A quality detection method for Herba Dioscoreae formula granules

Abstract

The present invention discloses a quality detection method for a herba purpurogenous grass formula granules, including property detection, extract determination, thin layer identification, characteristic spectrum determination, content determination, and moisture, particle size, filling volume difference, and microbial limit test. Characteristic spectrum determination includes: preparation of four reference substance solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin; preparation of test solution; detection. Determination of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin includes: preparation of four reference substance solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin; preparation of test solution; detection. Determination of the total content of kaempferol and quercetin includes: preparation of two reference substance solutions of quercetin and kaempferol; preparation of test solution; detection. The method of the present invention has the characteristics of high accuracy, high precision, stability, good reproducibility, good specificity, etc., and realizes the effective control of the quality of the herba purpurogenous grass formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection of traditional Chinese medicines, and more specifically, to a quality detection method for Herba Dicentrariae Herba Formula Granules. Background Art

[0002] Chinese medicine formula granules are made from single Chinese medicine pieces extracted and concentrated through one or more processes and are used for clinical prescriptions in traditional Chinese medicine. They have the advantages of being stable and controllable, safe and efficient, requiring no decoction, being easy to prepare, convenient to carry, and ready to take.

[0003] Dicentra has the effects of clearing away heat, detoxifying, promoting diuresis, and promoting blood circulation. It can be used to treat jaundice, dysentery, painful stranguria caused by sandstone, leucorrhea, edema, furuncle, swelling, trauma, and other diseases. At present, the quality control of Dicentra formula granules is still limited to the qualitative and quantitative analysis of a certain effective ingredient among its many components, and there is a lack of standard control methods that fully reflect the Dicentra formula granules. With the increasing use of this formula granule in clinical practice, a comprehensive, convenient, and controllable detection method is urgently needed to control the quality of Dicentra formula granules. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a quality detection method for the herbaceous purslane formula granules, which has the characteristics of high accuracy and precision, good stability and reproducibility, and good specificity, thereby realizing effective control of the quality of the herbaceous purslane formula granules.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A quality detection method for a herba purpurogenae formula granule, comprising property detection, extract determination, thin layer identification, characteristic spectrum determination, content determination, and moisture, particle size, filling volume difference, and microbial limit inspection, wherein the content determination comprises the total content determination of kaempferol and quercetin, the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin;

[0007] Characteristic profile determination includes the following steps:

[0008] (1) Prepare the neochlorogenic acid reference substance solution, chlorogenic acid reference substance solution, cryptochlorogenic acid reference substance solution, and rutin reference substance solution respectively;

[0009] (2) Prepare the test solution by taking the powder of the Dicentra purpurogena formula granule sample;

[0010] (3) Determination: Perform liquid chromatography on the above four reference substance solutions and the test sample solution respectively;

[0011] The determination of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin content includes the following steps:

[0012] (1) Prepare neochlorogenic acid reference solution, chlorogenic acid reference solution, cryptochlorogenic acid reference solution, and rutin reference solution respectively;

[0013] (2) Prepare the test solution by taking the powder of the Dicentra purpurogena formula granule sample

[0014] (3) Determination: The above four reference solutions and the test solution were subjected to liquid chromatography detection, and the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin content were calculated based on the obtained chromatograms;

[0015] The determination of the total content of kaempferol and quercetin includes the following steps:

[0016] (1) Prepare quercetin reference solution and kaempferol reference solution respectively;

[0017] (2) Prepare the test solution by taking the powder of the Dicentra purpurogena formula granule sample;

[0018] (3) Determination: The above two reference solutions and the test solution were subjected to liquid chromatography detection, and the total contents of kaempferol and quercetin were calculated based on the obtained chromatograms.

[0019] Furthermore, in the characteristic spectrum determination, the four reference substance solutions were all prepared with methanol as the solvent, and the concentrations of the four reference substance solutions were all 30 μg / ml; the specific preparation method of the test solution was as follows: 0.5 g of the powder of the Herba Dioscoreae formula granules sample was added with 25 ml of 50 wt% methanol, and the mixture was cooled after ultrasonic treatment, and then the lost weight was supplemented with 50 wt% methanol, shaken well, filtered, and the filtrate was obtained.

[0020] Further, the chromatographic conditions for characteristic spectrum determination are:

[0021] Chromatographic column: ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 μm; mobile phase: acetonitrile as mobile phase A, 0.1 wt% phosphoric acid as mobile phase B; gradient elution program: during 0-10 min, mobile phase A increased from 5% to 15%, and mobile phase B decreased from 95% to 85%; during 10-19 min, mobile phase A increased from 15% to 20%, and mobile phase B decreased from 85% to 80%; during 19-24 min, mobile phase A increased from 20% to 23%, and mobile phase B decreased from 1.5% to 2.0%. During the period of 24-26 minutes, the mobile phase A increased from 23% to 26%, and the mobile phase B decreased from 77% to 74%; during the period of 26-30 minutes, the mobile phase A increased from 26% to 40%, and the mobile phase B decreased from 74% to 60%; during the period of 30-34 minutes, the mobile phase A decreased from 40% to 5%, and the mobile phase B increased from 60% to 95%; detection wavelength: 354nm; flow rate: 0.3ml / min; column temperature: 40℃; injection volume: 1μl.

[0022] Furthermore, in the determination of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin content, the four reference solutions were all prepared with methanol as the solvent, and the concentrations of the four reference solutions were all 30 μg / ml; the specific preparation method of the test solution was as follows: 0.5 g of the sample powder of the Herba Dioscoreae formula granules was added with 25 ml of 50 wt % methanol, and the mixture was cooled after ultrasonic treatment, and then the lost weight was supplemented with 50 wt % methanol, shaken well, filtered, and the filtrate was obtained.

[0023] Furthermore, in the determination of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin, the chromatographic conditions were:

[0024] Chromatographic column: ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 μm; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B; gradient elution program: during 0-10 min, mobile phase A increased from 5% to 15%, and mobile phase B decreased from 95% to 85%; during 10-19 min, mobile phase A increased from 15% to 20%, and mobile phase B decreased from 85% to 80%; during 19-24 min, mobile phase A increased from 20% to 23%, and mobile phase B increased from 85% to 80%. 0% decreased to 77%; during 24-26 minutes, mobile phase A increased from 23% to 26%, and mobile phase B decreased from 77% to 74%; during 26-30 minutes, mobile phase A increased from 26% to 40%, and mobile phase B decreased from 74% to 60%; during 30-34 minutes, mobile phase A decreased from 40% to 5%, and mobile phase B increased from 60% to 95%; detection wavelength: 354nm; flow rate: 0.3ml / min; column temperature: 40℃; injection volume: 1μl.

[0025] Furthermore, in the determination of the total content of kaempferol and quercetin, the two reference solutions were prepared with methanol as the solvent, and the concentrations of the two reference solutions were both 30 μg / ml; the specific preparation method of the test solution was as follows: 0.5 g of the sample powder of the Herba Dioscoreae formula granules was taken, 25 ml of a mixed solution of methanol and 25 wt % hydrochloric acid in a volume ratio of 4:1 was added, the mixture was heated to reflux and then cooled, and then 50 wt % methanol was used to make up the lost weight, the mixture was shaken well, filtered, and the filtrate was taken to obtain the result.

[0026] Furthermore, in the determination of the total content of kaempferol and quercetin, the chromatographic conditions are:

[0027] Chromatographic column: ACQUITY UPLC BEH C18, 2.1mm×100mm, 1.7μm; mobile phase: methanol as mobile phase A, 0.1wt% phosphoric acid as mobile phase B; gradient elution program: during 0-30min, mobile phase A increased from 5% to 70%, and mobile phase B decreased from 95% to 30%; during 30-35min, mobile phase A decreased from 70% to 5%, and mobile phase B increased from 30% to 95%; detection wavelength: 330nm; flow rate: 0.3ml / min; column temperature: 35℃; injection volume: 1μl.

