A detection method of fushenning capsules

Abstract

The present application relates to a kind of detection methods of Fushenning Capsules, comprising the microscopic identification of the product, the thin layer identification of Anemarrhena, the thin layer identification of Gardenoside, the thin layer identification of Rhubarb, the thin layer identification of Phellodendron, the determination of Berberine Hydrochloride.The present application optimizes the thin layer identification method of Anemarrhena in Fushenning Capsules on the basis of original quality detection method.Through methodology investigation, the detection method is strong in specificity, good in durability, high in accuracy, good in stability, simple and fast in operation, improves the quality detection method of Fushenning Capsules, so that quality is better controlled, and further guarantees the personal safety of test personnel.

Description

Technical Field

[0001] This invention relates to the field of drug testing, specifically to a testing method for Fushenning capsules. Background Technology

[0002] Fushenning Capsules, a product of Guizhou Liangji Pharmaceutical Co., Ltd., function to clear damp-heat, benefit the kidneys and remove blood stasis. It is used for acute and chronic urinary tract infections, acute and chronic cystitis, and acute and chronic pyelonephritis caused by damp-heat accumulation, with symptoms such as frequent urination, urgency, painful urination, and lower back pain.

[0003] The original standard method used Anemarrhena asphodeloides as a reference and benzene-acetone (9:1) as the developing solvent, employing thin-layer chromatography to identify Anemarrhena asphodeloides in this product. The original standard method has the following drawbacks: 1. The processing procedures for the test sample and reference standard are complex, time-consuming, and inefficient. The entire process requires adding the test sample or reference standard to ethanol, heating and refluxing for 40 minutes to dissolve, filtering, adding hydrochloric acid, heating and refluxing for another hour, concentrating, adding water, extracting with benzene, evaporating the extract to dryness, dissolving the residue in benzene, and finally obtaining the test sample or reference standard solution; 2. Benzene was used in both the sample pretreatment process and the developing solvent. Benzene is a highly toxic organic compound, classified as a Group 1 carcinogen by the IARC (International Agency for Research on Cancer), posing a health threat to laboratory personnel.

[0004] To address the aforementioned issues, improve the quality standards of Fushenning Capsules, and protect the health of testing personnel, this invention, based on the original detection method for Fushenning Capsules and incorporating the standards for Anemarrhena asphodeloides in the 2020 edition of the Chinese Pharmacopoeia, conducted in-depth research and investigation to establish a detection method for Fushenning Capsules. This detection method optimizes the thin-layer chromatography identification method for Anemarrhena asphodeloides in Fushenning Capsules, enabling better control of product quality and further ensuring the personal safety of testing personnel. Summary of the Invention

[0005] The purpose of this invention is to provide a detection method for Fushenning capsules.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The prescription for Fushenning Capsules of this invention is as follows: Plantago seed 434g, Polygonum aviculare 130g, Phellodendron bark (salt) 196g, Anemarrhena rhizome (salt-processed) 130g, Rhubarb (processed) 44g, Gardenia fruit 130g, Leonurus japonicus 130g, Moutan bark 130g, Aconitum carmichaelii (processed) 44g, and Glycyrrhiza uralensis 44g.

[0008] The preparation method of the Fushenning Capsules of this invention is as follows: Take the above ten ingredients, including rhubarb, aconite, peony bark, and licorice, grind them into a fine powder, sift, and set aside. For the remaining six ingredients, including plantain seed, first decoct with 10 times the amount of water for 3 hours, then with 8 times the amount of water for 2 hours, and finally with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate under reduced pressure to a thick paste. Mix this paste with the above-mentioned powder, dry under reduced pressure, grind, sift through a 60-mesh sieve, and fill into capsules to make 1000 capsules.

[0009] The detection method for Fushenning described in this invention includes: microscopic identification of the product, thin-layer chromatography identification of Anemarrhena asphodeloides, thin-layer chromatography identification of geniposide, thin-layer chromatography identification of rhubarb, thin-layer chromatography identification of Phellodendron chinense, and determination of berberine hydrochloride content, as detailed below:

[0010] Microscopic identification: Take this product and observe it under a microscope: the fibers are bundled, with a diameter of 8-14 μm, thick walls, slightly lignified, and the surrounding thin-walled cells contain calcium oxalate crystals, forming crystal fibers (licorice); calcium oxalate crystals, cluster crystals are common, with large calcium oxalate cluster crystals, with a diameter of 20-160 μm, some even larger; there are many starch granules, single granules are round or polygonal, and compound granules are composed of 2-6 sub-granules (rhubarb).

[0011] The thin-layer chromatography identification of Anemarrhena asphodeloides is as follows: Take 5-10g of the contents of Fushenning capsules, add 5-15mL of ethanol, sonicate for 10-20min, and take the supernatant as the test solution; take 2g of Anemarrhena asphodeloides reference material and prepare a reference material solution in the same way; take smilax saponin reference standard and add methanol to prepare a reference standard solution containing 1mg per 1mL; perform the thin-layer chromatography test: take 2-4μL each of the reference standard solution and the reference material solution, and 6-10μL of the test solution, and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-ethyl acetate-acetone as the developing solvent, develop, remove, air dry, spray with 10%-30% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference material and the reference standard.

[0012] The thin-layer chromatography identification of geniposide is as follows: Take 0.5-1.5g of the contents of Fushenning capsules, add 10-30mL of methanol, sonicate for 10-30min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution; separately take geniposide reference standard, add methanol to prepare a solution containing 0.5mg per 1mL to obtain the reference solution; perform thin-layer chromatography, take 1-5μL each of the test solution and the reference solution, spot them separately on the same silica gel G thin-layer plate, use chloroform-methanol in a ratio of 7:2 as the developing solvent, develop, remove, air dry, spray with 5-10% sulfuric acid ethanol solution, and bake at 105℃ until the spots are clearly visible; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard;

[0013] The thin-layer chromatography identification of Phellodendron bark is as follows: Take 0.5-1.5g of the contents of Fushenning capsules, add 10-30mL of methanol, sonicate for 10-30min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution; separately take 0.2g of Phellodendron bark reference material, add 10-20mL of methanol, sonicate for 10-30min, filter, and take the filtrate to obtain the reference material solution; according to the thin-layer chromatography test, take 1-2μL of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate, use n-butanol-glacial acetic acid-water in a ratio of 7:2:1 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm; 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 material;

