Quality detection method of Linggui curcuma zedoary and liquorice preparation

Abstract

The invention provides a quality detection method of a Linggui curcuma zedoary and liquorice preparation. The quality detection method of the Linggui curcuma zedoary and liquorice preparation comprises a quality detection method of Linggui curcuma zedoary and liquorice granules and a quality detection method of Linggui curcuma zedoary and liquorice granule mixed powder. The quality detection method of the Linggui curcuma zedoary and liquorice preparation comprises the following steps: constructing a characteristic chromatogram by adopting a liquid chromatography method, and determining the content of cinnamic acid and glycyrrhizic acid, in the liquid chromatography method, acetonitrile is taken as a mobile phase A, a 0.1% formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to regulations; according to the quality detection method of the Linggui curcuma zedoary and liquorice preparation, the quality of the Linggui curcuma zedoary and liquorice preparation is detected through multiple indexes, the quality standard of the Linggui curcuma zedoary and liquorice preparation can be established, the quality of the Linggui curcuma zedoary and liquorice preparation is effectively controlled, and a solid foundation is laid for the stable quality of the Linggui curcuma zedoary and liquorice preparation.

Description

Technical Field

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

[0002] Ling Gui Shu Gan Tang, a Chinese medicine prescription, is a dehumidifying agent with the effects of warming yang, transforming phlegm, strengthening spleen and eliminating dampness. Among them, Poria cocos is the dried sclerotium of the Polyporaceae fungus Poria cocos (Schw.) Wolf. It is mostly dug from July to September, and the mud and sand are removed after digging. After being piled up to "sweat", it is spread out to dry on the surface, and then "sweated" again. After repeated several times until wrinkles appear and most of the internal moisture is lost, it is dried in the shade and called "Poria cocos pieces"; or fresh Poria cocos is cut according to different parts and dried in the shade, which are called "Poria cocos blocks" and "Poria cocos slices" respectively. Cassia twig is the dried young branch of Cinnamomum cassia Presl, a plant of 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. Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala Koidz., a plant of Asteraceae. In winter, when the lower leaves turn yellow and the upper leaves become brittle, the leaves are dug up, the sand is removed, the leaves are dried in the oven or in the sun, and the fibrous roots are removed. Licorice is the dried root and rhizome of the leguminous plant Glycyrrhiza uralensis Fisch. It is dug up in spring and autumn, the fibrous roots are removed, and the leaves are dried in the sun.

[0003] Due to the complexity of Chinese medicinal materials and the heterogeneity of medicinal materials, the quality control of benchmark samples is very difficult. For the quality research of benchmark samples, it is necessary to use specific identification and multi-component, overall quality evaluation indicators to characterize their quality. Nowadays, the yield of decoction pieces, transfer rate of index components, content determination, fingerprint / characteristic spectrum, etc. are mostly used as indicators for value transfer research to evaluate the quality correlation between 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 Ling Gui Shu Gan preparations. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and provide a quality detection method that comprehensively reflects the intrinsic quality of Ling Gui Shu preparations and better controls Ling Gui Shu Gan preparations.

[0005] Contents of the present invention include:

[0006] A quality detection method for Ling Gui Shu Gan preparations, the quality detection method for Ling Gui Shu Gan preparations comprising a quality detection method for Ling Gui Shu Gan granules and a quality detection method for Ling Gui Shu Gan granule mixed powder; the quality detection method for Ling Gui Shu Gan preparations comprising constructing a characteristic spectrum and determining the contents 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 provisions in Table a below;

[0007] Table a Gradient elution program

[0008]

[0009]

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

[0011] The step of constructing a characteristic spectrum using liquid chromatography comprises:

[0012] S11, preparation of reference solution: take appropriate amount of protocatechuic acid reference substance, apigenin liquiritin reference substance, liquiritin reference substance, formononetin reference substance, cinnamaldehyde reference substance, ammonium glycyrrhizinate reference substance and cinnamic acid reference substance, add methanol respectively to obtain;

[0013] S12, preparation of test solution: take an appropriate amount of Ling Gui Shu Gan preparation sample, add 50% methanol by volume, sonicate, filter and obtain the filtrate;

[0014] S13, aspirate 15 μl of the reference solution and the test solution, inject into the liquid chromatograph, and measure to obtain.

[0015] In step S12, 2.0 g of the Ling Gui Shu Gan preparation sample is taken, 100 ml of methanol is added, the ultrasonic power is 500 W, the frequency is 40 kHz, and the ultrasonic time is 30 min.

[0016] Furthermore, in step S11 of the quality detection method of the Ling Gui Shu Gan granules, methanol is added to prepare a solution containing 5 μg of protocatechuic acid, 40 μg of apigenin, 50 μg of liquiritin, 5 μg of formononetin, 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 of the Ling Gui Shu Gan granule mixed powder, methanol is added to prepare a solution containing 5 μg of protocatechuic acid, 5 μg of apigenin, 30 μg of liquiritin, 5 μg of formononetin, 50 μg of cinnamaldehyde, 60 μg of glycyrrhizic acid and 5 μg of cinnamic acid per 1 ml.

[0017] Furthermore, the steps of determining the contents of cinnamic acid and glycyrrhizic acid by liquid chromatography include:

[0018] S21, take appropriate amount of ammonium glycyrrhizinate reference substance and cinnamic acid reference substance, add methanol to prepare solutions respectively, and obtain;

[0019] S22, preparation of the test solution: take an appropriate amount of Ling Gui Shu Gan preparation sample, add 50% methanol by volume, sonicate, filter and obtain the filtrate;

[0020] S23, aspirate 15 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure to obtain.

[0021] In the S22, 2.0 g of the Ling Gui Shu Gan preparation sample is taken, 100 ml of methanol is added, the ultrasonic power is 500 W, the frequency is 40 kHz, and the ultrasonic time is 30 min.

[0022] Furthermore, in step S21 of determining the contents of cinnamic acid and glycyrrhizic acid in the mixed powder of Ling Gui Shu Gan granules, a solution containing 0.10 mg of glycyrrhizic acid and 0.02 mg of cinnamic acid per 1 ml is prepared; in step S21 of determining the contents of cinnamic acid and glycyrrhizic acid in the Ling Gui Shu Gan granules, a solution containing 0.05 mg of glycyrrhizic acid and 0.01 mg of cinnamic acid per 1 ml is prepared.

[0023] Furthermore, the quality inspection method of the Ling Gui Shu Gan preparation also includes the Poria cocos thin layer identification method, the Atractylodes macrocephala thin layer identification method and the Licorice thin layer identification method.

[0024] Further, the steps of the Poria cocos thin layer identification method include:

[0025] S31, take 10g of Lingguishugan preparation of this product, add 50ml of ether, ultrasonically treat 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, taking 1.0 g of Poria cocos control medicinal material, adding 50 ml of ether, ultrasonically treating for 10 min, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1 ml of methanol as the control medicinal material solution;

[0027] S33, take 20μl of the test solution and 2μl of the control drug solution, spot them respectively on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid solution with a volume ratio of 20:5:0.5 as the developing solvent, develop, take out, dry, spray with a mixed solution of 2% vanillin sulfuric acid solution and ethanol with a volume ratio of 4:1, heat at 105℃ until the spots are clearly colored, and inspect under fluorescent light.

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

[0029] S41, take 4g of Ling Gui Shu Gan preparation of this product, grind it into powder, add 20ml of n-butanol, ultrasonically treat for 30min, filter, wash the filtrate with water twice, 30ml each time, discard the water, evaporate the n-butanol solution, add 1ml of acetone to dissolve the residue, and use it as the test solution;

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

[0031] S43, take 10 μl of the control medicinal material solution and the test sample solution, respectively, and spot them on the same silica gel G thin layer plate, use chloroform-acetone solution with a volume ratio of 19:1 as the developing agent, develop, take out, dry, and examine under a 365nm wavelength ultraviolet lamp.

[0032] Further, the steps of the licorice thin layer identification method include:

[0033] S51, take 4g of Ling Gui Shu Gan preparation of this product, grind it into powder, add 40ml of methanol, ultrasonically treat for 30min, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, shake and extract twice with n-butanol saturated with water, 25ml each time, combine the extracts, wash with 30ml of water saturated with 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 control medicinal material, grind it, add 40ml of methanol, ultrasonically treat it for 30min, filter it, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, shake and extract it twice with n-butanol saturated with water, each time 25ml, combine the extracts, wash them with 30ml of water saturated with n-butanol, discard the water, evaporate the n-butanol to dryness, add 1ml of methanol to dissolve the residue, and prepare a control medicinal material solution;

[0035] S53, take 5μl of the control medicinal material solution and the test sample solution, respectively, and spot them 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 with a volume ratio of 15:1:1:2 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under a UV lamp with a wavelength of 365nm.

