A quality detection method of lingui zhuogan preparation

Abstract

This invention provides a quality testing method for Linggui Zhugan preparations, including methods for testing the quality of Linggui Zhugan granules and mixed powders of Linggui Zhugan granules. The method includes constructing characteristic chromatograms and determining the content of cinnamic acid and glycyrrhizic acid using liquid chromatography. The liquid chromatography uses acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution according to regulations. This method detects the quality of Linggui Zhugan preparations through multiple indicators, establishes quality standards for Linggui Zhugan preparations, and achieves effective quality control, laying a solid foundation for stable quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a quality detection method for a Lingguizhugan preparation. Background Art

[0002] Lingguizhugan Decoction, a traditional Chinese medicine formula, is a dampness-removing agent, with the effects of warming yang to transform fluid retention and strengthening the spleen to promote diuresis. Among them, Poria is the dried sclerotium of the fungus Poria cocos (Schw.) Wolf of the family Polyporaceae. It is usually dug out from July to September. After removing the sediment, it is piled up for "sweating", then spread out to dry on the surface, and then "sweated" again. After repeating several times until wrinkles appear and most of the internal moisture is lost, it is dried in the shade, which is called "Poria cubensis"; or the fresh Poria is cut according to different parts and dried in the shade, which are respectively called "Poria pieces" and "Poria slices". Ramulus Cinnamomi is the dried tender branch of Cinnamomum cassia Presl of the family Lauraceae. It is harvested in spring and summer, the leaves are removed, and it is dried in the sun, or sliced and dried in the sun. Rhizoma Atractylodis Macrocephalae is the dried rhizome of Atractylodes macrocephala Koidz. of the family Asteraceae. It is dug out when the lower leaves wither and the upper leaves become brittle in winter. After removing the sediment, it is dried by baking or in the sun, and then the fibrous roots are removed. Radix Glycyrrhizae is the dried root and rhizome of Glycyrrhiza uralensis Fisch. of the family Leguminosae. It is dug out in spring and autumn, the fibrous roots are removed, and it is dried in the sun.

[0003] Due to the complexity of the components of traditional Chinese medicines and the unevenness of the medicinal materials, it is very difficult to control the quality of reference samples. For the quality research of reference samples, specific identification and multi-component and overall quality evaluation indexes are required to characterize their quality. Nowadays, the extraction rate of decocted pieces, the transfer rate of index components, content determination, fingerprint / characteristic spectrum, etc. are mostly used as indexes for value transfer research to evaluate the quality correlation among samples, intermediates and preparations prepared according to ancient records. There is no relatively complete quality control method in the prior art to evaluate the quality of Lingguizhugan preparations. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a quality detection method that can comprehensively reflect the internal quality of Lingguizhu preparations and better control the quality of Lingguizhugan preparations.

[0005] The content of the present invention includes:

[0006] A quality testing method for Linggui Zhugan preparations, comprising a quality testing method for Linggui Zhugan granules and a quality testing method for Linggui Zhugan granule mixed powder; the quality testing method for Linggui Zhugan preparations includes constructing characteristic chromatograms and determining the content of cinnamic acid and glycyrrhizic acid using liquid chromatography, wherein the liquid chromatography uses acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution according to the specifications in Table a;

[0007] Table a. Gradient elution procedure

[0008]

[0009]

[0010] The flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 254 nm.

[0011] The steps for constructing characteristic chromatograms using liquid chromatography include:

[0012] S11, Preparation of reference solution: Take appropriate amounts of protocatechuic acid reference standard, apigenin glycyrrhizin reference standard, glycyrrhizin reference standard, gentianin reference standard, cinnamaldehyde reference standard, ammonium glycyrrhizate reference standard and cinnamic acid reference standard, and add methanol to each to obtain the solution.

[0013] S12, Preparation of the test solution: Take an appropriate amount of Linggui Zhugan preparation sample, add 50% methanol (volume concentration), sonicate, filter and collect the filtrate to obtain the solution;

[0014] S13, take 15 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0015] In step S12, 2.0g of the Linggui Zhugan preparation sample was taken, 100ml of methanol was added, and the ultrasonic power was 500W, the frequency was 40kHz, and the ultrasonic time was 30min.

[0016] Furthermore, in step S11 of the quality detection method for Linggui Zhugan granules, methanol is added to prepare a solution containing 5 μg of protocatechuic acid, 40 μg of apigenin, 50 μg of glycyrrhizin, 5 μg of gentianin, 50 μg of cinnamaldehyde, 100 μg of glycyrrhizic acid, and 20 μg of cinnamic acid per 1 ml; in step S11 of the quality detection method for Linggui Zhugan granule mixed powder, methanol is added to prepare a solution containing 5 μg of protocatechuic acid, 5 μg of apigenin, 30 μg of glycyrrhizin, 5 μg of gentianin, 50 μg of cinnamaldehyde, 60 μg of glycyrrhizic acid, and 5 μg of cinnamic acid per 1 ml.

[0017] Furthermore, the determination of cinnamic acid and glycyrrhizic acid content using liquid chromatography includes the following steps:

[0018] S21, take appropriate amounts of ammonium glycyrrhizate reference standard and cinnamic acid reference standard, add methanol to prepare solutions respectively, and the solution is obtained;

[0019] S22, Preparation of the test solution: Take an appropriate amount of Linggui Zhugan preparation sample, add 50% methanol (volume concentration), sonicate, filter and collect the filtrate to obtain the solution;

[0020] S23, inject 15 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0021] In S22, 2.0g of the Linggui Zhugan preparation sample was taken, 100ml of methanol was added, and the ultrasonic power was 500W, the frequency was 40kHz, and the ultrasonic time was 30min.

[0022] Furthermore, in step S21 of the determination of cinnamic acid and glycyrrhizic acid content in the Linggui Zhugan granule mixture powder, a solution containing 0.10 mg glycyrrhizic acid and 0.02 mg cinnamic acid per 1 ml is prepared; in step S21 of the determination of cinnamic acid and glycyrrhizic acid content in the Linggui Zhugan granules, a solution containing 0.05 mg glycyrrhizic acid and 0.01 mg cinnamic acid per 1 ml is prepared.

[0023] Furthermore, the quality testing methods for the Linggui Zhugan preparation also include thin-layer chromatography identification of Poria cocos, thin-layer chromatography identification of Atractylodes macrocephala, and thin-layer chromatography identification of Glycyrrhiza uralensis.

[0024] Furthermore, the steps of the thin-layer chromatography method for identifying Poria cocos include:

[0025] S31. Take 10g of the Linggui Zhugan preparation of this product, add 50ml of ether, sonicate for 10min, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0026] S32, take 1.0g of Poria cocos reference material, add 50ml of ether, sonicate for 10min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to make the reference material solution;

[0027] S33. Take 20 μl of the test solution and 2 μl of the reference herb solution and spot them separately on the same silica gel G thin-layer plate. Use a toluene-ethyl acetate-formic acid solution with a volume ratio of 20:5:0.5 as the developing solvent. Develop the plate, remove it, air dry it, spray it with a 2% vanillin-sulfuric acid solution and ethanol mixture with a volume ratio of 4:1, and heat it at 105℃ until the spots are clearly visible. Examine it under fluorescent light.

[0028] Furthermore, the steps of the Atractylodes macrocephala thin-layer identification method include:

[0029] S41. Take 4g of the Linggui Zhugan preparation of this product, grind it into a fine powder, add 20ml of n-butanol, sonicate for 30min, filter, wash the filtrate with water twice, 30ml each time, discard the water, evaporate the n-butanol solution to dryness, add 1ml of acetone to the residue to dissolve it, and use it as the test solution.

[0030] S42, take 0.5g of Atractylodes macrocephala reference material, add 15ml of n-butanol, sonicate for 30min, filter, wash the filtrate twice with 10ml of water each time, discard the water, evaporate the n-butanol solution to dryness, add 1ml of acetone to dissolve the residue, and use it as the reference material solution.

[0031] S43, take 10 μl each of the reference herb solution and the test sample solution and spot them separately on the same silica gel G thin layer plate. Use a chloroform-acetone solution with a volume ratio of 19:1 as the developing solvent, develop, remove, air dry, and examine under a 365 nm ultraviolet light.

[0032] Furthermore, the steps of the licorice thin-layer chromatography identification method include:

[0033] S51. Take 4g of the Linggui Zhugan preparation, grind it into a fine powder, add 40ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, extract twice with water-saturated n-butanol, 25ml each time, combine the extracts, wash with 30ml of water-saturated n-butanol, discard the water, evaporate the n-butanol to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0034] S52, take 0.5g of licorice reference material, grind it into a fine powder, add 40ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, extract twice with water-saturated n-butanol, 25ml each time, combine the extracts, wash with 30ml of water-saturated n-butanol, discard the water, evaporate the n-butanol to dryness, add 1ml of methanol to dissolve the residue, and prepare the reference material solution;

[0035] S53. Take 5 μl each of the reference herb solution and the test sample solution and spot them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Use ethyl acetate-formic acid-glacial acetic acid-water in a volume ratio of 15:1:1:2 as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under a 365nm ultraviolet lamp.

[0036] Furthermore, the quality testing method for the Linggui Zhugan preparation also includes appearance inspection, general inspection of granules, and extractives inspection.

[0037] Furthermore, the extract is examined using a hot leaching method with ethanol as the solvent.

[0038] The beneficial effects of this invention are:

[0039] (1) The Linggui Zhugan preparation underwent morphological examination, thin-layer chromatography identification, characteristic chromatogram determination, granule general rules inspection, extractive matter inspection, and content determination. Thin-layer chromatography identification was performed using thin-layer chromatography. Granule general rules inspection included checks on moisture, particle size, solubility, fill weight variation, and microbial limits. Extractive matter was determined using the hot extraction method. Characteristic chromatograms, cinnamic acid, and glycyrrhizic acid content determination were all performed using liquid chromatography. The quality testing methods were relatively comprehensive and complete.

