A quality testing method for anti-tuberculosis granules

Abstract

This invention discloses a quality testing method for an anti-tuberculosis granule, comprising: (1) preparing a test solution of the anti-tuberculosis granule and a reference herb solution of *Lysimachia christinae*; (2) spotting the test solution and the reference herb solution of *Lysimachia christinae* onto the same thin-layer plate, and performing thin-layer chromatography identification using a mixed solution of butyl acetate-formic acid-water as the developing solvent; wherein the volume ratio of butyl acetate:formic acid:water in the developing solvent is 15:4:1. The quality testing method for the anti-tuberculosis granule of this invention is a set of detection methods specifically for anti-tuberculosis granule products and has specificity for the effective components contained therein, realizing targeted detection of the quality of anti-tuberculosis granules, which is beneficial to ensuring product quality stability and improving the accuracy of quality control.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically, it relates to a quality testing method for an anti-tuberculosis granule. Background Technology

[0002] Anti-tuberculosis granules are a traditional Chinese medicine composed of dwarf tea, stemona root, mulberry bark, clematis root, five-finger peach, and bletilla rhizome. It has the effects of promoting blood circulation, removing blood stasis and promoting new tissue growth, and relieving phlegm and cough. It is used for infiltrative pulmonary tuberculosis with blood in the sputum.

[0003] The preparation method of the anti-tuberculosis granules is as follows: The above six raw medicinal materials are decocted twice with water, each time for 3 hours. The decoctions are combined, filtered, and the filtrate is concentrated into a thick paste. Ethanol is added to make the ethanol content reach 35-45%, stirred well, and allowed to stand for 24 hours. The filtrate is then filtered, concentrated into a thick paste, and an appropriate amount of sucrose powder is added. The mixture is then mixed well, made into granules or pressed into blocks, and dried to obtain the final product.

[0004] Currently, the quality standard for anti-tuberculosis granules refers to Volume 7 of "Traditional Chinese Medicine Compound Preparations," standard number WS3-B-1337-93. The identification method simply involves dividing the processed filtrate into three portions, adding potassium iodide test solution, potassium mercuric sulfonate test solution, and silicotungstic acid test solution to each portion respectively to generate precipitates for identification. The current quality identification standard for anti-tuberculosis granules lacks identification of its active ingredients, and the quality standard needs further improvement, as does the quality control of anti-tuberculosis granules.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a quality testing method for anti-tuberculosis granules. This method is specific to the effective ingredients contained in the anti-tuberculosis granules, realizes the quality testing of anti-tuberculosis granules, helps to ensure the stability of product quality, and also improves the accuracy of quality control.

[0007] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0008] The purpose of this invention is to provide a quality testing method for anti-tuberculosis granules, comprising:

[0009] (1) Preparation of test solution for anti-tuberculosis granules and reference medicinal material solution of *Lysimachia christinae*;

[0010] (2) The test solution and the reference medicinal material solution of *Sclerotium affine* were spotted on the same thin-layer plate and identified by thin-layer chromatography using a mixed solution of butyl acetate-formic acid-water as the developing solvent; the volume ratio of butyl acetate:formic acid:water in the developing solvent was 15:4:1.

[0011] This invention explores a method for thin-layer chromatography identification of *Chuanposhi* (a type of mineral) in anti-tuberculosis granules. The method uses a butyl acetate-formic acid-water mixture (15:4:1, v / v) as the developing solvent, resulting in good development. This method produces good separation, with clear, round spots, moderate shift values, and good reproducibility.

[0012] In a further embodiment, step (1) of the method for preparing the test solution of the antituberculosis granules includes:

[0013] (1) Take the anti-tuberculosis granules, add ethyl acetate, heat under reflux, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol;

[0014] (2) Evaporate the solution prepared in step (1) to dryness, dissolve the residue in water, extract with petroleum ether by shaking, discard the petroleum ether layer, extract the aqueous layer with ethyl acetate by shaking, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution.

[0015] Preferably, in step (2), when adding petroleum ether and shaking for extraction, the temperature is controlled at 30-60℃;

[0016] Preferably, in step (2), ethyl acetate is used for two shaken extractions, the ethyl acetate solutions are combined, and then evaporated to dryness.

[0017] In the preparation method of the test solution of the present invention, the ethyl acetate fraction is extracted and purified by petroleum ether extraction, which reduces interference, facilitates better separation, and yields a chromatogram with round and clear spots, and has good reproducibility.

[0018] Preferably, in step (1), the preparation of the *Smilax china* reference medicinal material solution using the method of preparing the test sample solution includes:

[0019] Take the reference herb *Scutellaria baicalensis*, add ethyl acetate, heat under reflux, filter, evaporate the filtrate to dryness, dissolve the residue in methanol; then evaporate the solution to dryness, dissolve the residue in water, extract with petroleum ether by shaking, discard the petroleum ether layer, extract the aqueous layer with ethyl acetate by shaking, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use this as the reference herb solution.

[0020] A further method involves developing the sample with a developing solvent, removing it, air-drying it, spraying it with aluminum trichloride solution, and examining it under a 365nm ultraviolet light.

[0021] Further measures include quality testing methods for anti-tuberculosis granules, such as:

[0022] (1) Preparation of test solution for anti-tuberculosis granules and reference medicinal material solution of Prunus pubescens;

[0023] (2) Apply the test sample solution and the solution of the medicinal material of Ficus hirta Vahl. var. palmatiloba (Merr.) S. S. Chang for control respectively on the same thin-layer plate, and use the mixed solution of n-hexane and ethyl acetate as the developing solvent for thin-layer chromatography identification; in the said developing solvent, the volume ratio of n-hexane to ethyl acetate is 4:1.5.

[0024] The present invention has explored a method for thin-layer chromatography identification of Ficus hirta Vahl. var. palmatiloba (Merr.) S. S. Chang in the anti-tuberculosis granules. Among them, using n-hexane-ethyl acetate with a volume ratio of 4:1.5 as the developing solvent has a good developing effect. This method has good separation effect, the spots are round, clear, the Rf value is moderate, and the reproducibility is good.

[0025] For a further solution, in step (1), the method for preparing the test sample solution of the anti-tuberculosis granules includes:

[0026] Take the anti-tuberculosis granules, grind them finely, add ethyl acetate, heat under reflux, filter, evaporate the filtrate to dryness, dissolve the residue in methanol to obtain the test sample solution;

[0027] Preferably, in step (1), take the control medicinal material of Cudrania cochinchinensis (Lour.) Kudo & Masam., and prepare the control medicinal material solution according to the same method as the test sample solution, including: <00......​​​​​​​​​​​​​​​​​​​​​In a further embodiment, step (1) of the method for preparing the test solution of the antituberculosis granules includes:

[0036] Take the anti-tuberculosis granules, grind them into a fine powder, add ammonia water to moisten them, add chloroform, reflux to extract, cool, filter, evaporate the filtrate to dryness, add methanol to dissolve the residue, and use it as the test solution.

[0037] The test solution prepared by the above method has less interference, which is conducive to better separation, obtaining a chromatogram with round and clear spots, and good reproducibility.

[0038] Preferably, a reference medicinal material, Stemona japonica, is taken and prepared into a reference medicinal material solution using the same method as the test solution, including:

[0039] Grind into a fine powder, moisten with ammonia, add chloroform, reflux to extract, cool, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use as a reference solution.

[0040] Preferably, reference solutions are prepared by taking upright Stemona japonica and opposite-leaved Stemona japonica reference materials, respectively.

[0041] Further measures include quality testing methods for anti-tuberculosis granules, such as:

[0042] (1) Preparation of test solution and reference solution of antituberculosis granules;

[0043] (2) Accurately inject the reference solution and the test solution into the liquid chromatograph and determine them according to the chromatographic conditions; judge the quality of the anti-tuberculosis granules by calculating the content of dwarf tea as bergenin;

[0044] Preferably, the chromatographic conditions include: using octadecylsilane-bonded silica gel as the packing material; using a methanol-water mixture as the mobile phase, with a methanol:water volume ratio of 15-19:85-81, preferably 17:83; using a detection wavelength of 273 nm; and having a theoretical plate number calculated based on the Bergenin peak that is not less than 3000.

[0045] This invention also explores a high-performance liquid chromatography method for the quantitative detection of *Achyranthes bidentata* in anti-tuberculosis granules. It explores suitable conditions such as mobile phase, chromatographic column, detection wavelength, theoretical plate number, and preparation of test sample and reference standard. The overall method has good separation effect, high sensitivity, and good accuracy.