[0014] The thin-layer chromatography identification of rhubarb is as follows: Take 1-5g of the contents of Fushenning capsules, add 10-40mL of methanol, sonicate for 20-40min, filter, take 10-15mL of the filtrate, evaporate to dryness, dissolve the residue in 10-15mL of water, add 2mL of hydrochloric acid, heat in a water bath for 30-60min, remove, extract with ether 1-3 times, 15-30mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 1mL of chloroform, and use as the test solution; separately take 0.1g of rhubarb reference material, and prepare the reference material solution using the same method. Take emodin reference standard and add chloroform to prepare a solution containing 0.1 mg per 1 mL as the reference solution; perform thin-layer chromatography test, take 2-8 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate, use the upper layer solution of petroleum ether-ethyl formate-formic acid in a ratio of 15:5:1 at 30-60℃ as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard;

[0015] The content of berberine hydrochloride was determined as follows:

[0016] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the column packing material; the mobile phase was 0.01-0.0125 mol / L sodium dodecyl sulfate: 0.01-0.0125 mol / L potassium dihydrogen phosphate solution-acetonitrile in a ratio of 25:25:50; the detection wavelength was 345 nm; the theoretical plate number calculated based on the berberine hydrochloride peak should not be less than 2500.

[0017] Preparation of reference solution: Accurately weigh an appropriate amount of berberine hydrochloride, add methanol to prepare a solution containing 0.04 g of berberine hydrochloride per 1 mL;

[0018] Preparation of test solution: Take the contents of Fushenning capsules (within the specified weight range), grind them into a fine powder, take 1-1.2g, weigh accurately, place in a stoppered conical flask, accurately add 20-25mL of hydrochloric acid-methanol solution at a ratio of 1:100, weigh, reflux for 0.5-1.5h, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution;

[0019] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0020] Preferably, the thin-layer chromatography identification of Anemarrhena asphodeloides according to the present invention is as follows: Take 5g of the contents of Fushenning capsules, add 10mL of ethanol, sonicate for 20min, and take the supernatant as the test solution; take 2g of Anemarrhena asphodeloides reference material and prepare a reference material solution in the same way; take smilax saponin reference standard and add methanol to prepare a reference standard solution containing 1mg per 1mL; perform thin-layer chromatography test: take 4μL each of the reference standard solution and the reference material solution, and 8μL of the test solution, and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-ethyl acetate-acetone as the developing solvent, develop, remove, air dry, spray with 20% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference material and the reference standard.

[0021] In the thin-layer chromatography identification of Anemarrhena asphodeloides described in this invention, the ratio of the developing solvent cyclohexane-ethyl acetate-acetone is 8:1:2.

[0022] In the thin-layer chromatography identification of Anemarrhena asphodeloides described in this invention, the ethanol used is anhydrous ethanol.

[0023] Preferably, the thin-layer chromatography identification of geniposide according to the present invention is as follows: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution. Separately, take geniposide reference standard, add methanol to obtain a solution containing 0.5mg per 1mL to obtain the reference solution. Perform thin-layer chromatography, take 4μL each of the test solution and the reference solution, and spot them separately on the same silica gel G thin-layer plate. Use chloroform-methanol in a ratio of 7:2 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and bake at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0024] Preferably, the thin-layer chromatography identification of Phellodendron bark according to the present invention is as follows: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol, and use as the test solution. Separately, take 0.2g of Phellodendron bark reference material, add 10mL of methanol, sonicate for 15min, filter, and use the filtrate as the reference material solution. Perform thin-layer chromatography, take 2μL of each of the above two solutions, and spot them separately on the same silica gel G thin-layer plate, using n-butanol-glacial acetic acid-water in a ratio of 7:2:1 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm. 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 material.

[0025] Preferably, the thin-layer chromatography identification of rhubarb according to the present invention is as follows: Take 3g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 30min, filter, take 10mL of the filtrate, evaporate to dryness, dissolve the residue in 10mL of water, add 2mL of hydrochloric acid, heat in a water bath for 30min, remove, extract twice with 20mL of ether each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1mL of chloroform, and use as the test solution. Take 0.1g of rhubarb reference material and prepare a reference material solution using the same method. Take rhein reference standard and add chloroform to prepare a solution containing 0.1mg per 1mL, as the reference solution. Perform thin-layer chromatography test, take 5μL of each of the above three solutions, spot them separately on the same silica gel G thin-layer plate, use the upper layer solution of petroleum ether-ethyl formate-formic acid in a ratio of 15:5:1 at 30-60℃ as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0026] Preferably, the determination of berberine hydrochloride content in the present invention is as follows:

[0027] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the column packing material; a mobile phase of 0.0125 mol / L sodium dodecyl sulfate-0.0125 mol / L potassium dihydrogen phosphate solution-acetonitrile in a ratio of 25:25:50 was used; the detection wavelength was 345 nm; and the theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 2500.

[0028] To prepare the reference solution, accurately weigh an appropriate amount of berberine hydrochloride and add methanol to prepare a solution containing 0.04 g of berberine hydrochloride per 1 mL.

[0029] To prepare the test solution, take the contents of the Fushenning capsules (with variations in fill weight), grind them into a fine powder, take 1.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of a 1:100 hydrochloric acid-methanol solution, weigh it, reflux for 1h, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0030] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0031] The conditions for ultrasonic treatment described in this invention are: power 200-300W, frequency 30-50kHz.

[0032] Preferably, the conditions for ultrasonic treatment according to the present invention are: power 250W and frequency 50kHz.

[0033] The beneficial effects of this invention are:

[0034] This invention optimizes the thin-layer chromatography method for identifying Anemarrhena asphodeloides in Fushenning capsules based on existing detection methods. Methodological studies show that this detection method is highly specific, robust, accurate, and stable, and is simple and quick to operate. It improves the quality control of Fushenning capsules, allowing for better product quality control and further ensuring the safety of laboratory personnel.

[0035] This invention has conducted extensive research on the thin-layer chromatography identification method for Anemarrhena asphodeloides: (1) By investigating the extraction solvents of the test sample, namely anhydrous ethanol, cyclohexane, and petroleum ether, it was found that the spots were clearest, the resolution met the requirements, and the Rf value was moderate when anhydrous ethanol was used as the extraction solvent. (2) By investigating the extraction time of the test sample, namely, 5 min, 10 min, and 20 min, it was found that the spots were clearest, the resolution met the requirements, and the Rf value was moderate when the ultrasonic extraction time was 20 min. (3) By investigating the developing solvent of thin-layer chromatography, it was found that the spots were clearest, the resolution met the requirements, and the Rf value was moderate when the developing solvent was cyclohexane-ethyl acetate-acetone (8:1:2). (4) By investigating the spotting volume of thin-layer chromatography, it was found that the spots were clearest when the spotting volume of the reference herb solution and the reference solution was 4 μL and the spotting volume of the test sample solution was 8 μL. Attached Figure Description

[0036] Figure 1 TLC spectrum of Anemarrhena asphodeloides in Fushenning capsules

[0037] Figure 2 TLC spectra with different extraction solvents

[0038] Figure 3 TLC images at different ultrasound times

[0039] Figure 4 TLC spectra with cyclohexane-acetone (10:1) as the developing solvent

[0040] Figure 5 TLC spectra with cyclohexane-ethyl acetate (8:2) as the developing solvent

[0041] Figure 6 TLC chromatograms were examined with different spot sizes.

