Quality detection method of Zhuang medicine Luojian Yubu Plaster

Abstract

The application discloses a quality detection method of Zhuang medicine Luojun Yuba Plaster, which comprises the following steps: adopting UPLC to determine the contents of main components in the Zhuang medicine Luojun Yuba Plaster, i.e.sinomenine, mao liangmianjiesu, fufenji, lüguansu, hujangju and longxuesu B; and adopting thin layer chromatography (TLC) to identify sinofengteng, fufenji, hujang, feilongzhangxue, longxuiezhi, rushan and meiyao in the Zhuang medicine Luojun Yuba Plaster. The quality detection method can accurately and quickly determine the contents of the main components in the Zhuang medicine Luojun Yuba Plaster and effectively identify the used medicinal materials. The quality detection method is simple and high in accuracy, can systematically and accurately evaluate the quality of the Zhuang medicine Luojun Yuba Plaster, is favorable for comprehensively controlling the overall quality of the Zhuang medicine Luojun Yuba Plaster, ensures curative effect, and provides an effective reference for quality control and evaluation of the Zhuang medicine Luojun Yuba Plaster.

Description

Technical Field

[0004] , ,

[0001] The present invention relates to a quality detection method for Zhuang medicine Luoyan Yu cataplasm, belonging to the technical field of drug detection. Background Art

[0002] Knee osteoarthritis (KOA) refers to chronic degenerative knee osteoarthritis that mostly occurs in middle-aged people and later, characterized by degeneration of knee joint cartilage and reactive hyperplasia of subchondral bone, featuring joint pain, deformation, and movement disorders, and is a clinically common disabling disease with a complex multi-factor pathogenesis. Currently, the clinical treatment methods for knee osteoarthritis are mainly divided into two categories: conservative treatment and surgical treatment. For early-stage KOA patients, conservative treatment is often the first choice; for some patients who meet the surgical indications, if there are factors such as advanced age and obesity that can significantly increase the postoperative risk, conservative treatment is preferred, and it is mainly drug treatment. Commonly used western medicines include analgesics, non-steroidal anti-inflammatory drugs, etc., and the administration route is mainly oral. Western medicines mainly control symptoms, cannot control the disease progression, and have large side effects. The new generation of cyclooxygenase-2 inhibitors can effectively reduce side effects, but there is still a risk of cardiovascular-related diseases. In addition, surgical treatment is expensive and risky, and some physical therapy methods are expensive or restricted by conditions such as region.

[0003] Knee osteoarthritis belongs to the category of arthralgia syndrome in traditional medicine treatment. It is mainly treated with drugs, emphasizing syndrome differentiation and treatment, mainly using wind-dampness dispelling and meridian-warming and cold-dispersing methods, supplemented by qi and blood tonifying and liver and kidney nourishing, effectively eliminating knee joint inflammation, thus achieving remarkable effects and ultimately playing a role in eradicating the root cause. The traditional medicine treatment of KOA has definite curative effects and the advantage of a lower recurrence rate after treatment compared with western medicine treatment. It has both the accumulation of predecessors' experience and a large number of modern research techniques. Therefore, based on the unique advantages of traditional medicine treatment of knee osteoarthritis, it is very necessary to search for and develop new drugs for treating knee osteoarthritis that are safe, inexpensive, and have definite curative effects.​​​​

[0005] Poultices are plasters made by uniformly mixing medicinal extracts, medicinal materials, or chemical drugs with a suitable hydrophilic matrix and then applying the mixture to a backing material. Poultices can directly act on the target site to exert therapeutic effects, avoiding the first-pass effect of the liver and gastrointestinal tract and the irritation of the gastrointestinal tract by the drug. They maintain a constant blood drug concentration for a longer period, reduce adverse drug reactions, and decrease the frequency of administration. They are convenient to use, can be repeatedly applied, and have good breathability. Zhuang medicine Luoqianyu plaster is a new dosage form of Zhuang medicine Luoqianyu formula developed by Guangxi University of Chinese Medicine. Zhuang medicine Luoqianyu plaster has many ingredients and complex components, and currently there is no relevant quality testing method. Establishing a single indicator component is too one-sided and difficult to accurately detect the quality of Zhuang medicine Luoqianyu plaster. Therefore, in order to more comprehensively control the quality of Zhuang medicine Luoqianyu plaster and ensure its efficacy, developing a quality testing method for Zhuang medicine Luoqianyu plaster is of great significance for controlling the overall quality of Zhuang medicine Luoqianyu plaster and ensuring the safety and effectiveness of its clinical use. Summary of the Invention

[0006] The purpose of this invention is to provide a quality testing method for Zhuang medicine Luoqianyu plaster. The method described can accurately and comprehensively test the quality of Zhuang medicine Luoqianyu plaster, which is beneficial to comprehensively control the overall quality of Zhuang medicine Luoqianyu plaster and ensure its efficacy.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A quality testing method for a traditional Chinese medicine, Luoqianyu poultice, includes methods for determining the content of sinomenine, zephyranthesin, and tetrandrine; methods for determining the content of chlorogenic acid, polygalactoside, and dracoside B; the methods for determining the content of sinomenine, zephyranthesin, and tetrandrine include the following steps:

[0009] (1) Preparation of test solution: Accurately weigh about 5g of Zhuang medicine Luoqianyu Babuji, place it in a stoppered Erlenmeyer flask, accurately take 25mL of chloroform with a pipette, sonicate for 1h, twice, combine the filtrates, place them in an evaporating dish to evaporate to dryness, make up to 5mL with methanol, shake well, centrifuge, and filter through a microporous membrane to obtain the test solution.

[0010] (2) Preparation of reference standard mixed solution: Accurately weigh appropriate amounts of sinomenine, styrax chinensis, and tetrandrine reference standards, dissolve them in methanol, and prepare reference standard stock solutions with concentrations of 2.330 mg / mL, 2.140 mg / mL, and 1.668 mg / mL, respectively; separately take appropriate amounts of the above sinomenine, styrax chinensis, and tetrandrine solutions, place them in the same volumetric flask, dilute with methanol, and prepare alkaloid reference standard mixed solution containing sinomenine, styrax chinensis, and tetrandrine concentrations of 0.240 mg / mL, 0.344 mg / mL, and 0.096 mg / mL, respectively.

[0011] (3) Preparation of negative control solution: Weigh out the other medicinal materials except for Qingfengteng, Feilongzhangxue and Fenfangji according to the prescription ratio to make Zhuang medicine Luoqianyu Babuji. Prepare negative sample solutions for Qingfengteng deficiency, Feilongzhangxue deficiency and Fenfangji deficiency according to the method under (1) Preparation of test solution.

[0012] (4) Chromatographic conditions and system suitability test: A Waters BEH C18 2.1 × 100 mm 1.7 µm column was used; the mobile phase was acetonitrile-0.1 mmol / L ammonium acetate solution, and the gradient elution conditions are shown in the table below. The flow rate was 0.2 mL / min; the detection wavelength was 280 nm; the column temperature was 40℃; and the injection volume was 3 µL.

[0013]

[0014] (5) Determination: Under the above chromatographic conditions, the mixed solution of reference standard, the negative control solution, and the sample test solution were injected into the ultra-high performance liquid chromatograph for analysis and determination. The DAD detector was used for detection. The results showed that the retention time and UV spectrum of the chromatographic peaks of sinomenine, styraxanthine, and tetrandrine were consistent with the retention time and UV spectrum of the corresponding chromatographic peaks in the sample. The separation of each peak was good. Other components in the sample did not interfere with the determination of these three index components. The theoretical plate number of the chromatographic column, calculated based on sinomenine, was not less than 2500. The content of each component was calculated.

[0015] The above-mentioned quality testing method for the Zhuang medicine Luoqianyu poultice, specifically the method for determining the content of chlorogenic acid, polygalactoside, and dracoside B, includes the following steps:

[0016] (1) Preparation of test solution: Accurately weigh about 5g of Zhuang medicine Luoqianyu Babuji and place it in a stoppered Erlenmeyer flask. Accurately take 25mL of methanol with a pipette, sonicate for 1.5h twice, combine and evaporate to dryness, make up to 25mL with methanol, shake well, centrifuge, and filter through a microporous membrane to obtain the test solution.

[0017] (2) Preparation of the reference standard mixed solution: Accurately weigh appropriate amounts of polygalactoside, chlorogenic acid, and dracosanol B reference standards, dissolve them in methanol, and prepare reference standard stock solutions with concentrations of 0.808 mg / mL, 0.990 mg / mL, and 0.866 mg / mL, respectively. Take appropriate amounts of the above polygalactoside, chlorogenic acid, and dracosanol B solutions, place them in the same volumetric flask, dilute with methanol, and prepare a reference standard mixed solution containing polygalactoside, chlorogenic acid, and dracosanol B concentrations of 0.035 mg / mL, 0.139 mg / mL, and 0.106 mg / mL, respectively.

[0018] (3) Preparation of negative control solution: Weigh out the other medicinal materials except for Acanthopanax senticosus, Polygonum cuspidatum and Dragon's Blood according to the prescription ratio to make Zhuang medicine Luoqianyu Babuji. Prepare negative sample solution lacking Acanthopanax senticosus, negative sample solution lacking Polygonum cuspidatum and negative sample solution lacking Dragon's Blood according to the method under (1) Preparation of test solution.