[0046] In a further step, the preparation method of the test solution of the antituberculosis granules in step (1) includes:

[0047] Take the anti-tuberculosis powder, grind it into a fine powder, sieve it, accurately weigh it, accurately add anhydrous methanol, weigh it, sonicate it, cool it, weigh it again, make up the lost weight with anhydrous methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0048] Preferably, the conditions for the ultrasonic treatment include: power 700-900W, frequency 30-50kHz, and ultrasonic treatment for 40-50 minutes; more preferably, power 800W, frequency 40kHz, and ultrasonic treatment for 45 minutes.

[0049] Preferably, in step (1), the method for preparing the reference solution includes: taking an appropriate amount of Bergenin reference standard, accurately weighing it, and adding anhydrous methanol to prepare a solution containing 0.2 mg per 1 ml.

[0050] Preferably, each 14g of antituberculosis powder contains not less than 6mg of bergenin from dwarf tea.

[0051] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0052] 1. The quality testing method for anti-tuberculosis granules of the present invention is a set of testing methods specifically for anti-tuberculosis granule products and has exclusive testing methods for the effective ingredients contained therein. It realizes targeted testing of the quality of anti-tuberculosis granules, which helps to ensure the stability of product quality and improves the accuracy of quality control.

[0053] 2. This invention has explored thin-layer chromatography identification methods for *Chuanposhi*, *Wuzhimaotao*, and *Baibu* in anti-tuberculosis granules, respectively, and obtained suitable developing solvent systems, preparation conditions for test samples and reference standards, etc., so that the sample interference is small, the overall separation effect of the method is good, the spots are round and clear, the specific shift value is moderate, and the reproducibility is good.

[0054] 3. This invention also explores a high-performance liquid chromatography method for the quantitative detection of *Achyranthes bidentata* in anti-tuberculosis granules. It explores suitable conditions such as mobile phase, chromatographic column, detection wavelength, theoretical plate number, and preparation of test sample and reference standard. The overall method has good separation effect, high sensitivity, and good accuracy.

[0055] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0056] The accompanying drawings, as part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0057] Figure 1 These are TLC images of *Ficus hirta* identified using different developing solvents in Experiment Example 1 of this invention;

[0058] Figure 2The TLC images of different brands of prefabricated boards used for the identification of *Ficus hirta* in Experiment Example 1 of this invention are shown.

[0059] Figure 3 In Experiment 1 of this invention, the identification of *Ficus hirta* was performed using TLC images at different temperatures and humidity levels;

[0060] Figure 4 In Experiment 1 of this invention, TLC images were obtained using different sample amounts for the identification of *Ficus hirta*.

[0061] Figure 5 These are the TLC images of four batches of samples from *Ficus hirta* identified in Experiment Example 1 of this invention;

[0062] Figure 6 These are TLC images of the stone identification using different developing solvents in Experiment Example 2 of this invention;

[0063] Figure 7 The TLC images of precast concrete panels from different brands were used for the identification of the penetrating stone in Experiment Example 2 of this invention.

[0064] Figure 8 In Experiment 2 of this invention, TLC images at different temperatures and humidity were used to identify the perforating stone.

[0065] Figure 9 In Experiment 2 of this invention, TLC images with different sample amounts were used for the identification of the perforating stone.

[0066] Figure 10 These are the TLC images of four batches of samples identified by the stone-piercing stone in Experiment Example 2 of this invention;

[0067] Figure 11 These are TLC chromatograms of Stemona japonica identified using different developing solvents in Experiment Example 3 of this invention;

[0068] Figure 12 The TLC images of different brands of prefabricated panels used for the identification of *Stemona japonica* in Experiment Example 3 of this invention are shown.

[0069] Figure 13 In Experiment 3 of this invention, the identification of Stemona japonica was performed using TLC images at different temperatures and humidity levels;

[0070] Figure 14 In Experiment 3 of this invention, TLC images with different sample amounts were used for the identification of Stemona japonica;

[0071] Figure 15 These are the TLC images of four batches of samples from the identification of Stemona japonica in Experiment Example 3 of this invention;

[0072] Figure 16 This is the full-wavelength ultraviolet absorption scan of Bergenin in Experiment Example 4 of this invention;

[0073] Figure 17This is the high-performance liquid chromatogram of the bergenin reference solution under the specificity item of Test Example 4 of this invention;

[0074] Figure 18 This is the high-performance liquid chromatogram of the negative control sample solution of *Tea dwarf tea* under the specificity item of Experiment Example 4 of this invention;

[0075] Figure 19 This is the high-performance liquid chromatogram of batch solution of test sample 190801 under the specificity item of Test Example 4 of this invention; wherein, Bergenin t R =18.993 R=14.595 T=1.101 n=7.879×10 3 ;

[0076] Figure 20 This is the standard curve diagram in Experiment Example 4 of this invention;

[0077] Figure 21 This is the high-performance liquid chromatogram of batch 190801 of the test sample solution in the scope item of Test Example 4 of this invention, wherein a is the high-performance liquid chromatogram of batch 190801 of the test sample solution (2→25, lower limit), and bergenin t R =18.981 R=13.057T=1.073 n=7.861×10 3 b is the high-performance liquid chromatogram of the test sample 190801 batch solution (6→25, upper limit), bergenin t R =18.953 R=6.799 T=1.125 n=8.585×10 3 ;

[0078] Figure 22 This is the chromatogram of the CAPCELL PAK MG C18 column used in Experimental Example 4 of this invention. a is the high-performance liquid chromatogram of the bergenin reference solution, and bergenin t... R =18.108 T=1.077 n=7.708×10 3 b is the high-performance liquid chromatogram of batch 190201 of the test sample, containing bergenin t. R =18.097 R=13.896 T=1.083 n=7.825×10 3 c is the high-performance liquid chromatogram of batch 190202 of the test sample solution, containing bergenin t. R =18.102 R=9.075 T=1.066 n=7.845×10 3 ;d is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t R =18.087 R=13.530T=1.066 n=7.948×10 3 ;

[0079] Figure 23 This is the chromatogram of the Phenomenex Luna C18 column used in Experimental Example 4 of this invention. 'a' is the high-performance liquid chromatogram of the bergenin reference solution, and 't' is the bergenin chromatogram. R =16.862 T=1.105 n=3.620×10 3 b is the high-performance liquid chromatogram of batch 190201 of the test sample, containing bergenin t. R =16.528 R=2.432 T=1.144 n=3.756×10 3 c is the high-performance liquid chromatogram of batch 190202 of the test sample solution, containing bergenin t. R =16.631 R=2.198 T=1.155 n=3.680×10 3 ;d is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t R =16.524 R=2.776 T=1.139 n=3.786×10 3 ;

[0080] Figure 24 This is the chromatogram of the Waters-Sunfire C18 column used in Experimental Example 4 of this invention. 'a' is the high-performance liquid chromatogram of the bergenin reference solution, and 't' is the bergenin chromatogram. R =17.123 T=1.108 n=6.664×10 3 b is the high-performance liquid chromatogram of batch 190201 of the test sample, containing bergenin t. R =17.128 R=4.286 T=1.132 n=6.974×10 3 c is the high-performance liquid chromatogram of batch 190202 of the test sample solution, containing bergenin t. R =17.127 R=4.859 T=1.138 n=6.971×10 3 ;d is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t R =17.039 R=4.473 T=1.137 n=7.027×10 3 ;

[0081] Figure 25 This is the high-performance liquid chromatography (HPLC) chromatogram of Example 4 of this invention, using methanol-water 15:85 as the mobile phase. In this chromatogram, 'a' is the HPLC chromatogram of the bergenin reference solution, and 't' is the bergenin chromatogram. R =24.253 T=0.982 n=1.040×10 4 b is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t.R =24.249 R=20.366 T=0.989 n=1.070×10 4 ;

[0082] Figure 26 This is the high-performance liquid chromatography (HPLC) chromatogram of the methanol-water 17:83 mobile phase in Experimental Example 4 of this invention, where a is the HPLC chromatogram of the bergenin reference solution, and bergenin t R =18.710 T=0.981 n=9.005×10 3 b is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t. R =18.671 R=6.851 T=0.991 n=9.317×10 3 ;