[0028] Further, the thin layer identification method comprises the following steps:

[0029] (1) Preparation of test solution: Take 1.0 g of Dicentra purslane granules, add 20 ml of 50 wt% ethanol, and after ultrasonic treatment, cool and filter. The filtrate is used as the test solution.

[0030] (2) Preparation of reference solution and reference medicinal material solution: Take rutin and chlorogenic acid reference substances respectively, add methanol to prepare rutin reference solution and chlorogenic acid reference solution with a concentration of 1 mg / ml respectively; take 1.0 g of Dicentra herba decoction pieces, and prepare Dicentra herba decoction piece solution according to the preparation method of the test solution;

[0031] (3) Determination: 5 μl of the test sample solution, 10 μl of the reference medicinal material solution, 3 μl of the rutin reference solution and 3 μl of the chlorogenic acid reference solution were respectively spotted on the same silica gel G thin layer plate, and the plate was developed using ethyl acetate-butanone-formic acid-water in a volume ratio of 8:3:1.5:1. The plate was taken out, dried, sprayed with 1 wt% aluminum chloride solution, and inspected under ultraviolet light.

[0032] Furthermore, the test methods for moisture, particle size, filling volume difference, and microbial limit are as follows: the samples of the herbaceous purslane formula granules are tested in accordance with the provisions of the General Chapter 0104 Granules of the 2020 edition of the Chinese Pharmacopoeia.

[0033] Furthermore, the extract determination method is: using ethanol as the solvent, the extract determination of the Herba Dioscoreae formula granules sample is carried out in accordance with the hot infusion method determination provisions under Item 2201 of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia.

[0034] Furthermore, the formula granules of the herb are prepared by the following method: take the herb slices of the herb, add water and boil them for multiple times, filter, concentrate the filtrate to a clear paste, dry the clear paste into a dry paste, add auxiliary materials with a mass of 0-1 times the mass of the dry paste, mix well, and granulate to obtain the product.

[0035] Furthermore, the auxiliary material is maltodextrin.

[0036] The beneficial effects of the present invention are:

[0037] The Herba Lysimachiae formula granules were tested through properties, thin layer identification, characteristic spectrum, inspection, extracts and the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin content, and the total content of kaempferol and quercetin. A comprehensive, convenient and controllable quality detection method for the Herba Lysimachiae formula granules was established, and effective control of the Herba Lysimachiae formula granules was achieved. This method has high accuracy and precision, good stability and reproducibility, and a certain degree of specificity, which is of great significance to ensuring the stability of the production quality of the Herba Lysimachiae formula granules and the effectiveness and safety of clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The granule properties of three batches of Dicentra purpurogena formula granule samples are shown in the figure;

[0039] Figure 2 This is the chromatogram obtained from the specificity test of the thin layer chromatography identification method of the Herba Dioscoreae formula granules;

[0040] Figure 3 This is the chromatogram obtained by the durability test of the Dicentra purpurogena formula granules at 4°C using the thin layer chromatography identification method;

[0041] Figure 4 This is the chromatogram obtained from the durability test of the Dicentra purpurogena formula granules at 30°C by the thin layer chromatography identification method;

[0042] Figure 5 This is the chromatogram obtained from the durability test of the Dicentra purpurogena formula granules under 35% humidity by the thin layer chromatography identification method;

[0043] Figure 6 This is the chromatogram obtained from the durability test of the Dicentra purpurogena formula granules under 75% humidity by the thin layer chromatography identification method;

[0044] Figure 7 This is the chromatogram obtained by using a thin layer plate purchased from Qingdao Ocean Chemical Co., Ltd. to conduct a thin layer identification test on the formula granules of Dicentra purpurogena.

[0045] Figure 8This is the chromatogram obtained by using the thin layer plate purchased from Qingdao Shuoyuan Silica Gel Technology Co., Ltd. to conduct a thin layer identification test of Dicentra purpurogena formula granules;

[0046] Fig. 9 This is the chromatogram obtained by using the thin layer plate purchased from Qingdao Xinchanglai Silica Gel Co., Ltd. to conduct a thin layer identification test on the formula granules of Dicentra purpurogena.

[0047] Fig.10 This is the chromatogram of the thin layer identification test of the Dicentra purpurogena formula granule sample;

[0048] Fig.11 The chromatogram of the mixed solution of the reference substance obtained to verify the positioning of the characteristic spectrum determination method of the Dicentra purpurogena formula granules and the system applicability;

[0049] Fig.12 The chromatogram of the test sample obtained to verify the positioning of the characteristic spectrum determination method and system suitability of the Dicentra purpurogena formula granules;

[0050] Fig.13 The blank excipient chromatogram obtained to verify the specificity of the characteristic spectrum determination method of the Herba Dioscoreae formula granules;

[0051] Fig.14 The chromatogram obtained to verify the column temperature durability at 30°C of the characteristic spectrum determination method of the Herba Dioscoreae formula granules;

[0052] Fig.15 The chromatogram obtained to verify the column temperature durability at 35°C of the characteristic spectrum determination method of the Herba Dioscoreae formula granules;

[0053] Fig.16 The chromatogram obtained to verify the column temperature durability of 45℃ for the characteristic spectrum determination method of Dicentra purpurogena formula granules;

[0054] Fig.17 The chromatogram obtained to verify the flow rate durability of the characteristic spectrum determination method of the Dicentra purpurogena formula granules at a flow rate of 0.25 ml / min;

[0055] Fig.18 The chromatogram obtained to verify the flow rate durability of the characteristic spectrum determination method of the Dicentra purpurogena formula granules at a flow rate of 0.35 ml / min;

[0056] Fig.19 The chromatogram obtained to verify the wavelength durability of the characteristic spectrum determination method of the Dicentra purpurogena formula granules at a wavelength of 249nm;

[0057] Fig. 20 The chromatogram obtained to verify the wavelength durability of the characteristic spectrum determination method of the Dicentra purpurogena formula granules at a wavelength of 259nm;

[0058] Fig.21The chromatogram obtained by using ACQUITY UPLC BEH C1 column to verify the column durability of the characteristic spectrum determination method of Dioscorea purpurogena formula granules;

[0059] Fig. 22 This is the chromatogram obtained by verifying the durability of the chromatographic column for the characteristic spectrum determination method of the Herba Dioscoreae formula granules using the MicroPulite HSS T3 chromatographic column;

[0060] Fig.23 This is the chromatogram obtained by personnel a to verify the intermediate precision of the determination method of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin in the Herba Dioscoreae formula granules;

[0061] Fig.24 This is the chromatogram obtained by personnel b to verify the intermediate precision of the determination method of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin in the Herba Dioscoreae Formula Granules;

[0062] Fig.25 This is the chromatogram obtained by person c to verify the intermediate precision of the determination method of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin in the Herba Dioscoreae Formula Granules;

[0063] Fig.26 The chromatogram of the mixed solution of the reference substance obtained to verify the determination method positioning and system suitability of the total content of quercetin and kaempferol in the Dicentra purpurogena formula granules;

[0064] Fig. 27 The chromatogram of the test solution obtained to verify the positioning and system suitability of the determination method for the total content of quercetin and kaempferol in the Dicentra purpurogena formula granules;

[0065] Fig.28 The blank excipient chromatogram was obtained to verify the specificity of the determination method for the total content of quercetin and kaempferol in the Dicentra purpurogena formula granules;

[0066] Fig.29 This is the chromatogram obtained by personnel a to verify the intermediate precision of the method for determining the total content of quercetin and kaempferol in Dicentra purslane formula granules;

