A quality detection method for Dongwan cough granules

Abstract

The present invention belongs to the field of traditional Chinese medicine technology, and is specifically related to a quality detection method for Dong Wan Zhi Ke granules. The quality detection method includes the determination of flavonoid standard fingerprints, and the determination adopts high performance liquid chromatography, wherein the preparation method of the test sample includes first dissolving the Dong Wan Zhi Ke granules in water, centrifuging, and taking the supernatant; then adjusting the supernatant pH value to 4-5.5, passing the supernatant through NKA-Ⅱ macroporous resin column, eluting with water and discarding, and then eluting with 45-55% ethanol aqueous solution, collecting the eluent. The present invention establishes the HPLC fingerprint analysis method of flavonoid components in different batches of Dong Wan Zhi Ke granules, and uses rutin as a reference peak to calibrate the relative retention time of other common fingerprint peaks and calculate the ratio of other common fingerprint peak areas, which provides a scientific basis for the quality control of Dong Wan Zhi Ke granules.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a quality detection method for Dongwan cough-relieving granules. Background Art

[0002] Dongwan Cough Relief Granules are composed of eight Chinese medicinal herbs: ephedra, pinellia, asarum, ginger, coltsfoot, aster, magnolia, and Xanthium sibiricum. They have the functions of dispelling wind and cold, promoting lung function and relieving cough, and can treat nasal congestion and promote lung function.

[0003] The formula of Dongwan cough granules is recorded in Chinese patent CN1103224C, which discloses a Chinese patent medicine for treating cold cough, which is made of the following components in parts by weight as raw materials: 10-50 parts each of ephedra, ginger, aster, coltsfoot, pinellia, magnolia, and Xanthium sibiricum, and 2-20 parts of asarum.

[0004] Traditional Chinese medicine fingerprint technology has been widely used in the quality control research of traditional Chinese medicines according to the characteristics of multi-component and multi-target effects of traditional Chinese medicines. It has been gradually promoted and applied in traditional Chinese medicine injections, but there are few research reports on its application in the quality standards of traditional Chinese medicine solid preparations. Compared with injections, the active ingredients of solid preparations are complex and diverse. The current quality standards of solid preparations are difficult to comprehensively and effectively control their quality. Therefore, the current quality standards of solid preparations need to be further improved. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a quality detection method for Dongwan Zhike Granules. The present invention conducts a fingerprint study of flavonoids in Dongwan Zhike Granules to improve product quality standards and better control the quality of solid preparations, and also provides a reference for research on improving the quality standards of solid preparations.

[0006] In order to achieve the purpose of the present invention, the technical solution adopted is as follows:

[0007] A quality detection method for Dongwan cough granules, the quality detection method comprising the determination of a flavonoid standard fingerprint spectrum, the determination adopting high performance liquid chromatography, wherein the preparation method of the test sample comprises the following steps:

[0008] (1) Dissolve Dongwan Cough Granules in water, centrifuge, and collect the supernatant;

[0009] (2) Adjust the pH value of the supernatant to 4-5.5, pass the supernatant through a macroporous resin column, elute with water and discard, then elute with 45-55% ethanol aqueous solution, and collect the eluate;

[0010] In the chromatographic conditions of the high performance liquid chromatography, mobile phase A is acetonitrile, and mobile phase B is 0.05-0.15% phosphoric acid aqueous solution.

[0011] Preferably, the loading flow rate of the supernatant in step (2) is 1-2.5 ml / min; the flavonoid concentration in the supernatant is 0.5-2 mg / ml, preferably 1-2 mg / ml; more preferably 1.5 mg / ml.

[0012] Preferably, the macroporous resin in step (2) is a NKA-II type macroporous resin.

[0013] Preferably, the amount of water used in step (2) is 4-6.5 times the column volume, preferably 5-6.5 times the column volume.

[0014] Preferably, the amount of the 45-55% ethanol aqueous solution in step (2) is 3-5 times the column volume.

[0015] Preferably, step (2) further comprises evaporating the eluent to dryness and dissolving the residue in methanol.

[0016] Preferably, in the high performance liquid chromatography method, the detection wavelength is 355-365 nm, the column temperature is 20-30° C., the chromatographic column is filled with octadecylsilane bonded silica gel, and the flow rate is 0.7-0.8 ml / min.