[0036] Furthermore, the quality inspection method of the Ling Gui Zhu Gan preparation also includes property inspection, general inspection of granules and extract inspection.

[0037] Furthermore, the extract inspection adopts a hot immersion method and uses ethanol as a solvent for measurement.

[0038] Beneficial effects of the present invention:

[0039] (1) Ling Gui Shu Gan preparations were subjected to property inspection, thin layer identification, characteristic spectrum determination, general item inspection for granules, extract inspection and content determination. Thin layer identification was carried out by thin layer chromatography; general item inspection for granules included inspection of water content, particle size, solubility, filling volume difference and microbial limit; extract was determined by hot leaching; characteristic spectrum, cinnamic acid and glycyrrhizic acid content were determined by liquid chromatography. The quality inspection methods were relatively comprehensive and complete;

[0040] (2) The quality detection method of the Ling Gui Shu Gan preparation of the present invention detects the quality of the Ling Gui Shu Gan preparation through multiple indicators, can establish a feasible quality standard for the Ling Gui Shu Gan preparation, achieve effective control of the quality of the Ling Gui Shu Gan preparation, and lay a solid foundation for the stability of the quality of the Ling Gui Shu Gan preparation;

[0041] (3) Using the chromatographic conditions of the present application for liquid phase analysis can obtain a characteristic spectrum with better separation and clearer color. The method has strong specificity, good stability, high precision, good reproducibility, and can attribute and identify the 9 common peaks in the chromatogram;

[0042] (4) The thin layer chromatography identification method of the present application was used to achieve thin layer identification of four medicinal materials, Poria cocos, cinnamon twig, Atractylodes macrocephala and Licorice, through three display methods. It was simple and fast, with clear spots and distinct layers;

[0043] (5) The content determination method of the present application can be used to measure the contents of representative cinnamic acid and glycyrrhizic acid in Ling Gui Shu Gan preparations, with strong specificity, good repeatability, good linearity, and good sample recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is the first method of identification of Poria cocos by thin layer chromatography; Figure 2 It is the second method of identification of Poria cocos by thin layer chromatography; Figure 3 It is a study on the development agent for TLC identification of Poria cocos; Figure 4 It is a study on the colorimetric identification of Poria cocos by thin layer chromatography; Figure 5 It is a study on the specificity of Poria cocos TLC identification; Figure 6 It is a survey of the amount of samples for TLC identification of Poria cocos; Figure 7 It is a test of durability of Poria cocos thin layer identification at different temperatures; Figure 8 It is a thin layer of Poria cocos to identify the durability test of different relative humidity; Fig. 9 It is a test of the durability of different thin-layer boards by using Poria cocos thin-layer to identify them; Fig.10 This is the TLC spectrum of Poria cocos in the granules; Fig.11 It is the first method of identification of cinnamon twig thin layer; Fig.12 It is the second identification method of cinnamon twig thin layer; Fig.13 It is the first method of identification of Atractylodes macrocephala thin layer; Fig.14 It is the second method of identification of Atractylodes macrocephala thin layer; Fig.15 It is a study on the development agent of Atractylodes macrocephala thin layer identification; Fig.16 It is a study on the colorimetric agent for the identification of Atractylodes macrocephala thin layer; Fig.17 It is an investigation on the specificity of thin-layer identification of Atractylodes macrocephala; Fig.18 It is a study on the amount of samples for identification of Atractylodes macrocephala in thin layer; Fig.19 It is a test of durability of Atractylodes macrocephala thin layer identification at different temperatures; Fig. 20 It is a test of durability of Atractylodes macrocephala thin layer identification at different relative humidity; Fig.21 It is a test of the durability of different thin-layer boards of Atractylodes macrocephala; Fig. 22 This is the TLC spectrum of Atractylodes macrocephala in the granules; Fig.23 It is the first method of identification of licorice by thin layer chromatography; Fig.24 It is the second method of identification of licorice by thin layer chromatography; Fig.25 It is a study on the development agent for TLC identification of licorice; Fig.26 It is a study on the colorimetric agent for TLC identification of licorice; Fig. 27 It is a study on the specificity of TLC identification of licorice; Fig.28 It is a survey of the amount of samples for the identification of licorice by thin layer chromatography; Fig.29 It is a test of the durability of licorice thin layer identification at different temperatures; Fig.30 It is a test of the durability of licorice thin layer identification at different relative humidity; Fig.31 It is a test of the durability of different thin-layer plates for identification of liquorice thin-layer; Fig.32 This is the TLC spectrum of licorice in the granules; Fig.33 It is the characteristic spectrum of Poria cocos, lack of Poria cocos, and granules; Fig.34 It is cinnamon twig, lack of cinnamon twig, characteristic spectrum of granules; Fig.35 It is the characteristic spectrum of Atractylodes macrocephala, lack of Atractylodes macrocephala, and granul Fig.36 It is the characteristic spectrum of licorice, lack of licorice, and granules; Fig.37 It is an overlay of characteristic spectra of three batches of particles; Fig.38 It is a comparison chart of particle characteristic chart. DETAILED DESCRIPTION

[0045] The preparation method of Ling Gui Shu Gan granule mixed powder is as follows: 1600g of Poria cocos, 1200g of cinnamon twig, 1200g of Atractylodes macrocephala and 800g of liquorice are soaked in 8 times water for 30min, decocted for 1h, and aromatic water is collected at the same time, the aromatic water is included in beta-cyclodextrin, the inclusion compound is dried at low temperature, and crushed into fine powder for standby use; the medicinal liquid is filtered, and the filtrate is concentrated under reduced pressure to an extract with a relative density of 1.03-1.08 (60°C) (the dry extract yield is 13.3%-16.2%), filtered, 5% of the amount of dextrin in the amount of the decoction piece is added and mixed, spray dried, and then an appropriate amount of inclusion compound fine powder and dextrin are added, mixed, and the product is obtained.

[0046] The preparation method of Ling Gui Shu Gan granules is as follows: 1600g of Poria cocos, 1200g of cinnamon twig, 1200g of Atractylodes macrocephala, and 800g of licorice are soaked in 8 times water for 30min, decocted for 1h, and aromatic water is collected at the same time, and the aromatic water is included in beta-cyclodextrin, and the inclusion compound is dried at low temperature and crushed into fine powder for standby use; the medicinal liquid is filtered, and the filtrate is concentrated under reduced pressure to an extract with a relative density of 1.03-1.08 (60°C) (the dry extract yield is 13.3%-16.2%), filtered, 5% of dextrin in the amount of the decoction piece is added to mix, spray dried, and then an appropriate amount of inclusion compound fine powder and dextrin are added, mixed, dry granulated, made into 1000g, and packaged.

[0047] 1. Characteristic investigation

[0048] Based on the appearance, properties and taste of three batches of large-scale samples, this product is light yellow to brownish yellow granules with a slight fragrance and sweet taste.

[0049] 2. Thin layer identification

[0050] 2.1 Thin layer chromatography identification of Lingguishugan granule mixed powder

[0051] (1) Take 10g of the powder of this product, add 50ml of ether, ultrasonically treat for 10min, filter, evaporate the filtrate, and add 1ml of methanol to dissolve the residue as the test solution. Take 1.0g of Poria cocos control medicinal material, add 50ml of ether, ultrasonically treat for 10min, filter, evaporate the filtrate, and add 1ml of methanol to dissolve the residue as the control medicinal material solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 20μl of the test solution and 2μl of the control medicinal material solution are taken and spotted on the same silica gel G thin layer plate, respectively, with toluene-ethyl acetate-formic acid (20:5:0.5) solution as the developing solvent, develop, take out, dry, spray with 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material.

[0052] (2) Take 2g of the powder of this product, add 20ml of n-butanol, ultrasonically treat for 30min, filter, wash the filtrate with water twice, 30ml each time, discard the water, evaporate the n-butanol solution, add 1ml of acetone to dissolve the residue, and use it as the test solution. Take 0.5g of the control herb of Atractylodes macrocephala, add 15ml of n-butanol, ultrasonically treat for 30min, filter, wash the filtrate with water twice, 10ml each time, discard the water, evaporate the n-butanol solution, and add 1ml of acetone to dissolve the residue, and use it as the control herb solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), take 10μl of each of the above two solutions and spot them on the same silica gel G thin layer plate, use chloroform-acetone (19:1) as the developing solvent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test product, at the corresponding position of the chromatogram of the control herb, a fluorescent spot of the same color appears.

[0053] (3) Take 2g of the powder of this product, add 40ml of methanol, ultrasonically treat for 30min, filter, evaporate the filtrate, add 20ml of water to the residue to dissolve, shake and extract twice with n-butanol saturated with water, 25ml each time, combine the extracts, wash with 30ml of water saturated with n-butanol, discard the water, evaporate the n-butanol, and dissolve the residue in 1ml of methanol as the test solution. Take another 0.5g of licorice control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 5μl of each of the above two solutions and spot them 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, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). In the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the control medicinal material chromatogram.