[0067] Fig.30 The chromatogram is obtained by personnel b to verify the intermediate precision of the determination method of the total content of quercetin and kaempferol in the Dicentra purslane formula granules;

[0068] Fig.31 This is the chromatogram obtained by person c to verify the intermediate precision of the determination method of the total content of quercetin and kaempferol in Dicentra purslane formula granules;

[0069] Fig.32 The chromatogram obtained to verify the column temperature durability at 35°C for the determination method of the total content of quercetin and kaempferol in Dicentra purpurogena formula granules;

[0070] Fig.33 The chromatogram obtained to verify the column temperature durability at 30°C for the determination method of the total content of quercetin and kaempferol in Dicentra purpurogena formula granules;

[0071] Fig.34 The chromatogram obtained to verify the column temperature durability at 40°C for the determination method of the total content of quercetin and kaempferol in Dicentra purpurogena formula granules;

[0072] Fig.35 The chromatogram obtained to verify the flow rate durability of the determination method of quercetin and kaempferol in Dicentra purpurogena formula granules at a flow rate of 0.27 ml / min;

[0073] Fig.36 The chromatogram obtained to verify the flow rate durability of the determination method of quercetin and kaempferol in Dicentra purpurogena formula granules at a flow rate of 0.33 ml / min;

[0074] Fig.37 The chromatogram obtained to verify the wavelength durability of the determination method of the total content of quercetin and kaempferol in Dicentra purpurogena formula granules at a wavelength of 225 nm;

[0075] Fig.38 The chromatogram obtained to verify the wavelength durability of the determination method of the total content of quercetin and kaempferol in Dicentra purpurogena formula granules at a wavelength of 235 nm;

[0076] Fig.39 This is the chromatogram obtained by verifying the durability of the chromatographic column for the determination of the total content of quercetin and kaempferol in Dicentra purslane formula granules using MicroPulite HSS T3 chromatographic column. DETAILED DESCRIPTION

[0077] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] Example 1 Preparation of Dicentra purslane Formula Granules

[0079] The preparation method of the formula granules of purslane is as follows: take purslane herb slices, add water and boil for several times, filter, concentrate the filtrate to a clear paste, dry the clear paste into a dry paste (the yield of the clear paste dried to a dry paste is 17%~32%), add maltodextrin, an auxiliary material, which is 0-1 times the mass of the dry paste, mix well, and granulate to obtain the product.

[0080] Take 3 portions of Herba Dioscoreae Decoction pieces, and prepare Herba Dioscoreae Formula Granules according to the above preparation method and the parameters shown in Table 1 (in Table 1, general decoction means first adding materials and then heating and decoction).

[0081] Table 1 Production data of Dioscorea foetida formula granules

[0082]

[0083] Example 2 Quality Testing of Dioscorea purpurogena Formula Granules

[0084] 1. Trait detection

[0085] In a brightly lit room, visually observe the color and shape of the three batches of Dioscorea purpurogena formula granule samples, smell them, and take a small amount to taste. The test results of the properties of the three batches of samples are shown in Table 2. Granule properties are shown in Table Figure 1 (The batch numbers from left to right in the picture are HBC-F-240830-01, HBC-F-240830-02, and HBC-F-240830-03).

[0086] Table 2 Properties of Dioscorea purpurogena formula granules

[0087]

[0088] Based on the observation of the properties of the three batches of samples and combining the research and test results of the two, the formula granules of the purslane grass were formulated to have the properties of brown-yellow to yellow-brown granules with a slight odor and light taste.

[0089] 2. Thin layer chromatography identification

[0090] 2.1 Detection Method

[0091] 2.1.1 Preparation of test solution: Take 1.0 g of Dicentra purslane granule sample, grind it into powder, add 20 ml of 50 wt% ethanol, ultrasonically treat it for 60 min, cool it, filter it, and take the filtrate as the test solution.

[0092] 2.1.2 Preparation of reference solution and reference medicinal material solution: Take rutin reference solution and chlorogenic acid reference solution respectively, add methanol to prepare rutin reference solution and chlorogenic acid reference solution with a concentration of 1 mg / ml respectively; prepare the Herba Dicentratae Herba solution according to the preparation method of the test solution.

[0093] 2.1.3 Determination: Pipette 5 μl of test solution, 10 μl of reference medicinal material solution, 3 μl of rutin reference solution and 3 μl of chlorogenic acid reference solution, respectively, and spot them on the same silica gel G thin layer plate, use ethyl acetate-butanone-formic acid-water with a volume ratio of 8:3:1.5:1 as the developing solvent, develop, take out, dry, spray with 1wt% aluminum chloride test solution, and inspect under 365nm ultraviolet light.

[0094] 2.2 Methodological validation

[0095] 2.2.1 Specificity test

[0096] Prepare the test solution of Herba Dioscoreae Formula Granules (Batch No.: HBC-F-240830-02) and Herba Dioscoreae Decoction Pieces Solution according to the method in "2.1.1". Take maltodextrin and prepare the negative sample solution according to the method in "2.1.1". Prepare the corresponding rutin reference solution and chlorogenic acid reference solution according to the method in "2.1.2".

[0097] 5 μl of the test solution and negative sample solution of the Dicentra purslane formula granules, 10 μl of the Dicentra purslane slice solution, and 3 μl of the two reference solution were respectively taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-butanone-formic acid-water (volume ratio 8:3:1.5:1) was used as the developing agent. After development, the plate was taken out, dried, sprayed with 1wt% aluminum chloride test solution, and examined under ultraviolet light (365nm). The thin layer chromatogram is shown in Figure 2 (From left to right in each figure, they correspond to: Herba Dioscoreae Decoction Pieces, Herba Dioscoreae Formula Granules Test Sample, Negative Sample, Rutin Reference Substance, Chlorogenic Acid Reference Substance). The results show that in the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference substance, there are fluorescent spots of the same color; in the chromatogram of the negative sample, at the position corresponding to the chromatogram of the reference substance, there are no fluorescent spots. Therefore, this thin layer chromatography identification method is specific.

[0098] 2.2.2 Durability test

[0099] According to the method in "2.1.1", the test solution of Herba Dioscoreae Formula Granules (Batch No.: HBC-F-240830-02) and the Herba Dioscoreae Decoction Pieces solution were prepared respectively. According to the method in "2.1.2", the reference solution of rutin and the reference solution of chlorogenic acid were prepared respectively.

[0100] 2.2.2.1 Temperature durability

[0101] 5 μl of the above-mentioned Dicentra formula granule test solution, 10 μl of Dicentra decoction piece solution, and 3 μl of each of the two reference solution were respectively taken and spotted on the same silica gel G thin layer plate; ethyl acetate-butanone-formic acid-water (volume ratio 8:3:1.5:1) was used as the developing solvent, and the plates were developed at 4°C and 30°C respectively, taken out, dried, sprayed with 1wt% aluminum chloride test solution, and examined under ultraviolet light (365nm). The thin layer chromatogram is shown in Figure 3~Figure 4 (From left to right in each figure, they correspond to: Herba Dioscoreae Decoction Pieces, Herba Dioscoreae Formula Granules Test Sample, Rutin Reference Sample, Chlorogenic Acid Reference Sample). The results showed that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there were fluorescent spots of the same color.

[0102] 2.2.2.2 Humidity durability

[0103] 5 μl of the above-mentioned Dicentra formula granule test solution, 10 μl of Dicentra decoction piece solution, and 3 μl of each of the two reference solution were respectively taken and spotted on the same silica gel G thin layer plate; ethyl acetate-butanone-formic acid-water (volume ratio 8:3:1.5:1) was used as the developing solvent, and the plates were developed under a humidity of 35% and 75%, respectively. The plates were taken out, dried, sprayed with 1wt% aluminum chloride test solution, and examined under ultraviolet light (365nm). The thin layer chromatogram is shown in Figure 5~Figure 6 (From left to right in each figure, they correspond to: Herba Dioscoreae Decoction Pieces, Herba Dioscoreae Formula Granules Test Sample, Rutin Reference Sample, Chlorogenic Acid Reference Sample). The results showed that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there were fluorescent spots of the same color.