[0017] Preferably, the gradient elution procedure in high performance liquid chromatography is as follows:

[0018] 0 min, mobile phase A: 13-14%, mobile phase B: 86-87%;

[0019] 15 min, mobile phase A: 14-16%, mobile phase B: 84-86%;

[0020] 45 min, mobile phase A: 28-32%, mobile phase B: 68-72%;

[0021] 60min, mobile phase A: 33-37%, mobile phase B: 63-67%.

[0022] Preferably, the quality detection method also includes thin layer chromatography identification of coltsfoot, wherein the preparation of the test solution includes: extracting the Dongwan cough granules with ethanol, filtering, evaporating the filtrate to dryness, adding water, extracting with ethyl acetate, combining the extracts, evaporating the extracts to dryness, and dissolving them in ethyl acetate to prepare the test solution.

[0023] Preferably, the ethanol extraction is water bath heating reflux extraction, and the number of extractions is 2-4 times.

[0024] Preferably, in the thin layer chromatography identification, the preparation of the reference medicinal material solution comprises: adding ethanol to the coltsfoot flower medicinal material, heating in a water bath and refluxing for extraction, filtering, evaporating the filtrate to dryness, and adding ethyl acetate for dissolution. The ethanol is commercially available conventional 95% ethanol.

[0025] Preferably, in the thin layer chromatography identification, the developing solvent is petroleum ether (60-90° C.)-ethyl acetate in a volume ratio of 4.5-5.5:1.

[0026] Preferably, the raw materials of the Dongwan cough granules include 10-50 parts of ephedra, 10-50 parts of ginger, 10-50 parts of aster, 10-50 parts of coltsfoot, 10-50 parts of pinellia, 10-50 parts of magnolia, 10-50 parts of xanthium, and 2-20 parts of asarum.

[0027] Another object of the present invention is to provide a fingerprint spectrum of the Dongwan Antitussive Granules obtained by the above-mentioned high performance liquid chromatography detection, wherein the fingerprint spectrum contains 12 common peaks.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) Flavonoids are widely found in plants and are a class of active substances with extensive medicinal value. Many flavonoid components have antitussive, expectorant, antiasthmatic and antibacterial activities. According to literature reports, the medicinal materials such as ephedra, coltsfoot, aster, xanthium, and magnolia in Dongwan cough granules contain rich flavonoids, mainly quercetin and its glycosides. Flavonoids are one of the main active ingredients of Dongwan cough granules and have significant therapeutic effects in treating cough. Therefore, the present invention selects flavonoid components as the evaluation index for quality control of Dongwan cough granules.

[0030] The present invention studies the HPLC fingerprint of flavonoid components in Dongwan Zhike granules. Due to the complex components of the compound preparation, directly using solvent extraction to prepare the test solution will cause large interference in the liquid phase spectrum and make it difficult to separate the peaks. The present invention uses macroporous adsorption resin to purify and enrich the flavonoid components in the prescription, explores the HPLC fingerprint conditions, establishes the HPLC fingerprint analysis method of flavonoid components in different batches of Dongwan Zhike granules, and uses rutin as a reference peak to calibrate the relative retention time of other common fingerprint peaks and calculate the ratio of other common fingerprint peak areas. At the same time, the "Chinese medicine chromatographic fingerprint similarity evaluation software system (2012A version)" is used to calculate the similarity of the HPLC fingerprint of flavonoid components of different batches of products, providing a scientific basis for the quality control of Dongwan Zhike granules.

[0031] (2) Since the ingredients of traditional Chinese medicine compound are complex and the fingerprint spectrum has great interference, the present invention adopts macroporous resin to purify flavonoid components. By optimizing the parameters in the macroporous resin purification method, the optimal preparation method of the flavonoid component test sample is determined; by optimizing the chromatographic conditions, the optimal chromatographic conditions are determined, and the high-performance liquid chromatography standard fingerprint spectrum of the flavonoid components in Dongwan Zhike Granules is established. The fingerprint spectrum is evaluated by using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Version A", and 12 common peaks are calibrated. The similarity of 10 batches of samples is 0.97-0.99. By comparing this product with the medicinal materials, the exclusive absorption peaks of ephedra, magnolia and Xanthium sibiricum are determined. Through methodological investigation, it is proved that the fingerprint spectrum has good precision, repeatability and stability, conforms to the establishment principle of traditional Chinese medicine fingerprint spectrum, and can be used for quality control of flavonoid components.