[0040] (2) The quality detection method of Linggui Zhugan preparation of the present invention detects the quality of Linggui Zhugan preparation through multiple indicators, which can establish feasible quality standards for Linggui Zhugan preparation, realize effective control of the quality of Linggui Zhugan preparation, and lay a solid foundation for the stability of the quality of Linggui Zhugan preparation.

[0041] (3) Using the chromatographic conditions of this application for liquid phase analysis, a more clearly separated and distinct characteristic chromatogram can be obtained. The method has strong specificity, good stability, high precision, and good reproducibility. It can assign and identify the nine common peaks in the chromatogram.

[0042] (4) Using the thin-layer chromatography identification method of this application, the thin-layer identification of four medicinal materials, namely Poria cocos, Cinnamomum cassia, Atractylodes macrocephala and Glycyrrhiza uralensis, was achieved through three display methods. It is simple, fast, and produces clear spots and distinct layers.

[0043] (5) The content determination method of this application can determine the content of representative cinnamic acid and glycyrrhizic acid in Linggui Zhugan preparation. It has strong specificity, good repeatability, good linearity and good recovery rate. Attached Figure Description

[0044] Figure 1 This is one of the thin-layer chromatography methods for identifying Poria cocos. Figure 2 This is the second method for thin-layer chromatography identification of Poria cocos; Figure 3 This study investigated the developing solvent for thin-layer chromatography identification of Poria cocos. Figure 4 This study investigates the colorimetric reagents used for thin-layer chromatography identification of Poria cocos. Figure 5 This study investigates the specificity of thin-layer chromatography for identifying Poria cocos. Figure 6 This study examines the amount of sample used for thin-layer chromatography identification of Poria cocos. Figure 7 This is a thin-layer chromatography test to identify the durability of Poria cocos at different temperatures; Figure 8 This is a thin-layer chromatography test to identify the durability of Poria cocos under different relative humidity conditions. Figure 9 This is a thin-layer chromatography test to identify the durability of different thin-layer plates for Poria cocos. Figure 10 This is the TLC spectrum of Poria cocos in the granules; Figure 11 This is one of the thin-layer chromatography identification methods for cinnamon twigs; Figure 12 This is the second method for identifying cinnamon twigs using thin-layer chromatography; Figure 13 This is one of the thin-layer chromatography identification methods for Atractylodes macrocephala; Figure 14 This is the second method for thin-layer identification of Atractylodes macrocephala; Figure 15 This study investigates the developing solvent for thin-layer chromatography identification of Atractylodes macrocephala. Figure 16 This study investigates the colorimetric reagents used for thin-layer chromatography identification of Atractylodes macrocephala. Figure 17 This study examines the specificity of thin-layer chromatography for the identification of Atractylodes macrocephala. Figure 18 This study examines the sample quantity for thin-layer chromatography identification of Atractylodes macrocephala. Figure 19 This is a thin-layer chromatography test to identify the durability of Atractylodes macrocephala at different temperatures; Figure 20 This is a thin-layer chromatography test for Atractylodes macrocephala to identify its durability under different relative humidity conditions. Figure 21 This is a test to distinguish the durability of different thin-layer plates of Atractylodes macrocephala; Figure 22 This is the TLC spectrum of Atractylodes macrocephala in the particles; Figure 23 This is one of the thin-layer chromatography methods for identifying licorice. Figure 24 This is the second method for identifying licorice using thin-layer chromatography. Figure 25 This study investigated the developing solvent for thin-layer chromatography identification of licorice. Figure 26 This study investigates the colorimetric reagent for thin-layer chromatography identification of licorice. Figure 27 This study investigates the specificity of licorice in thin-layer chromatography identification. Figure 28 This study investigates the amount of licorice sample used for thin-layer chromatography identification. Figure 29 This is a thin-layer chromatography test for licorice to determine its durability at different temperatures; Figure 30 This is a thin-layer chromatography test for the durability of licorice under different relative humidity conditions. Figure 31 This is a thin-layer chromatography test for licorice to differentiate the durability of different thin-layer plates; Figure 32 This is the TLC spectrum of licorice in the granules; Figure 33 It is Poria cocos, Poria cocos is missing, characteristic spectrum of granules; Figure 34 It is a cassia branch, lacking a cassia branch, and the characteristic spectrum of the granules; Figure 35 It is a characteristic spectrum of Atractylodes macrocephala, lacking Atractylodes macrocephala, and granul Figure 36 It is licorice, but licorice is lacking; characteristic spectrum of granules; Figure 37 It is a superimposed image of the characteristic spectra of three batches of particles; Figure 38 It is a particle characteristic spectrum comparison spectrum. Detailed Implementation

[0045] Preparation method of Linggui Zhugan Granule Mixture Powder: 1600g of Poria cocos, 1200g of Cinnamomum cassia, 1200g of Atractylodes macrocephala, and 800g of Glycyrrhiza uralensis are soaked in 8 times the amount of water for 30 minutes, decocted for 1 hour, and the aromatic water is collected at the same time. The aromatic water is encapsulated with beta-cyclodextrin, and the encapsulation mixture is dried at low temperature and pulverized into fine powder for later use. The decoction is filtered, and the filtrate is concentrated under reduced pressure to an extract with a relative density of 1.03-1.08 (60℃) (dry extract yield is 13.3%-16.2%). The extract is filtered, and 5% of the amount of dextrin added to the decoction pieces is added and mixed evenly. The mixture is spray-dried, and then an appropriate amount of the encapsulation mixture powder and dextrin are added and mixed evenly to obtain the final product.

[0046] Preparation method of Linggui Zhugan Granules: 1600g of Poria cocos, 1200g of Cinnamomum cassia, 1200g of Atractylodes macrocephala, and 800g of Glycyrrhiza uralensis are soaked in 8 times the amount of water for 30 minutes and decocted for 1 hour. Aromatic water is collected at the same time. The aromatic water is encapsulated with beta-cyclodextrin. The encapsulation mixture is dried at low temperature and pulverized into fine powder for later use. The decoction is filtered and the filtrate is concentrated under reduced pressure to an extract with a relative density of 1.03-1.08 (60℃) (dry extract yield is 13.3%-16.2%). The extract is filtered, and 5% of the amount of dextrin added to the decoction pieces is added and mixed evenly. The mixture is spray-dried, and then an appropriate amount of the fine powder of the encapsulation mixture and dextrin are added and mixed evenly. The mixture is then granulated by dry method to make 1000g, packaged, and the product is obtained.

[0047] 1. Characteristic Examination

[0048] Based on the appearance and taste description of three batches of samples, this product consists of light yellow to brownish-yellow granules; it has a slightly fragrant odor and a sweet taste.

[0049] 2. Thin-layer chromatography identification

[0050] 2.1 Thin-layer chromatography identification of the mixed powder of Linggui Zhugan granules

[0051] (1) Take 10g of the powder, add 50ml of ether, sonicate for 10min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 1.0g of Poria cocos reference material, add 50ml of ether, sonicate for 10min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to prepare the reference material solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), apply 20μl of the test solution and 2μl of the reference material solution separately to the same silica gel G thin-layer plate, develop using toluene-ethyl acetate-formic acid (20:5:0.5) solution as the developing solvent, remove, air dry, spray with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture, 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.

[0052] (2) Take 2g of the powder, add 20ml of n-butanol, sonicate for 30min, filter, wash the filtrate twice with 30ml of water each time, discard the water, evaporate the n-butanol solution to dryness, dissolve the residue in 1ml of acetone, and use this as the test solution. Separately, take 0.5g of Atractylodes macrocephala reference material, add 15ml of n-butanol, sonicate for 30min, filter, wash the filtrate twice with 10ml of water each time, discard the water, evaporate the n-butanol solution to dryness, dissolve the residue in 1ml of acetone, and use this as the reference material solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), apply 10μl of each of the above two solutions to the same silica gel G thin-layer plate, develop with chloroform-acetone (19:1) as the developing solvent, remove, air dry, and examine under ultraviolet light (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.

[0053] (3) Take 2g of the powder, add 40ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, extract twice with 25ml of water-saturated n-butanol each time, combine the extracts, wash with 30ml of water-saturated n-butanol, discard the aqueous solution, evaporate the n-butanol to dryness, add 1ml of methanol to dissolve the residue, and use as the test solution. Take 0.5g of licorice reference material and prepare a reference material solution in the same way. Perform thin-layer chromatography (General Chapter 0502 of Chinese Pharmacopoeia 2020 Edition), take 5μl of each of the above two solutions, and spot them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide test solution. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (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 medicinal material.

[0054] 2.2 Thin-layer chromatography identification method for Linggui Zhugan granules

[0055] 2.2.1 Poria cocos

[0056] ① Experimental instruments, reagents, and reagents

[0057] Thin-layer chromatograph (model: GoodLook-1000, Shanghai Kezhe Biochemical Technology Co., Ltd.); electronic balance (model: AL-204, asset number 000000518, Mettler Toledo Instruments Ltd.); ultrasonic cleaner (model: KM-500DB, Kunshan Meimei Ultrasonic Instruments Co., Ltd.); silica gel pre-prepared plates (G plate 200*100mm, Qingdao Haiyang Silica Gel Co., Ltd., batch number 202200822); silica gel pre-prepared plates (G plate 200*100mm, Qingdao Hailang Silica Gel Development Co., Ltd., batch number 20220901); methanol (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20220907); petroleum ether (30℃~60℃) (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 2). 0220211); Diethyl ether (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20201017); Toluene (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20210813); Vanillin (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number 20200225); Sulfuric acid (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20220811); Ethyl acetate (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20220606); Formic acid (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20210518); Ethanol (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20211204); Poria cocos reference material (China National Institutes for Food and Drug Control, batch number: 121117-201910).

[0058] ② Screening method for thin-layer chromatography identification of Poria cocos

[0059] I. Method 1

[0060] Preparation of test solution: Take 10g of this product, grind it into a fine powder, add 50ml of diethyl ether, sonicate for 10min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to obtain the test solution.