[0104] 2.2.2.3 Thin-layer boards from different manufacturers

[0105] 5 μl of the above-mentioned Dicentra purslane formula granule test solution, 10 μl of Dicentra purslane slice solution, and 3 μl of each of the two reference solution were respectively taken and spotted on three silica gel G thin layer plates from different manufacturers; ethyl acetate-butanone-formic acid-water (volume ratio 8:3:1.5:1) was used as the developing agent, developed, taken out, dried, sprayed with 1wt% aluminum chloride test solution, and examined under ultraviolet light (365nm). The thin layer chromatogram is shown in Figure 7~Figure 9 (From left to right in each figure, they correspond to: Herba Dioscoreae Decoction Pieces, Herba Dioscoreae Formula Granules Test Sample, Rutin Reference Substance, Chlorogenic Acid Reference Substance). The results showed that in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

[0106] 2.3 Sample testing

[0107] Three batches of Dicentra purslane formula granule samples were tested according to the thin layer chromatography detection method described in "2.1". The results showed that in the chromatograms of the three batches of test samples, fluorescent spots of the same color appeared at the corresponding positions of the chromatograms of the reference sample. Fig.10 (From left to right in the figure, they correspond to: Herba Lysimachiae decoction pieces, Herba Lysimachiae formula granules test sample with batch number HBC-F-240830-01, Herba Lysimachiae formula granules test sample with batch number HBC-F-240830-02, Herba Lysimachiae formula granules test sample with batch number HBC-F-240830-03, rutin reference substance, and chlorogenic acid reference substance).

[0108] 3. Inspection

[0109] According to the provisions of the 2020 edition of the Chinese Pharmacopoeia, General Chapter 0104 Granules, the moisture, particle size, filling volume difference, and microbial limit of three batches of Dicentra purslane formula granule samples were tested. The test results are shown in Table 3.

[0110] Table 3 Inspection results of the formula granules of the purslane grass

[0111]

[0112] 4. Extract

[0113] Using ethanol as the solvent, three batches of Herba Dioscoreae formula granules (batch numbers: HBC-F-240830-01, HBC-F-240830-02, HBC-F-240830-03) were tested by the hot infusion method under the alcohol-soluble extract determination method (General Rules 2201 of the 2020 edition of the Chinese Pharmacopoeia). The results are shown in Table 4.

[0114] Table 4 Results of extracts from Dicentra purpurogena formula granules

[0115]

[0116] 5. Feature Spectrum

[0117] 5.1 Detection Method

[0118] 5.1.1 Preparation of reference substance solutions: Using methanol as solvent, prepare neochlorogenic acid reference substance solution, chlorogenic acid reference substance solution, cryptochlorogenic acid reference substance solution, and rutin reference substance solution with a concentration of 30 μg / ml respectively.

[0119] 5.1.2 Preparation of test solution: Grind 0.5 g of the powder of the Dicentra purslane formula granule sample, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 50 wt% methanol, weigh it, and treat it with ultrasound (power 250 W, frequency 50 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 wt% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0120] 5.1.3 Determination: Perform liquid chromatography on the above four reference substance solutions and the test sample solution respectively.

[0121] The liquid chromatography conditions were:

[0122] Chromatographic column: ACQUITY UPLC BEH C18, 2.1mm×100mm, 1.7μm; detection wavelength: 354nm; flow rate: 0.3ml / min; column temperature: 40℃; injection volume: 1μl; mobile phase: acetonitrile as mobile phase A, 0.1wt% phosphoric acid as mobile phase B, elution according to the gradient specified in the following table:

[0123]

[0124] 5.2 Validation of characteristic profile detection methodology

[0125] 5.2.1 Positioning and system applicability

[0126] Take appropriate amount of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin reference substances, accurately weigh them, and add methanol to make new chlorogenic acid reference solution containing 446.9 μg of new chlorogenic acid per 1 ml, chlorogenic acid reference solution containing 303.9 μg of chlorogenic acid per 1 ml, cryptochlorogenic acid reference solution containing 539.4 μg of cryptochlorogenic acid per 1 ml, and rutin reference solution containing 468.7 μg of rutin per 1 ml. Accurately measure 1 ml of each of the four reference substance solutions of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin, place them in the same 10 ml volumetric flask, add methanol to the volume, shake well, and use them as the reference substance mixed solution.

[0127] Accurately weigh 0.5 g of the Herba Dioscoreae formula granules sample (batch number: HBC-F-240830-02), place in a stoppered conical flask, accurately add 25 ml of 50 wt% methanol, weigh the weight, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50 wt% methanol, shake well, filter, and take the filtrate as the test solution.

[0128] Accurately pipette 1 μl of the reference substance mixed solution and the test solution, inject into the high performance liquid chromatograph for determination, and record the chromatogram. Figure 11~Figure 12 As can be seen from the figure, in the chromatogram of the reference mixed solution, the retention time of neochlorogenic acid is 4.123min, the theoretical plate number is 6232, and the tailing factor is 1.06; the retention time of chlorogenic acid is 7.040min, the theoretical plate number is 28427, and the tailing factor is 1.10; the retention time of cryptochlorogenic acid is 7.973min, the theoretical plate number is 40954, and the tailing factor is 1.12; the retention time of rutin is 14.257min, the theoretical plate number is 133010, and the tailing factor is 1.13. In the chromatogram of the test solution, the retention time of neochlorogenic acid is 4.107min, the theoretical plate number is 10388, and the tailing factor is 1.16; the retention time of chlorogenic acid is 7.020min, the theoretical plate number is 31978, and the tailing factor is 1.11; the retention time of cryptochlorogenic acid is 7.967min, the theoretical plate number is 42880, and the tailing factor is 1.21; the retention time of rutin is 14.253min, the theoretical plate number is 126581, and the tailing factor is 1.06. Therefore, the system applicability of the characteristic spectrum detection method is good.

[0129] 5.2.2 Exclusivity

[0130] Weigh 0.5g of maltodextrin accurately and prepare a blank excipient solution according to the method of "5.1.2 Preparation of test solution". Accurately pipette 1μl and inject it into the HPLC, record the chromatogram, and examine whether the excipient has any effect. The results show that the excipient has no effect on the characteristic spectrum determination. The relevant chromatogram is shown in Fig.13 .

[0131] 5.2.3 Precision test

[0132] Weigh 0.5 g of the sample of Herba Dioscoreae (batch number: HBC-F-240830-02) accurately, and prepare the test solution according to the method of "5.1.2 Preparation of test solution". Accurately pipette 1 μl of the test solution and inject it into the HPLC, repeat 6 times, take the peak corresponding to the rutin reference peak as the S peak, calculate the relative retention time and RSD value of peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16 and peak 17 with the S peak, and the results are shown in Table 5. The results show that the method has good precision.

[0133] Table 5 Precision relative retention time results

[0134]

[0135] 5.2.4 Stability test

[0136] Weigh 1 g of the sample of Herba Dioscoreae (batch number: HBC-F-240830-02) accurately, and prepare the test solution according to the method of "5.1.2 Preparation of test solution". Accurately aspirate 1 μl of the test solution at 0, 2, 4, 8, 12, 18, and 24 h, and inject them into the HPLC, respectively. The peak corresponding to the rutin reference peak is the S peak, and the relative retention time and RSD value of peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16, and peak 17 with the S peak are calculated. The results are shown in Table 6. The results show that the test solution has good stability within 24 h.