[0032] (3) Thin layer chromatography is a simple, rapid and trace chromatography method that is widely used in the identification of compounds. The present invention investigated the spotting amount and development system of the thin layer chromatography identification method of coltsfoot in Dongwan Zhike Granules, and established a thin layer chromatography identification method of coltsfoot in this product. The results showed that this method is simple and feasible, and can well control the quality of Dongwan Zhike Granules, and can be included in the quality standard item. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The fingerprints of flavonoid components of 10 batches of Dongwan Zhike Granules;

[0034] Figure 2 HPLC spectra of Ephedra medicinal materials and samples; from top to bottom: Ephedra medicinal materials, negative samples lacking Ephedra, Dongwan cough granules;

[0035] Figure 3 It is the chromatogram of Comparative Example 6;

[0036] Figure 4 It is the chromatogram of Comparative Example 7;

[0037] Figure 5 It is the chromatogram of Comparative Example 8;

[0038] Figure 6 It is the chromatogram of Comparative Example 9;

[0039] Figure 7 It is the chromatogram of Comparative Example 10;

[0040] Figure 8 This is a TLC identification chromatogram of coltsfoot; 1 is the coltsfoot control medicinal material solution, 2 is the coltsfoot negative sample solution, and 3-5 are the test sample solutions. DETAILED DESCRIPTION

[0041] Example 1 Determination of total flavonoids fingerprint and methodological investigation

[0042] 1. Preparation method of test solution

[0043] Take 2.5g of Dongwan cough granules, weigh accurately, add 50ml of pure water, heat to dissolve, cool, centrifuge, take the supernatant (flavonoid concentration is about 1.5mg / ml), adjust pH=5, pass through NKA-Ⅱ macroporous resin (15ml), adjust the flow rate to 2.0ml / min, elute with 90ml of water and discard, then elute with 50ml of 50% ethanol aqueous solution, collect the eluate, evaporate to dryness, dissolve the residue with methanol to make up to 10ml, pass through 0.45μm microporous filter membrane, and prepare for measurement.

[0044] Preparation of reference solution: Take an appropriate amount of rutin reference substance dried to constant weight, accurately weigh it, and add 50% volume concentration of ethanol aqueous solution to make a solution containing 0.198 mg per 1 ml.

[0045] 2. Chromatographic conditions

[0046] Chromatographic column: Agilent SB-C18 (250mm×4.6mm, 5um); column temperature: 25℃; detection wavelength: 360nm, injection volume 10ul, running time 60min; flow rate 0.8ml / min, mobile phase: phase A (acetonitrile)-phase B (0.1% phosphoric acid aqueous solution), linear gradient elution, the elution program is as shown in Table 1.

[0047] Table 1 Mobile phase elution gradient

[0048]

[0049] 3. Methodological investigation

[0050] After determining the preparation method and chromatographic separation conditions of the test sample, a methodological validation study was conducted on the flavonoid fingerprint of Dongwan Cough Relief Granules. The selected evaluation indicators mainly included the following three aspects: precision, stability, and repeatability test.

[0051] 3.1 Precision test

[0052] The same test solution was taken and injected 6 times continuously. The results were determined according to the above chromatographic conditions. The number of peaks in the obtained HPLC spectrum was the same, the relative retention time RSD changes of all peaks were less than 1.81%, and the relative peak area RSD were less than 3%, which met the requirements of the fingerprint spectrum.

[0053] 3.2 Repeatability test

[0054] Take samples from the same batch number and prepare 5 test solutions according to the test solution preparation method. Determine under the above chromatographic conditions. The number of peaks in the obtained HPLC spectrum is the same, the relative retention time RSD changes of all peaks are less than 2.28%, and the relative peak area RSD are less than 3%, which meets the requirements of the fingerprint spectrum.

[0055] 3.3 Stability test

[0056] The same test solution was taken and measured at 0, 4, 8, 16, 32, and 48 h respectively. The number of peaks in the obtained HPLC spectrum was the same, the relative retention time RSD changes of all peaks were less than 1.98%, and the relative peak area RSD were less than 3%, which met the requirements of the fingerprint spectrum.