[0083] Figure 27 This is the high-performance liquid chromatography (HPLC) chromatogram of the methanol-water 19:81 mobile phase used in Experimental Example 4 of this invention, where a is the HPLC chromatogram of the bergenin reference solution, and bergenin t R =14.623 T=0.983 n=7.670×10 3 b is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t. R =14.609 R=5.222 T=0.985 n=7.955×10 3 ;

[0084] Figure 28 This is the high-performance liquid chromatogram (HPLC) of the column temperature 25°C in Experimental Example 4 of this invention, where a is the HPLC chromatogram of the bergenin reference solution, and bergenin t R =22.217 T=1.010 n=9.807×10 3 b is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t. R =22.216 R=18.888 T=1.004 n=1.003×10 4 ;

[0085] Figure 29 This is the high-performance liquid chromatogram (HPLC) of Example 4 of the present invention at a column temperature of 35°C, where a is the HPLC chromatogram of the bergenin reference solution, and bergenin t... R =15.818 T=0.949 n=7.983×10 3 b is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t. R =15.827 R=2.5322 T=0.963 n=8.360×10 3;

[0086] Figure 30 This is the high-performance liquid chromatography (HPLC) chromatogram of the sample in Example 4 of this invention at a flow rate of 0.9 ml / min, where a is the HPLC chromatogram of the bergenin reference solution, and bergenin t R =20.098 T=1.066 n=8.050×10 3 b is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t. R =20.114 R=13.574 T=1.063 n=8.014×10 3 ;

[0087] Figure 31 This is the high-performance liquid chromatography (HPLC) chromatogram of the sample in Example 4 of this invention at a flow rate of 1.1 ml / min, where a is the HPLC chromatogram of the bergenin reference solution, and bergenin t R =16.647 T=1.078 n=7.414×10 3 b is the high-performance liquid chromatogram of batch 190801 of the test sample, containing bergenin t. R =16.660 R=5.683 T=1.115 n=7.361×10 3 .

[0088] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0089] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0090] The formula and preparation method of the anti-tuberculosis granules are as follows:

[0091] [Prescription] 500g of *Dwarf Tea*, 200g of *Stemona japonica*, 100g of *Morus alba* root bark.

[0092] 200g of *Smilax china*, 200g of *Ficus hirta*, 200g of *Bletilla striata*

[0093] [Preparation] Boil the above six ingredients twice with water, 3 hours each time. Combine the decoctions, filter, and concentrate the filtrate to a thick paste. Add ethanol to make the ethanol content reach 35-45%, stir well, let stand for 24 hours, filter, concentrate the filtrate to a thick paste, add an appropriate amount of sucrose powder, mix well, make into granules or press into blocks, and dry to obtain the final product.

[0094] Quality Inspection of Anti-Tuberculosis Granules in Example 1

[0095] (1) Preparation of test solution: Take 37 g of the finished product content of Anti-Tuberculosis Granules, grind it finely, add 100 ml of ethyl acetate, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of methanol to obtain the test solution.

[0096] Preparation of control medicinal material solution: Take 2 g of Ficus hirta Vahl. control medicinal material (National Institutes for Food and Drug Control, batch number: 121486 - 201202), and prepare the control medicinal material solution in the same method as the test solution.

[0097] (2) Test according to the thin-layer chromatography method (General Rules 0502, Volume IV of Chinese Pharmacopoeia 2020 Edition).吸取供试品溶液和对照药材溶液各5~10μl,分别点于同一硅胶G薄层板上,以正己烷-乙酸乙酯(4:1.5)为展开剂,展开,取出,晾干,喷以10%氢氧化钾甲醇溶液,置紫外光灯(365nm)下检视。供试品色谱中,在与对照药材色谱相应的位置上,显相同颜色的荧光斑点,则合格,否则不合格。Take 5 - 10 μl each of the test solution and the control medicinal material solution, respectively spot them on the same silica gel G thin-layer plate, use n-hexane - ethyl acetate (4:1.5) as the developing agent, develop, take out, dry, spray with 10% potassium hydroxide methanol solution, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, if there are fluorescent spots of the same color at the corresponding positions as the chromatogram of the control medicinal material, it is qualified; otherwise, it is unqualified.

[0098] Quality Inspection of Anti-Tuberculosis Granules in Example 2

[0099] (1) Preparation of test solution: Take 37 g of the finished product content of Anti-Tuberculosis Granules, grind it finely, add 100 ml of ethyl acetate, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of methanol; then evaporate to dryness, dissolve the residue in 5 ml of water, shake and extract with 10 ml of petroleum ether (30 - 60 °C), discard the petroleum ether layer, extract the water layer with ethyl acetate by shaking twice, each time with 10 ml, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 2 ml of methanol to obtain the test solution.

[0100] Preparation of control medicinal material solution: Take 3 g of Cudrania cochinchinensis (Lour.) Kudo & Masam. control medicinal material, and prepare the control medicinal material solution in the same method as the test solution.

[0101] (2) Test according to the thin-layer chromatography method (General Rules 0502, Volume IV of Chinese Pharmacopoeia 2020 Edition).吸取上述供试品溶液和对照药材溶液各5~10μl,分别点于同一硅胶G薄层板上,以乙酸丁酯-甲酸-水(15:4:1)为展开剂,展开,取出,晾干,喷以三氯化铝试液,置紫外光灯(365nm)下检视。供试品色谱中,在与对照药材色谱相应的位置上,显相同颜色的荧光斑点,则合格,否则不合格。Take 5 - 10 μl each of the above-mentioned test solution and the control medicinal material solution, respectively spot them on the same silica gel G thin-layer plate, use butyl acetate - formic acid - water (15:4:1) as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, if there are fluorescent spots of the same color at the corresponding positions as the chromatogram of the control medicinal material, it is qualified; otherwise, it is unqualified.

[0102] Quality Inspection of Anti-Tuberculosis Granules in Example 3

[0103] (1) Preparation of test solution: Take 25 g of the finished product of Kanglechongji, grind it finely, moisten it with 10 ml of ammonia water, add 50 ml of chloroform, reflux for 1 hour, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0104] Preparation of control medicinal material solution: Take 1 g of Stemona sessilifolia (Miq.) Miq. as control medicinal material and 1 g of Stemona tuberosa Lour. as control medicinal material, and prepare the control medicinal material solution according to the same method as the test solution.

[0105] (2) Test according to the thin-layer chromatography method (General Principles 0502, Section IV of Chinese Pharmacopoeia 2020 Edition).吸取供试品溶液5μl~10μl、对照药材溶液各2μl,分别点于同一硅胶G薄层板上,以二甲苯-丙酮-甲醇(8:3:0.5)为展开剂,展开,取出,晾干,喷以稀碘化铋钾试液,日光下检视。供试品色谱中,在与对照药材色谱相应的位置上,显相同颜色的斑点,则合格,否则不合格。

[0106] Example 4 Quality Inspection of Kanglechongji

[0107] Determination was carried out according to the high performance liquid chromatography method (General Principles 0512, Section IV of Chinese Pharmacopoeia 2020 Edition).

[0108] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; methanol-water (17:83) as the mobile phase; the detection wavelength is 273 nm. The number of theoretical plates calculated with respect to the bergenin peak should be not less than 3000.

[0109] Preparation of reference solution: Take an appropriate amount of bergenin reference substance, weigh it accurately, and make a solution containing 0.2 mg per 1 ml with anhydrous methanol to obtain the reference solution.

[0110] Preparation of test solution: Take an appropriate amount of this product, mix well, grind it finely, sieve through a 60-mesh sieve, take about 4 g, weigh it accurately, accurately add 25 ml of anhydrous methanol, weigh, ultrasonically treat (power 800 W, frequency 40 kHz) for 45 minutes, cool, weigh again, make up the lost weight with anhydrous methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0111] Determination method: Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph for determination to obtain the result.

[0112] Each bag (14 g) of the Kanglechongji product contains Ardisia japonica (Thunb.) Blume calculated as bergenin (C 14 H 16 O9) not less than 6 mg.

[0113] Test Example 1 Screening of Conditions for Thin-Layer Chromatographic Identification of Ficus hirta Vahl

[0114] Preparation of the test solution: Take 37g of the contents of this product, grind it into a fine powder, add 100ml of ethyl acetate, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 2ml of methanol to obtain the test solution.