[0019] (4) Chromatographic conditions and system suitability test: Waters HSST3, 2.1 × 100 mm 1.8 µm column was used; the mobile phase was acetonitrile-0.1% acetic acid aqueous solution, the gradient elution conditions are shown in the table below, the flow rate was 0.3 mL / min; the detection wavelengths were 310 nm and 280 nm; the column temperature was 25 ℃; the injection volume was 2 µL;

[0020]

[0021] (5) Determination: Under these chromatographic conditions, the mixed solution of reference standard, the negative control solution, and the sample test solution were injected into the ultra-high performance liquid chromatograph for analysis. The results showed that the retention times and UV spectra of the chromatographic peaks of styrax glycoside, chlorogenic acid, and dragon's blood B were consistent with the retention times and UV spectra of the corresponding chromatographic peaks in the sample. The resolution of each peak was good, and there was no interference in the determination of the index components. The theoretical plate number of the chromatographic column, calculated based on chlorogenic acid, was not less than 2500. The content of each component was calculated.

[0022] The quality testing method for the Zhuang medicine Luoqianyu plaster also includes using thin-layer chromatography to identify Qingfengteng, Fenfangji, Huzhang, Feilongzhangxue, Longxuejie, Ruxiang, and Moyao in the Zhuang medicine Luoqianyu plaster, as detailed below:

[0023] (1) Thin-layer chromatographic identification of Qingfengteng and Fenfangji

[0024] Preparation of test solution: Take 5g of Zhuang medicine Luoqianyu poultice, sonicate with 20ml of methanol for 1h, filter, take 10ml and evaporate to dryness, make up to 5ml with 50% ethanol, centrifuge, extract with 20ml of chloroform, evaporate to dryness, and make up to 2ml with methanol to obtain the test solution; Separately, take 0.5g of Qingfengteng and 0.2g of Fenfangji reference materials and prepare Qingfengteng and Fenfangji reference material solutions in the same way; Preparation of reference solution: Take appropriate amounts of Qingteng alkaloid and Fenfangji alkaloid reference standards, and make up to 0.240mg / mL and 0.344mg / mL of Qingteng alkaloid and Fenfangji alkaloid reference standard solutions with methanol to obtain reference standard solutions with concentrations of 0.240mg / mL and 0.344mg / mL, respectively; Preparation of negative control solution: Weigh the remaining medicinal materials except Qingfengteng and Fenfangji alkaloid, prepare according to the prescription process, and then prepare according to the above test solution preparation conditions to obtain the negative control solution;

[0025] According to the thin-layer chromatography method, 20 µL of the test solution, 4 µL of the *Sinomenium acutum* and *Stephania tetrandra* reference medicinal material solutions, 1.6 µL of the reference solution, and 27 µL of the negative control solution were spotted separately onto the same silica gel G thin-layer plate. A toluene-ethyl acetate-methanol-water mixture with a volume ratio of 2:4:2:1 was placed at 10 °C for 12 h. The upper layer was used as the developing solvent and developed in a developing tank pre-saturated with concentrated ammonia solution for 20 min. The plate was then removed, dried, and sprayed with freshly prepared bismuth potassium iodide solution. At the corresponding positions of the spots on the chromatogram, the test sample, *Sinomenium acutum* reference medicinal material, *Stephania tetrandra* reference medicinal material, sinomenine reference standard, and stephania tetrandra alkaloid showed spots of the same color, and the negative control did not interfere.

[0026] (2) Thin-layer identification of blood from the palm of the dragon

[0027] Preparation of test solution: Prepare the test solution using the same method as described above for the thin-layer identification of *Sinomenium acutum* and *Stephania tetrandra*. Separately, take 0.3g of *Pteris vittata* (a type of herb) as a reference herb and prepare a *Pteris vittata* reference herb solution using the same method. Preparation of reference solution: Take an appropriate amount of *Zanthoxylum nitidum* (a type of herb) reference standard and dilute it to a concentration of 0.096 mg / mL with methanol to prepare a *Stephania tetrandra* (another type of herb) reference standard solution. Preparation of negative control solution: Weigh the remaining herbs except *Pteris vittata*, prepare them according to the prescription process, and then prepare the negative control solution according to the above test solution preparation conditions.

[0028] According to the thin-layer chromatography method, take 8 µL of the test solution, 6 µL of the reference medicinal material solution, 3 µL of the reference solution, and 8 µL of the negative control solution; spot them separately on the same silica gel G thin-layer plate. Incubate a toluene-ethyl acetate-methanol-water mixture with a volume ratio of 2:4:2:1 at 10℃ for 12 h. Take the upper layer as the developing solvent and develop it in a developing tank pre-saturated with concentrated ammonia solution for 20 min. Remove the plate, air dry it, and examine it under ultraviolet light at 365 nm. At the corresponding positions of the spots on the chromatogram, the test sample, the reference medicinal material of *Pteris vittata*, and the reference standard of *Zanthoxylum nitidum* show the same blue fluorescent spots, and the negative control has no interference.

[0029] (3) Thin-layer identification of Polygonum cuspidatum

[0030] Preparation of test solution: Weigh 5g of Zhuang medicine Luoqianyu poultice, add 30ml of methanol, sonicate for 1h, filter, evaporate to dryness, dilute to 5ml with methanol, centrifuge, and obtain the test solution; separately take 0.2g of Polygonum cuspidatum reference material and prepare a Polygonum cuspidatum reference material solution using the same method; Preparation of reference solution: Take an appropriate amount of Polygonum cuspidatum glycoside reference standard, dilute to a concentration of 0.035mg / mL with methanol, and prepare the Polygonum cuspidatum glycoside reference standard solution; Preparation of negative control solution: Weigh the remaining medicinal materials except Polygonum cuspidatum, prepare according to the prescription process, and then prepare according to the preparation conditions of the test solution, and use as the negative control. Solution; according to the thin-layer chromatography method, take 4µL of the test solution, 2µL of the reference medicinal material solution, 1.5µL of the reference solution, and 4µL of the negative control solution; spot them separately on the same high-performance silica gel G thin-layer plate, use chloroform-methanol-water with a volume ratio of 15:5:0.5 as the developing solvent, pre-saturate for 20 min, develop, remove, air dry, and examine under ultraviolet light at 365nm; at the corresponding positions of the spots on the chromatogram, the test sample, Polygonum cuspidatum reference medicinal material, and Polygonin reference standard show the same blue fluorescent spots, and the negative control standard has no interference;

[0031] (4) Thin-layer chromatographic identification of dragon's blood

[0032] Preparation of test solution: Weigh 5g of Zhuang medicine Luoqianyu poultice, add 30ml of methanol, sonicate for 1h, filter, evaporate to dryness, dilute to 5ml with methanol, centrifuge to obtain the test solution; separately take 0.5g of dragon's blood medicinal material, dissolve in methanol to obtain dragon's blood reference medicinal material solution; Preparation of negative control solution: Weigh the remaining medicinal materials except dragon's blood, prepare according to the prescription process, and then prepare according to the test solution preparation conditions to obtain the negative control solution; according to the thin layer chromatography method, take 8µL of test solution, 3µL of reference medicinal material solution, and 8µL of negative control solution; spot them separately on the same high-performance silica gel G thin layer plate, use chloroform-methanol-formic acid with a volume ratio of 19:1:0.1 as the developing solvent, pre-saturate for 20min, develop, remove, air dry, spray with 5% vanillin sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and at the corresponding positions of the spots on the chromatogram, the test sample and the dragon's blood reference medicinal material show the same color spot A, and the negative control has no interference;

[0033] (5) Thin-layer chromatography identification of frankincense

[0034] Preparation of test solution: Prepare the test solution using the same method as for the thin-layer identification of *Sinomenium acutum* and *Stephania tetrandra*. Separately, take 0.2 g of frankincense and prepare a frankincense reference solution using the same method. Preparation of negative control solution: Weigh the remaining medicinal materials except frankincense, prepare them according to the prescription process, and then prepare the negative control solution according to the test solution preparation conditions. Using thin-layer chromatography, apply 6 µL of the test solution, 1 µL of the reference solution, and 6 µL of the negative control solution to the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-formic acid (v / v) as the developing solvent, pre-saturate for 20 min, develop, remove, air dry, spray with 5% vanillin-sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. At the corresponding positions of the spots on the chromatogram, the test sample and frankincense reference solution show the same color spots, and the negative control solution shows no interference.

[0035] (6) Thin-layer chromatography identification of myrrh

[0036] Preparation of test solution: Prepare the test solution using the same method as for the thin-layer identification of *Sinomenium acutum* and *Stephania tetrandra*. Separately, take 0.3 g of myrrh and prepare a myrrh reference solution using the same method. Preparation of negative control solution: Weigh the remaining medicinal materials (excluding myrrh), prepare them according to the prescription process, and then prepare the negative control solution according to the test solution preparation conditions. Using thin-layer chromatography, apply 16 µL of the test solution, 1 µL of the reference solution, and 16 µL of the negative control solution to the same high-performance silica gel G thin-layer plate. Use a cyclohexane-chloroform-ethyl acetate solution (8:1:1 v / v) as the developing solvent, pre-saturate for 20 min, develop, remove, air dry, spray with 5% vanillin-sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. At the corresponding positions of the spots on the chromatogram, the test sample and myrrh reference solution show the same color spots, and the negative control solution does not interfere.

[0037] This invention relates to a quality testing method for Zhuang medicine Luoqianyu plaster. The prescription of the medicinal materials for the Zhuang medicine Luoqianyu plaster is as follows (by weight): 8.55 parts of *Houttuynia cordata*, 4.27 parts of *Sinomenium acutum*, 4.27 parts of *Stephania tetrandra*, 3.85 parts of *Lycopodium clavatum*, 4.27 parts of *Polygonum cuspidatum*, 2.14 parts of *Pterocarya stenoptera*, 2.14 parts of *Erythrina variegata*, 3.42 parts of *Saururus chinensis*, 3.42 parts of *Acanthopanax senticosus*, 3.42 parts of *Smilax china*, 3.42 parts of *Tetracentron sinense*, 2.14 parts of *Acer buergerianum*, 2.14 parts of *Cinnamomum camphora*, 2.14 parts of *Dalbergia odorifera*, 2.14 parts of *Pistacia chinensis*, 2.14 parts of *Pistacia chinensis*, 1.28 parts of *Euonymus fortunei*, 0.85 parts of frankincense, and 0.85 parts of myrrh. 1.28 parts of dragon's blood; the Zhuang medicine Luoqianyu Babu powder of the present invention is prepared by using the above-mentioned prescription of each medicinal material as raw material.

