Quality evaluation method of Reiyanning granules based on specific chromatogram

Abstract

The invention provides a method for evaluating the quality of Reiyanning granules based on a specific chromatogram. The method comprises the following steps: carrying out high performance liquid chromatography detection treatment on a test solution containing Reiyanning granules, constructing the specific chromatogram, and carrying out calibration treatment on a chromatographic peak of the specific chromatogram to obtain a characteristic peak; carrying out content determination treatment on the characteristic peak to obtain a content determination result; and obtaining a quality evaluation result of the Reiyanning granules according to the types and content determination results of the characteristic peaks, according to the method, the test sample of the Reiyanning granules is easily determined through the high performance liquid chromatography, the characteristic chromatogram of the Reiyanning granules is obtained, the quality of the Reiyanning granules is evaluated according to the types of the characteristic peaks and the content determination result of the characteristic peaks, a scientific basis is provided for quality control of the Reiyanning granules, and the method is suitable for popularization and application. According to the method, the quality of the Reiyanning granules can be monitored more accurately and comprehensively, the method is simple, the repeatability is good, and reference is provided for subsequent clinical application and research.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a quality evaluation method for Reyanning granules based on a characteristic spectrum. Background Art

[0002] Reyanning Granules are composed of dandelion, Polygonum cuspidatum, Patrinia serrata, and Scutellaria barbata. They have the effect of clearing heat and detoxifying. They have good curative effects on wind-heat cold, fever, sore throat, etc. caused by exogenous wind-heat and internal fire. In this prescription, dandelion tastes bitter and sweet, and is cold in nature. Bitterness can reduce and drain, and sweetness can detoxify. Coldness can clear heat and disperse stagnant qi, which is heat-clearing and detoxifying; Polygonum cuspidatum can clear heat and detoxify, and reduce damp-heat in the liver and gallbladder; Patrinia serrata can clear heat and detoxify, eliminate carbuncle and pus, remove blood stasis and relieve pain; Scutellaria barbata can clear heat and detoxify, and promote diuresis and reduce swelling. The latest research has found that Reyanning can effectively treat upper respiratory tract infections and acute tonsillitis, especially in the treatment of influenza A. Other studies have found that Reyanning can promote the conversion of nucleic acid to negative and recovery in asymptomatic and mild Omicron infected patients, and is a safe and effective treatment method.

[0003] Reyanning Granules are a traditional Chinese medicine preparation included in the 2020 edition of the Pharmacopoeia of the People's Republic of China. The standard uses thin layer chromatography to qualitatively identify caffeic acid, rhubarb and rhubarb, and HPLC (high performance liquid chromatography) is used to determine the content of rhubarb and rhubarb glycosides in rhubarb. There are few related quality control indicators. With the development of modern medicine and the continuous improvement of people's requirements for drug quality and efficacy, it has become very important to develop a scientific, accurate and repeatable quality evaluation method for Reyanning Granules. Summary of the invention

[0004] The present invention provides a quality evaluation method for Reyanning granules based on a characteristic spectrum, which is used to solve the problem that there is no accurate and repeatable quality evaluation method for Reyanning granules in the prior art.

[0005] The present invention provides a method for evaluating the quality of Reyanning granules based on a characteristic spectrum, comprising the following steps:

[0006] Performing high performance liquid chromatography detection on a test solution including Reyanning granules, constructing a characteristic spectrum, and calibrating the chromatographic peak of the characteristic spectrum to obtain a characteristic peak;

[0007] Performing content determination processing on the characteristic peak to obtain content determination results;

[0008] According to the types and content determination results of characteristic peaks, the quality evaluation results of the Reyanning Granules are obtained.

[0009] Compared with the prior art, the advantages of the present invention are that the test sample of the Reyanning granules can be easily measured by high performance liquid chromatography to obtain the characteristic spectrum of the Reyanning granules, and the quality of the Reyanning granules can be evaluated according to the types of characteristic peaks and the determination results of the content of the characteristic peaks, thereby providing a scientific basis for the quality control of the Reyanning granules, and being able to monitor the quality of the Reyanning granules more accurately and comprehensively, and the method is simple and has good repeatability, thereby providing a reference for subsequent clinical applications and research.

[0010] Furthermore, the quality evaluation result of the Reyanning Granules is obtained according to the type and content determination results of the characteristic peaks, including:

[0011] The types of characteristic peaks include chromatographic peaks of apigenin, polydatin, baicalin, caffeic acid, chlorogenic acid, quercetin, resveratrol, chicoric acid and luteolin.