[0115] Preparation of the reference herb solution: Take 2g of *Ficus hirta* (China National Institutes for Food and Drug Control, batch number: 121486-201202) as the reference herb and prepare the reference herb solution using the same method.

[0116] Preparation of negative control sample without five-finger peach: Take 2.5 prescription amounts of this product, without five-finger peach, extract and prepare according to the process method under

Preparation Method

[0117] Prepare a negative control solution using the same method as the proposed method for preparing the test solution.

[0118] Test according to thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502).

[0119] (1) Investigate the development effect of different developing agents.

[0120] Samples for each lane: 1-3. Test samples (batch numbers: 190201, 190202, 190801); 4. Prunus pubescens reference material; 5. Negative control without Prunus pubescens; A. Yellow fluorescent spots;

[0121] Chromatographic conditions: Silica gel G thin-layer pre-prepared plate, manufacturer: Sinopharm Group Co., Ltd., batch number 20171123, size: 10cm×10cm; spotting: round dots, spotting volume: 1~3.10μl, 4.5μl, 5.10μl; temperature: 25℃; relative humidity: 60%;

[0122] Developing solvents: ① n-hexane-ethyl acetate (4:1.5); ② n-hexane-butyl acetate (4:1); ③ cyclohexane-butyl acetate (4:1)

[0123] Color development and inspection: Spray with 10% potassium hydroxide methanol solution and inspect under ultraviolet light (365nm).

[0124] By comparing three developing solvents—n-hexane-ethyl acetate (4:1.5), n-hexane-butyl acetate (4:1), and cyclohexane-butyl acetate (4:1)—the results showed that n-hexane-ethyl acetate (4:1.5) provided the best development effect, good separation, and clear spots. See the chromatogram for details. Figure 1 .

[0125] (2) Examine the unfolding effect of precast panels from different brands

[0126] Samples for each lane: 1-3. Test sample (batch number: 190201, 190202, 190801), 4. *Ficus hirta* reference material, 5. Negative control without *Ficus hirta*, A. Yellow fluorescent spots

[0127] Chromatographic conditions: Silica gel G thin-layer pre-prepared plate, size: 10cm×10cm; ① Manufacturer: Sinopharm Group Co., Ltd., batch number: 20171123; ② Manufacturer: Qingdao Ocean Chemical Co., Ltd., batch number: 20170808; ③ Manufacturer: Qingdao Yuminyuan Silica Gel Reagent Factory, batch number: 20190723; Spotting: round dots, spotting volume: 1~3.10μl, 4.5μl, 5.10μl; Temperature: 25℃; Relative humidity: 60%;

[0128] Developing solvent: n-hexane-ethyl acetate (4:1.5)

[0129] Developing and inspecting: Spray with 10% potassium hydroxide methanol solution and inspect under ultraviolet light (365nm).

[0130] The development effects of different brands of pre-prepared plates were compared. The results showed that all spots were distinct and the separation was good, indicating that this thin-layer chromatography identification method is robust. See chromatograms for details. Figure 2 .

[0131] (3) Investigate the unfolding effect under different temperature and humidity conditions.

[0132] Samples for each lane: 1-3. Test sample (batch number: 190201, 190202, 190801), 4. *Ficus hirta* reference material, 5. Negative control without *Ficus hirta*, A. Yellow fluorescent spots

[0133] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, manufacturer: Sinopharm Group Co., Ltd., batch number 20171123, size: 10cm×10cm;

[0134] Spotting in round dots, sample volume: 1–3.10 μl, 4.5 μl, 5.10 μl;

[0135] Temperature and humidity for each group: ①t: 4℃, RH: 60%; ②t: 25℃, RH: 60%; ③t: 35℃, RH: 60%; ④t: 25℃, RH: 30%; ⑤t: 25℃, RH: 90%;

[0136] Developing solvent: n-hexane-ethyl acetate (4:1.5);

[0137] Developing and inspecting: Spray with 10% potassium hydroxide methanol solution and inspect under ultraviolet light (365nm).

[0138] The development effects under different temperature and humidity conditions were compared. The results showed that all spots were distinct and the separation was good, indicating that the thin-layer chromatography identification method has good robustness. See details. Figure 3 .

[0139] (4) Investigate the development effect of different spotting amounts of the test solution.

[0140] Samples for each lane: 1-2. Test sample, batch number: 190201; 3-4. Test sample, batch number: 190202; 5-6. Test sample, batch number: 190801

[0141] 7. Five-finger peach control material; 8. Negative control without five-finger peach; A. Yellow fluorescent spots.

[0142] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, manufacturer: Sinopharm Group Co., Ltd., batch number 20171123, size: 10cm×20cm;

[0143] Spotting in round shapes: 5 μl for 1, 3, 5, and 7; 10 μl for 2, 4, 6, and 8.

[0144] Temperature: 25℃; Relative humidity: 60%

[0145] Developing solvent: n-hexane-ethyl acetate (4:1.5)

[0146] Developing and inspecting: Spray with 10% potassium hydroxide methanol solution and inspect under ultraviolet light (365nm).

[0147] The development effects of different sample amounts were compared. The results showed that sample amounts of 5–10 μl produced clear chromatograms, which was considered the optimal sample amount. See chromatograms for details. Figure 4 .

[0148] (5) Batch repeatability test

[0149] Samples for each lane: 1-4. Test samples (batch numbers: 181101, 190201, 190202, 190801), 5. *Ficus hirta* reference material, 6. Negative control without *Ficus hirta*, A. Yellow fluorescent spots

[0150] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, manufacturer: Sinopharm Group Co., Ltd., batch number 20171123, size: 10cm×10cm;

[0151] Spotting in round dots, sample volume: 1–4.10 μl, 5.5 μl, 6.10 μl

[0152] Temperature: 20.1℃; Relative humidity: 72%

[0153] Developing solvent: n-hexane-ethyl acetate (4:1.5)

[0154] Developing and inspecting: Spray with 10% potassium hydroxide methanol solution and inspect under ultraviolet light (365nm).

[0155] Four batches of samples were tested according to the method in Example 1. The results showed that the thin-layer chromatography separation was good, the spots were round and clear, the specific migration value was moderate, and the reproducibility was good. See the chromatograms for details. Figure 5 .

[0156] In summary, the results of the above methods show that the thin-layer chromatography separation of Prunus armeniaca using the method in Example 1 is effective, with clear and distinct spots, good reproducibility and specificity, and no interference from the negative control. All four batches of samples tested using this method met the requirements.

[0157] Experimental Example 2: Screening of Thin-Layer Chromatographic Identification Conditions for Perforator

[0158] (1) Preparation of test solution: Take 37g of the contents of the antituberculosis granules, grind finely, add 100ml of ethyl acetate, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue; then evaporate to dryness, add 5ml of water to dissolve the residue, extract with 10ml of petroleum ether (30-60℃) by shaking, discard the petroleum ether layer, extract the aqueous layer with ethyl acetate twice by shaking, 10ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 2ml of methanol to dissolve the residue, and use as the test solution.

[0159] Preparation of reference medicinal material solution: Take 3g of *Stonecrops chuanpo* reference medicinal material (China National Institutes for Food and Drug Control, batch number: 121727-201601) and prepare the reference medicinal material solution using the same method.

[0160] Preparation of negative control sample lacking *Cyclocarya paliurus*: Take 2.5 times the prescribed amount of medicinal material, without adding *Cyclocarya paliurus*, and extract and prepare it according to the process method under the

Preparation Method

[0161] Test according to thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502).

[0162] (1) Investigate the development effect of different developing agents.

[0163] Samples for each lane: 1-3. Test sample (batch numbers: 190201, 190202, 190801), 4. *Cyclocarya paliurus* reference material, 5. Negative control without *Cyclocarya paliurus*, A. Blue-green fluorescent spots

[0164] Chromatographic conditions: Silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Yuminyuan Silica Gel Reagent Factory, batch number 20190723, size: 10cm×10cm;

[0165] Spotting in round shapes; sample volume: 1–5.5 μl; temperature: 23.2℃; relative humidity: 63%;

[0166] Developing solvents: ① Butyl acetate-formic acid-water (15:4:1), ② Ethyl acetate-formic acid-water (20:3:0.2),

[0167] ③ Dichloromethane-ethyl acetate-methanol-formic acid (3:4.5:1:0.5)

[0168] Color development and inspection: Spray with aluminum trichloride solution and inspect under ultraviolet light (365nm).