[0057] 3.4 Establishment of fingerprint

[0058] 3.4.1 Sample determination

[0059] Take 10 batches of Dongwan Zhike Granule samples (batch numbers: 20230306, 20230519, 20230512, 20230409, 20230426, 20230507, 20230712, 20230909, 20231012, 20231210), prepare according to the method under the test solution preparation method in Example 1, measure and record the fingerprint according to the chromatographic conditions of Example 1, rutin chromatographic peak (peak 9), compare the retention time, use the rutin chromatographic peak (peak 9) as the reference peak to compare the chromatogram, and determine that there are 12 common peaks. Calculate the relative retention time and relative peak area of ​​each common peak in the fingerprint of the 10 batches of samples. The results are shown in Table 2-3 below; The fingerprints of flavonoid components of 10 batches of Dongwan Zhike Granules are shown in Table 2-3 below. Figure 1 .

[0060] Table 2 Relative retention time of common peaks in flavonoid fingerprint

[0061]

[0062] Table 3 Relative peak areas of common peaks in flavonoid fingerprints

[0063]

[0064] 3.4.2 Similarity Calculation

[0065] The fingerprints of the flavonoid components of the above 10 batches of Dongwan Zhike Granules were analyzed using the similarity software of Chinese medicine chromatographic fingerprints. A comprehensive evaluation was performed using the "Chinese medicine chromatographic fingerprint similarity evaluation software system (2012A version)". The generated sample spectrum was used as the reference fingerprint spectrum, and the average value of the relative peak area of ​​the fingerprint spectrum of the 10 batches of samples was used as the reference fingerprint spectrum (R) template. According to the results of peak matching, the similarity of each sample was calculated by comparing the peak area of ​​all peaks of the fingerprint spectrum to be tested with the peak area on the reference template. The results are shown in Table 4.

[0066] Table 4 Similarity of 10 batches of samples

[0067]

[0068] Results: From the results in Table 4 above, we can see that the similarity values ​​of the 10 batches of samples are all above 0.97, indicating that they are similar overall. Therefore, the mode spectrum is established based on the average of the relative peak area and relative retention time of these 10 batches of samples. When using this method to analyze unknown samples, it is only necessary to measure the chromatographic fingerprint of the sample and calculate the similarity between the unknown sample and the common pattern to identify and evaluate the sample.

[0069] 3.5 Relationship between common peaks and medicinal materials

[0070] The present invention further conducts experimental research on the flavonoid components of Ephedra, Xanthium sibiricum and Magnolia officinalis, and compares them with the flavonoid component fingerprint of the granules to determine the sources of each peak in the fingerprint.

[0071] According to the sample preparation method of Example 1, flavonoid component test sample solutions of Dongwan Zhike Granules, original medicinal materials and negative samples lacking a single medicinal material were prepared respectively. The chromatograms of the flavonoid components of Dongwan Zhike Granules, original medicinal materials and negative samples lacking a single medicinal material were measured according to the chromatographic conditions in Example 1. The correlation between the common peaks in the fingerprint spectrum of Dongwan Zhike Granules and the peak attribution in the color spectrum of the original medicinal material was studied.

[0072] 3.5.1 Study on the correlation between the common peaks of the fingerprint of flavonoid components in Dongwan Zhike Granules and the fingerprint of the corresponding parts of Ephedra sinica

[0073] Take 2.5 g of Dongwan Zhike Granules, 2.5 g of negative samples lacking ephedra medicinal materials, and 0.84 g of ephedra medicinal materials (equivalent to the amount of ephedra medicinal materials contained in 2.5 g of granules), and prepare Dongwan Zhike Granules, negative samples lacking ephedra medicinal materials, and flavonoid component test solutions of ephedra medicinal materials according to the test solution preparation method of Example 1.