[0061] Preparation of the reference herb solution: Take 1g of Poria cocos reference herb, add 50ml of ether to dissolve, sonicate for 10min, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the reference herb solution.

[0062] Preparation of negative control solution: Take 10g of Poria cocos-deficient negative control powder and prepare a negative control solution using the same method.

[0063] Identification: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 20 μl of the test solution and 2 μl of the reference herb solution separately to the same silica gel G thin-layer plate. Develop the plate using toluene-ethyl acetate-formic acid (20:5:0.5) solution as the developing solvent. Remove the plate, air dry it, spray it with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture, and heat it at 105℃ until the spots are clearly visible. Examine the plate under fluorescent light.

[0064] Results (see) Figure 1 The results indicate that the test solution has spots of the same color at the corresponding positions as the control medicinal material solution, therefore the preparation method of the test solution can be included in the quality standard.

[0065] II. Method Two

[0066] Preparation of test solution: Take 6g of this product, grind it into a fine powder, add 20ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 2ml of chloroform to dissolve the residue, and use it as the test solution.

[0067] Preparation of the reference herb solution: Take 1.0g of Poria cocos reference herb, add 10ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 2ml of chloroform to dissolve the residue, and use it as the reference herb solution.

[0068] Preparation of negative control solution: Take 6g of Poria cocos negative powder, add 20ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 2ml of chloroform to dissolve the residue, and use it as negative control solution.

[0069] Identification: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 5 μl of the above test sample and 4 μl of the reference material and spot them separately on the same silica gel G thin-layer plate. Use petroleum ether (30℃~60℃)-diethyl ether (3:2) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm).

[0070] Results (see) Figure 2 The results indicate that the test solution did not show the same fluorescent spots at the corresponding positions as the reference solution and the reference medicinal material solution, therefore the preparation method of the test solution cannot be included in the quality standard.

[0071] III. Conclusion

[0072] Considering all factors, Method 1 is the best method for preparing the test solution.

[0073] ③ Investigation of the developing solvent

[0074] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 20 μl of the test solution and 2 μl of the reference herb solution from Method 1 above and spot them separately on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (20:5:0.5) solution as the developing solvent, develop, remove, air dry, spray with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixed solution, heat at 105℃ until the spots are clearly visible, and examine under fluorescent light.

[0075] Results (see) Figure 3 The results show that toluene-ethyl acetate-formic acid (20:5:0.5) is better than the other two developing solvents. Therefore, the developing solvent used in the original method is toluene-ethyl acetate-formic acid (20:5:0.5).

[0076] ④ Colorimetric reagent test

[0077] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 2 μl of the reference herb solution prepared by Method 1 above, 20 μl each of the test sample solution and the negative control herb solution lacking Poria cocos, and spot them separately on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (20:5:0.5) as the developing solvent, develop, remove, air dry, spray with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture or a 2% ferric chloride solution, heat at 105℃ until the spots are clearly visible, and examine under fluorescent light.

[0078] Results (see) Figure 4 The results showed that when the Poria cocos reference solution was sprayed with 2% ferric chloride solution as the colorimetric reagent, there were no corresponding spots between the Poria cocos reference solution and the test solution. However, when the Poria cocos reference solution was sprayed with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture, the Poria cocos reference solution and the test solution showed the same color spots at the corresponding positions. Therefore, the 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture was determined to be the colorimetric condition for this method.

[0079] ⑤ Specificity Examination

[0080] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 20 μl of the test solution and 2 μl of the reference herb solution from Method 1 above and spot them separately on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (20:5:0.5) solution as the developing solvent, develop, remove, air dry, spray with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixed solution, heat at 105℃ until the spots are clearly visible, and examine under fluorescent light.

[0081] Results (see) Figure 5 The results showed that the test solution exhibited spots of the same color at the corresponding positions as the control solution, and there was no interference from the negative control solution lacking Poria cocos, indicating that the method has good specificity.

[0082] ⑥ Sample quantity investigation

[0083] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) and spot 1 μl, 2 μl, and 3 μl of the reference herb solution prepared by Method I above, and 15 μl, 20 μl, and 25 μl of the test sample solution onto the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (20:5:0.5) as the developing solvent, develop, remove, air dry, spray with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture, heat at 105℃ until the spots are clearly visible, and examine under fluorescent light.

[0084] Results (see) Figure 6 The results showed that the spots were more obvious when the sample volume of the Poria cocos reference material solution was 2 μl and 3 μl, and the corresponding spots were fainter when the sample volume was 1 μl. The spots were weaker when the sample volume of the test sample solution was 15 μl, and the spots were clearer when the sample volume was 20 μl and 25 μl. Taking all factors into consideration, the sample volume of the Poria cocos reference material solution was determined to be 2 μl and the sample volume of the test sample solution was determined to be 20 μl.

[0085] ⑦ Durability test

[0086] The effects of different temperatures, humidity levels, and brands of thin-layer chromatography plates on the experimental results were investigated according to the proposed method.

[0087] I. Tests at different temperatures

[0088] According to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020), 2 μl of the reference herb solution and 20 μl of the test solution prepared by Method I above were spotted separately on the same silica gel G thin-layer plate. Toluene-ethyl acetate-formic acid (20:5:0.5) was used as the developing solvent, and the plates were developed at 25℃ and 4℃ respectively. The plates were removed, dried, and sprayed with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture. The plates were heated at 105℃ until the spots were clearly visible. The results were observed under fluorescent light.

[0089] Results (see) Figure 7 The results show that when developed at 25℃ and 4℃, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the spots are clear and the separation is good, indicating that the method is applicable in the range of 4℃ to 25℃ and has good durability.

[0090] II. Tests at different humidity levels

[0091] According to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020), 2 μl of the reference herb solution and 20 μl of the test solution prepared by Method I above were spotted separately onto the same silica gel G thin-layer plate. Toluene-ethyl acetate-formic acid (20:5:0.5) was used as the developing solvent, and the plate was developed at relative humidity of 32%, 58%, and 88%, respectively. The plate was then removed, dried, and sprayed with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture. The plate was heated at 105℃ until the spots were clearly visible. The results were observed under fluorescent light.

[0092] Results (see) Figure 8 The results show that when the test sample is developed under relative humidity conditions of 32%, 58%, and 88%, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the spots are clear and the separation is good. This indicates that the method is applicable in the range of relative humidity from 32% to 88% and has good durability.

[0093] III. Tests on thin-layer boards of different brands

[0094] The thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition) was used. 2 μl of the reference herb solution and 20 μl of the test solution prepared by Method 1 were spotted onto silica gel G thin-layer plates of different brands (Qingdao Hailang, Qingdao Haiyang, and self-made). The plates were developed using toluene-ethyl acetate-formic acid (20:5:0.5) as the developing solvent. The plates were then removed, dried, and sprayed with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture. The plates were heated at 105℃ until the spots were clearly visible. The results were observed under fluorescent light.

[0095] Results (see) Figure 9 The results show that when different brands of silica gel G thin-layer plates (Qingdao Hailang, Qingdao Haiyang, and self-made) are used, the test sample chromatogram shows the same main spot of the same color as the reference medicinal material chromatogram at the corresponding position. The spots are clear and the separation is good, indicating that the method is applicable to different brands of silica gel G thin-layer plates and has good durability.

[0096] ⑧ Determination of thin-layer chromatography identification method for Poria cocos

[0097] In summary, the thin-layer chromatography method for identifying Poria cocos in Linggui Zhugan granules is determined as follows: Take 10g of the product, grind it into a fine powder, add 50ml of ether, sonicate for 10min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to obtain the test solution. Separately, take 1.0g of Poria cocos reference material, add 50ml of ether, sonicate for 10min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to obtain the reference material solution. Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 20μl of the test solution and 2μl of the reference material solution separately to the same silica gel G thin-layer plate. Develop the plate using toluene-ethyl acetate-formic acid (20:5:0.5) solution as the developing solvent. Remove the plate, air dry, spray with a 2% vanillin-sulfuric acid solution-ethanol (4:1) mixture, and heat at 105℃ until the spots are clearly visible. Examine under fluorescent light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. The results are shown in [Figure number missing]. Figure 10 .

[0098] 2.2.2 Cinnamon Twig

[0099] ① Experimental instruments, reagents, and reagents

[0100] Electronic balance (model: T-214, Beijing Sartorius Instrument Systems Co., Ltd.); Electronic balance (model: XPE105, asset number KY2016028003, Mettler Toledo Instruments Co., Ltd.); Ethyl acetate (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20200509); Ethanol (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20200918); Chloroform (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20200907); 2,4-dinitrophenylhydrazine (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number 20200815); Cinnamon twig reference material (China National Institutes for Food and Drug Control, batch number: 121191-201605); Others are the same as in 2.2.1.

[0101] ② Screening of thin-layer chromatography identification method for cinnamon twigs

[0102] I. Method 1

[0103] Preparation of test solution: Take 0.5g of this product, grind it into a fine powder, add 10ml of ethanol, seal tightly, soak for 20min, shake occasionally, filter, and take the filtrate as the test solution.

[0104] Preparation of the reference herb solution: Take 0.5g of cinnamon twig reference herb and prepare the reference herb solution using the same method.

[0105] Preparation of negative control solution: Take 0.5g of negative control powder lacking cinnamon twig and prepare negative control solution using the same method.

[0106] Identification: Perform thin-layer chromatography (General Chapter 0502 of Chinese Pharmacopoeia 2020). Take 15 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use petroleum ether (60-90℃)-ethyl acetate (17:3) as the developing solvent, develop, air dry, spray with dinitrophenylhydrazine ethanol test solution, and examine under fluorescent light.

[0107] Results (see) Figure 11 The results showed that the test solution did not have the same orange-red spots at the corresponding positions as the control solution, therefore the preparation method of the test solution was not feasible.