[0012] Furthermore, the quality evaluation result of the Reyanning Granules is obtained according to the type and content determination results of the characteristic peaks, including:

[0013] The content of apigenin, polydatin, baicalin, caffeic acid, chlorogenic acid, quercetin, resveratrol, chicoric acid and luteolin was 0.15-0.17 mg·g -1 4.62~5.11mg·g -1 , 0.88~1.11mg·g -1 , 0.17~0.21mg·g -1 , 0.42~0.50mg·g -1 , 0.27~0.39mg·g -1 , 0.42~0.50mg·g -1 3.30~3.92mg·g -1 , 0.25~0.30mg·g -1 .

[0014] Furthermore, the content determination process adopts the external standard method.

[0015] Furthermore, a reference solution is also required in the high performance liquid chromatography detection process. The preparation method of the reference solution comprises the following steps:

[0016] Take polydatin, baicalin, chicoric acid, apigenin, caffeic acid, chlorogenic acid, quercetin, resveratrol, and luteolin reference substances, dissolve them in methanol, shake well, and prepare them to have mass concentrations of 400.90, 49.58, 156.86, 8.56, 12.56, 34.04, 12.00, 48.32, and 16.81 μg mL -1 of the reference solution.

[0017] Furthermore, the method for preparing the test solution comprises the following steps:

[0018] (1) According to the prescription ratio of Reyanning Granules, dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata are weighed respectively to obtain the prescription raw materials, add 3 to 5 times the weight of water of the prescription raw materials and reflux for 2 to 2.5 hours, then add 4 to 6 times the weight of water of the prescription raw materials and reflux for 1 to 1.5 hours, combine the decoctions, filter, concentrate and dry, and obtain a dry extract;

[0019] (2) Take 0.5 g (± 2%) of the ground dry extract and place it in a conical flask. Add 20-30 mL of 50-100% methanol solution and perform ultrasonic treatment at a power of 100 W and a frequency of 40 kHz for 20-60 min. After cooling, make up the loss with 50-100% methanol solution, pass through a membrane, and take the filtrate to obtain the test solution.

[0020] Further, the mass concentration of the methanol solution is 100%; and / or,

[0021] The ultrasonic treatment time is 40 to 45 minutes.

[0022] Furthermore, in the HPLC detection process, the chromatographic column was ShimNex CS C 18 (4.6×250nm, 5μm) chromatographic column.

[0023] Furthermore, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution; the gradient elution program is:

[0024] 0-40min: Fluidity A increases from 10% to 30% at a constant rate, and fluidity B decreases from 87% to 70% at a constant rate;

[0025] 40-50min: Fluidity A increases from 30% to 35% at a constant rate, and fluidity B decreases from 70% to 65% at a constant rate;

[0026] 50-60min: Fluidity A increases uniformly from 35% to 40%, and fluidity B decreases uniformly from 65% to 60%.

[0027] Furthermore, in the high performance liquid chromatography detection process, the column temperature is 30°C; and / or,

[0028] The detection wavelength is 330 nm; and / or,

[0029] The test sample flow rate is 1.0 ml / min; and / or,

[0030] The injection volume was 10 μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the high performance liquid chromatography superposition characteristic spectrum of 15 batches of Reyanning granule samples in a test example of the present invention;

[0032] Figure 2 It is a characteristic spectrum of a dandelion-deficient negative sample solution in a test example of the present invention;

[0033] Figure 3 It is a characteristic spectrum of a negative sample solution lacking Polygonum cuspidatum in a test example of the present invention;

[0034] Figure 4 It is a characteristic spectrum of a negative sample solution lacking Scutellaria barbata in a test example of the present invention;

[0035] Figure 5 This is a characteristic spectrum of a negative sample solution lacking Patrinia paniculata in a test example of the present invention;

[0036] Figure 6 It is a comparison chart of the characteristic peaks of the HPLC of the Reyanning granule sample in a test example of the present invention;

[0037] Figure 7 This is a cluster analysis diagram of 15 batches of Reyanning granule samples in a test example of the present invention;

[0038] Figure 8 This is a scree map of 15 batches of Reyanning granule samples in a test example of the present invention;

[0039] Fig. 9 This is a principal component analysis diagram of 15 batches of Reyanning Granule samples in a test example of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are 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.

[0041] The present invention provides a quality evaluation method of Reyanning granules based on a characteristic spectrum, comprising the following steps:

[0042] Performing high performance liquid chromatography detection on a test solution including Reyanning granules, constructing a characteristic spectrum, and calibrating the chromatographic peak of the characteristic spectrum to obtain a characteristic peak;

[0043] Performing content determination processing on the characteristic peak to obtain content determination results;

[0044] According to the types and content determination results of characteristic peaks, the quality evaluation results of the Reyanning Granules are obtained.