[0042] Figure 7 TLC spectra when using strip spotting.

[0043] Figure 8 TLC spectra under different temperature conditions

[0044] Figure 9 TLC spectra under different humidity conditions

[0045] Figure 10 TLC chromatograms of Anemarrhena asphodeloides from ten batches of Fushenning capsules Detailed Implementation

[0046] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0047] Example 1

[0048] Thin-layer chromatography identification of Anemarrhena asphodeloides: Take 5g of the contents of Fushenning capsules, add 10mL of anhydrous ethanol, sonicate for 20min, and use the supernatant as the test solution. Separately, take 2g of Anemarrhena asphodeloides reference material and prepare a reference material solution using the same method. Take smilax saponin reference standard and prepare a reference standard solution containing 1mg per 1mL using methanol. Perform thin-layer chromatography: Apply 4μL each of the reference standard solution and the reference material solution, and 8μL of the test solution separately to the same silica gel G thin-layer plate. Develop using cyclohexane-ethyl acetate-acetone in a ratio of 8:1:2. Remove, air dry, spray with 20% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0049] Thin-layer chromatography identification of geniposide: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution. Separately, prepare a reference solution by dissolving geniposide reference standard in methanol to obtain a solution containing 0.5mg per 1mL. Perform thin-layer chromatography, applying 4μL each of the test solution and reference solution separately to the same silica gel G thin-layer plate. Develop using a 7:2 ratio of chloroform-methanol as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and bake at 105℃ until the spots are clearly visible. The test sample chromatogram should show spots of the same color at the corresponding positions as the reference chromatogram.

[0050] Thin-layer chromatography identification of Phellodendron bark: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution. Separately, take 0.2g of Phellodendron bark reference material, add 10mL of methanol, sonicate for 15min, filter, and use the filtrate as the reference material solution. Perform thin-layer chromatography, applying 2μL of each of the above two solutions separately to the same silica gel G thin-layer plate, using a 7:2:1 ratio of n-butanol-glacial acetic acid-water as the developing solvent, develop, remove, air dry, and examine under a 365nm ultraviolet lamp. 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 material.

[0051] Thin-layer chromatography identification of rhubarb: Take 3g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 30min, filter, take 10mL of the filtrate, evaporate to dryness, dissolve the residue in 10mL of water, add 2mL of hydrochloric acid, heat in a water bath for 30min, remove, extract twice with 20mL of ether each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 1mL of chloroform, and use as the test solution. Take 0.1g of rhubarb reference material and prepare a reference material solution using the same method. Take rhein reference standard and add chloroform to prepare a solution containing 0.1mg per 1mL, as the reference solution. Perform thin-layer chromatography test, take 5μL of each of the above three solutions, spot them separately on the same silica gel G thin-layer plate, use the upper layer solution of petroleum ether-ethyl formate-formic acid in a ratio of 15:5:1 at 30-60℃ as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0052] Determination of berberine hydrochloride content:

[0053] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the column packing material; a mobile phase of 0.0125 mol / L sodium dodecyl sulfate-0.0125 mol / L potassium dihydrogen phosphate solution-acetonitrile in a ratio of 25:25:50 was used; the detection wavelength was 345 nm; and the theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 2500.

[0054] To prepare the reference solution, accurately weigh an appropriate amount of berberine hydrochloride and add methanol to prepare a solution containing 0.04 g of berberine hydrochloride per 1 mL.

[0055] To prepare the test solution, take the contents of the Fushenning capsules (with variations in fill weight), grind them into a fine powder, take 1.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of a 1:100 hydrochloric acid-methanol solution, weigh it, reflux for 1h, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0056] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0057] Test for aconitine: Take the contents of 30 capsules of Fushenning, place them in a stoppered conical flask, add 150 mL of ether, shake for 10 min, add 10 mL of ammonia test solution, shake for 30 min, let stand for 2 h, separate the ether solution, evaporate to dryness, dissolve the residue in anhydrous ethanol, transfer to a 2 mL volumetric flask, add anhydrous ethanol to the mark, shake well, and use as the test solution. Separately, take aconitine reference standard, dissolve in anhydrous ethanol to prepare a solution containing 1 mg per mL, and use as the reference solution. Perform thin-layer chromatography, take 20 μL of the test solution and 2 μL of the reference solution, and spot them separately on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-diethylamine in a ratio of 4:3:1 as the developing solvent, develop, remove, air dry, and spray with dilute bismuth potassium iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard or no spot should appear.

[0058] Example 2

[0059] Thin-layer chromatography identification of Anemarrhena asphodeloides: Take 8g of the contents of Fushenning capsules, add 5mL of anhydrous ethanol, sonicate for 5min, and use the supernatant as the test solution. Separately, take 2g of Anemarrhena asphodeloides reference material and prepare a reference material solution using the same method. Take smilax saponin reference standard and add methanol to prepare a reference standard solution containing 1mg per 1mL. Perform thin-layer chromatography: Apply 2μL each of the reference standard solution and the reference material solution, and 10μL of the test solution separately to the same silica gel G thin-layer plate. Develop using cyclohexane-ethyl acetate-acetone in a ratio of 8:2:1. Remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0060] Thin-layer chromatography identification of geniposide: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution. Separately, prepare a reference solution by dissolving geniposide reference standard in methanol to obtain a solution containing 0.5mg per 1mL. Perform thin-layer chromatography, applying 4μL each of the test solution and reference solution separately to the same silica gel G thin-layer plate. Develop using a 7:2 ratio of chloroform-methanol as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and bake at 105℃ until the spots are clearly visible. The test sample chromatogram should show spots of the same color at the corresponding positions as the reference chromatogram.

[0061] Thin-layer chromatography identification of Phellodendron bark: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution. Separately, take 0.2g of Phellodendron bark reference material, add 10mL of methanol, sonicate for 15min, filter, and use the filtrate as the reference material solution. Perform thin-layer chromatography, applying 2μL of each of the above two solutions separately to the same silica gel G thin-layer plate, using a 7:2:1 ratio of n-butanol-glacial acetic acid-water as the developing solvent, develop, remove, air dry, and examine under a 365nm ultraviolet lamp. 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 material.