[0108] II. Method Two

[0109] Preparation of test solution: Take 2g of this product, grind it into a fine powder, add 10ml of ether, soak for 30min, shake occasionally, filter, evaporate the filtrate to dryness, add 1ml of chloroform to dissolve the residue, and use it as the test solution.

[0110] Preparation of the reference herb solution: Take 2g of cinnamon twig reference herb and prepare the reference herb solution using the same method.

[0111] Preparation of negative control solution: Take 2g of Guizhiling negative control powder and prepare negative control solution using the same method.

[0112] Identification: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 15 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use petroleum ether (60℃~90℃)-ethyl acetate (17:3) as the developing solvent. Develop, remove, and air dry. Spray with vanillin sulfuric acid test solution and heat at 105℃ until the spots are clearly visible. Examine under fluorescent light.

[0113] Results (see) Figure 12 The results indicate that the test solution does not have the same color spots at the corresponding positions as the control herbal solution, therefore the preparation method of the test solution is not feasible.

[0114] III. Conclusion

[0115] Taking all the above into consideration, the identification of this medicinal material will not be included in the quality standards for the time being.

[0116] 2.2.3 Atractylodes macrocephala

[0117] ① Experimental instruments, reagents, and reagents

[0118] n-Butanol (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20200604); chloroform (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20200104); ethyl acetate (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20201124); n-hexane (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20200204); acetone (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20200805); Atractylodes macrocephala reference material (China National Institutes for Food and Drug Control, batch number: 120925-201611).

[0119] ② Screening of thin-layer chromatography identification method for Atractylodes macrocephala

[0120] I. Method 1

[0121] Preparation of test solution: Take 4g of this product, grind it into a fine powder, add 20ml of n-butanol, treat for 30min, filter, wash the filtrate twice with 30ml of water each time, discard the water, evaporate the n-butanol solution to dryness, add 1ml of acetone to dissolve the residue, and use it as the test solution.

[0122] Preparation of the reference herb solution: Take 0.5g of Atractylodes macrocephala reference herb, add 15ml of n-butanol, sonicate for 30min, filter, wash the filtrate twice with 10ml of water each time, discard the water, evaporate the n-butanol solution to dryness, add 1ml of acetone to dissolve the residue, and use it as the reference herb solution.

[0123] Preparation of negative control solution: Take 4g of negative control powder of Atractylodes macrocephala, add 20ml of n-butanol, treat for 30min, filter, wash the filtrate twice with 30ml of water each time, discard the water, evaporate the n-butanol solution to dryness, add 1ml of acetone to dissolve the residue, and use it as negative control solution.

[0124] Identification: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 10 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use chloroform-acetone (19:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0125] Results (see) Figure 13 The results show that the test solution exhibits fluorescent spots of the same color at the corresponding positions as the control medicinal material solution, therefore the preparation method of the test solution can be included in the quality standard.

[0126] II. Method Two

[0127] Preparation of test solution: Take 0.5g of this product, grind it into a fine powder, add 2ml of n-hexane, sonicate for 15min, filter, and take the filtrate as the test solution.

[0128] Preparation of the reference herb solution: Take 0.5g of Atractylodes macrocephala reference herb and prepare the reference herb solution using the same method.

[0129] Preparation of negative control solution: Take 0.5g of Atractylodes macrocephala negative control powder and prepare negative control solution using the same method.

[0130] Identification: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 10 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use petroleum ether (60℃~90℃)-ethyl acetate (50:1) as the developing solvent. Develop, remove, and air dry. Spray with 5% vanillin sulfuric acid test solution and heat at 105℃ until the spots are clearly visible. Examine under fluorescent light.

[0131] Results (see) Figure 14 The results indicate that the test solution does not have the same color spots at the corresponding positions as the control herbal solution, therefore the preparation method of the test solution is not feasible.

[0132] III. Conclusion: Considering all factors, Method 1 is the best method for preparing the test solution.

[0133] ③ Investigation of the developing solvent

[0134] The thin-layer chromatography method (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) was followed. 10 μl of each of the three solutions prepared in Method 1 above was spotted separately onto the same silica gel G thin-layer plate. Developing solvents were chloroform-acetone (19:1) and toluene-methanol (6:1). The plates were then removed, dried, and examined under ultraviolet light (365 nm). The results were compared.

[0135] Results (see) Figure 15 The results show that chloroform-acetone (19:1) is better than chloroform-acetone (19:1) as the developing solvent. Therefore, chloroform-acetone (19:1) is used as the developing solvent in the original method.

[0136] ④ Colorimetric reagent test

[0137] Perform the thin-layer chromatography test (General Chapter 0502 of Chinese Pharmacopoeia 2020 Edition). Take 10 μl of each of the three solutions prepared by Method I above and spot them separately on the same silica gel G thin-layer plate. Use chloroform-acetone (19:1) as the developing solvent, develop, remove, air dry, first examine under ultraviolet light (365 nm), then spray with 5% vanillin sulfuric acid test solution, heat at 105℃ until the spots are clearly visible, and examine under fluorescent light.

[0138] Results (see) Figure 16 The results showed that, using 5% vanillin-sulfuric acid solution as the colorimetric reagent, the spots of the Atractylodes macrocephala reference solution and the test solution were not uniform in color when viewed under sunlight. However, when viewed directly at 365 nm, the Atractylodes macrocephala reference solution and the test solution showed fluorescent spots of the same color at the corresponding positions. Therefore, it was determined that 365 nm should be used directly as the colorimetric condition for this method.

[0139] ⑤ Specificity Examination

[0140] Perform the thin-layer chromatography test (General Chapter 0502 of Chinese Pharmacopoeia 2020). Take 10 μl each of the reference herb solution, the test sample solution, and the negative control herb solution of Atractylodes macrocephala prepared by Method I above, and spot them separately on the same silica gel G thin-layer plate. Use chloroform-acetone (19:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0141] Results (see) Figure 17 The results showed that the test solution exhibited fluorescent spots of the same color at the corresponding positions as the control herbal solution, and there was no interference from the negative control solution lacking Atractylodes macrocephala, indicating that the method has good specificity.

[0142] ⑥ Sample quantity investigation

[0143] Perform the thin-layer chromatography test (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition). Take 5 μl, 10 μl, and 15 μl of the reference herb solution prepared by Method I above, and 5 μl, 10 μl, and 15 μl of the test sample solution, respectively, and spot them on the same silica gel G thin-layer plate. Use chloroform-acetone (19:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm) to observe the results.

[0144] Results (see) Figure 18 The results showed that the spots were relatively obvious when the spotting volume of the Atractylodes macrocephala reference material solution was 5 μl, 10 μl, and 15 μl. The spots were weaker when the spotting volume of the test sample solution was 5 μl, clearer when the spotting volume was 10 μl, and clearer but harder to spot when the spotting volume was 15 μl. Therefore, the spotting volume of both the Atractylodes macrocephala reference material solution and the test sample solution was determined to be 10 μl.

[0145] ⑦ Durability test: The effects of different temperatures, humidity levels and different brands of thin-layer chromatography plates on the test results were investigated according to the proposed method.

[0146] I. Tests at different temperatures

[0147] Perform the thin-layer chromatography test (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition). Take 10 μl each of the reference herb solution and the test solution prepared by Method I above and spot them separately on the same silica gel G thin-layer plate. Use chloroform-acetone (19:1) as the developing solvent and develop at 25℃ and 4℃ respectively. Remove the plate, air dry it, and examine it under ultraviolet light (365nm) to observe the results.

[0148] Results (see) Figure 19The results show that when developed at 25℃ and 4℃, the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the spots are clear and the separation is good, indicating that the method is applicable in the range of 4℃ to 25℃ and has good durability.

[0149] II. Tests at different humidity levels

[0150] Perform the thin-layer chromatography test (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition). Take 10 μl each of the reference herb solution and the test solution prepared by Method I above and spot them separately on the same silica gel G thin-layer plate. Use chloroform-acetone (19:1) as the developing solvent and develop at relative humidity of 32%, 58%, and 88%, respectively. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm) to observe the results.

[0151] Results (see) Figure 20 The results show that when the test sample was developed at relative humidity of 32%, 58%, and 88%, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the reference medicinal material. The spots were clear and the separation was good, indicating that the method is applicable in the range of relative humidity from 32% to 88% and has good durability.

[0152] III. Tests on thin-layer boards of different brands

[0153] The thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition) was used. 10 μl of the reference herb solution and the test solution prepared by Method I were spotted onto silica gel G thin-layer plates of different brands (Qingdao Hailang, Qingdao Haiyang, and self-made). The plates were developed using chloroform-acetone (19:1) as the developing solvent. After development, the plates were removed, dried, and examined under ultraviolet light (365 nm). The results were then observed.

[0154] Results (see) Figure 21 The results show that when different brands (Qingdao Hailang, Qingdao Haiyang, and self-made) of silica gel G thin-layer plates are used, the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the spots are clear and the separation is good. This indicates that the method is applicable to different brands of silica gel G thin-layer plates and has good durability.

[0155] ⑧ Determination of thin-layer chromatography identification method for Atractylodes macrocephala

[0156] In summary, the thin-layer chromatography method for detecting Atractylodes macrocephala in Linggui Zhugan granules is determined as follows: Take 4g of the product, grind it into a fine powder, add 20ml of n-butanol, sonicate for 30min, filter, wash the filtrate twice with 30ml of water each time, discard the water, evaporate the n-butanol solution to dryness, dissolve the residue in 1ml of acetone to obtain the test solution. Separately, take 0.5g of Atractylodes macrocephala reference material, add 15ml of n-butanol, sonicate for 30min, filter, wash the filtrate twice with 10ml of water each time, discard the water, evaporate the n-butanol solution to dryness, dissolve the residue in 1ml of acetone to obtain the reference material solution. Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) using the above-mentioned reference herb solution and test sample solution. Apply 10 μl each to the same silica gel G thin-layer plate, using chloroform-acetone (19:1) as the developing solvent. Develop, remove, air-dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference herb. The results are shown in the figure below. Figure 22 .