[0038] The beneficial effects of this invention are as follows:

[0039] The present invention provides a quality detection method for Zhuangyao Luoqianyu cataplasm. This method uses thin layer chromatography (TLC) to identify Sinomenium acutum, Stephania tetrandra, Polygonum cuspidatum, Toddalia asiatica, Dracaena cochinchinensis, Boswellia carterii, and Commiphora myrrha in Zhuangyao Luoqianyu cataplasm, and uses UPLC method to determine the contents of the main components sinomenine, nitidine chloride, tetrandrine, chlorogenic acid, polydatin, and dracorhodin B in Zhuangyao Luoqianyu cataplasm. The thin layer chromatography identification method used has the characteristics of strong specificity and good separation effect, and can effectively and accurately identify each medicinal material. By using the described content determination method, the contents of the main components in Zhuangyao Luoqianyu cataplasm can be accurately and rapidly determined. This quality detection method is simple and highly accurate, can systematically and accurately evaluate the quality of Zhuangyao Luoqianyu cataplasm. Using the described method can comprehensively and effectively control the overall quality of Zhuangyao Luoqianyu cataplasm, ensure the curative effect, and provide an effective reference basis for the quality control and evaluation of the finished product of Zhuangyao Luoqianyu cataplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Thin layer chromatography identification specificity investigation test thin layer chromatogram of Sinomenium acutum and Stephania tetrandra. In the figure, 1 - 3: 3 batches of test samples, 4: sinomenine reference substance, 5: tetrandrine reference substance, 6: Sinomenium acutum reference medicinal material, 7: Stephania tetrandra reference medicinal material, 8: Sinomenium acutum negative control, 9: Stephania tetrandra negative control;

[0041] Figure 2 Thin layer chromatography identification specificity investigation test thin layer chromatogram of Toddalia asiatica. In the figure, 1 - 3: 3 batches of test samples, 4: Toddalia asiatica reference medicinal material, 5: nitidine chloride reference substance, 6: negative control, A is a blue fluorescent spot;

[0042] Figure 3 Thin layer chromatography identification specificity investigation test thin layer chromatogram of Polygonum cuspidatum. In the figure, 1 - 3: 3 batches of test samples, 4: Polygonum cuspidatum reference medicinal material, 5: nitidine chloride reference substance, 6: negative control, A is a blue fluorescent spot;

[0043] Figure 4 Thin layer chromatography identification specificity investigation test thin layer chromatogram of Dracaena cochinchinensis. In the figure, 1 - 3: 3 batches of test samples, 4: Dracaena cochinchinensis reference medicinal material, 5: negative control;

[0044] Figure 5 Thin layer chromatography identification specificity investigation test thin layer chromatogram of Boswellia carterii. In the figure, 1 - 3: 3 batches of test samples, 4: Boswellia carterii reference medicinal material, 5: negative control;

[0045] Figure 6 Thin layer chromatography identification specificity investigation test thin layer chromatogram of Commiphora myrrha. In the figure, 1 - 3: 3 batches of test samples, 4: Commiphora myrrha reference medicinal material, 5: negative control;

[0046] Figure 7The UPLC chromatograms of the sample solution and the mixed solution of the Zhuang medicine Luoqianyu Babu powder were obtained by using the method for determining the content of sinomenine, zephyranthesin, and tetrandrine. In the figure, the sample solution (A) and the mixed solution of the reference standard (B) are: 1: sinomenine, 2: zephyranthesin, and 3: tetrandrine.

[0047] Figure 8 The UPLC chromatograms obtained by detecting the negative control solution using the methods for determining the content of sinomenine, styrax tinctoria, and tetrandrine are shown in the figure. In the figure, the negative control solution is missing sinomenine (C), missing styrax tinctoria blood (D), and missing tetrandrine (E).

[0048] Figure 9 The UPLC chromatogram of the test sample solution of Zhuang medicine Luoqianyu Babu was detected at 310 nm using the method for determining the content of chlorogenic acid, polydipsia glycoside and dragon's blood B. In the figure, 1: chlorogenic acid, 2: polydipsia glycoside.

[0049] Figure 10 UPLC chromatogram of chlorogenic acid and polydipsia glycoside mixed control solution detected at 310 nm, where 1: chlorogenic acid; 2: polydipsia glycoside;

[0050] Figure 11 UPLC chromatogram of the negative control solution lacking Polygonum cuspidatum at 310 nm, where 1 represents chlorogenic acid;

[0051] Figure 12 UPLC chromatogram of negative control solution lacking styrax at 310 nm, where 2 represents styrax glycoside;

[0052] Figure 13 UPLC chromatogram of the test solution of the Zhuang medicine Luoqianyu Babu at 280 nm. In the figure, 3: Dragon's Blood B;

[0053] Figure 14 UPLC chromatogram of dracoside B reference solution at 280 nm, where 3 represents dracoside B.

[0054] Figure 15 UPLC chromatogram of a negative solution of dragon's blood without blood at 280 nm. Detailed Implementation

[0055] Experimental materials

[0056] 1.1 Instruments: Waters HSST3 (2.1 × 100 mm 1.8 µm), Waters BEH C18 (2.1 × 100 mm 1.7 µm), and Syncronis C18 (2.1 × 100 mm 1.7 µm) columns; Agilent 1290 ultra-high performance liquid chromatograph (including quaternary pump, autosampler, column oven, and DAD detector); XS105 METTLER TOLEDO balance; SOP balance; 20 µL-200 µL pipettes and 0.5 µL-10 µL manual pipettes; Merck Millipak Express 40 pure water system (Milli-Q); and a constant temperature water bath.

[0057] 1.2 Materials: Tetrandrine (purity ≥94.6%), Zanthoxylum bungeanum extract (purity ≥98%), tetrandrine (purity ≥99.6%), chlorogenic acid (purity ≥96.8%), polygalactoside (purity ≥87.3%), and dracoside B (purity ≥99.9%) were all purchased from the China National Institutes for Food and Drug Control; tetrandrine (purity ≥98.7%) was purchased from Chengdu Mansite Biotechnology Co., Ltd.; Zanthoxylum bungeanum extract (batch number: RP190406, purity ≥98%)... %) Sinomenine (purity >98%) was purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd.; Sinomenium acutum, Stephania tetrandra, Polygonum cuspidatum, Polygonum multiflorum, Dragon's Blood, Boswellia carterii, and Commiphora myrrha reference materials were all purchased from the China National Institutes for Food and Drug Control; high-performance silica gel G, silica gel G (for thin-layer chromatography), acetonitrile (chromatographic grade: Fisher Scientific), methanol, ethyl acetate, formic acid, xylene, toluene, chloroform, n-hexane, cyclohexane, acetic acid, ammonia, and benzene were all of analytical grade, and water was ultrapure water.

[0058] Ten batches of Zhuang medicine Luoqianyu poultice (batch numbers: 20210119, 2021010901, 2021010902, 2021010903, 2021010904, 2021010905, 2021010906, 2021010907, 2021010908, 2021010909) were all prepared in-house. The preparation method of the Zhuang medicine Luoqianyu poultice is as follows:

[0059] (1) Take the following ingredients according to the prescribed amounts: 8.55 parts of *Lysimachia christinae*, 4.27 parts of *Sinomenium acutum*, 4.27 parts of *Stephania tetrandra*, 3.85 parts of *Lycopodium clavatum*, 4.27 parts of *Polygonum cuspidatum*, 2.14 parts of *Pterocarya stenoptera*, 2.14 parts of *Erythrina variegata*, 3.42 parts of *Saururus chinensis*, 3.42 parts of *Acanthopanax senticosus*, 3.42 parts of *Smilax china*, 3.42 parts of *Smilax china*, 3.42 parts of *Smilax china*, 2.14 parts of *Acer buergerianum*, 2.14 parts of *Cinnamomum camphora*, 2.14 parts of *Dalbergia odorifera*, 2.14 parts of *Gynostemma pentaphyllum*, 2.14 parts of *Aconitum carmichaelii*, 1.28 parts of *Euonymus fortunei*, 0.85 parts of frankincense (processed with vinegar), 0.85 parts of myrrh (processed with vinegar), and 1.28 parts of dragon's blood; and take 0.196 parts of borneol and 0.196 parts of menthol. 0.196 parts camphor, 0.5 parts Tween-80, 0.223 parts methyl salicylate, for later use;

[0060] (2) Take five medicinal materials: Manshanxiang, Shenjincao, Sifangmupi, Yinxiangpi, and Shanju, and steam distill them for 8 hours to obtain volatile oil for later use; mix the residue from which the volatile oil has been extracted with the remaining medicinal materials of the Zhuang medicine Luoqianyufang except for Longxuejie, Frankincense, and Myrrh, add 10 times the volume of 60% ethanol for reflux extraction for 90 minutes, extract twice, filter, and recover the ethanol from the filtrate under reduced pressure until there is no alcohol taste, concentrate to a relative density of 1.20~1.25 to obtain clear paste A; crush Frankincense, Myrrh, and Longxuejie, dissolve them with an appropriate amount of ethanol and water, add clear paste A and mix well to obtain thick paste B for later use; mix the volatile oil, borneol, menthol, camphor with Tween-80 and methyl salicylate evenly to obtain mixed liquid C;

[0061] (5) According to the weight ratio: NP-700: micronized silica gel: aluminum hydroxyl: carbomer 940: CMC-Na: glycerol: water: thick paste B = 1.6: 0.70: 0.80: 0.5: 0.5: 9: 10: 12 and 1% of the total matrix weight of EDTA-2Na; take each matrix component for later use; first take NP-700, micronized silica gel and aluminum hydroxyl and place them in glycerol to swell as phase A, dissolve EDTA-2Na in water, add carbomer 940 and CMC-Na to fully swell as phase B, mix phase B and phase A evenly to prepare the matrix, then add thick paste B and mixed solution C, stir with a mixer at 400r / min for 15 min, after stirring evenly, coat it on non-woven fabric, dry and shape it, add a cover, cut and package it to obtain the final product.

