb solution: Take Patrinia scabiosifolia reference herb, add water and heat under reflux, filter, concentrate the filtrate to near dryness, add methanol to the residue, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, evaporate the ethyl acetate to dryness, dissolve the residue in methanol to obtain the reference herb solution. (3) Take the reference medicinal material solution and the test solution and spot them on the same silica gel G thin layer plate. Develop the plate with cyclohexane-ethyl acetate-methanol-formic acid in a volume ratio of 6:4:0.5:0.

1. Remove the plate, air dry it, spray it with sulfuric acid ethanol solution, heat it until the spots are clearly visible, examine it, and compare the thin layer chromatograms. (4) When the chromatogram of the test sample shows spots of the same color at the corresponding position as the chromatogram of the reference medicinal material, and there are no yellow-brown spots but blue spots at an Rf value of about 0.2, it indicates that the test sample is a formula granule of Patrinia scabiosifolia; when the chromatogram of the test sample shows spots of the same color at the corresponding position as the chromatogram of the reference medicinal material, and there are yellow-brown spots at an Rf value of about 0.2 but no blue spots, it indicates that the test sample is a formula granule of Patrinia scabiosifolia.

9. The method according to claim 8, characterized in that, The temperature for unfolding in step (3) is 4~40℃.

10. The method according to claim 8, characterized in that, The humidity level during the unfolding process in step (3) is 18-88%.

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