[0054] 2.2 Thin layer chromatography identification method of Lingguishugan granules

[0055] 2.2.1 Poria

[0056] ①Testing instruments, reagents, and test drugs

[0057] Thin layer chromatograph (model: GoodLook-1000, Shanghai Kezhe Biochemical Technology Co., Ltd.); electronic balance (model: AL-204, asset number 000000518, Mettler-Toledo Instrument Co., Ltd.); ultrasonic cleaner (model: KM-500DB, Kunshan Meimei Ultrasonic Instrument Co., Ltd.); silica gel preformed plate (G plate 200*100mm, Qingdao Ocean Silica Gel Co., Ltd., batch number 202200822); silica gel preformed plate (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); 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 medicinal material (China Food and Drug Inspection Institute, batch number: 121117-201910).

[0058] ② Screening of TLC identification methods for Poria cocos

[0059] Ⅰ. Method 1

[0060] Preparation of test solution: Take 10g of the product, grind it into powder, add 50ml of ether, treat it with ultrasound for 10min, filter it, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

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

[0062] Preparation of negative control solution: Take 10 g of Poria cocos negative control powder and prepare negative control solution in the same way.

[0063] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 20 μl of the test sample solution and 2 μl of the control drug solution were respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) solution was used as the developing agent. After development, the plate was taken out, dried, and sprayed with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution. The plate was heated at 105°C until the spots were clearly colored and inspected under a fluorescent light.

[0064] Results (see Figure 1 ) showed that the test solution had spots of the same color at the corresponding positions of the control medicinal material solution, so the preparation method of the test solution can be included in the quality standard.

[0065] Ⅱ. Method 2

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

[0067] Preparation of control medicinal material solution: Take 1.0 g of Poria cocos control medicinal material, add 10 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 2 ml of chloroform to the residue to dissolve it, and use it as the control medicinal material solution.

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

[0069] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 5 μl of the test sample and 4 μl of the control medicinal material were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (30℃~60℃)-ether (3:2) was used as the developing agent. After development, the plate was taken out, dried, and examined under ultraviolet light (365nm).

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

[0071] III. Conclusion

[0072] Based on the above considerations, method 1 is the best method for preparing the test solution.

[0073] ③ Development agent investigation

[0074] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 20 μl of the test sample solution and 2 μl of the control drug solution in the above method were respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) solution was used as the developing solvent. The plate was developed, taken out, dried, sprayed with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, heated at 105°C until the spots were clearly colored, and inspected under a fluorescent light.

[0075] Results (see Figure 3 ) showed that among the two developing solvents mentioned above, toluene-ethyl acetate-formic acid (20:5:0.5) had a better effect, so the developing solvent used in the original method was toluene-ethyl acetate-formic acid (20:5:0.5).

[0076] ④ Color developer investigation

[0077] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2 μl of the control medicinal solution prepared by the above method 1, 20 μl of the test solution and the negative control medicinal solution lacking Poria cocos were respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) was used as the developing agent. After development, the plate was taken out, dried, sprayed with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution or a 2% ferric chloride solution, heated at 105°C until the spots were clearly colored, and inspected under a fluorescent light.

[0078] Results (see Figure 4 ) showed that when sprayed with 2% ferric chloride solution as the color developer, there were no corresponding spots between the Poria cocos control medicinal material solution and the test solution, while when sprayed with 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, the Poria cocos control medicinal material solution and the test solution showed spots of the same color at the corresponding positions. Therefore, 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution was determined as the color development condition of this method.

[0079] ⑤Specificity inspection

[0080] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 20 μl of the test sample solution and 2 μl of the control drug solution in the above method were respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) solution was used as the developing solvent. The plate was developed, taken out, dried, sprayed with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, heated at 105°C until the spots were clearly colored, and inspected under a fluorescent light.

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

[0082] ⑥ Sample quantity inspection

[0083] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 1μl, 2μl, and 3μl of the control medicinal material solution prepared in the above method 1 and 15μl, 20μl, and 25μl of the test solution were respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) was used as the developing solvent. The plate was developed, taken out, dried, sprayed with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, heated at 105°C until the spots were clearly colored, and inspected under a fluorescent light.

[0084] Results (see Figure 6 ) showed that when the spotting volume of the Poria control medicinal solution was 2μl and 3μl, the spots were more obvious, and when it was 1μl, the corresponding spots were lighter. When the spotting volume of the test solution was 15μl, the spots were weaker, and when the spotting volume was 20μl and 25μl, the spots were clearer. Taking comprehensive considerations, it was determined that the spotting volume of the Poria control medicinal solution was 2μl, and the spotting volume of the test solution was 20μl.

[0085] ⑦Durability test

[0086] According to the proposed method, the effects of different temperatures, humidity and different brands of thin layer chromatography plates on the test results were investigated.

[0087] Ⅰ. Tests at different temperatures

[0088] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2 μl of the control medicinal solution prepared by the above method 1 and 20 μl of the test solution were respectively taken and spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) was used as the developing solvent. The plates were developed at 25°C and 4°C, respectively. The plates were taken out, dried, sprayed with a mixed solution of 2% vanillin sulfuric acid solution-ethanol (4:1), heated at 105°C until the spots were clearly colored, and observed under a fluorescent light.

[0089] Results (see Figure 7 ) showed that when developed at 25℃ and 4℃, the test sample chromatogram showed spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and had good separation, indicating that this method is applicable in the range of 4℃ to 25℃ and has good durability.

[0090] Ⅱ. Tests at different humidity

[0091] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2 μl of the control medicinal material solution prepared by the above method 1 and 20 μl of the test sample solution were respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) was used as the developing solvent. The plates were developed at relative humidity of 32%, 58%, and 88%, respectively. The plates were taken out, dried, sprayed with a mixed solution of 2% vanillin sulfuric acid solution-ethanol (4:1), heated at 105°C until the spots were clearly colored, and observed under fluorescent light.

[0092] Results (see Figure 8 ) showed that when the relative humidity was 32%, 58% and 88%, the test sample chromatogram showed spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and had good separation, indicating that this method is applicable in the relative humidity range of 32% to 88% and has good durability.

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

[0094] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2 μl of the control medicinal material solution prepared by the above method 1 and 20 μl of the test sample solution were respectively spotted on silica gel G thin layer plates of different brands (Qingdao Hailang, Qingdao Ocean, homemade), and toluene-ethyl acetate-formic acid (20:5:0.5) was used as the developing agent. The plates were developed, taken out, dried, sprayed with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, heated at 105°C until the spots were clearly colored, and observed under a fluorescent light.

[0095] Results (see Fig. 9 ) showed that when different brands (Qingdao Hailang, Qingdao Ocean, homemade) of silica gel G thin layer plates were used, the test sample chromatogram showed main spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and the separation was good, indicating that this method is applicable to different brands of silica gel G thin layer plates and has good durability.

[0096] ⑧ Determination of TLC identification method of Poria cocos

[0097] In summary, the thin-layer identification method for testing Poria cocos in Lingguishugan granules is determined as follows: take 10g of this product, grind it, add 50ml of ether, ultrasonically treat it for 10min, filter it, evaporate the filtrate to dryness, and add 1ml of methanol to dissolve the residue as the test solution. Take another 1.0g of Poria cocos control medicinal material, add 50ml of ether, ultrasonically treat it for 10min, filter it, evaporate the filtrate to dryness, and add 1ml of methanol to dissolve the residue as the control medicinal material solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 20μl of the test solution and 2μl of the control medicinal material solution are taken and spotted on the same silica gel G thin layer plate, respectively, with toluene-ethyl acetate-formic acid (20:5:0.5) solution as the developing agent, develop it, take it out, dry it, spray it with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, heat it at 105°C until the spots are clearly colored, and inspect it under a fluorescent lamp. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material. Fig.10 .

[0098] 2.2.2 Cassia twig

[0099] ①Testing instruments, reagents, and test drugs

[0100] Electronic balance (model: T-214, Beijing Sartorius Instrument Systems Co., Ltd.); electronic balance (model: XPE105, asset number KY2016028003, Mettler-Toledo Instruments); 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 medicinal material (China Food and Drug Control Institutes, batch number: 121191-201605); others are the same as 2.2.1.

[0101] ② Screening of TLC identification methods for cinnamon twigs

[0102] Ⅰ. Method 1

[0103] Preparation of test solution: Take 0.5 g of the product, grind it into powder, add 10 ml of ethanol, seal it tightly, soak it for 20 minutes, shake it from time to time, filter it, and take the filtrate as the test solution.