[0157] 2.2.4 Licorice

[0158] ① Experimental instruments, reagents, and reagents

[0159] Diethyl ether (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20220507); ethyl acetate (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20220527); glacial acetic acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number 20220307); n-butanol (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 2020621); sodium hydroxide (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number 20220725); acetone (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20211120); chloroform (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number 20210912); licorice reference material (China National Institutes for Food and Drug Control, batch number: 120904-201604); other materials are the same as in 2.2.1.

[0160] ② Screening of Licorice Thin-Layer Chromatography Identification Method

[0161] I. Method 1

[0162] Preparation of the test solution: Take 4g of this product, grind it into a fine powder, add 40ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, extract twice with water-saturated n-butanol, 25ml each time, combine the extracts, wash with 30ml of water-saturated n-butanol, discard the aqueous solution, evaporate the n-butanol to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0163] Preparation of the reference herb solution: Take 0.5g of licorice reference herb and prepare the reference herb solution using the same method.

[0164] Preparation of negative control solution: Take 2g of licorice-deficient negative control powder and prepare a negative control solution using the same method.

[0165] Identification: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 5 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide test solution. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under ultraviolet light (365nm).

[0166] Results (see) Figure 23 The results show that the test solution exhibits fluorescent spots of the same color at the corresponding positions as the control medicinal material solution, therefore the preparation method of the test solution can be included in the quality standard.

[0167] II. Method Two

[0168] Preparation of the test solution: Take 4g of this product, grind it into a fine powder, add 40ml of ether, heat under reflux for 1 hour, filter, discard the ether liquid, add 30ml of methanol to the residue, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 40ml of water to dissolve the residue, extract with n-butanol 3 times, 20ml each time, combine the extracts, wash with water 3 times, discard the aqueous liquid, evaporate the n-butanol solution to dryness, add 5ml of methanol to dissolve the residue, and use this as the test solution.

[0169] Preparation of the reference herb solution: Take 1g of licorice reference herb and prepare the reference herb solution using the same method.

[0170] Preparation of negative control solution: Take 4g of licorice-deficient negative control powder and prepare a negative control solution using the same method.

[0171] Identification: Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Take 5 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide test solution. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under ultraviolet light (365nm).

[0172] Results (see) Figure 24 The results showed that the test solution had fluorescent spots of the same color at the corresponding positions as the control solution, but there was a tailing phenomenon, so the preparation method of the test solution was not feasible.

[0173] III. Conclusion: Considering all factors, Method 1 is the best method for preparing the test solution.

[0174] ③ Investigation of the developing solvent

[0175] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) by taking 5 μl of each of the three solutions prepared by Method I above and spotting them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) and chloroform-acetone (19:1) as developing solvents, respectively. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365 nm).

[0176] Results (see) Figure 25 The results show that, when using the two developing solvents mentioned above, ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) has better results. Therefore, the developing solvent in the original method is ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2).

[0177] ④ Colorimetric reagent test

[0178] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) using the following method: Apply 5 μl of each of the three solutions prepared by Method I above to the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Develop the plate using ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm). Then spray it with 10% sulfuric acid ethanol solution and heat it at 105 °C until the spots are clearly visible. Examine the plate under ultraviolet light (365 nm) and compare the results.

[0179] Results (see) Figure 26 The results showed that when sprayed with a 10% sulfuric acid ethanol solution and heated at 105°C until the spots were clearly visible, and then examined under a UV lamp (365nm), the licorice reference solution and the test solution showed fluorescent spots of the same color at the corresponding positions. However, when examined under 365nm, the spots of both the licorice reference solution and the test solution were not obvious. Therefore, the inspection conditions for this method were determined to be spraying with a 10% sulfuric acid ethanol solution, heating at 105°C until the spots were clearly visible, and then examining under a UV lamp (365nm).

[0180] ⑤ Specificity Examination

[0181] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) according to the method above. Take 5 μl of each of the three solutions prepared by Method I and spot them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide test solution. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm).

[0182] Results (see) Figure 27 The results showed that the test solution exhibited fluorescent spots of the same color at the corresponding positions as the control solution, and there was no interference from the negative control solution lacking licorice, indicating that the method has good specificity.

[0183] ⑥ Sample quantity investigation

[0184] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) and spot 2 μl, 5 μl, and 10 μl of the reference herb solution and the test solution prepared by Method I above onto the same silica gel G thin-layer plate. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and examine under ultraviolet light (365 nm).

[0185] Results (see) Figure 28 The results showed that the spots were relatively obvious when the sample volume of the licorice reference material solution was 2 μl, 5 μl, and 10 μl. The spots were weaker when the sample volume of the test sample solution was 2 μl, clearer when the sample volume was 5 μl, and clearer but harder to spot when the sample volume was 10 μl. Therefore, the sample volume of both the licorice reference material solution and the test sample solution was determined to be 5 μl.

[0186] ⑦ Durability test

[0187] The effects of different temperatures, humidity levels, and brands of thin-layer chromatography plates on the experimental results were investigated according to the proposed method.

[0188] I. Tests at different temperatures

[0189] Perform the thin-layer chromatography test (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition). Take 5 μl of the reference herb solution and the test solution prepared by Method I above and spot them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent and develop at 25℃ and 4℃ respectively. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly visible, and examine it under ultraviolet light (365nm) to observe the results.

[0190] Results (see) Figure 29 The results show that when developed at 25℃ and 4℃, the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the spots are clear and the separation is good, indicating that the method is applicable in the range of 4℃ to 25℃ and has good durability.

[0191] II. Tests at different humidity levels

[0192] According to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020), 5 μl each of the reference herb solution and the test sample solution prepared by Method I above were spotted separately onto the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing solvent, and the plates were developed at relative humidity of 32%, 58%, and 88%, respectively. The plates were removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under ultraviolet light (365 nm) and the results were observed.

[0193] Results (see) Figure 30 The results show that when the test sample was developed at relative humidity of 32%, 58%, and 88%, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the reference medicinal material. The spots were clear and the separation was good, indicating that the method is applicable in the range of relative humidity from 32% to 88% and has good durability.

[0194] III. Tests on thin-layer boards of different brands

[0195] Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition) according to the method above. Take 5 μl each of the reference herb solution and the test solution prepared by Method 1 above and spot them on silica gel G thin-layer plates prepared with 1% sodium hydroxide solution of different brands (Qingdao Hailang, Qingdao Haiyang, and self-made). Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, examine under ultraviolet light (365nm) and observe the results.

[0196] Results (see) Figure 31 The results show that when different brands of silica gel G thin-layer plates (Qingdao Ocean, Qingdao Hailang, and self-made) are used, the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions as the reference medicinal material chromatogram, and the spots are clear and the separation is good. This indicates that the method is applicable to different brands of silica gel G thin-layer plates and has good durability.

[0197] ⑧ Determination of thin-layer chromatography identification method for licorice

[0198] In summary, the thin-layer chromatography method for detecting licorice in Linggui Zhugan granules is determined as follows:

[0199] Take 4g of this product, grind it into a fine powder, add 40ml of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract twice with 25ml of water-saturated n-butanol each time, combine the extracts, wash with 30ml of water-saturated n-butanol, discard the aqueous solution, evaporate the n-butanol to dryness, dissolve the residue in 1ml of methanol, and use this as the test solution. Separately, take 0.5g of licorice reference material and prepare a reference material solution using the same method. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 General Chapter 0502), apply 5μl of each of the above two solutions to the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution, develop with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (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 medicinal material. The results are shown in [Figure number missing]. Figure 32 .

[0200] 3. Feature Map

[0201] 3.1 Experimental Conditions

[0202] (1) Materials

[0203] ① Instruments: LC-20AT high performance liquid chromatograph (Shimadzu Corporation); KM-500DB ultrasonic cleaner (Kunshan Meimei Ultrasonic Instrument Co., Ltd.); T-214 0.001 g electronic balance (Beijing Sartorius Instrument Systems Co., Ltd.); XPE105 0.001 g electronic balance (Mettler-Toledo Instruments Co., Ltd.); DK-98-Ⅱ electric thermostatic water bath (Tianjin Test Instrument Co., Ltd.); 101-2AB electric thermostatic drying oven (Shanghai Tianyuan Experimental Chemical Factory).

[0204] ② Reagent solutions: Ethanol was analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20211110); Methanol was analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20210108); Methanol was chromatographic grade (Tianjin Kemei Chemical Reagent Co., Ltd., batch number: 21083825); Acetonitrile was chromatographic grade (Tianjin Kemei Chemical Reagent Co., Ltd., batch number: 21053124); Pure water (China Resources Yibao Beverage Co., Ltd., batch number: 20210106); Formic acid was chromatographic grade (Tianjin Kemei Chemical Reagent Co., Ltd., batch number: 20210112); Phosphoric acid was analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20201108); Acetic acid was chromatographic grade (Tianjin Kemei Chemical Reagent Co., Ltd., batch number: 20201121).

[0205] ③ Sample

[0206] I. Prescription composition: Poria cocos 1600g, Cinnamomum cassia twig 1200g, Atractylodes macrocephala 1200g, Glycyrrhiza uralensis 800g.