[0062] All raw medicinal materials used in the experiment were identified by Professor Zhu Yilin of Guangxi University of Chinese Medicine. Information on the medicinal materials and samples is shown in Table 1.

[0063]

[0064]

[0065] 2. Thin-layer chromatography identification

[0066] 2.1 Thin-layer chromatography identification of *Sinomenium acutum* and *Stephania tetrandra*

[0067] 2.1.1 Preparation of test solution: Take 5g of Zhuang medicine Luoqianyu poultice, sonicate with 20ml of methanol for 1h, filter, take 10ml and evaporate to dryness, make up to 5ml with 50% ethanol, centrifuge, extract with 20ml of chloroform, evaporate to dryness, and make up to 2ml with methanol to obtain the test solution.

[0068] 2.1.2 Preparation of solutions of Qingfengteng and Fenfangji medicinal materials: Take 0.5g of Qingfengteng and 0.2g of Fenfangji reference medicinal materials respectively, and prepare them according to the conditions in "2.1.1".

[0069] 2.1.3 Preparation of reference solutions: Take appropriate amounts of sinomenine and tetrandrine reference standards respectively, and dilute with methanol to obtain sinomenine reference solutions with concentrations of 0.240 mg / mL and 0.344 mg / mL tetrandrine reference standards respectively.

[0070] 2.1.4 Preparation of negative control solution: Weigh out all medicinal materials except for Sinomenium acutum and all medicinal materials except for Stephania tetrandra, prepare according to the prescription process, and then prepare according to the conditions in "2.1.1" to obtain the negative control solution.

[0071] 2.1.5 Specificity Examination: Thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was performed. 20 µL of the test solution, 4 µL of the reference herb, 1.6 µL of the reference standard, and 27 µL of the negative control solution were spotted separately onto the same silica gel G thin-layer plate. The plate was incubated at 10°C for 12 h with toluene-ethyl acetate-methanol-water (2:4:2:1). The upper layer was used as the developing solvent and developed in a developing tank pre-saturated with concentrated ammonia solution for 20 min. The plate was then removed, air-dried, and sprayed with freshly prepared potassium bismuth iodide solution. See [link to relevant documentation]. Figure 1 .

[0072] 2.1.6 Determination Results: At the corresponding positions of the spots on the chromatogram, the test sample, *Sinomenium acutum* reference material, *Stephania tetrandra* reference material, sinomenine reference standard, and stephania tetrandra alkaloid showed spots of the same color, A and B. Furthermore, the separation was good, and the negative control showed no interference, indicating that the method has good specificity.

[0073] 2.1.7 Durability test: (1) Test under different developing agents and different gradients: Tests were conducted on thin films with different developing agents and different developing system conditions, as follows: Toluene-ethyl acetate-methanol-water (2:4:2.1) Cyclohexane-ethyl acetate-methanol-water (2:4:2.1) Toluene-ethyl acetate-methanol-water (4:4:2.1), the results showed that , , Good separation, no stratification of spots, but The shift value is better, therefore it is chosen. The optimal conditions were as follows. Under the toluene-ethyl acetate-methanol-water (2:4:2.1) system, the Zhuang medicine Luoqian Yubabu sample, Qingfengteng reference material, Fenfangji reference material, and Qingteng alkaloid reference and Fenfangji alkaloid all showed the same color spots A and B at the corresponding positions, indicating good separation and good system performance. (2) Investigation of different temperatures and humidity: The temperature and humidity conditions of the thin-layer developing solvent were investigated in the experiment. Under the selected conditions of toluene-ethyl acetate-methanol-water (2:4:2.1), the separation effect was good under the conditions of 4-10℃, room temperature of 30℃, and relative humidity of 30% and 70%.

[0074] 2.2 Thin-layer chromatography identification of blood from the palm of the dragon

[0075] 2.2.1 Preparation of test solution: Same as the test solution preparation method under item "2.1.1" for thin-layer identification of Sinomenium acutum.

[0076] 2.2.2 Preparation of Flying Dragon Palm Blood Medicinal Material Solution: Take 0.3g of Flying Dragon Palm Blood reference material and prepare it according to the conditions in “2.1.1”.

[0077] 2.2.3 Preparation of reference solution: Take an appropriate amount of fentanyl zeylanin reference standard and dilute it with methanol to a concentration of 0.096 mg / mL to obtain the fentanyl zeylanin reference solution.

[0078] 2.2.4 Preparation of negative control solution: Weigh all medicinal materials except for the blood of the dragon's palm, prepare according to the prescription process, and then prepare according to the conditions in "2.1.1" to obtain the negative control solution.

[0079] 2.2.5 Specificity Examination: Thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was performed. 8 µL of the test solution, 6 µL of the reference medicinal material, 3 µL of the reference standard, and 8 µL of the negative control solution were applied separately to the same silica gel G thin-layer plate. The plate was incubated at 10°C for 12 h with toluene-ethyl acetate-methanol-water (2:4:2:1). The upper layer was used as the developing solvent and developed in a developing tank pre-saturated with concentrated ammonia solution for 20 min. The plate was then removed, air-dried, and examined under UV light at 365 nm. The results showed... Figure 2 .

[0080] 2.2.6 Determination results: At the corresponding positions of the spots in the chromatogram, the test sample, the reference herb *Pteris vittata*, and the reference standard *Zanthoxylum nitidum* showed the same blue fluorescent spot A, and the separation was good. The negative control showed no interference, indicating that the method has good specificity.

[0081] 2.2.7 Durability test: (1) Test under different developing agents and different gradients: The test conditions were as described in section “2.1.7.1”. The results show that: Good separation, with no stratification of spots. , The spots were not separated, causing interference; therefore, [they were selected]. The optimal conditions were as follows. The Zhuang medicine Luoqianyuba plaster sample, the Feilongzhang blood reference material and the Maoliangmianzhen reference standard all showed the same blue fluorescent spot A at the corresponding positions, indicating good separation and that the system was good. (2) Investigation of different temperatures and humidity: The temperature and humidity conditions of the thin-layer developing solvent were investigated in the experiment. The selected conditions of toluene-ethyl acetate-methanol-water (2:4:2.1) were investigated at 4-10℃ and 30℃, and at relative humidity of 30% and 70%, and the separation effect was good in all cases.

[0082] 2.3 Thin-layer chromatographic identification of Polygonum cuspidatum

[0083] 2.3.1 Preparation of test solution: Weigh 5g of Zhuang medicine Luoqianyu poultice, add 30ml of methanol and sonicate for 1h, filter, evaporate to dryness, make up to 5ml with methanol, and centrifuge to obtain the test solution.

[0084] 2.3.2 Preparation of Polygonum cuspidatum medicinal material solution: Take 0.2g of Polygonum cuspidatum reference medicinal material and prepare it in the same way as the test sample solution to obtain the Polygonum cuspidatum reference medicinal material solution.

[0085] 2.3.3 Preparation of reference solution: Take an appropriate amount of polygalactoside reference standard and dilute it with methanol to a concentration of 0.035 mg / mL to obtain the polygalactoside reference solution.

[0086] 2.3.4 Preparation of negative control solution: Weigh the remaining medicinal materials except for Polygonum cuspidatum, prepare them according to the prescription process, and then prepare them according to the conditions in "2.3.1" to obtain the negative control solution.

[0087] 2.3.5 Specificity Examination: Following thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), apply 4 µL of the test solution, 2 µL of the reference medicinal material, 1.5 µL of the reference standard, and 4 µL of the negative control solution to the same high-performance silica gel G thin-layer plate. Use chloroform-methanol-water (15:5:0.5) as the developing solvent, pre-saturate for 20 min, develop, remove, air dry, and examine under UV light at 365 nm. See... Figure 3 .

[0088] 2.3.6 Determination results: At the corresponding positions of the spots on the chromatogram, the test sample, Polygonum cuspidatum reference material and Polygonum cuspidatum glycoside reference standard showed the same blue fluorescent spot A, and the separation was good and the negative control standard had no interference, indicating that the method has good specificity.

[0089] 2.3.7 Durability test: (1) Test under different developing agents and different gradients: Tests were conducted on thin films with different developing agents and different developing system conditions, as follows: chloroform-methanol-water (15:5:0.5) Ethyl acetate-methanol-water (15:5:0.5) The results showed that chloroform-methanol-water (15:10:0.5) was used. Good separation, good specific shift value, and no stratification of spots. , Poor separation; therefore, [the following was chosen] The optimal conditions were as follows. Under the chloroform-methanol-water (15:5:0.5) system, the Zhuang medicine Luoqian Yuba plaster sample, the Polygonum cuspidatum reference material and the Polygonum cuspidatum glycoside reference standard all showed the same blue fluorescent spot A at the corresponding positions, indicating good separation and that the system was good. (2) Investigation of different temperatures and humidity: The temperature and humidity conditions of the thin-layer developing solvent were investigated in the experiment. The selected conditions of chloroform-methanol-water (15:5:0.5) were investigated at 4-10℃ and 30℃, and at relative humidity of 30% and 70%, and the separation effect was good in all cases.