[0104] Preparation of reference medicinal material solution: Take 0.5 g of cinnamon twig reference medicinal material and prepare reference medicinal material solution in the same way.

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

[0106] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 15 μl of each of the three solutions mentioned above were spotted on the same silica gel G thin layer plate, and petroleum ether (60-90°C)-ethyl acetate (17:3) was used as the developing agent. After development, the plate was dried and sprayed with dinitrophenylhydrazine ethanol test solution, and inspected under a fluorescent light.

[0107] Results (see Fig.11 ) showed that the test solution did not have the same orange-red spots at the corresponding positions of the control medicinal material solution, so the test solution preparation method was not feasible.

[0108] Ⅱ. Method 2

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

[0110] Preparation of control medicinal material solution: Take 2 g of cinnamon twig control medicinal material and prepare the control medicinal material solution in the same way.

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

[0112] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 15 μl of each of the three solutions mentioned above were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (60℃~90℃)-ethyl acetate (17:3) was used as the developing agent. After development, the plate was taken out and dried, sprayed with vanillin sulfuric acid test solution, heated at 105℃ until the spots were clearly colored, and inspected under fluorescent light.

[0113] Results (see Fig.12 ) showed that the test solution did not have spots of the same color at the corresponding positions of the control medicinal material solution, so the method for preparing the test solution was 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] ①Testing instruments, reagents, and test drugs

[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 medicinal material (China Food and Drug Control Institutes, batch number: 120925-201611).

[0119] ② Screening of thin-layer identification methods of Atractylodes macrocephala

[0120] Ⅰ. Method 1

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

[0122] Preparation of control medicinal material solution: Take 0.5 g of Atractylodes macrocephala control medicinal material, add 15 ml of n-butanol, ultrasonically treat for 30 minutes, filter, wash the filtrate with water twice, 10 ml each time, discard the water, evaporate the n-butanol solution, add 1 ml of acetone to the residue to dissolve it, and use it as the control medicinal material solution.

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

[0124] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions mentioned above were spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0125] Results (see Fig.13 ) showed that the test solution showed fluorescent spots of the same color at the corresponding positions of the control medicinal material solution, so the preparation method of the test solution can be included in the quality standard.

[0126] Ⅱ. Method 2

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

[0128] Preparation of control medicinal material solution: Take 0.5 g of Atractylodes macrocephala control medicinal material and prepare the control medicinal material solution in the same way.

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

[0130] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions mentioned above were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (60℃~90℃)-ethyl acetate (50:1) was used as the developing agent. After development, the plate was taken out and dried, sprayed with 5% vanillin sulfuric acid test solution, heated at 105℃ until the spots were clearly colored, and inspected under a fluorescent light.

[0131] Results (see Fig.14 ) showed that the test solution did not have spots of the same color at the corresponding positions of the control medicinal material solution, so the method for preparing the test solution was not feasible.

[0132] III. Conclusion: Based on the above considerations, method 1 is the best method for preparing the test solution.

[0133] ③ Development agent investigation

[0134] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 General Rules 0502), 10 μl of each of the three solutions prepared in the above method 1 were taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) and toluene-methanol (6:1) were used as developing agents. The plates were developed, taken out, dried, and examined under ultraviolet light (365 nm). Compare the results.

[0135] Results (see Fig.15 ) showed that among the two developing solvents mentioned above, chloroform-acetone (19:1) had a better effect, so chloroform-acetone (19:1) was used as the developing solvent in the original method.

[0136] ④ Color developer investigation

[0137] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions prepared in the above method 1 were taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) was used as the developing agent. After development, the plate was taken out and dried. It was first examined under an ultraviolet lamp (365 nm), and then sprayed with 5% vanillin sulfuric acid test solution, heated at 105°C until the spots were clearly colored, and examined under a fluorescent lamp.

[0138] Results (see Fig.16 ) showed that when 5% vanillin sulfuric acid solution was used as the color developer, the spots of the Atractylodes macrocephala control solution and the test solution observed under sunlight were not uniform in color, but when directly observed at 365 nm, the Atractylodes macrocephala control solution and the test solution showed fluorescent spots of the same color at the corresponding positions, so 365 nm was determined to be directly used as the color development condition of this method.

[0139] ⑤Specificity inspection

[0140] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 10 μl of the control medicinal solution, the test sample solution, and the negative control medicinal solution lacking Atractylodes macrocephala prepared in the above method 1 were respectively spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) was used as the developing agent. The plate was developed, taken out, dried, and examined under an ultraviolet lamp (365 nm).

[0141] Results (see Fig.17 ) showed that the test solution showed fluorescent spots of the same color at the corresponding positions of the control medicinal material solution, and there was no interference from the negative control solution lacking Atractylodes macrocephala, indicating that the method had good specificity.

[0142] ⑥ Sample quantity inspection

[0143] According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 5μl, 10μl, and 15μl of the control medicinal material solution prepared in the above method 1 and 5μl, 10μl, and 15μl of the test sample solution were respectively spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365nm) to observe the results.

[0144] Results (see Fig.18 ) showed that the spots were obvious when the spotting volume of the Atractylodes macrocephala control medicinal solution was 5μl, 10μl, and 15μl, the spots were weaker when the spotting volume was 5μl, the spots were clearer when the spotting volume was 10μl, and the spots were clearer but not easy to spot when the spotting volume was 15μl. Therefore, it was determined that the spotting volume of the Atractylodes macrocephala control medicinal solution and the test solution were both 10μl.

[0145] ⑦ Durability test: According to the proposed method, the influence of different temperature, humidity and different brands of thin layer chromatography plates on the test results were examined respectively.

[0146] Ⅰ. Tests at different temperatures

[0147] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 10 μl of the control medicinal solution and the test solution prepared in the above method 1 were respectively taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) was used as the developing solvent. The plates were developed at 25°C and 4°C, respectively. The plates were taken out, dried, and examined under ultraviolet light (365 nm) to observe the results.

[0148] Results (see Fig.19) showed that when developed at 25℃ and 4℃, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and had good separation, indicating that this method is applicable in the range of 4℃ to 25℃ and has good durability.

[0149] Ⅱ. Tests at different humidity

[0150] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 10 μl of the control medicinal material solution and the test sample solution prepared in the above method 1 were respectively taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) was used as the developing solvent. The plates were developed at relative humidity of 32%, 58%, and 88%, respectively. The plates were taken out, dried, and examined under ultraviolet light (365 nm) to observe the results.

[0151] Results (see Fig. 20 ) showed that when carried out under relative humidity of 32%, 58% and 88%, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and had good separation, indicating that this method is applicable in the relative humidity range of 32% to 88% and has good durability.

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

[0153] According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of the control medicinal solution and the test solution prepared by the above method 1 were respectively spotted on silica gel G thin layer plates of different brands (Qingdao Hailang, Qingdao Ocean and homemade), and chloroform-acetone (19:1) was used as the developing agent. The plates were developed, taken out, dried, and examined under ultraviolet light (365 nm) to observe the results.

[0154] Results (see Fig.21 ) showed that when different brands (Qingdao Hailang, Qingdao Ocean and homemade) of silica gel G thin layer plates were used, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and the separation was good, indicating that this method is applicable to different brands of silica gel G thin layer plates and has good durability.

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

[0156] In summary, the thin-layer identification method for testing Atractylodes macrocephala in Lingguishugan granules is determined as follows: take 4g of this product, grind it into powder, add 20ml of n-butanol, ultrasonically treat it for 30min, filter it, wash the filtrate with water twice, 30ml each time, discard the water, evaporate the n-butanol solution, add 1ml of acetone to the residue to dissolve it as the test solution. Take another 0.5g of Atractylodes macrocephala control medicinal material, add 15ml of n-butanol, ultrasonically treat it for 30min, filter it, wash the filtrate with water twice, 10ml each time, discard the water, evaporate the n-butanol solution, and add 1ml of acetone to the residue to dissolve it as the control medicinal material solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 General Rules 0502), 10 μl of the above-mentioned control medicinal material solution and the test sample solution were taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (19:1) was used as the developing solvent. After development, the plate was taken out, dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, the same color fluorescent spot appeared at the corresponding position of the chromatogram of the control medicinal material. The results are shown in Fig. 22 .

[0157] 2.2.4 Licorice

[0158] ①Testing instruments, reagents, and test drugs

[0159] 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 medicinal material (China Food and Drug Control Institutes, batch number: 120904-201604); others are the same as 2.2.1.

[0160] ② Screening of TLC identification methods for licorice

[0161] Ⅰ. Method 1

[0162] Preparation of test solution: Take 4 g of the product, grind it into powder, add 40 ml of methanol, ultrasonically treat it for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, shake and extract twice with n-butanol saturated with water, 25 ml each time, combine the extracts, wash with 30 ml of water saturated with 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.