[0137] Table 6 Stability relative retention time results

[0138]

[0139] 5.2.5 Repeatability test

[0140] Weigh 6 portions of the same Herba Dioscoreae formula granules (batch number: HBC-F-240830-02) accurately, and prepare the test solution according to the method of "5.1.2 Preparation of test solution". Accurately pipette 1 μl of each test solution, inject into the HPLC, record the retention time of the common peak, take the peak corresponding to the rutin reference peak as the S peak, calculate the relative retention time and RSD value of peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16 and peak 17 with the S peak, and the results are shown in Table 7. The results show that the method has good repeatability.

[0141] Table 7 Repeatability relative retention time results

[0142]

[0143] 5.2.6 Durability test

[0144] 5.2.6.1 Column temperature durability

[0145] According to the specified chromatographic conditions, change the column temperature by ±5°C, take the sample of the Herba Dioscoreae formula granules (batch number: HBC-F-240830-02), and prepare the test solution according to the method under "5.1.2". Accurately pipette 1μl of the test solution and inject it into the high performance liquid chromatograph, record the retention time of the common peak, take the peak corresponding to the rutin reference peak as the S peak, calculate the relative retention time and RSD value of peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16 and peak 17 with the S peak, and the results are shown in Table 8. Related chromatograms are shown in Figure 14~Figure 16 .

[0146] Table 8 Column temperature durability test results of characteristic spectrum method

[0147]

[0148] 5.2.6.2 Flow rate durability

[0149] According to the specified chromatographic conditions, change the flow rate by ±0.05ml / min, take the sample of the Herba Dioscoreae formula granules (batch number: HBC-F-240830-02), prepare the test solution according to the method under "5.1.2 Preparation of test solution", accurately pipette 1μl of the test solution, inject it into the high performance liquid chromatograph, record the retention time of the common peak, take the peak corresponding to the rutin reference peak as the S peak, calculate the relative retention time and RSD value of peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16 and peak 17 with the S peak, and the results are shown in Table 9. Related chromatograms are shown in Figure 17-18 .

[0150] Table 9 Flow rate durability test results of characteristic spectrum method

[0151]

[0152] 5.2.6.3 Wavelength durability

[0153] According to the specified chromatographic conditions, change the wavelength ± 5nm, take the sample of the formula granules of the purslane grass (batch number: HBC-F-240830-02), prepare the test solution according to the method under "5.1.2 Preparation of test solution", accurately pipette 1μl of the test solution, inject it into the high performance liquid chromatograph, record the retention time of the common peak, take the peak corresponding to the rutin reference peak as the S peak, calculate the relative retention time and RSD value of peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16 and peak 17 with the S peak, and the results are shown in Table 10. Related chromatograms are shown in Figure 19~Figure 20 .

[0154] Table 10 Wavelength durability test results of characteristic spectrum method

[0155]

[0156] 5.2.6.4 Column durability

[0157] According to the specified chromatographic conditions, different types of chromatographic columns were selected, and the sample of the Herba Dioscoreae (batch number: HBC-F-240830-02) was taken. The test solution was prepared according to the method under "5.1.2 Preparation of Test Solution". 1 μl of the test solution was accurately aspirated and injected into the high performance liquid chromatograph. The retention time of the common peak was recorded. The peak corresponding to the rutin reference peak was taken as the S peak. The relative retention time and RSD value of peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16 and peak 17 with the S peak were calculated. The results are shown in Table 11. The relevant chromatograms are shown in Figure 21-22 .

[0158] Table 11 Characteristic spectrum method chromatographic column durability test results

[0159]

[0160] The results of the above durability tests showed that the flow rate, wavelength, and durability of the chromatographic column of this method all met the requirements for characteristic spectrum research.

[0161] 5.3 Sample testing

[0162] Three batches of Herba Dioscoreae formula granules (batch numbers: HBC-F-240830-01, HBC-F-240830-02, HBC-F-240830-03) were tested according to “5.1 Test Method”. The results are shown in Table 12.

[0163] Table 12 Relative retention time of characteristic spectra of Herba Dioscoreae formula granules

[0164]

[0165] 6. Determination of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid content

[0166] 6.1 Determination method

[0167] 6.1.1 Preparation of reference solution: Using methanol as solvent, prepare neochlorogenic acid reference solution, chlorogenic acid reference solution, cryptochlorogenic acid reference solution, and rutin reference solution with a concentration of 30 μg / ml.

[0168] 6.1.2 Preparation of test solution: Grind 0.5% of the powder of the Dicentra purslane formula granule sample, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 50wt% methanol, weigh it, ultrasonically treat it (power 250W, frequency 50kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50wt% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0169] 6.1.3 Determination: Accurately pipette 1 μl of each of the above four reference solution and test solution, inject into liquid chromatograph for determination, and calculate the content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin according to the obtained chromatogram;

[0170] The liquid chromatography conditions were:

[0171] Chromatographic column: ACQUITY UPLC BEH C18, 2.1mm×100mm, 1.7μm; detection wavelength: 354nm; flow rate: 0.3ml / min; column temperature: 40℃; injection volume: 1μl; mobile phase: acetonitrile as mobile phase A, 0.1wt% phosphoric acid as mobile phase B, elution according to the gradient specified in the following table:

[0172]

[0173] 6.2 Methodological Validation

[0174] 6.2.1 Positioning and system applicability

[0175] Same as item “5.2.1 Positioning and system suitability” for feature map detection.

[0176] 6.2.1 Exclusivity

[0177] Same as item “5.2.2 Specificity” of feature spectrum detection.

[0178] 6.2.3 Injection precision

[0179] Accurately pipette 1 μl of the reference substance mixed solution in item "6.2.1" and inject it into the HPLC, record the chromatogram, and measure the peak area. Repeat 6 times and calculate the RSD (%). The results are shown in Table 13-1, Table 13-2, Table 13-3, and Table 13-4. The results show that the method has good precision.

[0180] Table 13-1 Injection precision test results (new chlorogenic acid)

[0181]

[0182] Table 13-2 Injection precision test results (chlorogenic acid)

[0183]

[0184] Table 13-3 Injection precision test results (cryptochlorogenic acid)

[0185]

[0186] Table 13-4 Injection precision test results (rutin)

[0187]

[0188] 6.2.4 Stability test

[0189] Take 0.5g of the sample of the formula granules of the herb (batch number: HBC-F-240830-02), weigh it accurately, and prepare the test solution according to the method of "6.1.2 Preparation of the test solution". Accurately draw 1μl of the test solution and inject it into the liquid chromatograph, and inject it in sequence at 0, 2, 4, 8, 12, 18, and 24h, record the chromatogram, and calculate the average value and RSD value of the rutin peak area. The results are shown in Table 14-1, Table 14-2, Table 14-3, and Table 14-4. The results show that the test solution has good stability within 24h.

[0190] Table 14-1 Results of stability test of test solution for content determination (neochlorogenic acid)

[0191]

[0192] Table 14-2 Results of stability test of test solution for content determination (chlorogenic acid)

[0193]

[0194] Table 14-3 Stability test results of test solution for content determination (cryptochlorogenic acid)

[0195]

[0196] Table 14-4 Content determination test results of the test solution stability test (rutin)

[0197]

[0198] 6.2.5 Repeatability test

[0199] Take 6 portions of the same batch of Herba Dioscoreae formula granules (batch number: HBC-F-240830-02), accurately weigh about 0.5g of each portion, and prepare the test solution according to the method of "6.1.2 Preparation of test solution". Accurately take 1μl of each of the 6 test solutions and inject them into the liquid chromatograph for detection, and calculate the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid and the content of rutin. The results are shown in Table 15. The results show that this method has good repeatability.