[0074] The fingerprint was determined according to the chromatographic conditions of Example 1, and the chromatograms of Dongwan Zhike Granules, Ephedra medicinal materials, and Ephedra-deficient negative solution and the UV absorption spectra of the corresponding chromatographic peaks were compared to determine the ownership of the Ephedra medicinal materials with the common fingerprint peaks in the fingerprint. The results are shown in Table 1. Figure 2 Result: From Figure 2 The results show that the common peaks 5, 6, 7, 8 and 9 in the chromatogram of the original ephedra medicinal material appear in the corresponding positions of the Dongwan cough granules, among which peaks 5, 7 and 8 have no chromatographic peaks in the corresponding positions of the negative chromatogram of ephedra. It can be basically determined that peaks 5, 7 and 8 are derived from ephedra medicinal materials and are the exclusive absorption peaks of ephedra.

[0075] 3.5.2 Study on the correlation between the common fingerprint peaks of the flavonoid components of Dongwan Zhike Granules and the fingerprints of the corresponding parts of Magnolia officinalis

[0076] Take 2.5 g of Dongwan Zhike Granules, 2.5 g of negative sample lacking Xinyi medicinal materials, and 0.84 g of Xinyi medicinal materials (equivalent to the amount of Xinyi medicinal materials contained in 2.5 g of granules), and prepare Dongwan Zhike Granules, negative sample lacking Xinyi medicinal materials, and flavonoid component test solutions of Xinyi medicinal materials according to the test solution preparation method in Example 1.

[0077] The fingerprint was determined according to the chromatographic conditions determined in Example 1, and the chromatograms of Dongwan Zhike Granules, Xinyi medicinal materials, and Xinyi negative solution and the ultraviolet absorption spectra of the corresponding chromatographic peaks were compared to determine the Xinyi medicinal materials with common fingerprint peaks in the fingerprint.

[0078] The results showed that the common peaks 1, 3, and 11 in the chromatogram of magnolia medicinal materials appeared in the corresponding positions of Dongwan cough granules, among which peak 1 had no chromatographic peak in the corresponding position of the negative chromatogram of magnolia. It can be basically determined that peak 1 originated from magnolia medicinal materials and was the exclusive absorption peak of magnolia.

[0079] 3.5.3 Study on the correlation between the common fingerprint peaks of the flavonoid components of Dongwan Zhike Granules and the fingerprints of the corresponding parts of Xanthium sibiricum

[0080] Take 2.5 g of Dongwan Zhike Granules, 2.5 g of negative sample lacking Xanthium sibiricum medicinal materials, and 0.84 g of Xanthium sibiricum medicinal materials (equivalent to the amount of Xanthium sibiricum medicinal materials contained in 2.5 g of granules), and prepare Dongwan Zhike Granules, negative sample lacking Xanthium sibiricum medicinal materials, and flavonoid component test solutions of Xanthium sibiricum medicinal materials according to the test solution preparation method in Example 1.

[0081] The fingerprint was determined according to the chromatographic conditions and method of Example 1, and the chromatograms of Dongwan Zhike Granules, Xanthium sibiricum medicinal materials, and negative solutions lacking Xanthium sibiricum and the ultraviolet absorption spectra of the corresponding chromatographic peaks were compared to determine the Xanthium sibiricum medicinal materials that shared the fingerprint peaks in the fingerprint.

[0082] The results showed that the common peaks 2, 3, 4, 6, 10, 11, and 12 in the chromatogram of Xanthium sibiricum medicinal material appeared in the corresponding positions of Dongwan Cough Relief Granules, among which peak 2 had no chromatographic peak in the corresponding position of the negative chromatogram of Xanthium sibiricum. It can be basically determined that peak 2 originated from Xanthium sibiricum medicinal material and was the exclusive absorption peak of Xanthium sibiricum.

[0083] 3.6 Results and Discussion

[0084] 3.6.1 Preparation of flavonoid test solution

[0085] The invention investigates the preparation method of the test solution, investigates macroporous resins with different polarities, screens out NKA-II resin with relatively good purification effect, optimizes the relevant parameters of the NKA-II macroporous resin for purifying flavonoid test samples, and determines the best preparation method of the flavonoid test solution: taking about 2.5g of Dongwan cough granules, accurately weighing, adding 50ml of pure water, heating to dissolve, cooling, centrifuging, taking the supernatant, adjusting the pH value to 5, passing through an NKA-II macroporous adsorption resin column (15ml), adjusting the sample flow rate to 2.0ml / min, eluting with 90ml of water (discarding), eluting with 50ml of 50% ethanol, collecting the 50% ethanol eluate, evaporating to dryness in a water bath, and dissolving the residue with methanol to a fixed volume of 10ml to obtain the product.