[0062] Thin-layer chromatography identification of rhubarb: Take 3g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 30min, filter, take 10mL of the filtrate, evaporate to dryness, dissolve the residue in 10mL of water, add 2mL of hydrochloric acid, heat in a water bath for 30min, remove, extract twice with 20mL of ether each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 1mL of chloroform, and use as the test solution. Take 0.1g of rhubarb reference material and prepare a reference material solution using the same method. Take rhein reference standard and add chloroform to prepare a solution containing 0.1mg per 1mL, as the reference solution. Perform thin-layer chromatography test, take 5μL of each of the above three solutions, spot them separately on the same silica gel G thin-layer plate, use the upper layer solution of petroleum ether-ethyl formate-formic acid in a ratio of 15:5:1 at 30-60℃ as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0063] Determination of berberine hydrochloride content:

[0064] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the column packing material; a mobile phase of 0.0125 mol / L sodium dodecyl sulfate-0.0125 mol / L potassium dihydrogen phosphate solution-acetonitrile in a ratio of 25:25:50 was used; the detection wavelength was 345 nm; and the theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 2500.

[0065] To prepare the reference solution, accurately weigh an appropriate amount of berberine hydrochloride and add methanol to prepare a solution containing 0.04 g of berberine hydrochloride per 1 mL.

[0066] To prepare the test solution, take the contents of the Fushenning capsules (with variations in fill weight), grind them into a fine powder, take 1.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of a 1:100 hydrochloric acid-methanol solution, weigh it, reflux for 1h, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0067] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0068] Test for aconitine: Take the contents of 30 capsules of Fushenning, place them in a stoppered conical flask, add 150 mL of ether, shake for 10 min, add 10 mL of ammonia test solution, shake for 30 min, let stand for 2 h, separate the ether solution, evaporate to dryness, dissolve the residue in anhydrous ethanol, transfer to a 2 mL volumetric flask, add anhydrous ethanol to the mark, shake well, and use as the test solution. Separately, take aconitine reference standard, dissolve in anhydrous ethanol to prepare a solution containing 1 mg per mL, and use as the reference solution. Perform thin-layer chromatography, take 20 μL of the test solution and 2 μL of the reference solution, and spot them separately on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-diethylamine in a ratio of 4:3:1 as the developing solvent, develop, remove, air dry, and spray with dilute bismuth potassium iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard or no spot should appear.

[0069] Example 3

[0070] Thin-layer chromatography identification of Anemarrhena asphodeloides: Take 10g of the contents of Fushenning capsules, add 15mL of anhydrous ethanol, sonicate for 10min, and use the supernatant as the test solution. Separately, take 2g of Anemarrhena asphodeloides reference material and prepare a reference material solution using the same method. Take smilax saponin reference standard and add methanol to prepare a reference standard solution containing 1mg per 1mL. Perform thin-layer chromatography: Apply 4μL each of the reference standard solution and the reference material solution, and 6μL of the test solution separately to the same silica gel G thin-layer plate. Develop using cyclohexane-ethyl acetate-acetone in a ratio of 8:2:1. Remove, air dry, spray with 30% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0071] Thin-layer chromatography identification of geniposide: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution. Separately, prepare a reference solution by dissolving geniposide reference standard in methanol to obtain a solution containing 0.5mg per 1mL. Perform thin-layer chromatography, applying 4μL each of the test solution and reference solution separately to the same silica gel G thin-layer plate. Develop using a 7:2 ratio of chloroform-methanol as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and bake at 105℃ until the spots are clearly visible. The test sample chromatogram should show spots of the same color at the corresponding positions as the reference chromatogram.

[0072] Thin-layer chromatography identification of Phellodendron bark: Take 1g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 20min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1mL of methanol to obtain the test solution. Separately, take 0.2g of Phellodendron bark reference material, add 10mL of methanol, sonicate for 15min, filter, and use the filtrate as the reference material solution. Perform thin-layer chromatography, applying 2μL of each of the above two solutions separately to the same silica gel G thin-layer plate, using a 7:2:1 ratio of n-butanol-glacial acetic acid-water as the developing solvent, develop, remove, air dry, and examine under a 365nm ultraviolet lamp. 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 material.

[0073] Thin-layer chromatography identification of rhubarb: Take 3g of the contents of Fushenning capsules, add 20mL of methanol, sonicate for 30min, filter, take 10mL of the filtrate, evaporate to dryness, dissolve the residue in 10mL of water, add 2mL of hydrochloric acid, heat in a water bath for 30min, remove, extract twice with 20mL of ether each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 1mL of chloroform, and use as the test solution. Take 0.1g of rhubarb reference material and prepare a reference material solution using the same method. Take rhein reference standard and add chloroform to prepare a solution containing 0.1mg per 1mL, as the reference solution. Perform thin-layer chromatography test, take 5μL of each of the above three solutions, spot them separately on the same silica gel G thin-layer plate, use the upper layer solution of petroleum ether-ethyl formate-formic acid in a ratio of 15:5:1 at 30-60℃ as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0074] Determination of berberine hydrochloride content:

[0075] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the column packing material; a mobile phase of 0.0125 mol / L sodium dodecyl sulfate-0.0125 mol / L potassium dihydrogen phosphate solution-acetonitrile in a ratio of 25:25:50 was used; the detection wavelength was 345 nm; and the theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 2500.

[0076] To prepare the reference solution, accurately weigh an appropriate amount of berberine hydrochloride and add methanol to prepare a solution containing 0.04 g of berberine hydrochloride per 1 mL.

[0077] To prepare the test solution, take the contents of the Fushenning capsules (with variations in fill weight), grind them into a fine powder, take 1.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of a 1:100 hydrochloric acid-methanol solution, weigh it, reflux for 1h, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0078] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0079] Test for aconitine: Take the contents of 30 capsules of Fushenning, place them in a stoppered conical flask, add 150 mL of ether, shake for 10 min, add 10 mL of ammonia test solution, shake for 30 min, let stand for 2 h, separate the ether solution, evaporate to dryness, dissolve the residue in anhydrous ethanol, transfer to a 2 mL volumetric flask, add anhydrous ethanol to the mark, shake well, and use as the test solution. Separately, take aconitine reference standard, dissolve in anhydrous ethanol to prepare a solution containing 1 mg per mL, and use as the reference solution. Perform thin-layer chromatography, take 20 μL of the test solution and 2 μL of the reference solution, and spot them separately on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-diethylamine in a ratio of 4:3:1 as the developing solvent, develop, remove, air dry, and spray with dilute bismuth potassium iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard or no spot should appear.