[0200] Table 15 Results of repeatability test of content determination method

[0201]

[0202] 6.2.6 Sample recovery test

[0203] (a) Recovery test of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid:

[0204] Using methanol as solvent, prepare 446.9μg / ml new chlorogenic acid reference substance mother solution, 303.9μg / ml chlorogenic acid reference substance mother solution, and 539.4μg / ml cryptochlorogenic acid reference substance mother solution. Take 6 stoppered conical bottles, add 0.5ml of new chlorogenic acid reference substance mother solution, 2.5ml of chlorogenic acid reference substance mother solution, and 0.5ml of cryptochlorogenic acid reference substance mother solution to each conical bottle, and evaporate to dryness; then accurately weigh six portions of the formula granule sample of the purslane grass (batch number: HBC-F-240830-02), each portion is about 0.20g, and place them in each conical bottle, and accurately add 25ml of 50wt% methanol to each conical bottle, seal it, weigh it, ultrasonicate it for 30 minutes, cool it, weigh it, make up the lost weight with 50wt% methanol, shake it well, filter it, take the filtrate, and get six portions of test solution.

[0205] Accurately pipette 1 μl of each of the above test solution into the high performance liquid chromatograph, record the chromatogram, determine the peak area, and calculate the recovery rate according to the following formula. The results are shown in Table 16-1, Table 16-2, and Table 16-3. The results show that the recovery rate test results are good.

[0206]

[0207] Table 16-1 New chlorogenic acid recovery test results

[0208]

[0209] Table 16-2 Chlorogenic acid recovery test results

[0210]

[0211] Table 16-3 Results of the recovery test of chlorogenic acid

[0212]

[0213] (b) Rutin recovery test:

[0214] Use methanol as solvent to prepare 468.7μg / ml rutin reference substance mother solution. Take 6 stoppered conical flasks, add 1.5ml of rutin reference substance mother solution to each conical flask, evaporate to dryness, and then accurately weigh six portions of the Herba Dioscoreae formula granules sample (batch number: HBC-F-240830-02), each about 0.25g, and place them in each conical flask, accurately add 25ml of 50wt% methanol to each conical flask, stopper, weigh, ultrasonic for 30 minutes, cool, weigh, make up the lost weight with 50wt% methanol, shake well, filter, take the filtrate, and obtain six test solution.

[0215] Accurately pipette 1 μl of each of the above test solution into the high performance liquid chromatograph, record the chromatogram, determine the peak area, and calculate the recovery rate according to the following formula. The results are shown in Table 17. The results show that the recovery rate determination results of the six tests are between 90% and 108%, with an average value of 97.2% and an RSD of 2.26%, indicating that the recovery test results are good.

[0216]

[0217] Table 17 Rutin recovery test results

[0218]

[0219] 6.2.7 Intermediate precision test

[0220] The same batch of samples of Herba Dioscoreae formula granules (batch number: HBC-F-240830-02) were tested by different personnel, on different dates, and with different instruments. They were processed according to the law and their contents were determined. The results are shown in Table 18. The results show that the intermediate precision of this method is good. The relevant chromatograms are shown in Figure 23~Figure 25 .

[0221] Table 18 Intermediate precision test results

[0222]

[0223] 6.2.8 Durability test

[0224] 6.2.8.1 Column temperature durability

[0225] According to the specified chromatographic conditions, the column temperature was changed by ±5°C, and the sample of the Herba Dioscoreae (batch number: HBC-F-240830-02) was taken. The test solution was prepared according to the method under "6.1.2 Preparation of test solution" to determine the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin. The results are shown in Table 19.

[0226] Table 19 Column temperature durability test results of content determination method

[0227]

[0228] 6.2.8.2 Flow rate durability

[0229] According to the specified chromatographic conditions, the flow rate was changed by ±0.1 ml / min, and the sample of the Herba Dioscoreae (batch number: HBC-F-240830-02) was taken. The test solution was prepared according to the method under "6.1.2 Preparation of test solution" to determine the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin. The results are shown in Table 20.

[0230] Table 20 Results of flow rate durability test of content determination method

[0231]

[0232] 6.2.8.3 Wavelength durability

[0233] According to the specified chromatographic conditions, the wavelength was changed by ±5nm, and the sample of the Herba Dioscoreae (batch number: HBC-F-240830-02) was taken. The test solution was prepared according to the method under "6.1.2 Preparation of test solution" to determine the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin. The results are shown in Table 21.

[0234] Table 21 Results of wavelength durability test of content determination method

[0235]

[0236] 6.2.8.4 Column durability

[0237] According to the specified chromatographic conditions, different chromatographic columns were selected, and the sample of the Herba Dioscoreae Formula Granules (batch number: HBC-F-240830-02) was taken. The test solution was prepared according to the method under "6.1.2 Preparation of Test Solution" to determine the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin. The results are shown in Table 22.

[0238] Table 22 Chromatographic column test results

[0239]

[0240] The results of the above durability tests show that the column temperature, flow rate, wavelength and durability of the chromatographic column of this method all meet the requirements of content determination. The chromatograms related to column temperature, flow rate and wavelength durability can be found in the characteristic spectrum item, and the chromatograms related to chromatographic column durability can be found in the content intermediate precision item.

[0241] 6.3 Sample determination

[0242] Three batches of HBC-F-240830-01, HBC-F-240830-02, and HBC-F-240830-03 were taken and their contents were determined according to the law. The results are shown in Table 23.

[0243] Table 23 Content determination results of Dioscorea purpurogena formula granules

[0244]

[0245] 7. Determination of quercetin and kaempferol content

[0246] 7.1 Assay method

[0247] 7.1.1 Preparation of reference solution: Using methanol as solvent, prepare quercetin reference solution and kaempferol reference solution with a concentration of 30 μg / ml respectively.

[0248] 7.1.2 Preparation of test solution: Grind 0.5 g of the powder of the Dicentra purslane formula granule sample, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of a mixed solution of methanol-25% hydrochloric acid (methanol and 25wt% hydrochloric acid, volume ratio 4:1) and weigh it. Heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the solution.

[0249] 7.1.3 Determination: Accurately pipette 1 μl of each reference solution and test solution, inject into liquid chromatograph for determination, and calculate the total content of kaempferol and quercetin based on the obtained chromatogram;

[0250] The liquid chromatography conditions were:

[0251] Chromatographic column: ACQUITY UPLC BEH C18, 2.1mm×100mm, 1.7μm; detection wavelength: 330nm; flow rate: 0.3ml / min; column temperature: 35℃; injection volume: 1μl; mobile phase: methanol as mobile phase A, 0.1wt% phosphoric acid as mobile phase B; elution according to the gradient specified in the following table:

[0252]

[0253] 7.2 Methodological Validation

[0254] 7.2.1 Positioning and system applicability

[0255] Take appropriate amount of quercetin and kaempferol reference substances respectively, weigh accurately, add methanol to prepare a solution containing 732.5 μg of quercetin and 340.6 μg of kaempferol per 1 ml, which is used as the reference substance mixed solution.

[0256] Take appropriate amount of quercetin and kaempferol reference substances respectively, weigh them accurately, add methanol to prepare quercetin reference substance solution containing 732.5 μg of quercetin per 1 ml and kaempferol reference substance solution containing 340.6 μg of kaempferol per 1 ml, then accurately measure 1 ml of quercetin and 1 ml of kaempferol reference substance solution respectively, add them into the same 10 ml volumetric flask, add methanol to the volume, shake well, and use them as the reference substance mixed solution.

[0257] Accurately weigh 0.5 g of the Herba Dioscoreae formula granules sample (batch number: HBC-F-240830-02), place in a stoppered conical flask, accurately add 25 ml of a mixed solution of methanol-25% hydrochloric acid (volume ratio 4:1), weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate as the test solution.