[0169] Three developing solvents were compared: butyl acetate-formic acid-water (15:4:1), ethyl acetate-formic acid-water (20:3:0.2), and dichloromethane-ethyl acetate-methanol-formic acid (3:4.5:1:0.5). The results showed that butyl acetate-formic acid-water (15:4:1) provided the best developing effect. See chromatogram for details. Figure 6 .

[0170] (2) Examine the unfolding effect of precast panels from different brands

[0171] Samples for each lane: 1-3. Test sample (batch numbers: 190201, 190202, 190801), 4. *Cyclocarya paliurus* reference material, 5. Negative control without *Cyclocarya paliurus*, A. Blue-green fluorescent spots

[0172] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, size: 10cm×10cm;

[0173] ① Manufacturer: Qingdao Yuminyuan Silica Gel Reagent Factory, Batch No.: 20190723

[0174] ② Manufacturer: Sinopharm Group Co., Ltd., Batch No.: 20171123

[0175] ③ Manufacturer: Merck KGaA, Batch No.: HX73381626

[0176] Spotting in round shapes; sample volume: 1–5.5 μl; temperature: 23.2℃; relative humidity: 63%;

[0177] Developing solvent: Butyl acetate-formic acid-water (15:4:1)

[0178] Color development and inspection: Spray with aluminum trichloride solution and inspect under ultraviolet light (365nm).

[0179] The development effects of different brands of pre-prepared plates were compared. The results showed that all spots were distinct and the separation was good, indicating that this thin-layer chromatography identification method is robust. See chromatograms for details. Figure 7 .

[0180] (3) Investigate the unfolding effect under different temperature and humidity conditions.

[0181] Samples for each lane: 1-3. Test samples (batch numbers: 190201, 190202, 190801), 4. *Cyclocarya paliurus* reference material, 5. Negative control without *Cyclocarya paliurus*, A. Blue-green fluorescent spots

[0182] Chromatographic conditions: Silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Yuminyuan Silica Gel Reagent Factory, batch number 20190723, size: 10cm×10cm;

[0183] Spotting in round dots, sample volume: 1–5.5 μl

[0184] Temperature and humidity for each group: ①t: 4℃, RH: 60%; ②t: 25℃, RH: 60%; ③t: 35℃, RH: 60%; ④t: 25℃, RH: 30%; ⑤t: 25℃, RH: 90%;

[0185] Developing solvent: Butyl acetate-formic acid-water (15:4:1);

[0186] Color development and inspection: Spray with aluminum trichloride solution and inspect under ultraviolet light (365nm).

[0187] The development effects under different temperature and humidity conditions were compared. The results showed that all spots were distinct and the separation was good, indicating that the thin-layer chromatography identification method has good robustness. See chromatograms for details. Figure 8 .

[0188] (4) Investigate the development effect of different spotting amounts of the test solution.

[0189] Samples for each lane: 1-2. Test sample, batch number: 190201; 3-4. Test sample, batch number: 190202; 5-6. Test sample, batch number: 190801; 7. *Scutellaria baicalensis* control material; 8. Negative control without *Scutellaria baicalensis*, A. Blue-green fluorescent spots.

[0190] Chromatographic conditions: Silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Yuminyuan Silica Gel Reagent Factory, batch number 20190723, size: 10cm×20cm;

[0191] Spotting in round shapes: 5 μl for 1, 3, 5, and 7; 10 μl for 2, 4, 6, and 8.

[0192] Temperature: 25℃; Relative humidity: 60%;

[0193] Developing solvent: Butyl acetate-formic acid-water (15:4:1)

[0194] Color development and inspection: Spray with aluminum trichloride solution and inspect under ultraviolet light (365nm).

[0195] The development effects of different sample amounts were compared. The results showed that sample amounts of 5–10 μl produced clear chromatograms, which was considered the optimal sample amount. See chromatograms for details. Figure 9 .

[0196] (5) Batch repeatability test

[0197] Samples for each lane: 1-4. Test samples (batch numbers: 181101, 190201, 190202, 190801), 5. *Scutellaria baicalensis* reference material, 6. Negative control without *Scutellaria baicalensis*, A. Blue-green fluorescent spots

[0198] Chromatographic conditions: Silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Yuminyuan Silica Gel Reagent Factory, batch number 20190723, size: 10cm×10cm;

[0199] Spotting in round shapes, sample volume: 1–4.10 μl, 5.5 μl, 6.10 μl;

[0200] Temperature: 20.1℃; Relative humidity: 72%;

[0201] Developing solvent: Butyl acetate-formic acid-water (15:4:1);

[0202] Color development and inspection: Spray with aluminum trichloride solution and inspect under ultraviolet light (365nm).

[0203] Four batches of samples were tested using the above method. The results showed that thin-layer chromatography had good separation effect, with round and clear spots, moderate shift values, and good reproducibility. See chromatograms for details. Figure 10 .

[0204] The results, verified by the above methods, show that the thin-layer chromatography separation of *Scleroderma pentaphyllum* shown in Example 2 is effective, with clear and distinct spots, good reproducibility and specificity, and no interference from the negative control. All four batches of samples tested using this method met the requirements.

[0205] Experimental Example 3: Screening of Thin-Layer Chromatographic Identification Conditions for Stemona japonica

[0206] Preparation of test solution: Take 25g of the contents of this product, grind it into a fine powder, add 10ml of ammonia water to moisten it, add 50ml of chloroform, reflux and extract for 1 hour, cool, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0207] Preparation of reference medicinal material solutions: Take 1g of Stemona japonica upright (China National Institutes for Food and Drug Control, batch number: 121588-201502) and 1g of Stemona japonica opposite leaf (China National Institutes for Food and Drug Control, batch number: 121221-201604) and prepare reference medicinal material solutions respectively using the same method.

[0208] Preparation of negative control sample lacking Stemona: Take 2.5 times the prescription amount of this product, without Stemona, and extract and prepare it according to the process method under

Preparation Method

[0209] Test according to thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502).

[0210] (1) Investigate the development effect of different developing agents.

[0211] Samples for each lane: 1-3. Test samples (batch numbers: 181101, 190201, 190202), 4. Reference material of opposite-leaved Stemona japonica, 5. Reference material of erect Stemona japonica, 6. Negative control of Stemona japonica without Stemona japonica, A. Orange-yellow spots

[0212] Chromatographic conditions: Silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Ocean Chemical Co., Ltd., batch number 20170808, specifications: 10cm×10cm round spotting, spotting volume: 1~3.5μl, 4~5.2μl, 6.5μl; temperature: 20.1℃; relative humidity: 72%;

[0213] Developing solvents: ① xylene-acetone-methanol (8:3:0.5), ② n-hexane-chloroform-acetone (5:2:2), ③ cyclohexane-ethyl acetate-acetone-concentrated ammonia (6:4:4:1);

[0214] Developing color and examining: Spray with dilute potassium bismuth iodide test solution and examine under sunlight.

[0215] Three developing solvents were compared: xylene-acetone-methanol (8:3:0.5), n-hexane-chloroform-acetone (5:2:2), and cyclohexane-ethyl acetate-acetone-concentrated ammonia (6:4:4:1). The results showed that xylene-acetone-methanol (8:3:0.5) provided the best developing effect. See chromatogram for details. Figure 11 .

[0216] (2) Examine the unfolding effect of precast panels from different brands

[0217] Samples for each lane: 1-3. Test samples (batch numbers: 181101, 190201, 190202), 4. Reference material of opposite-leaved Stemona japonica, 5. Reference material of erect Stemona japonica, 6. Negative control of Stemona japonica without Stemona japonica, A. Orange-yellow spots

[0218] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, size: 10cm×10cm;

[0219] ① Manufacturer: Qingdao Ocean Chemical Co., Ltd., Batch No.: 20170808

[0220] ② Manufacturer: Sinopharm Group Co., Ltd., Batch No.: 20171123

[0221] ③ Manufacturer: Qingdao Yuminyuan Silica Gel Reagent Factory, Batch No.: 20190723

[0222] Spotting in round shapes; sample volume: 1–3.5 μl, 4–5.2 μl, 6.5 μl; temperature: 20.1℃; relative humidity: 72%.

[0223] Developing solvent: xylene-acetone-methanol (8:3:0.5)

[0224] Developing color and examining: Spray with dilute potassium bismuth iodide test solution and examine under sunlight.