[0207] II. Sample batch number

[0208] Table 13 Batch Numbers of Linggui Zhugan Granules, Reference Samples, and Herbal Pieces

[0209]

[0210] ④ Reference Standard Information

[0211] Cinnamic acid (Chengdu Kloma Biotechnology Co., Ltd., batch number CHB201212, purity 98%)

[0212] Cinnamaldehyde (China National Institutes for Food and Drug Control, batch number 110710-201221, purity 99.6%)

[0213] Protocatechuic acid (China National Institutes for Food and Drug Control, batch number 110809-201205, purity 99.9%)

[0214] Protocatechuic acid (China National Institutes for Food and Drug Control, batch number 110809-201906, purity 97.7%)

[0215] Celery glycyrrhizin (Chengdu Kloma Biotechnology Co., Ltd., batch number CHB180109, purity 98%)

[0216] Celery glycyrrhizin (Chengdu Kloma Biotechnology Co., Ltd., batch number CHB201126, purity 98%)

[0217] Glycyrrhizin (China National Institutes for Food and Drug Control, batch number 111610-201908, purity 95%)

[0218] Mangzhigan (Chengdu Kloma Biotechnology Co., Ltd., batch number CHB180129, purity 98%)

[0219] Mangzhigan (Chengdu Kloma Biotechnology Co., Ltd., batch number CHB201114, purity 98%)

[0220] Ammonium glycyrrhizate (China National Institutes for Food and Drug Control, batch number 110731-202021, purity 96.2%)

[0221] Ammonium glycyrrhizate (China National Institutes for Food and Drug Control, batch number 110731-202122, purity 94.4%)

[0222] (2) Investigation of chromatographic conditions

[0223] ① Selection of organic phase

[0224] The experiment investigated methanol-pure water and acetonitrile-pure water. When the organic phase was acetonitrile, the separation was better and the elution ability of acetonitrile was stronger, and components with lower polarity could be effectively eluted. Therefore, acetonitrile was chosen as the organic phase.

[0225] ② Investigation of the aqueous phase

[0226] The experiment investigated acetonitrile-pure water and different types of acidic solutions. The results showed that the characteristic spectra of different acids were not significantly different. Considering that phosphoric acid is an inorganic acid with poor miscibility with organic acids, and acetic acid is highly volatile, 0.1% formic acid solution was ultimately chosen as the aqueous phase. Next, the concentrations of formic acid (0.05%, 0.1%, and 0.2%) were investigated. The acid concentration had little effect on the separation, so 0.1% formic acid solution was selected as the aqueous phase.

[0227] The final determined mobile phase was acetonitrile-0.1% formic acid water, and the optimal separation effect was achieved by adjusting the ratio of the mobile phase.

[0228] ③ Selection of chromatographic column

[0229] The experiment examined three different chromatographic columns: LP-C18, InertSustain C18, and Agilent ZORBAX SB-C18. The chromatograms of the LP-C18 column showed poor peak resolution in the 30-35 min range, while the baseline of the Agilent ZORBAX SB-C18 column was not very stable. The InertSustain C18 column had higher resolution and better peak shape. Considering all factors, the InertSustain C18 column was selected.

[0230] ④ Optimization of separation conditions

[0231] Chromatographic column: InertSustain C18 (5μm, 4.6×250mm); mobile phase: acetonitrile-0.1% formic acid water; flow rate: 1.0ml / min; column temperature: 30℃; wavelength: 254nm; injection volume: 15μl; chromatographic separation conditions were optimized. Results showed that gradient 1 had excessively concentrated peaks in the 50min–65min range and contained solvent peaks in the chromatogram. Gradient 2 separated the chromatographic peaks in the 50min–65min range and discarded the solvent peaks. Gradient 3, based on gradient 2, optimized the separation of cinnamic acid and glycyrrhizic acid to meet the analytical requirements for content determination.

[0232] Table 2 Gradient 1

[0233]

[0234] Table 3 Gradient 2

[0235]

[0236] Table 4 (Table a) Gradient 3

[0237]

[0238] (3) Investigation of the preparation method of the test sample

[0239] ① Selection of extraction solvent

[0240] The experiment investigated three extraction solvents: methanol, ethanol, and pure water. It was found that ethanol had a lower extraction efficiency. Pure water had a better extraction efficiency than methanol, but considering that pure water was prone to bacterial growth in the sample, different ratios of methanol and water (20% methanol, 50% methanol, and 70% methanol) were also investigated. The results showed that the peak areas of these three extraction solvents, with glycyrrhizic acid as the reference peak, were 1463551, 1489521, and 1449843, respectively. The peak area was larger, the separation was better, and the baseline was flatter when the solvent was 50% methanol. Therefore, 50% methanol was selected as the extraction solvent.

[0241] ② Selection of extraction method and time

[0242] The experiment investigated two extraction methods: reflux and ultrasound. The results showed that the peak areas of the different extraction methods were not significantly different and the extraction efficiencies were similar. To make the experiment more convenient, ultrasound was chosen as the extraction method.

[0243] The study investigated three different ultrasound times: 20 min, 30 min, and 40 min. The results showed that the ultrasound time had little effect on the peak shape. The peak areas of glycyrrhizic acid at different ultrasound times were 1322564, 1498521, and 1216253, respectively. The peak area was larger at 30 min, so 30 min was ultimately chosen as the ultrasound time.

[0244] ③ Selection of extraction solvent amount

[0245] The experiment investigated the use of 50% methanol as extraction solvent at concentrations of 1g-25ml, 1g-50ml, 1g-100ml, 2g-50ml, and 2g-100ml. The peak areas of glycyrrhizic acid for the first three were 2567697, 1266012, and 538836, respectively. The results showed that the peak area of ​​1g-25ml was the largest, but it was not chosen for the spiked recovery experiment. Considering the uniformity of the particles, the experiment was expanded to include 2g. The difference in content between 25 times and 50 times the amount of solvent was not significant, so 100ml of solvent was chosen for 2g of particles.

[0246] Table 5. Investigation of different solvent dosages for Linggui Zhugan Granules

[0247]

[0248] (4) Determination of chromatographic conditions and extraction methods

[0249] Based on preliminary exploration, the optimal chromatographic conditions and extraction method were determined as follows:

[0250] Optimal chromatographic conditions: Column: InertSustain C18 (5μm, 4.6×250mm); mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid solution, gradient shown in Table a; flow rate: 1.0ml / min; column temperature: 30℃; wavelength: 254nm; injection volume: 15μl.

[0251] Preparation of the test solution: Take about 2g of this product, grind it into a fine powder, weigh it accurately, place it in a stoppered conical flask, accurately add 100ml of 50% methanol, sonicate (power 500W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0252] Preparation of reference solution: Take appropriate amounts of protocatechuic acid reference standard, apigenin glycyrrhizin reference standard, glycyrrhizin reference standard, gentianin reference standard, cinnamaldehyde reference standard, ammonium glycyrrhizate reference standard and cinnamic acid reference standard, accurately weigh them, place them in volumetric flasks, add methanol to prepare a solution containing 5 μg protocatechuic acid, 5 μg apigenin glycyrrhizin, 30 μg glycyrrhizin, 5 μg gentianin, 50 μg cinnamaldehyde, 60 μg glycyrrhizic acid and 5 μg cinnamic acid per ml.

[0253] (5) Methodological investigation

[0254] ① Repeatability test

[0255] Six samples of the same test sample (batch number: 201101) were prepared, and test solutions were injected separately. The samples were measured according to the method, and the results were recorded and integrated. Peak 8 (glycyrrhizic acid) was used as the reference peak. The relative retention time and relative peak area were calculated. The results are shown in Tables 6 and 7. The RSD% values ​​of the relative retention time and relative peak area were less than 3%, indicating that the method has good repeatability.

[0256] Table 6. Repeatability data (relative retention time) of Linggui Zhugan Granules

[0257]

[0258] Table 7. Repeatability data (relative peak area) of Linggui Zhugan Granules

[0259]

[0260] ②Precision test

[0261] Take one sample of the same test sample (batch number: 201101), prepare a test solution, inject the sample 6 times consecutively, record the results, and use peak 8 (glycyrrhizic acid) as the reference peak to calculate the relative retention time and relative peak area. The results are shown in Tables 8 and 9. The RSD% values ​​of the relative retention time and relative peak area are less than 5%, indicating that the method has good precision.

[0262] Table 8. Precision data of Linggui Zhugan granules (relative retention time)

[0263]

[0264] Table 9. Precision data (relative peak area) of Linggui Zhugan Granules

[0265]

[0266] ③Stability test

[0267] One sample from the same batch of test samples (batch number: 201101) was used to prepare a test solution. The sample was injected at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, and 24h. The chromatograms were recorded and integrated. Peak 8 (glycyrrhizic acid) was used as the reference peak. The relative retention time and relative peak area were calculated. The results are shown in Tables 10 and 11. The RSD% of the relative retention time and relative peak area was less than 5%. The experiment showed that the test solution had good stability within 24h.

[0268] Table 10. Stability data (relative retention time) of Linggui Zhugan Granules

[0269]

[0270] Table 11. Stability data (relative peak area) of Linggui Zhugan Granules

[0271]

[0272] ④ Specificity test

[0273] Prepare a test solution using Linggui Zhugan Granules (batch number 220901). Following the same formulation and preparation method as the Linggui Zhugan Granules reference sample, prepare negative samples lacking licorice and cinnamon twig. Prepare negative sample solutions using the same method as the test solution. Determine the chromatograms under the above chromatographic conditions. Plot chromatograms of ammonium glycyrrhizate reference standard, cinnamic acid reference standard, cinnamaldehyde reference standard, the Linggui Zhugan Granules test sample, and the negative sample. The test sample chromatogram shows peaks with the same retention times as the reference standards, and the negative sample shows no interference. The experiment demonstrates that this method has good specificity.

[0274] (6) Research on Peak Identification

[0275] ① Confirmation with reference standard

[0276] The reference standard was used to identify each chromatographic peak in the characteristic chromatogram. The preparation of the reference standard solution is described in the preparation of the reference solution. The peaks were injected separately according to the optimal chromatographic conditions and identified by the reference standard.

[0277] ②Chromatographic peak identification

[0278] The nine common peaks from three batches of particles were assigned and identified. Figures 33-36 Peaks 1 and 5 belong to cinnamon twig; peaks 2-4 and 6-9 belong to licorice.

[0279] Table 12 Peak Attribution and Identification

[0280]

[0281] (7) Establishment and evaluation of the characteristic spectrum of Linggui Zhugan Granules

[0282] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in Table a; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on glycyrrhizic acid, should be no less than 3000.