[0258] Accurately pipette 1 μl of the reference substance mixed solution and the test solution respectively, inject into the high performance liquid chromatograph for determination, and record the chromatogram. The relevant chromatogram is shown in Figure 26-27 . As can be seen from the figure, the retention time of quercetin in the chromatogram of the reference mixed solution is 20.387min, the theoretical plate number is 123765, and the tailing factor is 1.22; the retention time of kaempferol is 23.173min, the theoretical plate number is 156176, and the tailing factor is 1.19. The retention time of quercetin in the chromatogram of the test solution is 20.367min, the theoretical plate number is 113113, and the tailing factor is 1.17; the retention time of kaempferol is 23.163min, the theoretical plate number is 158955, and the tailing factor is 1.21. This shows that the system has good applicability.

[0259] 7.2.2 Exclusivity

[0260] Weigh 0.5g of maltodextrin accurately and prepare a blank excipient solution according to the method in "7.1.2 Preparation of test solution". Accurately pipette 1μl and inject it into the HPLC, record the chromatogram, and examine whether the excipient has any effect. See the relevant chromatogram for details. Fig.28 The results showed that the excipients had no effect on the characteristic spectrum determination.

[0261] 7.2.3 Injection precision

[0262] Accurately pipette 1 μl of the reference substance mixed solution under "7.2.1 Positioning and system suitability" and inject it into the HPLC, record the chromatogram, and measure the peak area. Repeat 6 times and calculate the RSD (%). The results are shown in Table 24. The results show that the method has good precision.

[0263] Table 24 Injection precision test results

[0264]

[0265] 7.2.4 Stability test

[0266] Take 0.5g of the sample of the Herba Dioscoreae (batch number: HBC-F-240830-02), weigh it accurately, and prepare the test solution according to the method of "7.1.2 Preparation of test solution". Accurately draw 1μl of the test solution and inject it into the liquid chromatograph, and inject it in sequence at 0, 2, 4, 8, 12, 18, and 24h, record the chromatogram, and calculate the average and RSD values ​​of the peak areas of quercetin and kaempferol. The results are shown in Table 25. The results show that the test solution has good stability within 24h.

[0267] Table 25 Results of stability test of test solution for content determination

[0268]

[0269] 7.2.5 Repeatability test

[0270] Take 6 portions of the same batch of Herba Dioscoreae formula granules (batch number: HBC-F-240830-02), accurately weigh 0.5g of each portion, prepare the test solution according to the method of "7.1.2 Preparation of test solution", accurately pipette 1μl of each of the 6 test solutions and inject them into the liquid chromatograph for detection, and calculate the total content of quercetin and kaempferol. The results are shown in Table 26. The results show that this method has good repeatability.

[0271] Table 26 Results of repeatability test of content determination method

[0272]

[0273] 7.2.6 Sample recovery test

[0274] Using methanol as solvent, prepare 732.5μg / ml quercetin reference substance mother solution and 340.6μg / ml kaempferol reference substance mother solution. Take 6 stoppered conical bottles, add 0.5ml of quercetin reference substance mother solution and 0.7ml of kaempferol reference substance mother solution to each conical bottle, evaporate to dryness; then accurately weigh six portions of the Dicentra purslane formula granule sample (batch number: HBC-F-240830-02), each portion is about 0.25g, and put them into 6 stoppered conical bottles, and accurately add 25ml of methanol-25% hydrochloric acid (volume ratio 4:1) mixed solution to each conical bottle, weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, take the filtrate, and obtain six test solution.

[0275] Accurately pipette 1 μl of each of the above test solution into the high performance liquid chromatograph, record the chromatogram, determine the peak area, and calculate the recovery rate according to the following formula. The results are shown in Table 27 and Table 28. The results show that the recovery test results are good.

[0276]

[0277] Table 27 Quercetin recovery test results

[0278]

[0279] Table 28 Kaempferol recovery test results

[0280]

[0281] 7.2.7 Intermediate precision test

[0282] The same batch of samples of Dioscorea purpurogena formula granules (batch number: HBC-F-240830-02) were tested by different personnel, on different dates, and with different instruments. They were processed according to the law and the total contents of quercetin and kaempferol were determined. The results are shown in Table 29. The results show that the intermediate precision of this method is good. The relevant chromatograms are shown in Figure 29~Figure 31 .

[0283] Table 29 Intermediate precision test results

[0284]

[0285] 7.2.8 Durability test

[0286] 7.2.8.1 Column temperature durability

[0287] According to the specified chromatographic conditions, the column temperature was changed by ±5°C, and the sample of Dicentra purpurogena formula granules (batch number: HBC-F-240830-02) was taken, and the test solution was prepared according to the method under "7.1.2 Preparation of test solution" to determine the total content of quercetin and kaempferol. The results are shown in Table 30. The relevant chromatograms are shown in Figure 32~Figure 34 .

[0288] Table 30 Column temperature durability test results of content determination method

[0289]

[0290] 7.2.8.2 Flow rate durability

[0291] According to the specified chromatographic conditions, the flow rate was changed by ±0.03 ml / min, and the sample of Dicentra purpurogena formula granules (batch number: HBC-F-240830-02) was taken, and the test solution was prepared according to the method under "7.1.2 Preparation of test solution" to determine the total content of quercetin and kaempferol. The results are shown in Table 31. The relevant chromatogram is shown in Figure 35-36 .

[0292] Table 31 Results of flow rate durability test of content determination method

[0293]

[0294] 7.2.8.3 Wavelength durability

[0295] According to the specified chromatographic conditions, the wavelength was changed by ±5nm, and the sample of Dicentra purpurogena formula granules (batch number: HBC-F-240830-02) was taken. The test solution was prepared according to the method under "7.1.2 Preparation of test solution" to determine the total content of quercetin and kaempferol. The results are shown in Table 32. The relevant chromatogram is shown in Figure 37-38 .

[0296] Table 32 Results of wavelength durability test of content determination method

[0297]

[0298] 7.2.8.4 Column durability

[0299] According to the specified chromatographic conditions, different chromatographic columns were selected, and the sample of Dicentra purpurogena formula granules (batch number: HBC-F-240830-02) was taken. The test solution was prepared according to the method under "7.1.2 Preparation of test solution" to determine the total content of quercetin and kaempferol. The results are shown in Table 33. The relevant chromatograms are shown in Fig.39 .

[0300] Table 33 Chromatographic column test results

[0301]

[0302] The results of the above durability tests showed that the column temperature, flow rate, wavelength and durability of the chromatographic column of this method all met the requirements for content determination.

[0303] 7.3 Sample determination

[0304] Three batches of Herba Dioscoreae formula granules (HBC-F-240830-01, HBC-F-240830-02, and HBC-F-240830-03) were taken and the total contents of quercetin and kaempferol were determined according to the law. The results are shown in Table 34.

[0305] Table 34 Content determination results of Dioscorea purpurogena formula granules

[0306]

[0307] In the present invention, the rutin reference product is provided by China Food and Drug Inspection Institutes, batch number: 100080-202012, with a content of 91.6%; the neochlorogenic acid reference product is provided by Shanghai Shidande Standard Technology Service Co., Ltd., batch number: ST06230120, with a content of 98.0%; the chlorogenic acid reference product is provided by China Food and Drug Inspection Institutes, batch number: 110753-202119, with a content of 96.3%; the cryptochlorogenic acid reference product is provided by Shanghai Shidande Standard Technology Service Co., Ltd., batch number: ST07850120, with a content of 98.0%; the quercetin reference product is provided by China Food and Drug Inspection Institutes, batch number: 100081-201610, with a content of 99.1%; the kaempferol reference product is provided by China Food and Drug Inspection Institutes, batch number: 110861-202214, with a content of 97.4%.