[0163] Preparation of control medicinal material solution: Take 0.5 g of licorice control medicinal material and prepare the control medicinal material solution in the same way.

[0164] Preparation of negative control solution: Take 2 g of licorice negative control powder and prepare negative control solution in the same way.

[0165] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 5 μl of each of the three solutions mentioned above were respectively spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide test solution, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. After development, the plate was taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm).

[0166] Results (see Fig.23 ) showed that the test solution showed fluorescent spots of the same color at the corresponding positions of the control medicinal material solution, so the preparation method of the test solution can be included in the quality standard.

[0167] Ⅱ. Method 2

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

[0169] Preparation of control medicinal material solution: Take 1 g of licorice control medicinal material and prepare the control medicinal material solution in the same way.

[0170] Preparation of negative control solution: Take 4 g of licorice negative control powder and prepare negative control solution in the same way.

[0171] Identification: According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 5 μl of each of the three solutions mentioned above were respectively spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide test solution, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. After development, the plate was taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm).

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

[0173] III. Conclusion: Based on the above considerations, method 1 is the best method for preparing the test solution.

[0174] ③ Development agent investigation

[0175] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 5 μl of each of the three solutions prepared in the above method 1 were taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide test solution, using ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) and chloroform-acetone (19:1) as developing agents, respectively. After development, the plate was taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm).

[0176] Results (see Fig.25 ) showed that ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was the best developing solvent, so ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing solvent in the original method.

[0177] ④ Color developer investigation

[0178] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 5 μl of each of the three solutions prepared in the above method 1 were taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide test solution, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. The plate was developed, taken out, and dried. First, it was examined under an ultraviolet lamp (365 nm), and then sprayed with 10% ethanolic sulfuric acid solution. The plate was heated at 105°C until the spots were clearly colored, and then examined under an ultraviolet lamp (365 nm) to compare the results.

[0179] Results (see Fig.26 ) showed that when sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and examined under an ultraviolet lamp (365nm), the licorice control medicinal material solution and the test solution showed fluorescent spots of the same color at the corresponding positions; while when examined at 365nm, the licorice control medicinal material solution and the test solution had unclear spots. Therefore, spraying with 10% ethanolic sulfuric acid solution, heating at 105°C until the spots were clearly colored, and examining under an ultraviolet lamp (365nm) was determined as the inspection condition of this method.

[0180] ⑤Specificity inspection

[0181] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 5 μl of each of the three solutions prepared in the above method 1 were taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide test solution, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm).

[0182] Results (see Fig. 27 ) showed that the test solution showed fluorescent spots of the same color at the corresponding positions of the control medicinal material solution, and there was no interference from the negative control solution lacking licorice, indicating that the method had good specificity.

[0183] ⑥ Sample quantity inspection

[0184] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2 μl, 5 μl, and 10 μl of the control medicinal material solution and the test sample solution prepared in the above method 1 were respectively spotted on the same silica gel G thin layer plate, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and examined under ultraviolet light (365 nm).

[0185] Results (see Fig.28 ) showed that the spots were obvious when the sampling volume of the licorice control medicinal solution was 2μl, 5μl, and 10μl, the spots were weak when the sampling volume of the test solution was 2μl, the spots were clearer when the sampling volume was 5μl, and the spots were clearer but not easy to spot when the sampling volume was 10μl. Therefore, it was determined that the sampling volume of the licorice control medicinal solution and the test solution was 5μl.

[0186] ⑦Durability test

[0187] According to the proposed method, the effects of different temperatures, humidity and different brands of thin layer chromatography plates on the test results were investigated.

[0188] Ⅰ. Tests at different temperatures

[0189] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 5 μl of the control medicinal material solution prepared in the above method 1 and the test sample solution were respectively taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide test solution, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. The plates were developed at 25°C and 4°C, respectively. The plates were taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and observed under ultraviolet light (365nm) to observe the results.

[0190] Results (see Fig.29 ) showed that when developed at 25℃ and 4℃, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and had good separation, indicating that this method is applicable in the range of 4℃ to 25℃ and has good durability.

[0191] Ⅱ. Tests at different humidity

[0192] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 5 μl of the control medicinal material solution and the test sample solution prepared in the above method 1 were respectively taken and spotted on the same silica gel G thin layer plate prepared with 1% sodium hydroxide test solution, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. The plates were developed at relative humidity of 32%, 58%, and 88%, respectively. The plates were taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and observed under ultraviolet light (365nm) to observe the results.

[0193] Results (see Fig.30 ) showed that when carried out under relative humidity of 32%, 58% and 88%, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and had good separation, indicating that this method is applicable in the relative humidity range of 32% to 88% and has good durability.

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

[0195] According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 5 μl of the control medicinal material solution and the test sample solution prepared by the above method 1 were respectively taken and spotted on silica gel G thin layer plates of different brands (Qingdao Hailang, Qingdao Ocean, homemade) prepared with 1% sodium hydroxide test solution, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. The plates were developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm) to observe the results.

[0196] Results (see Fig.31 ) showed that when different brands (Qingdao Ocean, Qingdao Hailang, homemade) of silica gel G thin layer plates were used, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions in the control medicinal material chromatogram, and the spots were clear and the separation was good, indicating that this method is applicable to different brands of silica gel G thin layer plates and has good durability.

[0197] ⑧ Determination of TLC Identification Method for Licorice

[0198] Based on the above considerations, the TLC identification method for testing licorice in Lingguishugan granules is determined as follows:

[0199] Take 4g of this product, grind it, add 40ml of methanol, ultrasonically treat it for 30min, filter it, evaporate the filtrate, add 20ml of water to dissolve the residue, shake and extract it twice with n-butanol saturated with water, 25ml each time, combine the extracts, wash with 30ml of water saturated with n-butanol, discard the water, evaporate n-butanol, and dissolve the residue in 1ml of methanol as the test solution. Take another 0.5g of licorice control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 5μl of each of the above two solutions and spot them 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 agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material. Fig.32 .

[0200] 3. Feature Spectrum

[0201] 3.1 Experimental conditions

[0202] (1) Materials

[0203] ①Instruments: LC-20AT high performance liquid chromatograph (Shimadzu Corporation); KM-500DB ultrasonic cleaner (Kunshan Meimei Ultrasonic Instrument Co., Ltd.); T-214 1 / 10,000 electronic balance (Beijing Sartorius Instrument System Co., Ltd.); XPE105 1 / 100,000 electronic balance (Mettler-Toledo Instrument Co., Ltd.); DK-98-Ⅱ electric constant temperature water bath (Tianjin Test Instrument Co., Ltd.); 101-2AB electric constant temperature blast drying oven (Shanghai Tianyuan Experimental Chemical Factory).

[0204] ② Reagent solution: ethanol is of analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20211110); methanol is of analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20210108); methanol is of chromatographic grade (American Tiandi Co., Ltd., batch number: 21083825); acetonitrile is of chromatographic grade (American Tiandi Co., Ltd., batch number: 21053124); pure water (China Resources C'estbon Beverage Co., Ltd., batch number: 20210106); formic acid is of chromatographic grade (Tianjin Comeo Chemical Reagent Co., Ltd., batch number: 20210112); phosphoric acid is of analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 20201108); acetic acid is of chromatographic grade (Tianjin Comeo Chemical Reagent Co., Ltd., batch number: 20201121).

[0205] ③Sample

[0206] Ⅰ. Prescription composition: 1600g Poria cocos, 1200g cinnamon twig, 1200g Atractylodes macrocephala, 800g licorice.

[0207] Ⅱ. Sample batch number

[0208] Table 13 Batch numbers of Lingguishugan granules, reference samples and decoction pieces

[0209]

[0210] ④ Reference material information

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

[0212] Cinnamaldehyde (China Food and Drug Inspection Institute, batch number 110710-201221, purity 99.6%)

[0213] Protocatechuic acid (China Food and Drug Inspection Institute, batch number 110809-201205, purity 99.9%)

[0214] Protocatechuic acid (China Food and Drug Inspection Institute, batch number 110809-201906, purity 97.7%)

[0215] Licorice glycoside (Chengdu Kroma Biotechnology Co., Ltd., batch number CHB180109, purity 98%)

[0216] Licorice glycoside (Chengdu Kroma Biotechnology Co., Ltd., batch number CHB201126, purity 98%)

[0217] Liquorice glycoside (China Food and Drug Inspection Institute, batch number 111610-201908, purity 95%)

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

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

[0220] Ammonium glycyrrhizinate (China Food and Drug Inspection Institute, batch number 110731-202021, purity 96.2%)

[0221] Ammonium glycyrrhizinate (China Food and Drug Inspection Institute, batch number 110731-202122, purity 94.4%)

[0222] (2) Investigation of chromatographic conditions

[0223] ① Selection of organic phase

[0224] Methanol-pure water and acetonitrile-pure water were investigated in the experiment. When the organic phase was acetonitrile, the separation was better, and acetonitrile had a stronger elution ability, so the components with less polarity could be effectively eluted. Therefore, acetonitrile was selected as the organic phase.