[0283] Preparation of reference solutions: Take appropriate amounts of protocatechuic acid reference standard, apigenin reference standard, glycyrrhizin reference standard, gentianin reference standard, cinnamaldehyde reference standard, ammonium glycyrrhizate reference standard and cinnamic acid reference standard, accurately weigh them, and add methanol to prepare solutions containing 5 μg of protocatechuic acid, 5 μg of apigenin reference standard, 30 μg of glycyrrhizin, 5 μg of gentianin, 50 μg of cinnamaldehyde, 60 μg of glycyrrhizic acid and 5 μg of cinnamic acid per ml.

[0284] Preparation of the test solution: Take about 2g of this product, grind it into a fine powder, weigh it accurately, place it in a stoppered conical flask, add 100ml of 50% methanol accurately, sonicate (power 500W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0285] Determination method: Accurately pipette 15 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0286] Three batches of particulate samples were tested according to the above-mentioned detection method. The results are shown in the figure. Figures 37-38The chromatogram of the test sample should show 9 characteristic peaks, of which 7 peaks should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the glycyrrhizic acid reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak. The relative retention times should be within ±10% of the specified values, which are 0.21 (peak 1), 0.49 (peak 2), 0.50 (peak 3), 0.71 (peak 4), 0.81 (peak 5), 0.87 (peak 6), 0.95 (peak 7), and 1.03 (peak 9).

[0287] Table 133 shows the relative retention times of the characteristic chromatograms of Linggui Zhugan Granules.

[0288]

[0289]

[0290] Table 143 shows the relative peak areas of the characteristic chromatograms of Linggui Zhugan Granules.

[0291]

[0292] 4. Inspection

[0293] In accordance with the requirements of General Chapter 0104 Granules in the 2020 edition of the Chinese Pharmacopoeia, the particle size, moisture content, solubility, fill weight variation and microbial limits of this product were tested.

[0294] (1) Particle size

[0295] According to the particle size determination method (Chinese Pharmacopoeia 2020 Edition, General Chapter 0982, Method II, Double Sieve Method), the sum of particles that cannot pass through sieve No. 1 and particles that can pass through sieve No. 5 shall not exceed 15%. The verification test results of three batches of this product are shown in Table 15, and all meet the requirements.

[0296] Table 15. Results of Particle Size Examination for Linggui Zhugan Granules

[0297]

[0298] (2)Moisture

[0299] According to the moisture determination method (General Chapter 0832, Method II, Chinese Pharmacopoeia 2020 Edition), the moisture content of traditional Chinese medicine granules shall not exceed 8%. The verification test results of three batches of this product are shown in Table 16, and all meet the requirements.

[0300] Table 16. Moisture content of Linggui Zhugan Granules

[0301]

[0302] (3) Solubility

[0303] According to the requirements of General Chapter 0104 of the 2020 edition of the Chinese Pharmacopoeia, take one bag of the test sample (10g for multi-dose packaging), add 200ml of hot water, stir for 5 minutes, and observe immediately. It should be completely dissolved without any burnt residue or other foreign matter. The validation test results of three batches of this product showed that all samples dissolved completely without any burnt residue or other foreign matter, and all met the requirements.

[0304] (4) Content difference

[0305] Take 10 bags of the test sample, remove the packaging, and accurately weigh the contents of each bag. Calculate the fill weight of each bag and the average fill weight, with a fill weight variation limit of ±5%. The results of the three batches of validation tests for this product all met the requirements.

[0306] (5) Microbial limits

[0307] According to General Chapter 0104 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the microbial limit test for non-sterile products, namely the microbial count method (General Chapter 1105 of the 2020 edition of the Chinese Pharmacopoeia) and the control bacteria test method (General Chapter 1106 of the 2020 edition of the Chinese Pharmacopoeia), and the microbial limit standard for non-sterile drugs (General Chapter 1107 of the 2020 edition of the Chinese Pharmacopoeia), stipulates that the total number of aerobic bacteria should not exceed 10. 3 cfu / g, total mold and yeast count not greater than 10 2 CFU / g, Escherichia coli not detectable. The test results for three batches of this product are shown in Table 17, and all meet the requirements.

[0308] Table 17 Microbiological Examination Results

[0309]

[0310] 5. Extract

[0311] 5.1 Extract of the mixed powder of Linggui Zhugan granules

[0312] The extract should be determined by hot extraction method under the alcohol-soluble extractives determination method (General Chapter 2201, Chinese Pharmacopoeia 2020 Edition), using ethanol as the solvent, and the extractives should not be less than 10.0%.

[0313] 5.2 Extract of Linggui Zhugan Granules

[0314] Take an appropriate amount of this product, grind it into a fine powder, take 2g, weigh it accurately, add 100ml of ethanol accurately, and determine it according to the hot extraction method of the alcohol-soluble extractives determination method (General Chapter 2201 of Chinese Pharmacopoeia 2020). Ethanol is used as the solvent. According to the lower limit of the extractives range of the reference sample, it is 10.93%. The addition of excipients may affect the extractives of the product.

[0315] The formulation is as follows: Poria cocos 1600g, Cinnamomum cassia twig 1200g, Atractylodes macrocephala 1200g, Glycyrrhiza uralensis 800g, totaling 4800g. The yield of the dry extract is between 13.3% and 16.2%, which means the weight of the dry extract is between 638.4g and 777.6g. The maximum amount of excipients is 1000-638.4=361.6g, and the amount of excipients ranges from 22.2% to 36.2%. Based on the proposed leachate range (10.93%–20.31%) of the reference sample of this product, the leachate range per 1g of particles can be calculated as: (1-0.362)×10.93%=5.84%, (1-0.222)×20.31%=15.8%. Considering that the leachate from the excipients themselves accounts for a certain proportion, and in conjunction with the test data of three batches of production samples, the leachate limit is appropriately increased, and the proposed leachate of this product is not less than 9.0%.

[0316] Table 18 Results of Leachate Extract

[0317]

[0318] 6. Content determination

[0319] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0320] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A; and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table a above; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on glycyrrhizic acid, should not be less than 3000.

[0321] 6.1 Content Detection of Linggui Zhugan Granule Mixture Powder

[0322] Preparation of reference solutions: Accurately weigh appropriate amounts of ammonium glycyrrhizate reference standard and cinnamic acid reference standard, and add methanol to prepare solutions containing 0.10 mg glycyrrhizic acid and 0.02 mg cinnamic acid per 1 ml. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207).

[0323] Preparation of the test solution: Weigh 1.0 g of the powder accurately, place it in a stoppered conical flask, add 100 ml of 70% methanol accurately, weigh the solution, sonicate (500 W power, 40 kHz frequency) for 30 min, cool, weigh the solution again, make up the weight loss with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0324] Assay: Accurately pipette 15 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0325] The proposed yield range for this product is 13.3%–16.2%, with an average yield of 14.77%. After adding excipients before and after drying to make up the weight, and based on the proposed specification of 11.5g / bag, taken three times a day, one bag each time, this is equivalent to adding excipients to make up to 34.50g of extract from 165.6g of medicinal slices. 165.6g of medicinal slices yields 24.46g of dry extract at an average yield of 14.77%, which, when made up to 34.50g, is equivalent to a content dilution of 24.46 / 34.5 = 0.71. Therefore, based on the reference sample of this product, the proposed content range is as follows: Calculated on a dried basis, the content of cinnamic acid (C9H8O2) is 0.53mg / g–0.98mg / g, and the content of glycyrrhizic acid (C9H8O2) is 0.53mg / g–0.98mg / g. 42 H 62 O 16 The content is calculated to be 5.20 mg / g to 9.65 mg / g.

[0326] 6.2 Content Detection of Linggui Zhugan Granules

[0327] The content determination index components are consistent with those of the reference sample, namely glycyrrhizic acid, cinnamic acid, and cinnamaldehyde.

[0328] (1) Material and Sample Information

[0329] ① Instruments

[0330] LC-20AT High Performance Liquid Chromatograph (Shimadzu Corporation)

[0331] KM-500DB Ultrasonic Cleaner (Kunshan Meimei Ultrasonic Instrument Co., Ltd.)

[0332] T-214 Type 0.01% Electronic Balance (Beijing Sartorius Instrument Systems Co., Ltd.)

[0333] XPE105 electronic balance with a precision of 0.00001 g / dL (Mettler-Toledo Instruments Ltd.)

[0334] DK-98-Ⅱ Electric Thermostatic Water Bath (Tianjin Tester Instrument Co., Ltd.)

[0335] 101-2AB Electric Heating Constant Temperature Drying Oven (Shanghai Tianyuan Experimental Chemical Factory)

[0336] ② Reagents

[0337] Ethanol was of analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20211110).

[0338] Methanol was of analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20210108).

[0339] Methanol was of chromatographic grade (Tex Corporation, USA, batch number: 21083825).

[0340] Acetonitrile was of chromatographic grade (Tex Corporation, USA, lot number: 21053124).

[0341] Pure water (China Resources Yibao Beverage Co., Ltd., batch number: 20210106)

[0342] Formic acid was of chromatographic grade (Tianjin Kemeio Chemical Reagent Co., Ltd., batch number: 20210112).

[0343] Phosphoric acid was of analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20201108).

[0344] Acetic acid was of chromatographic grade (Tianjin Kemeio Chemical Reagent Co., Ltd., batch number: 20201121).

[0345] ③ Sample and prescription information

[0346] Poria cocos 1600g, Cinnamomum cassia twig 1200g, Atractylodes macrocephala 1200g, Glycyrrhiza uralensis 800g.

[0347] Table 19. Batch numbers of Linggui Zhugan Granules and Herbal Pieces

[0348]

[0349] ④ Reference Standard Information

[0350] Cinnamic acid (Chengdu Kloma Biotechnology Co., Ltd., batch number CHB201212, purity 98%)

[0351] Cinnamaldehyde (China National Institutes for Food and Drug Control, batch number 110710-201221, purity 99.6%)

[0352] Ammonium glycyrrhizate (China National Institutes for Food and Drug Control, batch number 110731-202021, purity 96.2%)

[0353] Ammonium glycyrrhizate (China National Institutes for Food and Drug Control, batch number 110731-202122, purity 94.4%)

[0354] (2) Establishment of content determination method

[0355] The characteristic chromatographic determination method was adopted, and the determination was carried out according to the high performance liquid chromatography method (General Chapter 0512 of Chinese Pharmacopoeia 2020).