[0080] To verify the effectiveness of this invention, the inventors conducted a series of experiments, some of which are excerpted below:

[0081] 1. Test materials

[0082] Samples: Fushenning Capsules, batch numbers 20200401, 20200402, 20200501, 20210201, 20210402, 20210701, 20210702, 20210703, provided by Guizhou Ruifeng Pharmaceutical Co., Ltd.; batch number 220903 provided by Jilin Jichun Pharmaceutical Co., Ltd.; batch numbers 20230606, 2023060701, 2023060702 provided by Guizhou Liangji Pharmaceutical Co., Ltd.

[0083] Reference standards and reference medicinal materials: Smilax glabra saponin reference standard (batch number: 110744-201711) and Anemarrhena asphodeloides reference medicinal material (batch number: 121070-201806) were provided by the China National Institutes for Food and Drug Control.

[0084] Reference herbs for herbs lacking Anemarrhena asphodeloides: Weigh out the prescribed amounts of rhubarb, aconite, peony bark, and licorice, grind them into fine powder, sift them, and set aside. For the remaining five herbs except Anemarrhena asphodeloides, decoct them for 3 hours with 10 times the amount of water for the first time, 2 hours with 8 times the amount of water for the second time, and 1 hour with 8 times the amount of water for the third time. Combine the decoctions, filter them, concentrate the filtrate under reduced pressure to a thick paste, mix it with the above-mentioned powder, dry it under reduced pressure, grind it, and pass it through a 60-mesh sieve to obtain the final product.

[0085] Reagents: All reagents used were of analytical grade.

[0086] Silicone sheet: Silicone G sheet (Qingdao Shenghai Fine Silicone Chemical Co., Ltd., 0.20~0.25mm)

[0087] 2. Screening of Thin-Layer Chromatography Identification Method for Anemarrhena asphodeloides in Fushenning Capsules

[0088] 2.1 Method Source

[0089] Previously, our company's thin-layer chromatography method for identifying Anemarrhena asphodeloides in the testing project for Fushenning capsules was as follows:

[0090] Take 4g of the contents of this product, add 20mL of ethanol, heat under reflux for 40min, filter, take 10mL of the filtrate, add 1mL of hydrochloric acid, heat under reflux for 1h, concentrate to about 5mL, add 10mL of water, gently extract with 20mL of benzene, evaporate the extract to dryness, dissolve the residue in 2mL of benzene, and use this as the test solution. Separately, take 2g of Anemarrhena asphodeloides reference material and prepare a reference material solution using the same method. Perform thin-layer chromatography, applying 7μL each of the test solution and the reference material solution to the same silica gel G thin-layer plate, using benzene-acetone (9:1) as the developing solvent, develop, remove, air dry, spray with 20% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible.

[0091] In the above methods, using Anemarrhena asphodeloides as a control, the processing procedures for the test sample and control are complex, time-consuming, and inefficient. Furthermore, the use of benzene in the experiment is detrimental to the health of the experimenters. To address these issues, and considering the properties of Fushenning capsules, a thin-layer chromatography method for identifying Anemarrhena asphodeloides is investigated, as follows: 2.1.1 Solution Preparation

[0092] Preparation of test solution: Accurately weigh 5g of the contents of this product, place it in a 25mL Erlenmeyer flask, add 10mL of anhydrous ethanol, sonicate for 20min, and take the supernatant as the test solution.

[0093] Preparation of the reference herb solution: Accurately weigh 2g of Anemarrhena asphodeloides reference herb, place it in a 25mL Erlenmeyer flask, add 10mL of anhydrous ethanol, sonicate for 20min, and take the supernatant as the reference herb solution.

[0094] Preparation of reference solution: Accurately weigh 0.05 g of smilax saponin reference standard and place it in a 50 mL volumetric flask. Dissolve it in methanol and dilute to volume to obtain a reference solution containing 1 mg per 1 mL.

[0095] Preparation of negative solution for anemarrhena asphodeloides: Accurately weigh 5g of negative sample for anemarrhena asphodeloides, place it in a 25mL Erlenmeyer flask, add 10mL of anhydrous ethanol, sonicate for 20min, and take the supernatant as negative solution for anemarrhena asphodeloides.

[0096] 2.1.2 Thin-layer chromatography identification of Anemarrhena asphodeloides

[0097] According to the thin-layer chromatography method, take 2 μL each of the reference solution and the reference medicinal material solution prepared under section 2.1.1, and 4 μL of the test solution and the negative solution for Anemarrhena asphodeloides deficiency, and spot them separately on the same silica gel G thin-layer plate. Use cyclohexane:ethyl acetate:acetone (8:1:2) as the developing solvent, develop, remove, air dry, spray with 20% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible.

[0098] 2.1.3 Results

[0099] The TLC spectrum of Anemarrhena asphodeloides in Fushenning capsules is shown below. Figure 1 The room temperature was 20℃ and the relative humidity was 75%. Silica gel G plates manufactured by Qingdao Shenghai Fine Silica Gel Chemical Co., Ltd. were used. The thin-layer sampling sequence was: 1-control medicinal material solution, 2-control solution, 3-test solution (batch number 20230606), 4-test solution (batch number 2023060701), 5-test solution (batch number: 2023060702), 6-Anemarrhena asphodeloides negative solution.

[0100] 2.2 Methodological Examination

[0101] 2.2.1 Investigation of extraction solvent

[0102] Prepare the test solution and reference medicinal material solution according to the method in section 2.1.1. Accurately weigh the contents of this product and the reference medicinal material, and use 10 mL each of anhydrous ethanol, cyclohexane and petroleum ether as extraction solvents, respectively. Under the same conditions, perform thin-layer identification of Anemarrhena asphodeloides according to the method in section 2.1.2.

[0103] 2.2.2 Examination of Ultrasound Time

[0104] Anhydrous ethanol was used as the extraction solvent. The test solution and the reference herb solution were prepared according to the method in section 2.1.1. The ultrasonic time was set to 5 min, 10 min and 20 min respectively. Other conditions remained unchanged. The thin-layer identification of Anemarrhena asphodeloides was performed according to the method in section 2.1.2.

[0105] 2.2.3 Development of the developing solvent

[0106] Anhydrous ethanol was used as the extraction solvent. The test solution and the reference herb solution were prepared according to the method in section 2.1.1. The ultrasonic time was 20 min. The developing solvents were set as cyclohexane-ethyl acetate-acetone (8:1:2), cyclohexane-acetone (10:1), and cyclohexane-ethyl acetate (8:2). Other conditions remained unchanged. Thin-layer identification of Anemarrhena asphodeloides was performed according to the method in section 2.1.2.