[0090] 2.4 Thin-layer chromatography identification of dragon's blood

[0091] 2.4.1 Preparation of test solution: Weigh 5g of Zhuang medicine Luoqianyu poultice, add 30ml of methanol and sonicate for 1h, filter, evaporate to dryness, make up to 5ml with methanol, centrifuge to obtain the solution.

[0092] 2.4.2 Preparation of Dragon's Blood Herbal Solution: Take 0.5g of Dragon's Blood Herbal Material and dissolve it in methanol to obtain the solution.

[0093] 2.4.3 Preparation of negative control solution: Weigh all the medicinal materials except for dragon's blood, prepare them according to the prescription process, and then prepare them according to the conditions in "2.4.1" to obtain the negative control solution.

[0094] 2.4.4 Specificity Examination: Thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was performed. 8 µL of the test solution, 3 µL of the reference herb, and 8 µL of the negative control solution were spotted separately onto the same high-performance silica gel G thin-layer plate. A chloroform-methanol-formic acid (19:1:0.1) solution was used as the developing solvent. The plate was pre-saturated for 20 min, developed, removed, and air-dried. The plate was then sprayed with 5% vanillin-sulfuric acid ethanol solution and heated at 105℃ until the spots were clearly visible. Figure 4 .

[0095] 2.4.5 Determination results: At the corresponding positions of the spots in the chromatogram, the test sample and the reference material of dragon's blood showed the same color spot A, and the separation was good. The negative control showed no interference, indicating that the method has good specificity.

[0096] 2.4.6 Durability Test

[0097] 2.4.6.1 Investigation with Different Developing Solvents and Gradients: The thin-layer chromatography (TLC) was investigated using different developing solvents and different development system conditions as follows: ① chloroform-methanol-formic acid (19:1:0.1) ② ethyl acetate-methanol-formic acid (19:1:0.1) ③ chloroform-methanol-formic acid (19:2:0.1). The results showed that ① and ③ had good separation, but ① had better specific migration values ​​and no spot stratification, while ② had poorer separation. Therefore, ① was selected as the optimal condition. In the chloroform-methanol-formic acid (19:1:0.1) system, both the Zhuang medicine Luoqianyu poultice sample and the dragon's blood medicinal material showed the same color spots, indicating good separation and a good system performance.

[0098] 2.4.6.2 Investigation under different temperatures and humidity: The temperature and humidity conditions of the thin-layer developing solvent were investigated in the experiment. The selected condition of chloroform-methanol-formic acid (19:1:0.1) was investigated under the conditions of 4-10℃, room temperature of 30℃, and relative humidity of 30% and 70%. The separation effect was good in all cases.

[0099] 2.5 Thin-layer chromatography identification of frankincense

[0100] 2.5.1 Preparation of test solution: Same as the test solution preparation method under item "2.1.1" for thin-layer identification of Sinomenium acutum.

[0101] 2.5.2 Preparation of frankincense reference solution: Take 0.2g of frankincense and prepare it in the same way as the test sample to obtain the frankincense reference solution.

[0102] 2.5.3 Preparation of negative control solution: Weigh all medicinal materials except frankincense, prepare according to the prescription process, and then prepare according to the conditions in "2.1.1" to obtain the negative control solution.

[0103] 2.5.4 Specificity Examination: Thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was performed. 6 µL of the test solution, 1 µL of the reference herb, and 6 µL of the negative control solution were spotted separately onto the same silica gel G thin-layer plate. Cyclohexane-ethyl acetate-formic acid (9:1.5:0.1) was used as the developing solvent. The plate was pre-saturated for 20 min, developed, removed, and air-dried. The plate was then sprayed with 5% vanillin-sulfuric acid ethanol solution and heated at 105℃ until the spots were clearly visible. Figure 5 .

[0104] 2.5.5 Determination results: At the corresponding positions of the spots on the chromatogram, the test sample of Zhuang medicine Luoqianyubabu and the reference material of frankincense showed the same color spot A, and the separation was good. The negative control showed no interference, indicating that the method has good specificity.

[0105] 2.5.6 Durability Test: (1) Tests with different developing solvents and gradients: The thin-layer chromatography was tested with different developing solvents and different developing system conditions as follows: ① Cyclohexane-ethyl acetate-formic acid (9:1.5:0.1) ② Benzene-ethyl acetate-formic acid (9:1.5:0.1) ③ Cyclohexane-ethyl acetate-formic acid (9:3:0.1). The results showed that ①, ②, and ③ had good separation, but ① had the best ratio shift value, so ① was selected as the best condition. In cyclohexane-ethyl acetate-formic acid (9:1.5:0.1), the Zhuang medicine Luoqianyu poultice sample and the frankincense reference material both showed the same color spot A at the corresponding positions, indicating good separation and that the system was good. (2) Investigation of different temperatures and humidity: The temperature and humidity conditions of the thin-layer developing solvent were investigated in the experiment. The selected conditions of cyclohexane-ethyl acetate-formic acid (9:1.5:0.1) were investigated at 4-10℃ and 30℃ at room temperature and 30% and 70% relative humidity. The separation effect was good in all cases.

[0106] 2.6 Thin-layer chromatography identification of myrrh

[0107] 2.6.1 Preparation of test solution: Same as the test solution preparation method under item "2.1.1" for thin-layer identification of Sinomenium acutum.

[0108] 2.6.2 Preparation of myrrh reference solution: Take about 0.3g of myrrh and prepare it in the same way as the test sample to obtain the myrrh reference solution.

[0109] 2.6.3 Preparation of negative control solution: Weigh all medicinal materials except myrrh, prepare according to the prescription process, and then prepare according to the conditions in "2.1.1" to obtain the negative control solution.

[0110] 2.6.4 Specificity Examination: Following thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 16 µL of the test solution, 1 µL of the reference herb, and 16 µL of the negative control solution were spotted separately onto the same high-performance silica gel G thin-layer plate. Cyclohexane-chloroform-ethyl acetate (8:1:1) was used as the developing solvent, pre-saturated for 20 min, developed, removed, and air-dried. The plate was then sprayed with 5% vanillin-sulfuric acid ethanol solution and heated at 105℃ until the spots were clearly visible. Figure 6 .

[0111] 2.6.5 Determination results: At the corresponding positions of the spots on the chromatogram, the test sample of Zhuang medicine Luoqianyubabu and the reference herb showed the same color spot A, and the separation was good. The negative control showed no interference, indicating that the method has good specificity.

[0112] 2.6.6 Durability test: (1) Tests with different developing solvents and gradients: The thin-layer chromatography was tested with different developing solvents and different developing system conditions as follows: ① Cyclohexane-chloroform-ethyl acetate (8:1:1) ② Benzene-chloroform-ethyl acetate (8:1:1) ③ Cyclohexane-chloroform-ethyl acetate (8:1:2). The results showed that ①, ②, and ③ had good separation, but ① had the best specific shift value, so ① was selected as the best condition. In cyclohexane-chloroform-ethyl acetate (8:1:1), the Zhuang medicine Luoqianyu poultice sample and myrrh control material showed the same color spots at the corresponding positions, indicating good separation, which shows that the system is good. (2) Investigation of different temperatures and humidity: The temperature and humidity conditions of the thin-layer developing solvent were investigated in the experiment. The selected conditions of cyclohexane-chloroform-ethyl acetate (8:1:1) were investigated at 4-10℃ and 30℃ at room temperature and 30% and 70% relative humidity. The separation effect was good in all cases.

[0113] 3. Determination of the content of multiple components in Zhuang medicine Luoqianyu poultice

[0114] 3.1 Determination of the content of quinine, sesquiterpenoid, and tetrandrine

[0115] 3.1.1 Investigation of the preparation method of the test solution: The solvent volume, ultrasonic time and ultrasonic times were investigated by experiments, and the solvent volume was determined to be 25 mL, the ultrasonic time was 1 h and the ultrasonic times were 2.

[0116] 3.1.2 Solution Preparation

[0117] 3.1.2.1 Preparation of test solution: Accurately weigh about 5g of Zhuang medicine Luoqianyubabu powder and place it in a stoppered Erlenmeyer flask. Accurately pipette 25mL of chloroform, sonicate for 1h twice, combine the filtrates, place them in an evaporating dish to evaporate to dryness, dilute to 5mL with methanol, shake well, centrifuge, and filter through a microporous membrane to obtain the test solution.

[0118] 3.1.2.2 Preparation of the mixed reference solution: Accurately weigh appropriate amounts of sinomenine, zeaxanthin, tetrandrine, and reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 2.330 mg / mL, 2.140 mg / mL, and 1.668 mg / mL, respectively. Separately, take appropriate amounts of the above sinomenine, zeaxanthin, and tetrandrine solutions, place them in the same volumetric flask, and dilute with methanol to prepare a mixed alkaloid reference solution containing sinomenine, zeaxanthin, and tetrandrine concentrations of 0.240 mg / mL, 0.344 mg / mL, and 0.096 mg / mL, respectively.

[0119] 3.1.2.3 Preparation of negative control solutions: Weigh out the medicinal materials of Zhuang medicine Luoqianyubabuji except for Qingfengteng, Feilongzhangxue, and Fenfangji according to the prescription ratio, and prepare them according to the method in section "3.1.2.1" to make negative sample solutions for Qingfengteng deficiency, Feilongzhangxue deficiency, and Fenfangji deficiency respectively.