[0225] ② Investigation of water phase

[0226] The experiment investigated acetonitrile-pure water and different types of acid water. The results showed that the characteristic spectra of different types of acids were slightly different. Considering that phosphoric acid is an inorganic acid and has poor solubility with organic acids, and acetic acid is highly volatile, 0.1% formic acid water was finally selected as the water phase. Secondly, the concentration of formic acid (0.05%, 0.1% and 0.2%) was investigated. The concentration of acid had little effect on the separation, so 0.1% formic acid water was selected as the water phase.

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

[0228] ③Selection of chromatographic column

[0229] In the experiment, three different chromatographic columns were investigated, namely LP-C18, InertSustain C18 and Agilent ZORBAX SB-C18. The chromatogram of the LP-C18 column showed that the peak separation between 30min and 35min was poor, while the baseline of the chromatogram of the Agilent ZORBAXSB-C18 column was not very stable. The InertSustain C18 column had higher separation and better peak shape. After comprehensive consideration, the InertSustain C18 column was selected.

[0230] ④Optimization of separation conditions

[0231] Chromatographic column: InertSustain C18 (5μm, 4.6×250mm); mobile phase: acetonitrile-0.1% formic acid water; volume flow rate: 1.0ml / min; column temperature: 30℃; wavelength: 254nm; injection volume: 15μl; optimization of chromatographic separation conditions, the results show that the peak of gradient 1 at 50min~65min is too concentrated and the chromatogram contains solvent peak. Gradient 2 separates the chromatographic peak of 50min~65min and discards the solvent peak; Gradient 3 optimizes the separation of cinnamic acid and glycyrrhizic acid based on gradient 2 to meet the analytical requirements of 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] ①Choice of extraction solvent

[0240] The experiment investigated three extraction solvents: methanol, ethanol and pure water. It was found that the extraction efficiency of ethanol as an extraction solvent was lower; pure water had better extraction efficiency than methanol, but considering that pure water was used as a solvent, the test sample was prone to bacteria growth. Therefore, different proportions of methanol and water (20% methanol, 50% methanol and 70% methanol) were 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. When the solvent was 50% methanol, the peak area was larger, the separation was better, and the baseline was flatter, so 50% methanol was selected as the extraction solvent.

[0241] ②Choice of extraction method and time

[0242] The experiment investigated two extraction methods: reflux and ultrasound. The results showed that the peak areas of different extraction methods were not much different and the extraction efficiency was similar. In order to make the experiment more convenient, ultrasound was selected as the extraction method.

[0243] Three different ultrasonic times of 20 min, 30 min and 40 min were investigated. The results showed that the ultrasonic time had little effect on the peak shape. The peak areas of glycyrrhizic acid at different ultrasonic times were 1322564, 1498521 and 1216253, respectively. The peak area at 30 min of ultrasonic treatment was larger, so 30 min was finally selected as the ultrasonic time.

[0244] ③Choice of extraction solvent dosage

[0245] The experiment investigated 1g-25ml, 1g-50ml, 1g-100ml, 2g-50ml and 2g-100ml of 50% methanol as the extraction solvent. The peak areas of glycyrrhizic acid in the first three were 2567697, 1266012 and 538836 respectively. The results showed that 1g-25ml had the largest peak area, but considering the spike recovery experiment, it was not selected; considering the uniformity of the particles, 2g was added for investigation. The content difference between 25 times and 50 times the solvent dosage was not large, so 100ml of solvent was selected for 2g particles.

[0246] Table 5 Investigation on the dosage of different solvents in Lingguishugan granules

[0247]

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

[0249] According to the preliminary conditions, the best chromatographic conditions and extraction methods are determined as follows:

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

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

[0252] Preparation of reference solution: Take appropriate amount of protocatechuic acid reference substance, apiosyl liquiritin reference substance, liquiritin reference substance, formononetin reference substance, cinnamic aldehyde reference substance, ammonium glycyrrhizate reference substance and cinnamic acid reference substance, weigh them accurately, place them in volumetric flasks respectively, add methanol to make a solution containing 5μg of protocatechuic acid, 5μg of apiosyl liquiritin, 30μg of liquiritin, 5μg of formononetin, 50μg of cinnamic aldehyde, 60μg of glycyrrhizic acid and 5μg of cinnamic acid per 1ml.

[0253] (5) Methodological investigation

[0254] ① Repeatability test

[0255] Take 6 portions of the same test sample (batch number: 201101), prepare test sample solutions and inject them separately, determine according to the law, record and integrate, take Peak 8 (glycyrrhizic acid) as the reference peak, calculate the relative retention time and relative peak area, the results are shown in Table 6 and Table 7, the relative retention time and relative peak area RSD% value is less than 3%, the results show that this method has good repeatability.

[0256] Table 6 Repeatability data of Lingguishugan granules (relative retention time)

[0257]

[0258] Table 7 Repeatability data of Lingguishugan granules (relative peak area)

[0259]

[0260] ②Precision test

[0261] Take one portion of the same test sample (batch number: 201101) to prepare the test sample solution, inject it 6 times continuously, record it, take Peak 8 (glycyrrhizic acid) as the reference peak, calculate the relative retention time and relative peak area. The results are shown in Tables 8 and 9. The RSD% value of the relative retention time and relative peak area is less than 5%. The experiment shows that this method has good precision.

[0262] Table 8 Precision data of Lingguishugan granules (relative retention time)

[0263]

[0264] Table 9 Precision data of Lingguishugan granules (relative peak area)

[0265]

[0266] ③Stability test

[0267] Take one portion of the same batch of test samples (batch number: 201101) to prepare test sample solutions, and inject them at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, and 24h respectively. Record the chromatogram and integrate it. Take Peak 8 (glycyrrhizic acid) as the reference peak to calculate the relative retention time and relative peak area. The results are shown in Tables 10 and 11. The RSD% of the relative retention time and relative peak area is less than 5%. The experiment shows that the test sample solution has good stability within 24h.

[0268] Table 10 Stability data of Lingguishugan granules (relative retention time)

[0269]

[0270] Table 11 Stability data of Lingguishugan granules (relative peak area)

[0271]

[0272] ④Specificity test

[0273] Take Ling Gui Shu Gan Granule (batch number 220901) and prepare the test solution. According to the Ling Gui Shu Gan Granule benchmark sample prescription and preparation method, prepare negative samples lacking licorice and cinnamon twigs, and prepare negative sample solutions in the same way as the test solution preparation method. According to the above chromatographic conditions, draw chromatograms of ammonium glycyrrhizinate reference substance, cinnamic acid reference substance, cinnamaldehyde reference substance, Ling Gui Shu Gan Granule test sample and its negative sample. In the test sample chromatogram, a chromatographic peak with the same retention time as the reference substance is presented, and there is no interference with the negative sample. The experiment shows that this method has good specificity.

[0274] (6) Peak identification research

[0275] ①Confirmation of reference material

[0276] Use reference substances to identify each chromatographic peak in the characteristic spectrum. The preparation of reference substance solution refers to the preparation of reference substance solution. Inject samples separately according to the optimal chromatographic conditions and identify them through reference substances.

[0277] ②Chromatographic peak identification

[0278] The 9 common peaks of the three batches of particles were assigned and identified ( Figure 33-Figure 36 ), among which peaks 1 and 5 belong to cassia twig; peaks 2 to 4 and 6 to 9 belong to liquorice.

[0279] Table 12 Peak attribution and identification

[0280]

[0281] (7) Establishment and evaluation of the characteristic spectrum of Lingguishugan granules

[0282] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel is used as filler, acetonitrile is used as mobile phase A, 0.1% formic acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in Table a; the flow rate is 1.0 ml per minute; the column temperature is 30°C; the detection wavelength is 254 nm. The theoretical plate number calculated based on glycyrrhizic acid should be no less than 3000.

[0283] Preparation of reference solution: Take appropriate amount of protocatechuic acid reference substance, apiosyl liquiritin reference substance, liquiritin reference substance, formononetin reference substance, cinnamic aldehyde reference substance, ammonium glycyrrhizate reference substance and cinnamic acid reference substance, weigh them accurately, and add methanol to make solutions containing 5μg of protocatechuic acid, 5μg of apiosyl liquiritin, 30μg of liquiritin, 5μg of formononetin, 50μg of cinnamic aldehyde, 60μg of glycyrrhizic acid and 5μg of cinnamic acid per 1ml.