[0107] 2.2.4 Investigation of different sample sizes

[0108] Anhydrous ethanol was used as the extraction solvent. The test solution and the reference herb solution were prepared according to the method in section 2.1.1. The ultrasonic time was 20 min. The spotting volume of the reference herb solution and the reference solution was set to 2 μL and 4 μL, respectively, and the spotting volume of the test solution was set to 6 μL, 8 μL and 10 μL, respectively. Other conditions remained unchanged. The sample was developed in cyclohexane-ethyl acetate-acetone (8:1:2) as the developing solvent according to the method in section 2.1.2. The thin-layer identification of Anemarrhena asphodeloides was performed according to the method in section 2.1.2.

[0109] 2.3 Results and Analysis

[0110] 2.3.1 Effect of different extraction solvents on the thin-layer chromatography identification of Anemarrhena asphodeloides

[0111] The test solution and reference herb solution were prepared according to the method described in section 2.1.1. Thin-layer chromatography (TLC) was performed on Anemarrhena asphodeloides using 10 mL each of anhydrous ethanol, cyclohexane, and petroleum ether as extraction solvents. The TLC spotting order was: 1-reference herb solution (extracted with anhydrous ethanol), 2-reference herb solution (extracted with cyclohexane), 3-reference herb solution (extracted with petroleum ether), 4-reference solution, 5-test solution (extracted with anhydrous ethanol). The results are shown in Table 1. Figure 2 .

[0112] Table 1. Effects of different extraction solvents on the thin-layer chromatography identification of Anemarrhena asphodeloides.

[0113]

[0114] As shown in Table 1, the samples treated with anhydrous ethanol as the extraction solvent had the clearest spots, met the separation requirements, and had a moderate Rf value. Therefore, anhydrous ethanol was chosen as the extraction solvent.

[0115] 2.3.2 Effect of different ultrasound times on thin-layer identification of Anemarrhena asphodeloides

[0116] Anhydrous ethanol was used as the extraction solvent. Test solution and reference herb solution were prepared according to the method described in section 2.1.1. Ultrasonication times were set to 5 min, 10 min, and 20 min, respectively. Thin-layer chromatography (TLC) identification of Anemarrhena asphodeloides was performed. The TLC spotting sequence was: 1-Reference herb solution (20 min), 2-Reference herb solution (10 min), 3-Reference herb solution (5 min), 4-Reference solution, 5-Test solution (5 min). The results are shown in Table 2. Figure 3 .

[0117] Table 2. Effects of different ultrasound times on thin-layer identification of Anemarrhena asphodeloides.

[0118]

[0119] As shown in Table 2, the sample treated with ultrasound for 20 minutes had the clearest spots, met the separation requirements, and had a moderate Rf value. Therefore, the ultrasound time was selected as 20 minutes.

[0120] 2.3.3 Effects of different developing solvents on the thin-layer identification of Anemarrhena asphodeloides

[0121] Anhydrous ethanol was used as the extraction solvent. The test solution and reference herb solution were prepared according to the method described in section 2.1.1. The ultrasonic time was 20 min. The developing solvents were set as cyclohexane-ethyl acetate-acetone (8:1:2), cyclohexane-acetone (10:1), and cyclohexane-ethyl acetate (8:2). Thin-layer chromatography identification of Anemarrhena asphodeloides was performed, and the results are shown in Table 3. Figure 3-5 ,in Figure 4 , Figure 5 The thin-layer sampling sequence is as follows: 1- control medicinal material solution (extracted with anhydrous ethanol), 2- control medicinal material solution (extracted with cyclohexane), 3- control medicinal material solution (extracted with petroleum ether), 4- reference solution, 5- test solution (extracted with anhydrous ethanol).

[0122] Table 3. Effects of different developing solvents on the thin-layer chromatography identification of Anemarrhena asphodeloides.

[0123]

[0124] As shown in Table 3, the developing solvent is cyclohexane-ethyl acetate-acetone (8:1:2). The spots are clear, the resolution meets the requirements, and the Rf value is moderate. Therefore, cyclohexane-ethyl acetate-acetone (8:1:2) is selected as the developing solvent.

[0125] 2.3.4 Effect of different spotting amounts on the thin-layer identification of Anemarrhena asphodeloides

[0126] Anhydrous ethanol was used as the extraction solvent. The test solution and reference herb solution were prepared according to the method described in section 2.1.1. The sonication time was 20 min. The spotting volumes of the reference herb solution and reference solution were set to 2 μL and 4 μL, respectively, and the spotting volumes of the test solution were set to 6 μL, 8 μL, and 10 μL, respectively. Other conditions remained unchanged. The solution was developed in cyclohexane-ethyl acetate-acetone (8:1:2) as the developing solvent according to the method described in section 2.1.2 for thin-layer chromatography identification of Anemarrhena asphodeloides. The spotting sequence was as follows: 1-4 μL of reference herb solution, 2-2 μL of reference herb solution, 3-2 μL of reference solution, 4-4 μL of reference solution, 5-10 μL of test solution, 6-8 μL of test solution, 7-6 μL of test solution. The results showed that the spots were clearest when the spotting volumes of the reference herb solution and reference solution were 4 μL, and the spotting volume of the test solution was 8 μL. Therefore, this method was adopted.

[0127] 3. Methodological Validation

[0128] 3.1 Specificity Experiment

[0129] The test solution, reference herb solution, and reference solution were prepared according to the method described in section 2.1.1. Anhydrous ethanol was used as the extraction solvent, and the mixture was sonicated for 20 min. The mixture was then developed in cyclohexane-ethyl acetate-acetone (8:1:2) as the developing solvent according to the method described in section 2.1.2 for thin-layer chromatography identification of Anemarrhena asphodeloides. The results are shown in [Figure 1]. Figure 1 .

[0130] 3.2 Durability Assessment

[0131] 3.2.1 Comparison of different spotting methods

[0132] The test solution, reference herb solution, and reference standard solution were prepared according to the method described in section 2.1.1. Anhydrous ethanol was used as the extraction solvent, and the samples were sonicated for 20 min. Strip and spot sampling were performed respectively according to the method described in section 2.1.2. The samples were developed in cyclohexane-ethyl acetate-acetone (8:1:2) for thin-layer chromatography identification of Anemarrhena asphodeloides. The sampling order was 1-reference herb solution, 2-reference standard solution, 3-test solution (batch number 20230606), 4-test solution (batch number 2023060701), and 5-test solution (batch number 2023060702). The results showed no significant difference in separation efficiency between strip and spot sampling. (See attached table for details.) Figure 7 , Figure 1 .