[0045] The quality evaluation method of Reyanning granules based on characteristic spectrum provided by the present invention can easily measure the test sample of Reyanning granules by high performance liquid chromatography to obtain the characteristic spectrum of Reyanning granules, and can evaluate the quality of Reyanning granules according to the types of characteristic peaks and the measurement results of the contents of characteristic peaks, so as to provide a scientific basis for the quality control of Reyanning granules, and can monitor the quality of Reyanning granules more accurately and comprehensively, and the method is simple and has good repeatability, so as to provide a reference for subsequent clinical application and research.

[0046] The present invention does not limit the preparation method of the test solution, and optionally, comprises the following steps:

[0047] (1) According to the prescription ratio of Reyanning Granules, dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata are weighed respectively to obtain the prescription raw materials, add 3 to 5 times the weight of water of the prescription raw materials and reflux for 2 to 2.5 hours, then add 4 to 6 times the weight of water of the prescription raw materials and reflux for 1 to 1.5 hours, combine the decoctions, filter, concentrate and dry, and obtain a dry extract;

[0048] (2) Take 0.5 g (± 2%) of the ground dry extract and place it in a conical flask. Add 20-30 mL of 50-100% methanol solution and perform ultrasonic treatment at a power of 100 W and a frequency of 40 kHz for 20-60 min. After cooling, make up the loss with 50-100% methanol solution, pass through a membrane, and take the filtrate to obtain the test solution.

[0049] Furthermore, the inventors found that when anhydrous methanol was used as the extractant, the peak shapes of the components on the chromatogram were ideal, and the number of characteristic peaks was also large, indicating that the extraction effect was good; the chromatographic information when the extraction time was 20 minutes was inferior to that of 40 minutes, and the chromatographic information at 60 minutes was not much different from that at 40 minutes. Therefore, the mass concentration of the methanol solution was selected to be 100%; the ultrasonic treatment time was 40 to 45 minutes.

[0050] It can be understood that a reference solution is also required in the high performance liquid chromatography detection process. The present invention does not limit the preparation method of the reference solution. Optionally, the method includes the following steps:

[0051] Take polydatin, baicalin, chicoric acid, apigenin, caffeic acid, chlorogenic acid, quercetin, resveratrol, and luteolin reference substances, dissolve them in methanol, shake well, and prepare them to have mass concentrations of 400.90, 49.58, 156.86, 8.56, 12.56, 34.04, 12.00, 48.32, and 16.81 μg mL -1 of the reference solution.

[0052] Reyanning Granules is a traditional Chinese medicine compound preparation composed of 4 medicinal materials, with complex ingredients and greatly different chemical properties. In this experiment, the test solution was scanned at 190-400nm. The results showed that at a wavelength of 330nm, the chromatographic peak information of Reyanning Granules was the richest, the baseline was stable, and the 9 known components had a good response. The present invention also tested WondaSiL C 18 -WR (4.6×250nm, 5μm), Sepax GP-C 18 (4.6 × 250 nm, 5 μm) and ShimNex CS C 18 The influence of three chromatographic columns (4.6×250nm, 5μm) on the chromatograms was found that ShimNex CS C 18 (4.6×250nm, 5μm) chromatographic column, the separation effect between the chromatographic peak of the component to be measured and the adjacent peaks is good, the peak shape is sharp, and has good symmetry.

[0053] Therefore, in one embodiment, the HPLC column is ShimNex CS C 18 (4.6×250nm, 5μm) chromatographic column; column temperature is 30℃; detection wavelength is 330nm; sample flow rate is 1.0ml / min; injection volume is 10μL.

[0054] In one embodiment, the mobile phase A of the high performance liquid chromatography is acetonitrile, and the mobile phase B is a 0.1% phosphoric acid aqueous solution; the gradient elution program is: 0-40 min: the fluidity A increases uniformly from 10% to 30%, and the fluidity B decreases uniformly from 87% to 70%; 40-50 min: the fluidity A increases uniformly from 30% to 35%, and the fluidity B decreases uniformly from 70% to 65%; 50-60 min: the fluidity A increases uniformly from 35% to 40%, and the fluidity B decreases uniformly from 65% to 60%.

[0055] Furthermore, the quality evaluation result of the Reyanning Granules is obtained according to the type and content determination results of the characteristic peaks, including:

[0056] The types of characteristic peaks need to include the chromatographic peaks of the nine key components: apigenin, polydatin, baicalin, caffeic acid, chlorogenic acid, quercetin, resveratrol, chicoric acid and luteolin;

[0057] The content of apigenin, polydatin, baicalin, caffeic acid, chlorogenic acid, quercetin, resveratrol, chicoric acid and luteolin was 0.15-0.17 mg·g -1 4.62~5.11mg·g -1 , 0.88~1.11mg·g-1 , 0.17~0.21mg·g -1 , 0.42~0.50mg·g -1 , 0.27~0.39mg·g -1 , 0.42~0.50mg·g -1 3.30~3.92mg·g -1 , 0.25~0.30mg·g -1 , and determined that the quality of Reyanning Granules met the standards.