[0120] 3.1.3 Chromatographic conditions and system suitability test: A Waters BEH C18 (2.1×100 mm 1.7µm) column was used; the mobile phase was acetonitrile (B)-0.1 mmol / L ammonium acetate solution (D), gradient elution is shown in Table 2, flow rate: 0.2 mL / min; detection wavelength: 280 nm; column temperature: 40℃; injection volume: 3 µL.

[0121] Under these chromatographic conditions, reference solutions and sample solutions were injected and analyzed using DAD. The results showed that the retention times and UV spectra of the chromatographic peaks of sinomenine, buergerianin, and tetrandrine were consistent with those of the corresponding peaks in the samples. The peak resolution was good, and other components in the samples did not interfere with the determination of these three indicators. The theoretical plate number of the chromatographic column, calculated based on sinomenine, was not less than 2500. The UPLC chromatograms of the test solution, the mixed solution of reference solution, and the negative control solution of the Zhuang medicine Luoqianyu poultice are shown below. Figures 7-8 .

[0122]

[0123] 3.1.4 Methodological Examination

[0124] 3.1.4.1 Linearity Range Study: Accurately weigh appropriate amounts of sinomenine, zeaxanthin, and tetrandrine reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 2.330 mg / L, 2.140 mg / mL, and 1.668 mg / mL, respectively. Separately, take appropriate amounts of the above sinomenine, zeaxanthin, and tetrandrine solutions, place them in the same volumetric flask, and dilute with methanol to prepare a mixed alkaloid reference solution containing sinomenine, zeaxanthin, and tetrandrine concentrations of 0.240 mg / mL, 0.344 mg / mL, and 0.096 mg / mL, respectively. Using an autosampler, precisely sample 0.3, 1, 2, 3, 4, 5, and 6 µL respectively, and determine the chromatographic conditions under section "3.1.3". Record the peak area of ​​each index component. Plot the peak area (Y) as the ordinate and the injection volume (µg) (X) as the abscissa. The linear regression equations for quinine are: Y = 2897.2x + 24.368 r = 0.9999; quinquefoliin Y = 2284.3x + 35.920 r = 0.9999; and tetrandrine Y = 2263.6x + 1.096r=0.9999; The results show that the peak areas of sinomenine, styrax tinctoria, and tetrandrine have a good linear relationship with the injection amount (µg) in the ranges of 0.072µg~1.144µg, 0.1032µg~2.0064µg, and 0.0288µg~0.576µg, respectively (n=7).

[0125] 3.1.4.2 Precision test: Accurately pipette 3µL of the mixed reference solution under “3.1.2.2” and inject it into the ultra-high performance liquid chromatograph. Measure the chromatographic conditions under “3.1.3”. After six consecutive injections, the RSDs of sinomenine, styraxanthine, and tetrandrine were 0.2%, 0.5%, and 0.1%, respectively, all less than 3%, indicating good instrument precision.

[0126] 3.1.4.3 Stability Test: Accurately weigh 5g of Zhuang medicine Luoqianyu Babuji (batch 20210119), prepare the test solution according to the method in section “3.1.2.1”, and determine the chromatographic conditions in section “3.1.3”. The RSDs of sinomenine, buergerianin and tetrandrine were 2.3%, 1.9% and 1.7% respectively, all less than 3%, indicating that the sample solution was stable within 24 hours.

[0127] 3.1.4.4 Repeatability Test: Accurately weigh 5g of Zhuang medicine Luoqianyu poultice (batch 20210119), prepare the test solution according to the method in section “3.1.2.1”, determine the content according to the chromatographic conditions in section “3.1.3”, record the corresponding peak area, and calculate the corresponding content. The results show that the average contents of sinomenine, sesquiterpenoid, and tetrandrine are 0.1408mg / g, 0.2058mg / g, and 0.0668mg / g, respectively, with RSDs of 2.8%, 2.7%, and 2.1%, respectively, indicating that the method has good repeatability.

[0128] 3.1.4.5 Spiking Recovery Test: Approximately 2.5g of Zhuang medicine Luoqianyu poultice (batch number 20210119) (containing 0.1408mg / g, 0.2058mg / g, and 0.0668mg / g of sinomenine, zephyranthesin, and tetrandrine, respectively) was accurately weighed in 9 portions. Based on a 1:0.5 ratio of the original sample amount to the reference standard, 0.1761mg of sinomenine, 0.2574mg of zephyranthesin, and 0.0668mg / g of tetrandrine were accurately taken from the reference standard stock solution. Prepare three aliquots of reference solutions containing 0.0836 mg of tetrandrine; precisely prepare three aliquots of reference solutions containing 0.3522 mg of sinomenine, 0.5147 mg of sesquiterpenoid, and 0.1671 mg of tetrandrine stock solutions, respectively, at a 1:1 ratio of the original sample amount to the reference standard; precisely prepare three aliquots of reference solutions containing 0.5279 mg of sinomenine, 0.7742 mg of sesquiterpenoid, and 0.2509 mg of tetrandrine stock solutions, respectively, at a 1:1.5 ratio of the original sample amount to the reference standard; place each aliquot in a stoppered triangular container, precisely add 25 mL of chloroform, sonicate for 1 hour twice, combine the filtrates, evaporate to dryness, dilute to 5 mL with methanol, shake well, and filter through a microporous membrane to obtain the final product. The chromatographic conditions were followed as described in "3.1.3". The peak areas were measured, and the recoveries of each component were calculated. The results showed that the recoveries of sinomenine, buergerianin, and tetrandrine ranged from 95.2% to 102.6%, 95.7% to 104.1%, and 95.2% to 104.9%, respectively, with average recoveries of 97.8%, 99.7%, and 98.6%, and RSDs of 2.7%, 2.5%, and 3.8%, respectively. The recoveries were all within the range of 95% to 105%, which meets the relevant pharmacopoeia requirements, indicating that the method is accurate.

[0129] 3.2 Determination of the content of chlorogenic acid, polygalactoside, and dracoside B

[0130] 3.2.1 Investigation of the preparation method of the test solution: The solvent volume, ultrasonic time and ultrasonic times were investigated first. The solvent volume was determined to be 25 mL, the ultrasonic time to be 1.5 h and the ultrasonic times to be 2.

[0131] 3.2.2 Solution Preparation

[0132] 3.2.2.1 Preparation of test solution: Accurately weigh about 5g of Zhuang medicine Luoqianyubabuji and place it in a stoppered Erlenmeyer flask. Accurately add 25mL of methanol using a pipette, sonicate for 1.5h twice, combine the solutions and evaporate to dryness. Make up the volume with methanol to 25mL, shake well, centrifuge, and filter through a microporous membrane to obtain the test solution.

[0133] 3.2.2.2 Preparation of the mixed reference solution: Accurately weigh appropriate amounts of polygalactoside, chlorogenic acid, and dracosanol B reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 0.808 mg / mL, 0.990 mg / mL, and 0.866 mg / mL, respectively. Separately, take appropriate amounts of the above polygalactoside, chlorogenic acid, and dracosanol B solutions, place them in the same volumetric flask, and dilute with methanol to prepare a mixed reference solution containing polygalactoside, chlorogenic acid, and dracosanol B concentrations of 0.035 mg / mL, 0.139 mg / mL, and 0.106 mg / mL, respectively.

[0134] 3.2.2.3 Preparation of negative control solutions: Weigh out the medicinal materials of Zhuang medicine Luoqianyubabuji except for Sanjiapi, Huzhang, and Longxuejie according to the prescription ratio, and prepare them according to the method in section "3.2.2.1" to make negative sample solutions for Sanjiapi deficiency, Huzhang deficiency, and Longxuejie deficiency, respectively.

[0135] 3.2.3 Chromatographic conditions and system suitability test: A Waters HSST3 (2.1 × 100 mm 1.8 µm) column was used; the mobile phase was acetonitrile (B) - 0.1% acetic acid aqueous solution (D), gradient elution is shown in Table 3, flow rate: 0.3 mL / min; detection wavelength: 310 nm and 280 nm; column temperature: 25 ℃; injection volume: 2 µL.

[0136] Under these chromatographic conditions, the reference solution and sample solution were injected and analyzed, respectively. The retention times and UV spectra of the chromatographic peaks of polygalactosidase, chlorogenic acid, and dracosanol B were consistent with those of the corresponding peaks in the sample. The peak resolution was good, and there was no interference in the determination of the indicator components. The theoretical plate number of the chromatographic column, calculated based on chlorogenic acid, was not less than 2500. The UPLC chromatograms of the test solution, mixed reference solution, and negative control solution of the Zhuang medicine Luoqianyu poultice are shown below. Figures 9-15 .

[0137]

[0138] 3.2.4 Methodological Examination

[0139] 3.2.4.1 Linearity Range Study: Accurately weigh appropriate amounts of polygalactoside, chlorogenic acid, and dracosanol B reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 0.808 mg / mL, 0.990 mg / mL, and 0.866 mg / mL, respectively. Separately, take appropriate amounts of the above polygalactoside, chlorogenic acid, and dracosanol B solutions, place them in the same volumetric flask, and dilute with methanol to prepare a mixed reference solution containing polygalactoside, chlorogenic acid, and dracosanol B concentrations of 0.035 mg / mL, 0.139 mg / mL, and 0.106 mg / mL, respectively. Using an autosampler, precisely sample 0.2, 0.5, 1, 2, 3, 4, and 5 µL respectively, and determine the chromatographic conditions under section "3.2.3". Record the peak area of ​​each index component. Plot the peak area (Y) as the ordinate and the injection volume (µg) (X) as the abscissa to obtain the linear regression equations for chlorogenic acid: Y = 7470.4x + 2.576, r = 0.9999; for polygalactoside: Y = 12672x - 9.877, r = 0.9999; and for dracosan (BY = 10439x - 9.479). r=0.9999, the results showed that chlorogenic acid, polygalactosidase, and dracosanol B had a good linear relationship with the injection amount (µg) in the ranges of 0.007µg~0.175µg, 0.0258µg~0.645µg, and 0.0318µg~0.636µg, respectively (n=7).