[0225] The development effects of different brands of pre-prepared plates were compared. The results showed that all spots were distinct and the separation was good, indicating that this thin-layer chromatography identification method is robust. See chromatograms for details. Figure 12 .

[0226] (3) Investigate the unfolding effect under different temperature and humidity conditions.

[0227] Samples for each lane: 1-3. Test samples (batch numbers: 181101, 190201, 190202), 4. Reference material of opposite-leaved Stemona japonica, 5. Reference material of erect Stemona japonica, 6. Negative control of Stemona japonica without Stemona japonica, A. Orange-yellow spots

[0228] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Ocean Chemical Co., Ltd., batch number 20170808, size: 10cm×10cm;

[0229] Spotting in round dots, sample volume: 1–3.5 μl, 4–5.2 μl, 6.5 μl;

[0230] Temperature and humidity for each group: ①t: 4℃, RH: 60%; ②t: 25℃, RH: 60%; ③t: 35℃, RH: 60%; ④t: 25℃, RH: 30%; ⑤t: 25℃, RH: 90%;

[0231] Developing solvent: xylene-acetone-methanol (8:3:0.5);

[0232] Developing color and examining: Spray with dilute potassium bismuth iodide test solution and examine under sunlight.

[0233] The development effects under different temperature and humidity conditions were compared. The results showed that all spots were distinct and the separation was good, indicating that the thin-layer chromatography identification method has good robustness. See chromatograms for details. Figure 13 .

[0234] (4) Investigate the development effect of different spotting amounts of the test solution.

[0235] Samples for each lane: 1-2. Test sample, batch number: 181101; 3-4. Test sample, batch number: 190201; 5-6. Test sample, batch number: 190202; 7. Opposite-leaved Stemona japonica reference material; 8. Erect Stemona japonica reference material; 9. Negative control with Stemona japonica absent; A. Orange-yellow spots.

[0236] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Ocean Chemical Co., Ltd., batch number 20170808, size: 10cm×20cm;

[0237] Spotting: 5 μl for samples 1, 3, and 5; 10 μl for samples 2, 4, 6, and 9; 2 μl for samples 7 and 8; Temperature: 16.8℃; Relative humidity: 46%.

[0238] Developing solvent: xylene-acetone-methanol (8:3:0.5);

[0239] Developing color and examining: Spray with dilute potassium bismuth iodide test solution and examine under sunlight.

[0240] The development effects of different sample amounts were compared. The results showed that sample amounts of 5–10 μl produced clear chromatograms, which was considered the optimal sample amount. See chromatograms for details. Figure 14 .

[0241] (5) Batch repeatability test

[0242] Samples for each lane: 1-4. Test samples (batch numbers: 181101, 190201, 190202, 190801), 5. Reference material of opposite-leaved Stemona japonica, 6. Reference material of erect Stemona japonica, 7. Negative control of Stemona japonica, A. Orange-yellow spots

[0243] Chromatographic conditions: silica gel G thin-layer pre-prepared plate, manufacturer: Qingdao Ocean Chemical Co., Ltd., batch number 20170808, size: 10cm×10cm;

[0244] Spotting in round shapes; spotting volume: 1–4.5 μl, 5–6.2 μl, 7.5 μl; temperature: 16.9℃; relative humidity: 73%;

[0245] Developing solvent: xylene-acetone-methanol (8:3:0.5);

[0246] Developing color and examining: Spray with dilute potassium bismuth iodide test solution and examine under sunlight.

[0247] Four batches of samples were tested using the method described above. The results showed that thin-layer chromatography had good separation effect, with round and clear spots, moderate shift values, and good reproducibility. See chromatograms for details. Figure 15 .

[0248] The above methods were verified, and the results showed that the thin-layer chromatography of Stemona japonica had good separation effect, clear and obvious spots, good reproducibility and specificity, and no interference from the negative control. All four batches of samples were tested according to this method and met the requirements.

[0249] Experimental Example 4: Screening of High Performance Liquid Chromatography Conditions for Dwarf Tea

[0250] I. Instruments and Reagents

[0251] Waters e2695 high-performance liquid chromatograph; Waters 2998PDA detector. Numerical control ultrasonic cleaner, model KQ-800DE, Kunshan Ultrasonic Instrument Co., Ltd. Electronic balance, model XP205, METTLER TOLEDO. Electronic balance, model XP404S, METTLER TOLEDO.

[0252] Methanol was chromatographically pure, water was ultrapure water, and all other reagents were analytically pure. Bergenin reference standard (batch number: 111532-201604, content 94.1%) was provided by the China National Institutes for Food and Drug Control for content determination.

[0253] II. Methodological Investigation and Results

[0254] 1. Chromatographic conditions

[0255] 1.1 Column: The column is packed with octadecylsilane-bonded silica gel.

[0256] 1.2 Selection of mobile phase: Methanol-water (17:83) was used as the mobile phase.

[0257] 1.3 Selection of detection wavelength

[0258] The mobile phase was methanol-water (17:83). A bergenin reference solution (concentration 0.2085 mg / ml) was injected into the liquid chromatograph for determination. Data were acquired using a diode matrix detector in the wavelength range of 200–400 nm. Spectral analysis of the chromatographic peaks showed that the maximum absorption wavelength of bergenin was 272.3 nm; therefore, 273 nm was selected as the detection wavelength. The maximum absorption spectrum of the chromatographic peak is shown below. Figure 16 .

[0259] 1.4 Determination of the number of theoretical plates

[0260] Three different brands of chromatographic columns were used: Column 1: CAPCELL PAK MG C18 5μm 4.6×250mm; Column 2: Phenomenex Luna C18 5μm 250×4.60mm; and Column 3: Waters-Sunfire C18 5μm 4.6×250mm. Analysis was performed using a high-performance liquid chromatograph (HPLC) to investigate the theoretical plate number and resolution. The analytical results using a Waters e2695 HPLC system are shown in Table 1, and the chromatograms are shown in [Figure 1]. Figure 22 d, 23d, 24d.

[0261] Table 1 Determination of the number of theoretical plates

[0262]

[0263] According to the experimental results, when the theoretical plate number N is greater than 3000, the resolution between bergenin and its adjacent chromatographic peaks can meet the technical requirements for content determination. The theoretical plate number should be no less than 3000 based on bergenin.

[0264] 2. Specificity test

[0265] Preparation of negative control sample lacking *Lysimachia foetida*: Take 2.5 times the prescription amount of this product, without adding *Lysimachia foetida*, and extract and prepare it according to the process method under [Preparation Method] to obtain the finished product, which is the negative control sample lacking *Lysimachia foetida*. The preparation of the reference standard, negative control standard, and test solution is as described in Example 4.

[0266] Negative interference test: The mobile phase was methanol-water (17:83). The reference standard, negative control sample, and test sample solution were injected into the liquid chromatograph for determination. The results showed that the negative control sample of *Gnaphalium affine* showed no absorption peak at the corresponding position of the bergenin reference standard peak, indicating no interference from the negative control. The chromatogram is shown below. Figures 17-19 .

[0267] 3. Preparation of the test solution

[0268] 3.1 Investigation of Extraction Solvent: Take an appropriate amount of this product, grind it finely, pass it through a 60-mesh sieve, and take about 4g (batch number: 190801). Accurately weigh it and make four parallel portions. For each pair of portions, accurately add 25ml of anhydrous methanol and 25ml of anhydrous ethanol, weigh them, shake well, and sonicate (power 800W, frequency 40KHz) for 45 minutes. After cooling, weigh them again. Make up the weight loss with anhydrous methanol and anhydrous ethanol respectively, shake well, filter, and take the filtrate to obtain the test solution. Inject it into the liquid chromatograph and calculate the content of bergenin. The results are shown in Table 2. The experimental results show that anhydrous methanol is a high-content extractant, so anhydrous methanol is selected as the extraction solvent.

[0269] Table 2. Experimental results of the extraction solvent investigation

[0270]

[0271] 3.2 Investigation of Extraction Method: Take an appropriate amount of this product, mix well, grind finely, pass through a 60-mesh sieve, take about 4g (batch number: 190801), accurately weigh, make 8 parallel portions, accurately add 25ml of anhydrous methanol, weigh, shake well, and treat each pair with ultrasound (power 800W, frequency 40KHz) for 45 minutes, heat under reflux for 2 hours, 2.5 hours, and 3 hours respectively, cool, weigh again, make up the weight loss with anhydrous methanol, shake well, filter, take the filtrate to obtain the test solution, inject into the liquid chromatograph, calculate the content of bergenin, and the determination results are shown in Table 3. The experimental results show that ultrasound for 45 minutes is the extraction method with a higher content, therefore, ultrasound for 45 minutes is selected as the extraction method.