[0308] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A quality detection method for Herba Dioscoreae formula granules, characterized in that: It includes property detection, extract determination, thin layer identification, characteristic spectrum determination, content determination, and moisture, particle size, loading difference, and microbial limit inspection. The content determination includes the total content determination of kaempferol and quercetin and the total content determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin. The Herba Dioscoreae formula granules are prepared by the following method: taking Herba Dioscoreae decoction pieces, adding water to boil for multiple times, filtering, concentrating the filtrate to a clear paste, drying the clear paste to a dry paste, adding auxiliary material maltodextrin, mixing, and granulating to obtain; Characteristic profile determination includes the following steps: (1) Prepare the neochlorogenic acid reference substance solution, chlorogenic acid reference substance solution, cryptochlorogenic acid reference substance solution, and rutin reference substance solution respectively; (2) Prepare a test solution by taking the powder of the sample of the formula granules of the Dicentra purslane. The specific preparation method of the test solution is as follows: take 0.5 g of the sample powder of the formula granules of the Dicentra purslane, add 25 ml of 50 wt% methanol, ultrasonically treat, cool, and then add 50 wt% methanol to make up the lost weight, shake well, filter, and take the filtrate to obtain the solution; (3) Determination: Perform liquid chromatography on the neochlorogenic acid reference substance solution, chlorogenic acid reference substance solution, cryptochlorogenic acid reference substance solution, rutin reference substance solution, and test solution respectively; The chromatographic conditions for characteristic spectrum determination are: Chromatographic column: ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 μm; Mobile phase: acetonitrile as mobile phase A, 0.1wt% phosphoric acid as mobile phase B; Gradient elution program: during 0-10 min, mobile phase A increased from 5% to 15%, and mobile phase B decreased from 95% to 85%; during 10-19 min, mobile phase A increased from 15% to 20%, and mobile phase B decreased from 85% to 80%; during 19-24 min, mobile phase A increased from 20% to 23%, and mobile phase B decreased from 80% to 77%; during 24-26 min, mobile phase A increased from 23% to 26%, and mobile phase B decreased from 77% to 74%; during 26-30 min, mobile phase A increased from 26% to 40%, and mobile phase B decreased from 74% to 60%; during 30-34 min, mobile phase A decreased from 40% to 5%, and mobile phase B increased from 60% to 95%; Detection wavelength: 354nm; Flow rate: 0.3ml / min; Column temperature: 40°C; Injection volume: 1 μl; The determination of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin content includes the following steps: (1) Prepare neochlorogenic acid reference solution, chlorogenic acid reference solution, cryptochlorogenic acid reference solution, and rutin reference solution respectively; (2) Prepare a test solution by taking the powder of the sample of the formula granules of the Dicentra purslane. The specific preparation method of the test solution is as follows: take 0.5 g of the sample powder of the formula granules of the Dicentra purslane, add 25 ml of 50 wt% methanol, ultrasonically treat, cool, and then add 50 wt% methanol to make up the lost weight, shake well, filter, and take the filtrate to obtain the solution; (3) Determination: Perform liquid chromatography on the neochlorogenic acid reference solution, chlorogenic acid reference solution, cryptochlorogenic acid reference solution, rutin reference solution, and test solution, respectively, and calculate the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin based on the obtained chromatograms; In the determination of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin, the chromatographic conditions were: Chromatographic column: ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 μm; Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B; Gradient elution program: during 0-10 min, mobile phase A increased from 5% to 15%, and mobile phase B decreased from 95% to 85%; during 10-19 min, mobile phase A increased from 15% to 20%, and mobile phase B decreased from 85% to 80%; during 19-24 min, mobile phase A increased from 20% to 23%, and mobile phase B decreased from 80% to 77%; during 24-26 min, mobile phase A increased from 23% to 26%, and mobile phase B decreased from 77% to 74%; during 26-30 min, mobile phase A increased from 26% to 40%, and mobile phase B decreased from 74% to 60%; during 30-34 min, mobile phase A decreased from 40% to 5%, and mobile phase B increased from 60% to 95%; Detection wavelength: 354nm; Flow rate: 0.3ml / min; Column temperature: 40°C; Injection volume: 1 μl; The determination of the total content of kaempferol and quercetin includes the following steps: (1) Prepare quercetin reference solution and kaempferol reference solution respectively; (2) Prepare the test solution by taking the powder of the sample of the formula granule of the Dicentra purslane. The specific preparation method of the test solution is as follows: take 0.5 g of the powder of the formula granule of the Dicentra purslane, add 25 ml of a mixed solution of methanol and 25 wt% hydrochloric acid in a volume ratio of 4:1, heat to reflux and then cool, then add 50 wt% methanol to make up the lost weight, shake well, filter, and take the filtrate to obtain the solution; (3) Determination: Perform liquid chromatography on the quercetin reference solution, kaempferol reference solution and test solution, respectively, and calculate the total content of kaempferol and quercetin based on the obtained chromatograms; In the determination of the total content of kaempferol and quercetin, the chromatographic conditions are: Chromatographic column: ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 μm; Mobile phase: methanol as mobile phase A, 0.1wt% phosphoric acid as mobile phase B; Gradient elution program: During 0-30 min, mobile phase A increased from 5% to 70%, and mobile phase B decreased from 95% to 30%; during 30-35 min, mobile phase A decreased from 70% to 5%, and mobile phase B increased from 30% to 95%; Detection wavelength: 330nm; Flow rate: 0.3ml / min; Column temperature: 35°C; Injection volume: 1 μl; The thin layer identification method includes the following steps: (1) Preparation of test solution: Take 1.0 g of Dicentra purslane granules, add 20 ml of 50 wt% ethanol, and after ultrasonic treatment, cool and filter. The filtrate is used as the test solution. (2) Preparation of reference solution and reference medicinal material solution: Take rutin and chlorogenic acid reference substances respectively, add methanol to prepare rutin reference solution and chlorogenic acid reference solution with a concentration of 1 mg / ml respectively; take 1.0 g of Dicentra herba decoction pieces, and prepare Dicentra herba decoction piece solution according to the preparation method of the test solution; (3) Determination: 5 μl of the test sample solution, 10 μl of the reference medicinal material solution, 3 μl of the rutin reference solution and 3 μl of the chlorogenic acid reference solution were respectively spotted on the same silica gel G thin layer plate, and the plate was developed using ethyl acetate-butanone-formic acid-water in a volume ratio of 8:3:1.5:

1. The plate was taken out, dried, sprayed with 1 wt% aluminum chloride solution, and inspected under ultraviolet light.

2. The quality inspection method of the Herba Dicentrariae granules according to claim 1, characterized in that: In the characteristic spectrum determination, the four reference substance solutions were all prepared with methanol as solvent, and the concentrations of the four reference substance solutions were all 30 μg / ml.

3. The quality inspection method of the Herba Dioscoreae formula granules according to claim 1, characterized in that: In the determination of the total content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin content, the four reference solutions were all prepared with methanol as solvent, and the concentrations of the four reference solutions were all 30 μg / ml.

4. The quality inspection method of the Herba Dioscoreae formula granules according to claim 1, characterized in that: In the determination of the total content of kaempferol and quercetin, the two reference solutions were prepared with methanol as solvent, and the concentrations of the two reference solutions were both 30 μg / ml.

5. The quality inspection method of the Herba Dioscoreae formula granules according to claim 1, characterized in that: The test methods for moisture, particle size, filling volume difference, and microbial limit are as follows: Test the samples of the herbaceous purslane formula granules in accordance with the provisions of the General Chapter 0104 Granules of the 2020 edition of the Chinese Pharmacopoeia.

6. The quality inspection method of the Herba Dicentrariae granules according to claim 1, characterized in that: The extract determination method is: using ethanol as the solvent, the extract determination of the Herba Dioscoreae formula granules sample is carried out in accordance with the hot infusion method determination provisions under Item 2201 of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia.

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