[0356] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A; and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table a; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on glycyrrhizic acid, should not be less than 3000.

[0357] Preparation of reference solutions: Accurately weigh appropriate amounts of ammonium glycyrrhizate reference standard and cinnamic acid reference standard, and dissolve them separately in methanol to prepare solutions containing 0.05 mg of glycyrrhizic acid and 0.01 mg of cinnamic acid per 1 ml. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207)

[0358] Preparation of the test solution: Take about 2g of this product, grind it into a fine powder, weigh it accurately, place it in a stoppered conical flask, accurately add 100ml of 50% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30min, cool it, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0359] Assay: Accurately pipette 15 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0360] This product, calculated on a dried basis, contains 0.53 mg / g to 0.98 mg / g of cinnamic acid (C9H8O2) and glycyrrhizic acid (C... 42 H 62 O 16 The content is calculated to be 5.20 mg / g to 9.65 mg / g.

[0361] (3) Methodological investigation of content determination

[0362] ①Specificity research

[0363] Prepare a test solution using Linggui Zhugan granules (batch number 220901). Prepare negative samples lacking licorice and cinnamon twig according to the Linggui Zhugan granules prescription and preparation method. Prepare negative sample solutions using the same method as the test solution. Determine the chromatograms under the above chromatographic conditions. Plot chromatograms of ammonium glycyrrhizate reference standard, cinnamic acid reference standard, cinnamaldehyde reference standard, Linggui Zhugan granules test sample, and negative sample. The test sample chromatogram shows peaks with the same retention times as the reference standards, and the negative sample shows no interference. The experiment demonstrates that this method has good specificity.

[0364] ② Repeatability test

[0365] Six samples of the same test sample (batch number: 221001) were taken, and test solutions were prepared and injected separately. The content was determined according to the method. Peak 6 (cinnamaldehyde) was used as the reference peak, and the content was calculated. The results are shown in Table 20. The RSD% value of the content was less than 3%, which indicates that the method has good repeatability.

[0366] Table 20. Repeatability data for Linggui Zhugan Granules (cinnamaldehyde)

[0367]

[0368] ③ Linear range test

[0369] Accurately weigh appropriate amounts of ammonium glycyrrhizate, cinnamic acid, and cinnamaldehyde standards, place them in volumetric flasks, and dilute to volume with methanol. Use the above solutions as standard stock solutions, dilute and determine them sequentially, record the chromatograms and integrate them. Calculate the peak areas using cinnamic acid, glycyrrhizic acid, and cinnamaldehyde as reference peaks. The results are shown in Table 21.

[0370] Table 21 Linearity and Range

[0371]

[0372] ④ Spiking recovery test

[0373] Six portions of the same sample with pre-determined content were weighed accurately (batch number 220901, glycyrrhizic acid content was 6.35 mg / g, cinnamic acid content was 0.52 mg / g, and cinnamaldehyde content was 2.68 mg / g in batch number 20230520). Appropriate amounts of reference standards were added to each sample, and the solution was prepared according to the test solution preparation method. The results are shown in Table 22. The experiment shows that the recovery rate of this method is good.

[0374] Table 22 Sampling and Recovery Data

[0375]

[0376]

[0377] (4) Content of indicative components in Linggui Zhugan Granules

[0378] The indicator components in the particles were determined using the above-described chromatographic conditions.

[0379] The average glycyrrhizic acid content of the three batches of granules was 9.15 mg / g, with an actual range of 9.12-9.19 mg / g; the average cinnamic acid content was 1.00 mg / g, with an actual range of 1.00-1.00 mg / g; and the average cinnamaldehyde content was 0.92 mg / g, with an actual range of 0.89-0.94 mg / g.

[0380] Table 23 Content of Indicative Components in Linggui Zhugan Granules

[0381]

[0382] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0383] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A quality testing method for a preparation of Linggui Zhugan, characterized in that, The quality detection method of the Linggui Zhugan preparation is the quality detection method of Linggui Zhugan granules; the quality detection method of the Linggui Zhugan preparation includes constructing characteristic chromatograms by liquid chromatography and determining the content of cinnamic acid and glycyrrhizic acid. The liquid chromatography uses acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution according to the provisions in Table a. Table a. Gradient elution program The flow rate was 1.0 ml per minute; the column temperature was 30 ℃; and the detection wavelength was 254 nm. The steps for constructing characteristic chromatograms using liquid chromatography include: S11, Preparation of reference solution: Take appropriate amounts of protocatechuic acid reference standard, apigenin glycyrrhizin reference standard, glycyrrhizin reference standard, gentianin reference standard, cinnamaldehyde reference standard, ammonium glycyrrhizate reference standard and cinnamic acid reference standard, and add methanol to each to obtain the solution. S12, Preparation of the test solution: Take an appropriate amount of Linggui Zhugan preparation sample, add 50% methanol (volume concentration), sonicate, filter and collect the filtrate to obtain the solution; S13, inject 15 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result; The chromatographic column used in the liquid chromatography method was an InertSustain C18, 5 μm, 4.6 × 250 mm.

2. The quality testing method for the Linggui Zhugan preparation as described in claim 1, characterized in that, In step S11 of the quality detection method for Linggui Zhugan granules, methanol is added to prepare a solution containing 5 μg of protocatechuic acid, 40 μg of apigenin glycyrrhizin, 50 μg of glycyrrhizin, 5 μg of gentianin, 50 μg of cinnamaldehyde, 100 μg of glycyrrhizic acid and 20 μg of cinnamic acid per ml.

3. The quality testing method for the Linggui Zhugan preparation as described in claim 1, characterized in that, The determination of cinnamic acid and glycyrrhizic acid content using liquid chromatography includes the following steps: S21, take appropriate amounts of ammonium glycyrrhizate reference standard and cinnamic acid reference standard, add methanol to prepare solutions respectively, and the solution is obtained; S22, Preparation of the test solution: Take an appropriate amount of Linggui Zhugan preparation sample, add 50% methanol (volume concentration), sonicate, filter and collect the filtrate to obtain the solution; S23, inject 15 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

4. The quality testing method for the Linggui Zhugan preparation as described in claim 1, characterized in that, The quality testing methods for the Linggui Zhugan preparation also include thin-layer chromatography identification of Poria cocos, thin-layer chromatography identification of Atractylodes macrocephala, and thin-layer chromatography identification of Glycyrrhiza uralensis.

5. The quality testing method for the Linggui Zhugan preparation as described in claim 4, characterized in that, The steps of the thin-layer chromatography method for identifying Poria cocos include: S31. Take 10 g of the Linggui Zhugan preparation of this product, add 50 ml of ether, sonicate for 10 min, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution. S32, take 1.0 g of Poria cocos reference material, add 50 ml of ether, sonicate for 10 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to prepare the reference material solution; S33. Take 20 μl of the test solution and 2 μl of the reference herb solution and spot them separately on the same silica gel G thin-layer plate. Use a toluene-ethyl acetate-formic acid solution with a volume ratio of 20:5:0.5 as the developing solvent. Develop, remove, and air dry. Spray with a mixture of 2% vanillin-sulfuric acid solution and ethanol with a volume ratio of 4:

1. Heat at 105 °C until the spots are clearly visible and examine under fluorescent light.

6. The quality testing method for the Linggui Zhugan preparation as described in claim 4, characterized in that, The steps of the thin-layer chromatography identification method for Atractylodes macrocephala include: S41. Take 4 g of the Linggui Zhugan preparation of this product, grind it into a fine powder, add 20 ml of n-butanol, sonicate for 30 min, filter, wash the filtrate twice with 30 ml of water each time, discard the water, evaporate the n-butanol solution to dryness, add 1 ml of acetone to the residue to dissolve it, and use it as the test solution. S42, take 0.5 g of Atractylodes macrocephala reference material, add 15 ml of n-butanol, sonicate for 30 min, filter, wash the filtrate twice with 10 ml of water each time, discard the water, evaporate the n-butanol solution to dryness, add 1 ml of acetone to dissolve the residue, and use it as the reference material solution. S43, take 10 μl each of the reference herb solution and the test sample solution and spot them separately on the same silica gel G thin-layer plate. Use a chloroform-acetone solution with a volume ratio of 19:1 as the developing solvent, develop, remove, air dry, and examine under a 365 nm ultraviolet light.

7. The quality testing method for the Linggui Zhugan preparation as described in claim 4, characterized in that, The steps of the licorice thin-layer chromatography identification method include: S51. Take 4 g of the Linggui Zhugan preparation, grind it into a fine powder, add 40 ml of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, extract twice with water-saturated n-butanol, 25 ml each time, combine the extracts, wash with 30 ml of water-saturated n-butanol, discard the water, evaporate the n-butanol to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution. S52, take 0.5 g of licorice reference material, grind it into a fine powder, add 40 ml of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, extract twice with water-saturated n-butanol, 25 ml each time, combine the extracts, wash with 30 ml of water-saturated n-butanol, discard the water, evaporate the n-butanol to dryness, add 1 ml of methanol to dissolve the residue, and prepare the reference material solution; S53. Take 5 μl each of the reference herb solution and the test sample solution and spot them separately on the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Use ethyl acetate-formic acid-glacial acetic acid-water (volume ratio 15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under a 365 nm ultraviolet lamp.

8. The quality testing method for the Linggui Zhugan preparation as described in claim 1, characterized in that, The quality testing methods for the Linggui Zhugan preparation also include appearance inspection, general inspection of granules, and extractives inspection.

9. The quality testing method for the Linggui Zhugan preparation as described in claim 8, characterized in that, The extract was tested using a hot leaching method with ethanol as the solvent.

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