[0133] 3.2.2 Comparison of different temperatures

[0134] The test solution, reference herb solution, and reference standard solution were prepared according to the method described in section 2.1.1. Anhydrous ethanol was used as the extraction solvent, and the mixture was sonicated for 20 min. Following the method described in section 2.1.2, the mixture was developed in cyclohexane-ethyl acetate-acetone (8:1:2) as the developing solvent for thin-layer chromatography identification of Anemarrhena asphodeloides. The sample spotting order was: 1-reference herb solution, 2-reference standard solution, 3-test solution (batch number 20230606), 4-test solution (batch number 2023060701), 5-test solution (batch number 2023060702). The results showed no significant difference in separation efficiency under different temperature conditions. (See attached table). Figure 8 A is the TLC spectrum under the conditions of 10℃ and 75% relative humidity, and B is the TLC spectrum under the conditions of 35℃ and 75% relative humidity.

[0135] 3.2.3 Comparison of different humidity levels

[0136] The test solution, reference herb solution, and reference solution were prepared according to the method described in section 2.1.1. Anhydrous ethanol was used as the extraction solvent, and the mixture was sonicated for 20 min. Following the method described in section 2.1.2, the mixture was developed in cyclohexane-ethyl acetate-acetone (8:1:2) as the developing solvent for thin-layer chromatography identification of Anemarrhena asphodeloides. The sample spotting order was: 1-reference herb solution, 2-reference solution, 3-test solution (batch number 20230606), 4-test solution (batch number 2023060701), 5-test solution (batch number 2023060702). The results showed no significant difference in separation efficiency under different relative humidity conditions. (See attached table). Figure 9 A is the TLC spectrum under the conditions of 25℃ and 45% relative humidity, and B is the TLC spectrum under the conditions of 25℃ and 88% relative humidity.

[0137] 3.3 Reproducibility Test

[0138] The test solution, reference herb solution, and reference solution were prepared according to the method described in section 2.1.1. Anhydrous ethanol was used as the extraction solvent, and the samples were sonicated for 20 min. Ten batches of Fushenning capsules were subjected to thin-layer chromatography identification according to the method described in section 2.1.2. The sampling order was as follows: 1- test solution (batch number 20200401), 2- test solution (batch number 20200402), 3- test solution (batch number 20200501), 4- test solution 5-Test solution (batch number 20210201), 6-Reference medicinal material solution, 7-Reference solution, 8-Test solution (batch number 20210701), 9-Test solution (batch number 20210702), 10-Test solution (batch number 20210703), 11-Test solution (batch number 220903), 12-Test solution (batch number 20230606). The results showed that in the chromatograms of the ten batches of Fushenning capsules, spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal material and the reference solution, indicating good reproducibility. See [link to results]. Figure 10 .

Claims

1. A method for detecting Fushenning capsules, wherein the formula and preparation method of Fushenning capsules are as follows: ; Take rhubarb, aconite, peony bark, and licorice, grind them into fine powder, sift them, and set aside. Take the remaining six ingredients, including plantain seed, add 10 times the amount of water and decoct for 3 hours the first time, add 8 times the amount of water and decoct for 2 hours the second time, and add 8 times the amount of water and decoct for 1 hour the third time. Combine the decoctions, filter them, concentrate the filtrate under reduced pressure to a thick paste, mix it with the above fine powder, dry it under reduced pressure, grind it, pass it through a 60-mesh sieve, and fill it into capsules to make 1000 capsules. The detection methods for the Fushenning capsules are as follows: microscopic identification of the product, thin-layer chromatography identification of Anemarrhena asphodeloides, thin-layer chromatography identification of geniposide, thin-layer chromatography identification of rhubarb, thin-layer chromatography identification of Phellodendron chinense, and determination of berberine hydrochloride content, as detailed below: Microscopic identification: Take this product and observe it under a microscope: the fibers are bundled, 8-14 μm in diameter, with thick walls and slight lignification. The surrounding thin-walled cells contain calcium oxalate crystals, forming crystal fibers. This is the microscopic characteristic of licorice. Calcium oxalate crystals and clusters are common, including large calcium oxalate clusters with a diameter of 20-160 μm, and some are even larger. There are many starch granules. Single granules are round or polygonal, and compound granules are composed of 2-6 sub-granules. This is the microscopic characteristic of rhubarb. The thin-layer chromatography identification of Anemarrhena asphodeloides is as follows: Take 5-10 g of the contents of Fushenning capsules, add 5-15 mL of ethanol, sonicate for 5-20 min, and take the supernatant as the test solution; take 2 g of Anemarrhena asphodeloides reference material and prepare a reference material solution in the same way; take smilax saponin reference standard and add methanol to prepare a reference standard solution containing 1 mg per 1 mL; perform the thin-layer chromatography test: take 2-4 μL each of the reference standard solution and the reference material solution, and 6-10 μL of the test solution, and spot them separately on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-acetone in a ratio of 8:1:2 as the developing solvent, develop, remove, air dry, spray with 10%-30% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference material and the reference standard. The thin-layer chromatography identification of geniposide is as follows: Take 0.5-1.5 g of the contents of Fushenning capsules, add 10-30 mL of methanol, sonicate for 10-30 min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1 mL of methanol to obtain the test solution; separately take geniposide reference standard, add methanol to prepare a solution containing 0.5 mg per 1 mL to obtain the reference solution; according to the thin-layer chromatography method, take 1-5 μL of each of the test solution and the reference solution, and spot them separately on the same silica gel G thin-layer plate, using chloroform-methanol in a ratio of 7:2 as the developing solvent, develop, remove, air dry, spray with 5-10% sulfuric acid ethanol solution, and bake at 105℃ until the spots are clearly visible; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard; The thin-layer chromatography identification of Phellodendron bark is as follows: Take 0.5-1.5 g of the contents of Fushenning capsules, add 10-30 mL of methanol, sonicate for 10-30 min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1 mL of methanol to obtain the test solution; separately take 0.2 g of Phellodendron bark reference material, add 10-20 mL of methanol, sonicate for 10-30 min, filter, and take the filtrate to obtain the reference material solution; according to the thin-layer chromatography test, take 1-2 μL of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate, use n-butanol-glacial acetic acid-water in a ratio of 7:2:1 as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 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 material. The thin-layer chromatography identification of rhubarb is as follows: Take 1-5 g of the contents of Fushenning capsules, add 10-40 mL of methanol, sonicate for 20-40 min, filter, take 10-15 mL of the filtrate, evaporate to dryness, dissolve the residue in 10-15 mL of water, add 2 mL of hydrochloric acid, heat in a water bath for 30-60 min, remove, extract with ether 1-3 times, 15-30 mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 1 mL of chloroform, and use as the test solution; separately take 0.1 g of rhubarb reference material, and prepare a reference material solution using the same method; then take rhein reference standard, add chloroform to prepare a solution containing 0.1 mg per 1 mL, and use as the reference solution; perform the thin-layer chromatography test, taking 2-8 μL of each of the above three solutions. μL was spotted onto the same silica gel G thin-layer plate. The upper layer of a petroleum ether-ethyl formate-formic acid solution at 30-60℃ in a ratio of 15:5:1 was used as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard. The content of berberine hydrochloride was determined as follows: Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the column packing material; the mobile phase was sodium dodecyl sulfate solution-potassium dihydrogen phosphate solution-acetonitrile in a ratio of 25:25:50, with the concentrations of sodium dodecyl sulfate solution and potassium dihydrogen phosphate solution both ranging from 0.01 to 0.0125 mol / L; the detection wavelength was 345 nm; the theoretical plate number, calculated based on the berberine hydrochloride peak, should not be less than 2500. Preparation of reference solution: Accurately weigh an appropriate amount of berberine hydrochloride, add methanol to prepare a solution containing 0.04 g of berberine hydrochloride per 1 mL; Preparation of test solution: Take the contents of Fushenning capsules (within the specified weight range), grind them into a fine powder, take 1-1.2 g, weigh accurately, place in a stoppered conical flask, accurately add 20-25 mL of hydrochloric acid-methanol solution at a ratio of 1:100, weigh, reflux for 0.5-1.5 h, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution; Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