[0272] Table 3. Experimental results of the extraction method investigation

[0273]

[0274] 3.3 Investigation of Solvent Dosage: Take an appropriate amount of this product, mix well, grind finely, and pass through a 60-mesh sieve. Take approximately 4g (batch number: 190801), accurately weigh it, and make four parallel portions. For each of two portions, accurately add 25ml and 50ml of anhydrous methanol, weigh them, shake well, and sonicate (power 800W, frequency 40KHz) for 45 minutes. After cooling, weigh them again, replenish the lost weight with anhydrous methanol, shake well, filter, and take the filtrate to obtain the test solution. Inject the solution into a liquid chromatograph and calculate the content of bergenin. The results are shown in Table 4. Based on the experimental results, the solvent dosage was selected as 25ml.

[0275] Table 4 Results of the test on solvent usage

[0276]

[0277] 3.4 Investigation of Extraction Time: Take an appropriate amount of this product, mix well, grind finely, pass through a 60-mesh sieve, take about 4g (batch number: 190801), accurately weigh, make 6 parallel portions, accurately add 25ml of anhydrous methanol, weigh, shake well, and sonicate each pair (power 800W, frequency 40KHz) for 30 minutes, 45 minutes, and 60 minutes respectively. Cool, weigh again, make up the weight loss with anhydrous methanol, shake well, filter, take the filtrate to obtain the test solution, inject into the liquid chromatograph, calculate the content of bergenin, and the results are shown in Table 5. Based on the experimental results, the extraction time was selected as 45 minutes.

[0278] Table 5 Results of the experiment on extraction time

[0279]

[0280]

[0281] In summary, the preparation method of the test solution is as follows: Take an appropriate amount of this product, mix well, grind finely, pass through a 60-mesh sieve, take about 4g, accurately weigh it, accurately add 25ml of anhydrous methanol, weigh it, sonicate (power 800W, frequency 40KHz) for 45 minutes, cool, weigh it again, replenish the lost weight with anhydrous methanol, shake well, filter, and take the filtrate.

[0282] 4. Examination of linear range

[0283] 4.1 Preparation of reference solution: Accurately weigh 26.8 mg of bergenin reference standard, place it in a 25 ml volumetric flask, add anhydrous methanol to dissolve and dilute to the mark, shake well to obtain bergenin reference solution (1.0088 mg / ml).

[0284] 3.2 Determination of Standard Curve: Accurately pipette 0.5 ml, 0.5 ml, 1 ml, 2 ml, and 5 ml of bergenin reference solution into 100 ml, 10 ml, 10 ml, 10 ml, and 10 ml volumetric flasks, respectively. Dilute to the mark with anhydrous methanol and mix well to obtain reference solutions of different concentrations. Inject 10 μl of each of the above reference solutions under the chromatographic conditions of Example 4. Plot a standard curve with the injection volume (μg) of the reference solution as the abscissa and the average peak area as the ordinate. The results are shown in Table 6. Figure 20 .

[0285] Table 6 Results of the determination of Bergenin standard curve

[0286]

[0287] The regression equation is Y = 1.39 × 10 7 X + 6.05 × 10 4 r = 0.999531.

[0288] The results showed that when the injection amount of Bergenin reference standard was in the range of 0.0504–10.0880 μg, there was a good linear relationship between the injection amount and the peak area.

[0289] 5. Precision test

[0290] Take this product (batch number: 190801), prepare the test solution according to the method in Example 4, and inject it six times consecutively under the chromatographic conditions of Example 4. Calculate the content of bergenin, and the results are shown in Table 7. The table shows that the RSD of the test solution measured on the same instrument is <3%, and the precision meets the experimental requirements.

[0291] Table 7 Precision Test Results

[0292]

[0293] 6. Repeatability test

[0294] Six samples from the same batch (batch number: 190801) were accurately weighed and measured in parallel according to the method in Example 4. Test solutions were prepared, and the content of bergenin was determined. The results are shown in Table 8. The experimental results indicate that this method has good reproducibility.

[0295] Table 8 Results of Repeatability Tests

[0296]

[0297] 7. Spiking recovery test

[0298] Accurately weigh 19.58 mg of bergenin reference standard, place it in a 250 ml volumetric flask, add anhydrous methanol to dissolve and dilute to the mark, shake well to obtain bergenin reference standard solution (0.0737 mg / ml).

[0299] Accurately weigh 2g of a sample (batch number: 190801) with a known content (bergenin content 12.5344mg / bag), prepare six parallel samples, and accurately add 25ml of anhydrous methanol solution of bergenin to each sample. Prepare the test solution according to the method in Example 4, inject it into the liquid chromatograph for analysis, and calculate the recovery rate using the following formula. The average recovery rate was 104.31%, with an RSD of 1.83%. The results are shown in Table 9. The experimental results indicate that the recovery rate is between 90% and 108%, and the recovery is good. The content determination method in Example 4 is feasible.

[0300]

[0301] Table 9 Results of Bergenin Spiking Recovery Test

[0302]

[0303] 8. Scope

[0304] Take samples from the same batch (batch number: 190801), and sample according to the content determination method in Example 4. Take 0.5 times the amount of the sample as the content range for the lower limit of concentration, and take 1.5 times the amount of the sample as the content range for the upper limit of concentration. Prepare 6 samples in total, 2 samples for each concentration, and inject them into the liquid chromatograph. See the chromatogram for details. Figure 17 , 1921a and 21b. The injection content and sample content of bergenin were calculated, and the results are shown in Table 10. The experimental results show that the injection content of bergenin in the test sample is within the lower and upper limits of 0.7774 to 2.3347 μg, which meets the requirements of linear range. The determination results all meet the analytical requirements and there is no significant difference.

[0305] Table 10 Results of Bergenin Range Determination

[0306]

[0307]

[0308] 9. Durability test

[0309] 9.1 Stability test:

[0310] The test solution (batch number: 190801) was injected at 10 μl intervals at time intervals of 0, 1, 2, 6, 12, and 24 hours according to the chromatographic conditions of Example 4. The content of bergenin was calculated, and the results are shown in Table 11. The table shows that the RSD of bergenin in the test solution was 0.32% within 24 hours. The experimental results indicate that the test solution yields relatively stable results within 24 hours.

[0311] Table 11 Stability Test Results

[0312]

[0313] 9.2 Investigation of different chromatographic columns:

[0314] Three batches of this product samples (batch numbers: 190201, 190202, and 190801) were taken. Test solutions were prepared according to the content determination method in Example 4. Three different brands of chromatographic columns were used for testing: column 1: CAPCELL PAK MG C18 5μm 4.6×250mm; column 2: Phenomenex Luna C18 5μm 250×4.60mm; and column 3: Waters-Sunfire C18 5μm 4.6×250mm. The content of bergenin was calculated, and the results are shown in Table 12. The results show that the determination results all met the analytical requirements, and there were no significant differences. See the chromatograms for details. Figures 22-24 .

[0315] Table 12 Results of the investigation of different brands of chromatographic columns

[0316]

[0317] 9.3 Investigation of different mobile phase ratios:

[0318] Take samples from the same batch (batch number: 190801), and prepare test solutions according to the content determination method in Example 4. Inject these solutions into the liquid chromatograph using mobile phase ratios 1: methanol-water (17:83), 2: methanol-water (15:85), and 3: methanol-water (19:81), respectively. See the chromatograms for details. Figures 25-27 The content of bergenin was calculated, and the results are shown in Table 13. The experimental results show that the determination results met the analytical requirements for different mobile phase ratios, and there were no significant differences.

[0319] Table 13 Experimental results for different mobile phase ratios

[0320]

[0321] 9.4 Investigation at different column temperatures:

[0322] Samples from the same batch (batch number: 190801) were used. Test solutions were prepared according to the content determination method in Example 4. Column temperatures were set to 25℃, 30℃, and 35℃, and the solutions were injected into the liquid chromatograph. The content of bergenin was calculated, and the results are shown in Table 14. The experimental results show that the determination results met the analytical requirements when the column temperature fluctuated within the range of 25–35℃, and there were no significant differences. See the chromatogram for details. Figures 28-29 .