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

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

[0286] Take 3 batches of particle samples and test them according to the above test method. The results are shown in Figure 37-38. There should be 9 characteristic peaks in the chromatogram of the test sample, of which 7 peaks should correspond to the retention time of the corresponding reference peak of the reference substance. The peak corresponding to the glycyrrhizic acid reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time should be within the range of ±10% of the specified value. The specified value is 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), 1.03 (peak 9).

[0287] Table 133 Relative retention time of characteristic spectrum of Pi Ling Gui Shu Gan granules

[0288]

[0289]

[0290] Table 143 Relative peak area of ​​characteristic spectrum of Pi Ling Gui Shu Gan granules

[0291]

[0292] 4. Inspection

[0293] According to the requirements of General Chapter 0104 Granules of the 2020 Edition of the Chinese Pharmacopoeia, the particle size, moisture, solubility, filling volume difference and microbial limit of this product were inspected.

[0294] (1) Particle size

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

[0296] Table 15 Ling Gui Shu Gan Granules Particle Size Test Results

[0297]

[0298] (2)Moisture

[0299] According to the moisture determination method (Method 2 of General Rules 0832 of the 2020 edition of the Chinese Pharmacopoeia), the moisture content of Chinese herbal granules shall not exceed 8%. The results of the verification test of three batches of this product are shown in Table 16, all of which meet the requirements.

[0300] Table 16 Ling Gui Shu Gan Granules Moisture Test Results

[0301]

[0302] (3) Solubility

[0303] According to the requirements of General Chapter 0104 of the 2020 edition of the Chinese Pharmacopoeia, take 1 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, and there should be no foreign matter such as charred dust. The three batches of verification and inspection results of this product were all dissolved, and no foreign matter such as charred dust was found, which met the requirements.

[0304] (4) Difference in loading volume

[0305] Take 10 bags of the test product, remove the packaging, accurately weigh the weight of each bag, calculate the amount of each bag and the average amount, and the difference limit of the amount is ±5%. The verification test results of three batches of this product all meet the requirements.

[0306] (5) Microbial limits

[0307] According to the General Rules 0104 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the total number of aerobic bacteria is not more than 10 according to the microbial limit inspection of non-sterile products: microbial count method (General Rules 1105 of the 2020 edition of the Chinese Pharmacopoeia) and control bacteria inspection method (General Rules 1106 of the 2020 edition of the Chinese Pharmacopoeia) and the microbial limit standard of non-sterile drugs (General Rules 1107 of the 2020 edition of the Chinese Pharmacopoeia). 3 cfu / g, total number of molds and yeasts is not more than 10 2 cfu / g, Escherichia coli shall not be detected. The test results of three batches of this product are shown in Table 17, all of which meet the requirements.

[0308] Table 17 Microbiological test results

[0309]

[0310] 5. Extract

[0311] 5.1 Extracts from Lingguishugan Granule Mixed Powder

[0312] According to the hot infusion method under the alcohol-soluble extract determination method (General Rule 2201 of the 2020 edition of the Chinese Pharmacopoeia), using ethanol as the solvent, the extract should not be less than 10.0%.

[0313] 5.2 Extracts of Lingguishugan Granules

[0314] Take an appropriate amount of this product, grind it into powder, take 2g, weigh it accurately, add 100ml of ethanol accurately, and determine it according to the hot infusion method of the alcohol-soluble extract determination method (General Rules 2201 of the 2020 edition of the Chinese Pharmacopoeia), using ethanol as the solvent. The lower limit of the extract range of the benchmark sample is 10.93%. The addition of excipients may affect the extract of the product.

[0315] The preparation prescription is: 1600g of Poria cocos, 1200g of cinnamon twig, 1200g of Atractylodes macrocephala, and 800g of licorice, a total of 4800g. The dry extract yield ranges from 13.3% to 16.2%, that is, the dry extract weight is 638.4g to 777.6g, the maximum amount of auxiliary materials is 1000-638.4=361.6, and the amount of auxiliary materials ranges from 22.2% to 36.2%. If the extractable range (10.93% to 20.31%) is proposed based on the benchmark sample of this product, the extractable range per 1g of granules can be calculated as: (1-0.362)×10.93%=5.84%, (1-0.222)×20.31%=15.8%. Taking into account that the extractables of the auxiliary materials themselves account for a certain proportion, and combined with the test data of three batches of production samples, the extractable limit is appropriately increased, and it is proposed that the extractable content of this product should not be less than 9.0%.

[0316] Table 18 Extraction results

[0317]

[0318] 6. Content Determination

[0319] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0320] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to the provisions in Table a above; flow rate 1.0 ml per minute; column temperature 30°C; detection wavelength 254 nm. The theoretical plate number calculated based on glycyrrhizic acid should be no less than 3000.

[0321] 6.1 Content detection of Lingguishugan granule mixed powder

[0322] Preparation of reference solution: Take appropriate amount of ammonium glycyrrhizic acid reference and cinnamic acid reference, weigh accurately, add methanol to make a solution containing 0.10 mg of glycyrrhizic acid and 0.02 mg of cinnamic acid per 1 ml. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizic acid / 1.0207).

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

[0324] Determination method: Accurately pipette 15μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0325] The proposed extract yield of this product is in the range of 13.3% to 16.2%, and the average extract yield is 14.77%. After adding auxiliary materials before drying and after drying to make up the weight, according to the proposed specification of 11.5g / bag, three times a day, one bag at a time, it is equivalent to 165.6g of extract extracted from the slices and the auxiliary materials are added to 34.50g. 165.6g of slices are obtained according to the average extract yield of 14.77%, and the dry extract is 24.46g, which is enough to make up to 34.50g, which is equivalent to the content dilution of 24.46 / 34.5=0.71. Therefore, the content range is proposed according to the benchmark sample of this product, and the intermediate content range is finally proposed: this product is calculated as a dry product, with cinnamic acid (C9H8O2), the content is 0.53mg / g~0.98mg / g, and glycyrrhizic acid (C 42 H 62 O 16 ) is calculated, and the content is 5.20mg / g~9.65mg / g.

[0326] 6.2 Content Detection of Lingguishugan 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] ①Instrument

[0330] LC-20AT high performance liquid chromatograph (Shimadzu Corporation)

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

[0332] T-214 1 / 10,000 electronic balance (Beijing Sartorius Instrument Systems Co., Ltd.)

[0333] XPE105 1 / 100,000 electronic balance (Mettler-Toledo Instrument Co., Ltd.)

[0334] DK-98-Ⅱ Electric Constant Temperature Water Bath (Tianjin Test Instrument Co., Ltd.)

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

[0336] ②Reagents

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

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

[0339] Methanol was chromatographically pure (United States Tiandi Co., Ltd., batch number: 21083825)

[0340] Acetonitrile was chromatographically pure (United States Tiandi Co., Ltd., batch number: 21053124)

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

[0342] Formic acid was chromatographically pure (Tianjin Komiou Chemical Reagent Co., Ltd., batch number: 20210112)

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

[0344] Acetic acid was chromatographically pure (Tianjin Komiou Chemical Reagent Co., Ltd., batch number: 20201121)

[0345] ③Sample and prescription information

[0346] 1600g Poria cocos, 1200g cinnamon twig, 1200g Atractylodes macrocephala, and 800g licorice.

[0347] Table 19 Batch number of Lingguishugan granules and slices

[0348]

[0349] ④ Reference material information

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

[0351] Cinnamaldehyde (China Food and Drug Inspection Institute, batch number 110710-201221, purity 99.6%)

[0352] Ammonium glycyrrhizinate (China Food and Drug Inspection Institute, batch number 110731-202021, purity 96.2%)

[0353] Ammonium glycyrrhizinate (China Food and Drug Inspection Institute, batch number 110731-202122, purity 94.4%)

[0354] (2) Establishment of content determination method

[0355] The characteristic spectrum determination method was adopted and determined according to the high performance liquid chromatography method (General Rules 0512 of the 2020 Edition of the Chinese Pharmacopoeia).

[0356] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to the provisions in Table a; flow rate 1.0 ml per minute; column temperature 30°C; detection wavelength 254 nm. The theoretical plate number calculated based on glycyrrhizic acid should be no less than 3000.

[0357] Preparation of reference solution: Take appropriate amount of ammonium glycyrrhizinate reference substance and cinnamic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.05 mg of glycyrrhizic acid and 0.01 mg of cinnamic acid per 1 ml. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207)

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

[0359] Determination method: Accurately pipette 15μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0360] This product is calculated on a dry basis, with a content of 0.53mg / g to 0.98mg / g in terms of cinnamic acid (C9H8O2), and a content of 0.53mg / g to 0.98mg / g in terms of glycyrrhizic acid (C 42 H 62 O 16 ) is calculated, and the content is 5.20mg / g~9.65mg / g.