[0140] 3.2.4.2 Precision test: Accurately pipette 2µL of the mixed reference solution under section “3.2.2.2” and inject it into the ultra-high performance liquid chromatograph. Measure according to the chromatographic conditions in section “3.2.3”. Inject 6 times consecutively. The results show that the RSD values ​​of the peak areas of chlorogenic acid, polygalactoside, and dracosanol B are 0.4%, 0.1%, and 0.1%, respectively. The RSD values ​​are all less than 3%, indicating that the instrument has good precision.

[0141] 3.2.4.3 Stability Test: Accurately weigh 5g of Zhuang medicine Luoqianyu poultice (batch 20210119), prepare the test solution according to the method in section “3.2.2.1”, and determine the chromatographic conditions according to “3.2.3”. The results showed that the RSD values ​​of the peak areas of chlorogenic acid, polygalactoside, and dracoside B were 0.9%, 1.0%, and 1.9%, respectively, and the RSD values ​​were all less than 3%, indicating that the sample solution was stable within 24 hours.

[0142] 3.2.4.4 Repeatability Test: Accurately weigh 5g of Zhuang medicine Luoqianyu poultice (batch 20210119), prepare the test solution according to the method in section “3.2.2.1”, determine the content according to the chromatographic conditions in section “3.2.3”, record the corresponding peak area, and calculate the corresponding content. The results show that the average contents of chlorogenic acid, polygalactoside, and dracosanol B are 0.2045mg / g, 0.6907mg / g, and 0.1731mg / g, respectively, with RSDs of 1.2%, 0.8%, and 0.9%, respectively. The RSD values ​​are all less than 3%, indicating that the method has good repeatability.

[0143] 3.2.4.5 Spiking Recovery Test: Take approximately 2.5g of Zhuang medicine Luoqianyu poultice (batch 20210119) (chlorogenic acid, polygalactoside, and dracoside B, with contents of 0.2045mg / g, 0.6907mg / g, and 0.4567 mg / g, respectively), in 9 portions. Accurately weigh each portion. Precisely take 3 portions each of the reference solution containing 0.2556mg chlorogenic acid, 0.8635mg polygalactoside, and 0.5709mg dracoside B, respectively, at a 1:0.5 ratio of the original sample amount to the reference standard. Precisely take 3 portions each of the reference standard stock solution containing 0.5113mg chlorogenic acid, 1.7269mg polygalactoside, and dracoside B, respectively, at a 1:1 ratio of the original sample amount to the reference standard. Three aliquots of the reference solution containing 1.1418 mg were prepared. Three aliquots of the reference solution containing 0.7669 mg chlorogenic acid, 2.5904 mg polydipsia glycoside, and 1.7127 mg dracosanol B were accurately taken from the original sample and reference solution at a ratio of 1:1.5. Each aliquot was placed in a stoppered triangular container, and 25 mL of methanol was accurately added. The mixture was sonicated for 1.5 h twice. The filtrate was evaporated to dryness, and the volume was adjusted to 25 mL with methanol. The mixture was shaken well, centrifuged, and filtered through a microporous membrane. The chromatographic conditions were as described in "3.2.3". The peak areas were measured, and the recoveries of each component were calculated. The recoveries of chlorogenic acid, polydipsia glycoside, and dracosanol B ranged from 92.2% to 101.0%, 92.1% to 102.1%, and 95.4% to 103.3%, respectively, all within the range of 92% to 105%, which meets the relevant pharmacopoeia requirements.

[0144] 3.3 Sample Determination

[0145] Take two portions (approximately 5g) of each from 10 batches of Zhuang medicine Luoqianyu poultice, with batch numbers (20210119, 2021010901, 2021010902, 2021010903, 2021010904, 2021010905, 2021010906, 2021010907, 2021010908, 2021010909) and prepare test solutions according to the methods described in sections “3.1.2.1” and “3.2.2.1”. Then proceed to section “3.1.3”. For the determination of chromatographic conditions under item “3.2.3”, the test sample and the mixed reference solution were accurately pipetted into the ultra-high performance liquid chromatograph, and their peak areas were measured. The contents of each component were calculated. The average contents of sinomenine, styracin, tetrandrine, chlorogenic acid, styracin, and dracoside B in the sample are shown in Table 4.

[0146]

Claims

1. A quality testing method for a traditional Chinese medicine plaster containing *Luoqianyu*, characterized in that... The method includes methods for determining the content of sinomenine, taurenzin, and tetrandrine, and methods for determining the content of chlorogenic acid, polygalactosidase, and dracosanol B; the method for determining the content of sinomenine, taurenzin, and tetrandrine includes the following steps: (1) Preparation of test solution: Accurately weigh 5g of Zhuang medicine Luoqianyu Babuji, place it in a stoppered triangular flask, accurately take 25mL of chloroform with a pipette, sonicate for 1h, twice, combine the filtrates, place them in an evaporating dish to evaporate to dryness, make up to 5mL with methanol, shake well, centrifuge, and filter through a microporous membrane to obtain the test solution. (2) Preparation of reference standard mixed solution: Accurately weigh appropriate amounts of sinomenine, styrax chinensis, and tetrandrine reference standards, dissolve them in methanol, and prepare reference standard stock solutions with concentrations of 2.330 mg / mL, 2.140 mg / mL, and 1.668 mg / mL, respectively; separately take appropriate amounts of the above sinomenine, styrax chinensis, and tetrandrine solutions, place them in the same volumetric flask, dilute with methanol, and prepare alkaloid reference standard mixed solution containing sinomenine, styrax chinensis, and tetrandrine concentrations of 0.240 mg / mL, 0.344 mg / mL, and 0.096 mg / mL, respectively. (3) Preparation of negative control solution: Weigh out the other medicinal materials except for Qingfengteng, Feilongzhangxue and Fenfangji according to the prescription ratio to make Zhuang medicine Luoqianyu Babuji. Prepare negative sample solutions for Qingfengteng deficiency, Feilongzhangxue deficiency and Fenfangji deficiency according to the method under (1) Preparation of test solution. (4) Chromatographic conditions and system suitability test: Waters BEH C18, 2.1×100 mm 1.7µm column was used; the mobile phase was acetonitrile-0.1 mmol / L ammonium acetate solution, the gradient elution conditions are shown in the table below, the flow rate was 0.2 mL / min; the detection wavelength was 280 nm; Column temperature: 40℃; Injection volume: 3 µL; (5) Determination: Under the above chromatographic conditions, the mixed solution of reference standard, the negative control solution, and the sample test solution were injected into the ultra-high performance liquid chromatograph for analysis and determination. The DAD detector was used for detection. The results showed that the chromatographic peak retention time and UV spectrum of sinomenine, styraxanthine, and tetrandrine were consistent with the corresponding chromatographic peak retention time and UV spectrum in the sample. The peak separation was good. Other components in the sample did not interfere with the determination of these three index components. The theoretical plate number of the chromatographic column was not less than 2500 based on sinomenine. The content of each component was calculated. The medicinal material prescription for the Zhuang medicine Luoqianyu Babuji is as follows (by weight): 8.55 parts of *Houttuynia cordata*, 4.27 parts of *Sinomenium acutum*, 4.27 parts of *Stephania tetrandra*, 3.85 parts of *Lycopodium clavatum*, 4.27 parts of *Polygonum cuspidatum*, 2.14 parts of *Pterocarya stenoptera*, 2.14 parts of *Erythrina variegata*, 3.42 parts of *Saururus chinensis*, 3.42 parts of *Acanthopanax senticosus*, 3.42 parts of *Smilax china*, 3.42 parts of *Smilax china*, 3.42 parts of *Smilax china*, 2.14 parts of *Acer buergerianum*, 2.14 parts of *Cinnamomum camphora*, 2.14 parts of *Dalbergia odorifera*, 2.14 parts of *Piper kadsura*, 1.28 parts of *Euonymus fortunei*, 0.85 parts of frankincense, 0.85 parts of myrrh, and 1.28 parts of *Dracaena cochinchinensis*.

2. The quality testing method for the Zhuang medicine Luoqianyu poultice according to claim 1, characterized in that, The method for determining the content of chlorogenic acid, polygalactoside, and dracoside B includes the following steps: (1) Preparation of test solution: Accurately weigh 5g of Zhuang medicine Luoqianyu Babuji, place it in a stoppered triangular flask, accurately take 25mL of methanol with a pipette, sonicate for 1.5h twice, combine and evaporate to dryness, make up to 25mL with methanol, shake well, centrifuge, filter through a microporous membrane to obtain the test solution; (2) Preparation of reference standard mixed solution: Accurately weigh appropriate amounts of styrax glycoside, chlorogenic acid and dracosanol B reference standards, dissolve them in methanol, and prepare reference standard stock solutions with concentrations of 0.808 mg / mL, 0.990 mg / mL and 0.866 mg / mL respectively; Take appropriate amounts of the above solutions of polygalactoside, chlorogenic acid, and dracoside B, place them in the same volumetric flask, dilute with methanol, and prepare a mixed solution of reference standards containing 0.035 mg / mL, 0.139 mg / mL, and 0.106 mg / mL of polygalactoside, chlorogenic acid, and dracoside B, respectively. (3) Preparation of negative control solution: Weigh out the other medicinal materials except for Acanthopanax senticosus, Polygonum cuspidatum and Dragon's Blood according to the prescription ratio to make Zhuang medicine Luoqianyu Babuji. Prepare negative sample solution lacking Acanthopanax senticosus, negative sample solution lacking Polygonum cuspidatum and negative sample solution lacking Dragon's Blood according to the method under (1) Preparation of test solution. (4) Chromatographic conditions and system suitability test: A Waters HSST3 2.1 × 100 mm 1.8 µm column was used; the mobile phase was acetonitrile-0.1% acetic acid aqueous solution, and the gradient elution conditions are shown in the table below. The flow rate was 0.3 mL / min; the detection wavelengths were 310 nm and 280 nm; the column temperature was 25 ℃; and the injection volume was 2 µL. (5) Determination: Under these chromatographic conditions, the mixed solution of reference standard, the negative control solution, and the sample test solution were injected into the ultra-high performance liquid chromatograph for analysis. The results showed that the retention times and UV spectra of the chromatographic peaks of styrax glycoside, chlorogenic acid, and dragon's blood B were consistent with the retention times and UV spectra of the corresponding chromatographic peaks in the sample. The resolution of each peak was good, and there was no interference in the determination of the index components. The theoretical plate number of the chromatographic column, calculated based on chlorogenic acid, was not less than 2500. The content of each component was calculated.