2. The detection method according to claim 1, characterized in that, The thin-layer chromatography identification of Anemarrhena asphodeloides was performed as follows: 5 g of the contents of Fushenning capsules were added to 10 mL of ethanol and sonicated for 20 min. The supernatant was used as the test solution. 2 g of Anemarrhena asphodeloides reference material was prepared as a reference material solution using the same method. Smilax china reference standard was prepared by adding methanol to a solution containing 1 mg per mL. Thin-layer chromatography was performed as follows: 4 μL each of the reference solution and the reference material solution, and 8 μL of the test solution were spotted onto the same silica gel G thin-layer plate. A cyclohexane-ethyl acetate-acetone solution with a ratio of 8:1:2 was used as the developing solvent. The plate was developed, removed, dried, sprayed with 20% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as those in the chromatograms of the reference material and the reference standard.

3. The detection method according to any one of claims 1 or 2, characterized in that, The ethanol is anhydrous ethanol.

4. The detection method according to claim 1, characterized in that, The thin-layer chromatography identification of geniposide is as follows: Take 1 g of the contents of Fushenning capsules, add 20 mL of methanol, sonicate for 20 min, cool, filter, evaporate the filtrate to near dryness, dissolve the residue in 1 mL of methanol to obtain the test solution; separately take geniposide reference standard, add methanol to obtain a solution containing 0.5 mg per 1 mL to obtain the reference solution; perform thin-layer chromatography, take 4 μL each of the test solution and the reference solution, spot them separately on the same silica gel G thin-layer plate, use chloroform-methanol in a ratio of 7:2 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and bake at 105℃ until the spots are clearly visible; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

5. The detection method according to claim 1, characterized in that, The thin-layer chromatography identification of Phellodendron bark was performed as follows: 1 g of the contents of Fushenning capsules was added to 20 mL of methanol, sonicated for 20 min, cooled, filtered, and the filtrate was evaporated to near dryness. The residue was dissolved in 1 mL of methanol to obtain the test solution. Separately, 0.2 g of Phellodendron bark reference material was added to 10 mL of methanol, sonicated for 15 min, filtered, and the filtrate was used as the reference material solution. Thin-layer chromatography was performed, and 2 μL of each of the two solutions were spotted onto the same silica gel G thin-layer plate. A 7:2:1 ratio of n-butanol-glacial acetic acid-water was used as the developing solvent. The plate was developed, removed, dried, and examined under a 365 nm UV lamp. Fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the test sample and the reference material.

6. The detection method according to claim 1, characterized in that, The thin-layer chromatography identification of rhubarb was performed as follows: 3 g of the contents of Fushenning capsules were added to 20 mL of methanol, sonicated for 30 min, filtered, and 10 mL of the filtrate was evaporated to dryness. The residue was dissolved in 10 mL of water, and then 2 mL of hydrochloric acid was added. The mixture was heated in a water bath for 30 min, removed, and extracted twice with 20 mL of ether each time. The ether extracts were combined, evaporated to dryness, and the residue was dissolved in 1 mL of chloroform to obtain the test solution. Separately, 0.1 g of rhubarb reference material was prepared as a reference material solution using the same method. Rhein reference standard was prepared by adding chloroform to a solution containing 0.1 mg per mL, which was used as the reference solution. Thin-layer chromatography was performed by applying 5 μL of each of the three solutions to the same silica gel G plate. The upper layer of a petroleum ether-ethyl formate-formic acid solution at 30–60 °C (ratio 15:5:1) was used as the developing solvent. The plate was then removed, dried, and placed under ultraviolet light for 365°C. When examined at nm, fluorescent spots of the same color appear at the corresponding positions in the chromatograms of the test sample and the reference medicinal material.

7. The detection method according to claim 1, characterized in that, The content of berberine hydrochloride was determined as follows: Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the column packing material; the mobile phase was 0.0125 mol / L sodium dodecyl sulfate solution-0.0125 mol / L potassium dihydrogen phosphate solution-acetonitrile in a ratio of 25:25:50; the detection wavelength was 345 nm; the theoretical plate number calculated based on the berberine hydrochloride peak should not be less than 2500. Preparation of reference solution: Accurately weigh an appropriate amount of berberine hydrochloride, add methanol to prepare a solution containing 0.04 g of berberine hydrochloride per 1 mL; Preparation of test solution: Take the contents of Fushenning capsules with varying fill weights, grind them into a fine powder, take 1.2 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of hydrochloric acid-methanol solution with a ratio of 1:100, weigh it, reflux for 1 h, cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution; Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

8. The detection method according to any one of claims 1, 2, 4, 5, or 6, characterized in that, The conditions for ultrasonic treatment are: power 200-300 W, frequency 30-50 kHz.

9. The detection method according to claim 8, characterized in that, The conditions for the ultrasonic treatment are: power 250 W, frequency 50 kHz.

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