[0086] 3.6.2 Determination of chromatographic conditions

[0087] Due to the complex composition of traditional Chinese medicine compound and the large interference of fingerprint spectrum, macroporous resin was used to purify flavonoid components. The optimal preparation method of flavonoid component test sample was determined by screening the parameters in the macroporous resin purification method; the optimal chromatographic conditions were determined by screening and optimization of chromatographic conditions, and the HPLC standard fingerprint spectrum of flavonoid components in Dongwan Zhike Granules was established. The fingerprint spectrum was evaluated by "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Version A", and 12 common peaks were calibrated. The similarity of 10 batches of samples was 0.97-0.99. Through comparative study of this product with medicinal materials, the exclusive absorption peaks of ephedra, magnolia and Xanthium sibiricum were determined. After methodological investigation, it was proved that the fingerprint spectrum had good precision, repeatability and stability, which was in line with the establishment principles of Chinese medicine fingerprint spectrum and could be used for quality control of flavonoid components.

[0088] Comparative Example 1 Screening of different types of macroporous resins

[0089] Weigh 15 ml of each of the treated AB-8, NKA-Ⅱ, X-5, and HPD-100 macroporous resins accurately and place them in a stoppered bottle, add 45 ml of the sample solution (2.0 mg / ml), shake well, and oscillate in an oscillator (25°C, 120 r / min) for 1 hour and overnight. After each resin is saturated with adsorption, filter it and take the filtrate to measure the total flavonoid content, and calculate the static specific adsorption amount and adsorption rate. Accurately add 45 ml of 95% ethanol to each saturated resin, oscillate it in an oscillator for 8 hours until desorption equilibrium is reached, take the supernatant to measure the total flavonoid content, and calculate the desorption rate according to the following formula. The results are shown in Table 5.

[0090] Adsorption amount per gram = (C0V0-C1V1) / W;

[0091] Adsorption rate = (C0V0-C1V1) / C0V0×100%;

[0092] Desorption rate (P) = C2V2 / (C0V0-C1V1) × 100%;

[0093] Where C0: sample mass concentration (g / L);

[0094] C1: mass concentration of residual liquid after adsorption (g / L);

[0095] C2: mass concentration of desorption liquid;

[0096] V0: volume of sample loaded (ml);

[0097] V1: volume of the remaining solution after adsorption (ml);

[0098] V2: desorption liquid volume (ml);

[0099] W: resin mass (g).

[0100] Table 5 Adsorption and desorption results of total flavonoids from Dongwan Zhike Granules by different types of macroporous resins

[0101]

[0102] Results: It can be seen from Table 5 that the adsorption rate and desorption rate of the macroporous resin NKA-Ⅱ are higher than those of other resins. Considering the adsorption and desorption capacity of different types of macroporous resins for total flavonoids, NKA-Ⅱ macroporous resin was selected to purify and enrich the total flavonoids in Dongwan Zhike Granules.

[0103] Comparative Example 2 Investigation of sample concentration

[0104] Take 4 portions of Dongwan Zhike Granule extract, each 50 ml with concentrations of 0.5 mg / ml, 1.5 mg / ml, 2 mg / ml, and 2.5 mg / ml, adjust the pH value to 5, and load them onto the same macroporous resin column at a flow rate of 2 ml / min, collect the flowing liquid, and measure the adsorption rate. The results are shown in Table 6.

[0105] Table 6 Effect of sample concentration on adsorption

[0106]

[0107] Results: As shown in Table 6, the adsorption rate was the highest when the loading mass concentration was 1.5 mg / ml, so 1.5 mg / ml was selected as the loading concentration.

[0108] Comparative Example 3 Investigation of sample loading flow rate

[0109] Four portions of Dongwan Zhike Granules extract with a total flavonoids concentration of 2.0 mg / ml were taken, and each portion was 50 ml. They were added into four macroporous resin columns (glass columns, inner diameter of about 1.5 cm, column length of about 10 cm, wet packing) at flow rates of 1.0 ml / min, 2.0 ml / min, 3.0 ml / min, and 4.0 ml / min, respectively. Dynamic adsorption experiments were carried out to determine the adsorption rate. The results are shown in Table 7.