[0323] Table 14 Comparison Test Results at Different Column Temperatures

[0324]

[0325] 9.5 Investigation of different flow velocities:

[0326] Samples from the same batch (batch number: 190801) were used. Test solutions were prepared according to the content determination method in Example 4. Flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min were set and injected into the liquid chromatograph. The content of bergenin was calculated. The results are shown in Table 15. Flow rate affects resolution and retention time; see chromatograms for details. Figures 30-31 The results showed that flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min all exhibited good separation capabilities, and the measurement results met the analytical requirements without significant differences, indicating that small changes in flow rate do not affect the effectiveness of the method.

[0327] Table 15 Comparison of test results at different flow velocities

[0328]

[0329] 10. Sample Determination

[0330] The content of bergenin in four batches of samples was determined according to the content determination method in Example 4. The results are shown in Table 16.

[0331] Table 16 Results of the tests on 4 batches of samples

[0332]

[0333] 11. Transfer rate

[0334] The content of bergenin in the finished product made from the medicinal material was determined according to the content determination method in Example 4, and the transfer rate was calculated. The determination results are shown in Table 17.

[0335] Table 17 Investigation on the Transfer Rate of *Tea dwarf tea* Medicinal Materials

[0336]

[0337] 12. Establishment of content limits

[0338] The average transfer rate of the four batches of samples tested above was 62.74%. The 2020 edition of the Pharmacopoeia, Part I, stipulates that the amount of bergenin in *Gnaphalium affine* should not be less than 0.50%. After adjusting for the average transfer rate and the minimum content of bergenin in *Gnaphalium affine*, the theoretical content of bergenin in each bag of this product should not be less than 7.8 mg. Considering fluctuations due to production processes, transfer rates, and other factors, the minimum theoretical content (7.8 mg) is reduced by 20%. Therefore, the content limit for this product is proposed as follows: "Each bag of this product contains bergenin (C...) in *Gnaphalium affine*." 14 H 16 (O9) The content shall not be less than 6.0mg.

[0339] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can explore the scope of the present invention without departing from its technical solution.

[0340] When the above-mentioned technical content can be modified or altered to form equivalent embodiments, any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of the present invention.

Claims

1. A quality testing method for an anti-tuberculosis granule, the granule being composed of *Dwarf Tea*, *Stemona japonica*, *Morus alba* bark, *Smilax china*, *Ficus hirta*, and *Bletilla striata*, characterized in that... Quality testing methods include: 1) Preparation of the test solution for the anti-tuberculosis granules and the reference herb solution of *Ligusticum striatum*; The method for preparing the test solution of the antituberculosis granules includes: S1, take the anti-tuberculosis granules, add ethyl acetate, heat to reflux, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol; S2, evaporate the solution prepared in S1 to dryness, dissolve the residue in water, extract with petroleum ether by shaking, discard the petroleum ether layer, extract the aqueous layer with ethyl acetate by shaking, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol, and use it as the test solution. Take the reference medicinal material *Stone-piercing Stone* and prepare a reference medicinal material solution using the same method as the test solution; 2) Spot the test solution and the reference herb solution of *Scutellaria baicalensis* onto the same thin-layer plate, and use a mixed solution of butyl acetate-formic acid-water as the developing solvent for thin-layer chromatography identification; the volume ratio of butyl acetate:formic acid:water in the developing solvent is 15:4:

1. After developing with a developing solvent, remove the sample, air dry it, spray it with aluminum trichloride solution, and examine it under ultraviolet light.

2. The quality inspection method according to claim 1, characterized in that, In step S2, when adding petroleum ether and shaking for extraction, the temperature is controlled at 30–60°C.

3. The quality inspection method according to claim 1, characterized in that, In step S2, the ethyl acetate was shaken twice, the ethyl acetate extracts were combined, and then evaporated to dryness.

4. The quality inspection method according to claim 1, characterized in that, After spraying with aluminum trichloride test solution, examine under a 365nm ultraviolet lamp.

5. The quality inspection method according to any one of claims 1-4, characterized in that, Also includes: (1) Preparation of test solution for anti-tuberculosis granules and reference medicinal material solution of Prunus pubescens; (2) The test solution and the reference medicinal material solution of Prunus pedunculata were spotted on the same thin layer plate and identified by thin layer chromatography using a mixed solution of n-hexane and ethyl acetate as the developing solvent; the volume ratio of n-hexane to ethyl acetate in the developing solvent was 4:1.

5.

6. The quality inspection method according to claim 5, characterized in that, In step (1), the method for preparing the test solution of the antituberculosis granules includes: Take the anti-tuberculosis granules, grind them into a fine powder, add ethyl acetate, heat under reflux, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use it as the test solution.

7. The quality inspection method according to claim 5, characterized in that, In step (1), take the five-finger peach reference material and prepare the reference material solution in the same way as the test solution.

8. The quality inspection method according to claim 5, characterized in that, In step (2), after developing with a developing solvent, the product is taken out, dried, sprayed with 10% potassium hydroxide methanol solution, and examined under a 365nm ultraviolet lamp.

9. The quality inspection method according to any one of claims 1-4, characterized in that, Also includes: (1) Preparation of test solution for anti-tuberculosis granules and reference herb solution of Stemona japonica; (2) The test solution and the reference herb solution were spotted on the same thin-layer plate and identified by thin-layer chromatography using a mixed solution of xylene-acetone-methanol as the developing solvent; the volume ratio of xylene:acetone:methanol in the developing solvent was 8:3:0.

5.

10. The quality inspection method according to claim 9, characterized in that, After developing with a developing solvent, remove the sample, air dry it, spray it with dilute potassium bismuth iodide solution, and examine it under sunlight.

11. The quality inspection method according to claim 9, characterized in that, In step (1), the method for preparing the test solution of the antituberculosis granules includes: Take the anti-tuberculosis granules, grind them into a fine powder, add ammonia water to moisten them, add chloroform, reflux to extract, cool, filter, evaporate the filtrate to dryness, add methanol to dissolve the residue, and use it as the test solution.

12. The quality inspection method according to claim 9, characterized in that, Take the reference herb Stemona japonica and prepare a reference herb solution using the same method as the test sample solution.

13. The quality inspection method according to claim 9, characterized in that, Reference solutions were prepared from upright Stemona japonica and opposite-leaved Stemona japonica, respectively.

14. The quality inspection method according to any one of claims 1-4, characterized in that, Also includes: (1) Preparation of test solution and reference solution for anti-tuberculosis granules; (2) Accurately pipette the reference solution and the test solution into the liquid chromatograph and perform the determination according to the chromatographic conditions; The quality of the anti-tuberculosis powder was determined by calculating the content of geranium in dwarf tea.

15. The quality inspection method according to claim 14, characterized in that, Chromatographic conditions included: octadecylsilane-bonded silica gel as the packing material; a methanol-water mixture as the mobile phase with a methanol:water volume ratio of 15-19:85-81; a detection wavelength of 273 nm; and a theoretical plate number calculated based on the Bergenin peak of not less than 3000.

16. The quality inspection method according to claim 15, characterized in that, The volume ratio of methanol to water is 17:

83.

17. The quality inspection method according to claim 14, characterized in that, In step (1), the preparation method of the test solution of the antituberculosis granules includes: Take the anti-tuberculosis powder, grind it into a fine powder, sieve it, accurately weigh it, accurately add anhydrous methanol, weigh it again, sonicate it, cool it, weigh it again, replenish the lost weight with anhydrous methanol, shake it well, filter it, and take the filtrate to obtain the product.

18. The quality inspection method according to claim 17, characterized in that, The conditions for the ultrasonic treatment include: power 700-900W, frequency 30-50kHz, and ultrasonic treatment for 40-50 minutes.

19. The quality inspection method according to claim 18, characterized in that, The conditions for the ultrasonic treatment include: power 800W, frequency 40kHz, and ultrasonic treatment for 45 minutes.

20. The quality inspection method according to claim 14, characterized in that, In step (1), the preparation method of the reference solution includes: taking an appropriate amount of Bergenin reference standard, accurately weighing it, and adding anhydrous methanol to prepare a solution containing 0.2 mg per 1 ml.

21. The quality inspection method according to claim 14, characterized in that, Each 14g dose of anti-tuberculosis powder contains no less than 6mg of bergenin from ground tea.

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