3. The quality testing method for the Zhuang medicine Luoqianyu poultice according to claim 2, characterized in that, The quality testing method also includes using thin-layer chromatography to identify Qingfengteng, Fenfangji, Huzhang, and Feilongzhangxue in the Zhuang medicine Luoqianyu Babuji, as detailed below: (1) Thin-layer chromatographic identification of Qingfengteng and Fenfangji Preparation of test solution: Take 5g of Zhuang medicine Luoqianyu poultice, sonicate with 20ml of methanol for 1h, filter, take 10ml and evaporate to dryness, make up to 5ml with 50% ethanol, centrifuge, extract with 20ml of chloroform, evaporate to dryness, and make up to 2ml with methanol to obtain the test solution; Separately, take 0.5g of Qingfengteng and 0.2g of Fenfangji reference materials and prepare Qingfengteng and Fenfangji reference material solutions in the same way; Preparation of reference solution: Take appropriate amounts of Qingteng alkaloid and Fenfangji alkaloid reference standards, and make up to 0.240mg / mL and 0.344mg / mL of Qingteng alkaloid and Fenfangji alkaloid reference standard solutions with methanol to obtain reference standard solutions with concentrations of 0.240mg / mL and 0.344mg / mL, respectively; Preparation of negative control solution: Weigh the remaining medicinal materials except Qingfengteng and Fenfangji alkaloid, prepare according to the prescription process, and then prepare according to the above test solution preparation conditions to obtain the negative control solution; According to the thin-layer chromatography method, 20 µL of the test solution, 4 µL of the *Sinomenium acutum* and *Stephania tetrandra* reference medicinal material solutions, 1.6 µL of the reference solution, and 27 µL of the negative control solution were spotted separately onto the same silica gel G thin-layer plate. A toluene-ethyl acetate-methanol-water mixture with a volume ratio of 2:4:2:1 was placed at 10 °C for 12 h. The upper layer was used as the developing solvent and developed in a developing tank pre-saturated with concentrated ammonia solution for 20 min. The plate was then removed, dried, and sprayed with freshly prepared bismuth potassium iodide solution. At the corresponding positions of the spots on the chromatogram, the test sample, *Sinomenium acutum* reference medicinal material, *Stephania tetrandra* reference medicinal material, sinomenine reference standard, and stephania tetrandra alkaloid showed spots of the same color, and the negative control did not interfere. (2) Thin-layer identification of blood from the palm of the dragon Preparation of test solution: Prepare the test solution using the same method as described above for the thin-layer identification of *Sinomenium acutum* and *Stephania tetrandra*. Separately, take 0.3g of *Pteris vittata* (a type of herb) as a reference herb and prepare a *Pteris vittata* reference herb solution using the same method. Preparation of reference solution: Take an appropriate amount of *Zanthoxylum nitidum* (a type of herb) reference standard and dilute it to a concentration of 0.096 mg / mL with methanol to prepare a *Stephania tetrandra* (another type of herb) reference standard solution. Preparation of negative control solution: Weigh the remaining herbs except *Pteris vittata*, prepare them according to the prescription process, and then prepare the negative control solution according to the above test solution preparation conditions. According to the thin-layer chromatography method, take 8 µL of the test solution, 6 µL of the reference medicinal material solution, 3 µL of the reference solution, and 8 µL of the negative control solution; spot them separately on the same silica gel G thin-layer plate. Incubate a toluene-ethyl acetate-methanol-water mixture with a volume ratio of 2:4:2:1 at 10℃ for 12 h. Take the upper layer as the developing solvent and develop it in a developing tank pre-saturated with concentrated ammonia solution for 20 min. Remove the plate, air dry it, and examine it under ultraviolet light at 365 nm. At the corresponding positions of the spots on the chromatogram, the test sample, the reference medicinal material of *Pteris vittata*, and the reference standard of *Zanthoxylum nitidum* show the same blue fluorescent spots, and the negative control has no interference. (3) Thin-layer identification of Polygonum cuspidatum Preparation of test solution: Weigh 5g of Zhuang medicine Luoqianyu poultice, add 30ml of methanol, sonicate for 1h, filter, evaporate to dryness, dilute to 5ml with methanol, centrifuge, and obtain the test solution; separately take 0.2g of Polygonum cuspidatum reference material and prepare a Polygonum cuspidatum reference material solution using the same method; Preparation of reference solution: Take an appropriate amount of Polygonum cuspidatum glycoside reference standard, dilute to a concentration of 0.035mg / mL with methanol, and prepare the Polygonum cuspidatum glycoside reference standard solution; Preparation of negative control solution: Weigh the remaining medicinal materials except Polygonum cuspidatum, prepare according to the prescription process, and then prepare according to the preparation conditions of the test solution, and use as the negative control. Solution; according to the thin-layer chromatography method, take 4µL of the test solution, 2µL of the reference medicinal material solution, 1.5µL of the reference solution, and 4µL of the negative control solution; spot them separately on the same high-performance silica gel G thin-layer plate, use chloroform-methanol-water with a volume ratio of 15:5:0.5 as the developing solvent, pre-saturate for 20 min, develop, remove, air dry, and examine under ultraviolet light at 365nm; at the corresponding positions of the spots on the chromatogram, the test sample, Polygonum cuspidatum reference medicinal material, and Polygonum cuspidatum glycoside reference standard show the same blue fluorescent spots, and the negative control standard has no interference.

4. The quality testing method for the Zhuang medicine Luoqianyu poultice according to claim 3, characterized in that, The quality testing method also includes using thin-layer chromatography to identify dragon's blood, frankincense, and myrrh in the Zhuang medicine Luoqianyu poultice, as detailed below: (4) Thin-layer chromatographic identification of dragon's blood Preparation of test solution: Weigh 5g of Zhuang medicine Luoqianyu poultice, add 30ml of methanol, sonicate for 1h, filter, evaporate to dryness, dilute to 5ml with methanol, centrifuge to obtain the test solution; separately take 0.5g of dragon's blood medicinal material, dissolve in methanol to obtain dragon's blood reference medicinal material solution; Preparation of negative control solution: Weigh the remaining medicinal materials except dragon's blood, prepare according to the prescription process, and then prepare according to the test solution preparation conditions to obtain the negative control solution; according to the thin layer chromatography method, take 8µL of test solution, 3µL of reference medicinal material solution, and 8µL of negative control solution; spot them separately on the same high-performance silica gel G thin layer plate, use chloroform-methanol-formic acid with a volume ratio of 19:1:0.1 as the developing solvent, pre-saturate for 20min, develop, remove, air dry, spray with 5% vanillin sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and at the corresponding positions of the spots on the chromatogram, the test sample and the dragon's blood reference medicinal material show the same color spot A, and the negative control has no interference; (5) Thin-layer chromatography identification of frankincense Preparation of test solution: Prepare the test solution using the same method as for the thin-layer identification of *Sinomenium acutum* and *Stephania tetrandra*. Separately, take 0.2 g of frankincense and prepare a frankincense reference solution using the same method. Preparation of negative control solution: Weigh the remaining medicinal materials except frankincense, prepare them according to the prescription process, and then prepare the negative control solution according to the conditions for preparing the test solution. According to thin-layer chromatography, apply 6 µL of the test solution, 1 µL of the reference solution, and 6 µL of the negative control solution to the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-formic acid (v / v) as the developing solvent, pre-saturate for 20 min, develop, remove, air dry, spray with 5% vanillin-sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. At the corresponding positions of the spots on the chromatogram, the test sample and frankincense reference solution show the same color spots, and the negative control solution shows no interference. (6) Thin-layer chromatography identification of myrrh Preparation of test solution: Prepare the test solution using the same method as for the thin-layer identification of *Sinomenium acutum* and *Stephania tetrandra*. Separately, take 0.3 g of myrrh and prepare a myrrh reference solution using the same method. Preparation of negative control solution: Weigh the remaining medicinal materials (excluding myrrh), prepare them according to the prescription process, and then prepare the negative control solution according to the test solution preparation conditions. Using thin-layer chromatography, apply 16 µL of the test solution, 1 µL of the reference solution, and 16 µL of the negative control solution to the same high-performance silica gel G thin-layer plate. Use a cyclohexane-chloroform-ethyl acetate solution (8:1:1 v / v) as the developing solvent, pre-saturate for 20 min, develop, remove, air dry, spray with 5% vanillin-sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. At the corresponding positions of the spots on the chromatogram, the test sample and myrrh reference solution show the same color spots, and the negative control solution shows no interference.

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