[0110] Table 7 Effect of sample flow rate on adsorption rate of total flavonoids in Dongwanzhike granules

[0111]

[0112] Comparative Example 4 Investigation of pH Value of the Drug Solution on the Column

[0113] Take 4 portions of Dongwan cough granules extract, 50 ml each, with a total flavonoids concentration of 2.0 mg / ml, adjust the pH value to 5, 6, 7, and 8, and load them on the same macroporous resin column at a flow rate of 2.0 ml / min for dynamic adsorption experiments. Collect the effluent and determine the adsorption rate. The results are shown in Table 8.

[0114] Table 8 Effect of pH value of the column solution on its adsorption

[0115]

[0116] Results: When the pH was 6, the adsorption rate dropped sharply. Considering the effects of acid on chemical composition, resin service life and adsorption efficiency, the optimal adsorption pH was determined to be 5.

[0117] Comparative Example 5 Investigation of Eluent Ethanol Concentration

[0118] The treated resin (15 ml) was loaded on the column, and 50 ml of the supernatant of each Dongwan Zhike granule was adjusted to pH 5. The sample was adsorbed at a flow rate of 2 ml / min, and eluted with 90 ml of water and then with ethanol-water solution with a volume concentration of less than 100 ml. The eluate was collected, and the total flavonoids content in the water wash and the eluate was determined. The desorption rate was calculated. The results are shown in Table 9.

[0119] Table 9

[0120]

[0121] Comparative Example 6

[0122] The difference between this comparative example and Example 1 is that the mobile phase is different, acetonitrile-water, and the rest is the same. Figure 3 .

[0123] Comparative Example 7

[0124] The difference between this comparative example and Example 1 is that the mobile phase is different, which is methanol-0.1% phosphoric acid water, and the rest is the same. Figure 4 .

[0125] Comparative Example 8

[0126] The difference between this comparative example and Example 1 is that the mobile phase is different, that is, methanol-water, and the rest is the same. Figure 5 .

[0127] The results show that the number of peaks increases after adding phosphoric acid, and the separation is better. Acetonitrile has a stronger elution ability than methanol, and the number of peaks is more, so acetonitrile-0.1% phosphoric acid is selected as the mobile phase for elution.

[0128] Comparative Example 9

[0129] The difference between this comparative example and Example 1 is that the gradient elution procedure is different, as shown in Table 10 below, and the chromatogram is shown in Figure 6 .

[0130] Table 10 Mobile phase gradient

[0131]

[0132] Comparative Example 10

[0133] The difference between this comparative example and Example 1 is that the gradient elution procedure is different, as shown in Table 11 below, and the chromatogram is shown in Figure 7 .

[0134] Table 11 Mobile phase gradient

[0135]

[0136] Example 2 Study on Thin Layer Chromatography Identification of Coltsfoot Flos in Granules

[0137] 1.1 Preparation of test solution

[0138] Take 5g of Dongwan cough granules, grind them into powder, add 50ml of ethanol, heat in a water bath and reflux to extract for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, shake and extract with ethyl acetate for 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 0.5ml of ethyl acetate to dissolve the residue, and use it as the test solution.

[0139] 1.2 Preparation of negative sample solution lacking coltsfoot

[0140] Take the remaining medicinal materials except coltsfoot flower to prepare coltsfoot flower negative granule sample, and prepare coltsfoot flower negative sample solution according to "1.1 Preparation of test solution".

[0141] 1.3 Preparation of control medicinal material solution

[0142] Take 1 g of coltsfoot flower control medicinal material, add 20 ml of ethanol, heat in a water bath and reflux for extraction for 1 h, filter, evaporate the filtrate to dryness, add 0.5 ml of ethyl acetate to the residue to dissolve it, and use it as the control medicinal material solution.

[0143] 1.4 Selection of solvent system

[0144] The solvent systems of petroleum ether (60~90℃)-acetone (6:1) and petroleum ether (60~90℃)-ethyl acetate (5:1) were tested respectively. The results showed that petroleum ether (60~90℃)-ethyl acetate (5:1) as the system solvent system had good chromatographic separation, large spot capacity and good repeatability of relative spot positions.