[0361] (3) Investigation of content determination methodology

[0362] ① Specificity research

[0363] Take Ling Gui Shu Gan Granule (batch number 220901) and prepare the test solution. According to the prescription and preparation method of Ling Gui Shu Gan Granule, prepare negative samples lacking licorice and cinnamon twig, and prepare negative sample solution by the same method as the test solution preparation method. According to the above chromatographic conditions, draw the chromatograms of ammonium glycyrrhizinate reference substance, cinnamic acid reference substance, cinnamaldehyde reference substance, Ling Gui Shu Gan Granule test sample and its negative sample. In the chromatogram of the test sample, a chromatographic peak with the same retention time as the reference substance is presented, and there is no interference with the negative sample. The experiment shows that this method has good specificity.

[0364] ② Repeatability test

[0365] Take 6 portions of the same test sample (batch number: 221001), prepare test sample solutions and inject them separately, determine them according to the law, take Peak 6 (cinnamaldehyde) as the reference peak, calculate the content, and the results are shown in Table 20. The RSD% value of the content is less than 3%. The results show that this method has good repeatability.

[0366] Table 20 Repeatability data of Lingguishugan granules (cinnamaldehyde)

[0367]

[0368] ③Linear range test

[0369] Take appropriate amount of ammonium glycyrrhizinate, cinnamic acid, and cinnamaldehyde standard respectively, weigh accurately, place in a volumetric flask, and add methanol to make up to volume. Take the above solutions as standard stock solutions, dilute and measure in sequence, record and integrate the chromatograms, and use cinnamic acid, glycyrrhizic acid, and cinnamaldehyde as reference peaks to calculate the peak areas. The results are shown in Table 21.

[0370] Table 21 Linearity and range

[0371]

[0372] ④ Sample recovery test

[0373] Weigh 6 portions of the same sample with determined content (batch number 220901, glycyrrhizic acid content is 6.35 mg / g, cinnamic acid content is 0.52 mg / g, batch number 20230520 cinnamaldehyde content is 2.68 mg / g), accurately weigh, add appropriate amount of reference substance respectively, treat according to the test sample solution preparation method, and determine according to the law. The results are shown in Table 22. The experiment shows that: this method has a good sample recovery rate.

[0374] Table 22 Sample recovery data

[0375]

[0376]

[0377] (4) Content of index components in Lingguishugan granules

[0378] The index components in the particles were measured using the above-mentioned chromatographic conditions.

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

[0380] Table 23 Content of index components of Lingguishugan granules

[0381]

[0382] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

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

Claims

1. A quality detection method for Ling Gui Shu Gan preparation, characterized in that: The quality detection method of the Ling Gui Shu Gan preparation includes a quality detection method of Ling Gui Shu Gan granules and a quality detection method of Ling Gui Shu Gan granule mixed powder; the quality detection method of the Ling Gui Shu Gan preparation includes constructing a characteristic spectrum and determining the contents of cinnamic acid and glycyrrhizic acid by 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 provisions in Table a below; Table a Gradient elution program The flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 254 nm; The step of constructing a characteristic spectrum using liquid chromatography comprises: S11, preparation of reference solution: take appropriate amount of protocatechuic acid reference substance, apigenin liquiritin reference substance, liquiritin reference substance, formononetin reference substance, cinnamaldehyde reference substance, ammonium glycyrrhizinate reference substance and cinnamic acid reference substance, add methanol respectively to obtain; S12, preparation of test solution: take an appropriate amount of Ling Gui Shu Gan preparation sample, add 50% methanol by volume, sonicate, filter and obtain the filtrate; S13, aspirate 15 μl of the reference solution and the test solution, inject into the liquid chromatograph, and measure to obtain.

2. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 1, characterized in that: In step S11 of the quality detection method of the Lingguishugan granules, methanol is added to prepare a solution containing 5 μg of protocatechuic acid, 40 μg of apigenin, 50 μg of liquiritin, 5 μg of formononetin, 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 of the Lingguishugan granule mixed powder, methanol is added to prepare a solution containing 5 μg of protocatechuic acid, 5 μg of apigenin, 30 μg of liquiritin, 5 μg of formononetin, 50 μg of cinnamaldehyde, 60 μg of glycyrrhizic acid and 5 μg of cinnamic acid per 1 ml.

3. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 1, characterized in that: The liquid chromatography method is used to determine the contents of cinnamic acid and glycyrrhizic acid, and the steps include: S21, take appropriate amount of ammonium glycyrrhizinate reference substance and cinnamic acid reference substance, add methanol to prepare solutions respectively, and obtain; S22, preparation of test solution: take an appropriate amount of Ling Gui Shu Gan preparation sample, add 50% methanol by volume, sonicate, filter and obtain the filtrate; S23, aspirate 15 μl of the reference solution and the test solution, inject into the liquid chromatograph, and measure to obtain.

4. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 3, characterized in that: In step S21, the cinnamic acid and glycyrrhizic acid content of the Ling Gui Shu Gan granule mixed powder is determined by preparing a solution containing 0.10 mg of glycyrrhizic acid and 0.02 mg of cinnamic acid per 1 ml; in step S21, the cinnamic acid and glycyrrhizic acid content of the Ling Gui Shu Gan granule is determined by preparing a solution containing 0.05 mg of glycyrrhizic acid and 0.01 mg of cinnamic acid per 1 ml.

5. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 1, characterized in that: The quality detection method of the Ling Gui Shu Gan preparation also includes a Poria thin layer identification method, a Atractylodes macrocephala thin layer identification method and a Licorice thin layer identification method.

6. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 5, characterized in that: The steps of the Poria cocos thin layer identification method include: S31, take 10g of Lingguishugan preparation of this product, add 50ml of ether, ultrasonically treat for 10min, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; S32, taking 1.0 g of Poria cocos control medicinal material, adding 50 ml of ether, ultrasonically treating for 10 min, filtering, evaporating the filtrate to dryness, and dissolving the residue in 1 ml of methanol as the control medicinal material solution; S33, take 20μl of the test solution and 2μl of the control drug solution, spot them respectively on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid solution with a volume ratio of 20:5:0.5 as the developing solvent, develop, take out, dry, spray with a mixed solution of 2% vanillin sulfuric acid solution and ethanol with a volume ratio of 4:1, heat at 105℃ until the spots are clearly colored, and inspect under fluorescent light.

7. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 5, characterized in that: The steps of the Atractylodes macrocephala thin layer identification method include: S41, take 4g of Ling Gui Shu Gan preparation of this product, grind it into powder, add 20ml of n-butanol, ultrasonically treat for 30min, filter, wash the filtrate with water twice, 30ml each time, discard the water, evaporate the n-butanol solution, add 1ml of acetone to dissolve the residue, and use it as the test solution; S42, take 0.5 g of Atractylodes macrocephala control medicinal material, add 15 ml of n-butanol, ultrasonically treat for 30 min, filter, wash the filtrate with water twice, 10 ml each time, discard the water, evaporate the n-butanol solution, add 1 ml of acetone to the residue to dissolve it, and use it as the control medicinal material solution; S43, take 10 μl of the control medicinal material solution and the test sample solution, respectively, and spot them on the same silica gel G thin layer plate, use chloroform-acetone solution with a volume ratio of 19:1 as the developing agent, develop, take out, dry, and examine under a 365nm wavelength ultraviolet lamp.

8. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 5, characterized in that: The steps of the liquorice thin layer identification method include: S51, take 4g of Ling Gui Shu Gan preparation of this product, grind it into powder, add 40ml of methanol, ultrasonically treat for 30min, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, shake and extract twice with n-butanol saturated with water, 25ml each time, combine the extracts, wash with 30ml of water saturated with 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; S52, take 0.5g of licorice control medicinal material, grind it, add 40ml of methanol, ultrasonically treat it for 30min, filter it, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, shake and extract it twice with n-butanol saturated with water, each time 25ml, combine the extracts, wash them with 30ml of water saturated with n-butanol, discard the water, evaporate the n-butanol to dryness, add 1ml of methanol to dissolve the residue, and prepare a control medicinal material solution; S53, take 5μl of the control medicinal material solution and the test sample solution, respectively, and spot them 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 with a volume ratio of 15:1:1:2 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under a UV lamp with a wavelength of 365nm.

9. The quality detection method of Ling Gui Shu Gan preparation as claimed in claim 1, characterized in that: The quality inspection method of the Ling Gui Zhu Gan preparation also includes property inspection, general inspection of granules and extraction inspection.

10. The quality detection method of Ling Gui Shu Gan preparation according to any one of claims 1 to 9, characterized in that: The extractable material is tested by hot immersion method using ethanol as solvent.

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