[0145] 1.5 Selection of sample volume

[0146] Accurately pipette 5µl, 10µl, and 15µl of the control medicinal material solution and the test solution, respectively, and spot them on the same silica gel GF254 thin layer plate with sodium carboxymethyl cellulose as the adhesive, and use petroleum ether (60~90℃)-ethyl acetate (5:1) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm). The results showed that the spots were clear and easy to distinguish when the coltsfoot flower control medicinal material solution was 10µl and the Dongwan Zhike granule test solution was 15µl. Therefore, the spotting volume of the coltsfoot flower control medicinal material solution was 10µl and the spotting volume of the Dongwan Zhike granule test solution was 15µl.

[0147] 1.6 TLC identification

[0148] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 10µl of coltsfoot control medicinal material solution, 15μl of test solution and coltsfoot negative control solution were taken and spotted on the same silica gel GF254 thin layer plate with sodium carboxymethyl cellulose as adhesive, and petroleum ether (60~90℃)-ethyl acetate (5:1) was used as the developing agent. After development, it was taken out, dried, and inspected under ultraviolet light (365nm). In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material, and there was no interference with the negative. The results are shown in Figure 8 .

[0149] The invention establishes a thin layer chromatography identification method for coltsfoot flower in Dongwan Zhike granules. The results show that the method is simple and feasible, can well control the quality of Dongwan Zhike granules, and can be included in the quality standard item.

[0150] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A quality detection method for Dongwan cough granules, characterized in that: The quality detection method includes the determination of the standard fingerprint of flavonoids, and the determination adopts high performance liquid chromatography, wherein the preparation method of the test sample includes the following steps: (1) Dissolve Dongwan Cough Granules in water, centrifuge, and collect the supernatant; (2) adjusting the pH value of the supernatant to 4-5.5, passing the supernatant through a macroporous resin column, eluting with water and discarding it, then eluting with a 45-55% ethanol aqueous solution by volume, and collecting the eluate; In the HPLC chromatographic conditions, mobile phase A was acetonitrile and mobile phase B was 0.05-0.15% phosphoric acid aqueous solution; The macroporous resin in step (2) is a NKA-II type macroporous resin; In the HPLC method, the detection wavelength is 355-365 nm, the column temperature is 20-30°C, the chromatographic column is filled with octadecylsilane bonded silica gel, and the flow rate is 0.7-0.8 ml / min; The gradient elution program was as follows: 0 min, mobile phase A: 13–14%, mobile phase B: 86–87%; 15 min, mobile phase A: 14-16%, mobile phase B: 84-86%; 45 min, mobile phase A: 28-32%, mobile phase B: 68-72%; 60min, mobile phase A: 33-37%, mobile phase B: 63-67%.

2. The quality inspection method according to claim 1, characterized in that: The loading flow rate of the supernatant in step (2) is 1-2.5 ml / min; the flavonoid concentration in the supernatant is 1-2 mg / ml.

3. The quality inspection method according to claim 1, characterized in that: The amount of water used in step (2) is 5-6.5 times the column volume.

4. The quality inspection method according to claim 1, characterized in that: The amount of the 45-55% ethanol aqueous solution used in step (2) is 3-5 times the column volume.

5. The quality inspection method according to claim 1, characterized in that: Step (2) further comprises evaporating the eluent to dryness and dissolving the residue in methanol.

6. The quality inspection method according to claim 1, characterized in that: The quality inspection method also includes thin layer chromatography identification of coltsfoot flower, wherein the preparation of the test solution includes: adding ethanol to the Dongwan cough granules and heating them in a water bath to reflux for extraction, filtering, evaporating the filtrate to dryness, adding water, extracting with ethyl acetate 2-4 times, combining the extracts, evaporating to dryness, and dissolving them in ethyl acetate to prepare the test solution.

7. The quality inspection method according to claim 6, characterized in that: In the thin layer chromatography identification, the preparation of the reference medicinal material solution comprises adding ethanol to the coltsfoot flower medicinal material, heating in a water bath for reflux extraction, filtering, evaporating the filtrate to dryness, and adding ethyl acetate for dissolution.

8. The quality inspection method according to claim 6, characterized in that: In the thin layer chromatography identification, the developing solvent is petroleum ether-ethyl acetate in a volume ratio of 4.5-5.5:1.

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