[0058] When the Reyanning Granules were subjected to HPLC testing under the above-mentioned chromatographic conditions, the characteristic peaks of the above-mentioned 9 key components were obtained and the content was determined to be within the above-mentioned range, and it was determined that the quality of the Reyanning Granules met the standards.

[0059] In one embodiment, the content determination process adopts the external standard method.

[0060] In one embodiment, the relative retention time and relative peak area of ​​each common characteristic peak are calculated with the chicoric acid chromatographic peak as a reference. As a result, the RSD values ​​of the relative retention time of the test solution injected continuously for multiple times are all <3%, and the RSD values ​​of the relative peak area are all <2%, indicating that the precision of the quality evaluation method provided by the present invention is good.

[0061] In one embodiment, the relative retention time and relative peak area of ​​each common characteristic peak are calculated with reference to the chicoric acid chromatographic peak. The results show that the RSD values ​​of the relative retention time of the test samples placed at room temperature for 0, 2, 4, 8, 12, and 24 hours are all less than 2%, and the RSD values ​​of the relative peak area are all less than 1%, indicating that the quality evaluation method provided by the present invention has good stability.

[0062] In one embodiment, the relative retention time and relative peak area of ​​each common peak are calculated with the chicoric acid chromatographic peak as a reference. As a result, the RSD values ​​of the relative retention time of the test solution in multiple parallel tests are all <2%, and the RSD values ​​of the relative peak area are all <3%, indicating that the quality evaluation method provided by the present invention has good repeatability.

[0063] Hereinafter, a quality evaluation method of Reyanning Granules based on characteristic spectrum provided by the present invention is introduced in detail through specific test examples.

[0064] Test Example 1

[0065] (1) Preparation of mixed reference solution: Take polydatin, baicalin, chicoric acid, apigenin, caffeic acid, chlorogenic acid, quercetin, resveratrol, and luteolin reference substances, dissolve them in methanol, shake well, and prepare them to have mass concentrations of 400.90, 49.58, 156.86, 8.56, 12.56, 34.04, 12.00, 48.32, and 16.81 μg mL, respectively.-1 of reference solution;

[0066] (2) Preparation of test solution: According to the prescription ratio of Reyanning Granules, dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata were weighed out from 15 batches of medicinal material samples to obtain the prescription raw materials, and water 8 times the mass of the prescription raw materials was added to reflux for 2 h, and then filtered, concentrated and dried to obtain a dry extract;

[0067] Take 0.5g of the ground dry extract and place it in a conical flask, add 25mL of 50-100% methanol solution, and perform ultrasonic treatment at a power of 100W and a frequency of 40kHz for 20-60min. After cooling, make up the loss with 50-100% methanol solution, pass through a membrane, and take the filtrate to obtain 15 batches of test sample solutions, recorded as S1-S15;

[0068] (3) Constructing characteristic spectrum: The test solutions S1 to S15 were subjected to high performance liquid chromatography and the chromatograms were recorded; Chromatographic conditions: The chromatographic column was ShimNex CS C 18 (4.6×250nm, 5μm) chromatographic column; column temperature 30℃; detection wavelength 330nm; sample flow rate 1.0ml / min; injection volume 10μL; mobile phase A acetonitrile, mobile phase B 0.1% phosphoric acid aqueous solution; gradient elution program: 0-40min: fluidity A increases from 10% to 30% at a constant rate, fluidity B decreases from 87% to 70% at a constant rate; 40-50min: fluidity A increases from 30% to 35% at a constant rate, fluidity B decreases from 70% to 65% at a constant rate; 50-60min: fluidity A increases from 35% to 40% at a constant rate, fluidity B decreases from 65% to 60% at a constant rate;

[0069] (4) The above chromatograms were imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), and the chromatogram of the sample (No. S2) was used as the reference spectrum to generate the superimposed spectrum and the reference spectrum (R) of the 15 batches of samples. The results showed that 18 common characteristic peaks were calibrated for the 15 batches of Reyanning Granules, and 9 common peaks were identified. The results are shown in Figure 1 Taking the chicoric acid peak as the reference peak, the similarity between the S1 to S15 samples and the control characteristic spectra was calculated. The similarity of each batch of samples was 0.989 to 0.999, all above 0.980, indicating that different batches of Reyanning Granules had good quality consistency.

[0070] Test Example 2

[0071] (1) Preparation of mixed reference solution: same as step (1) in Experimental Example 1;

[0072] (2) Preparation of test solution: same as step (2) in Experimental Example 1;

[0073] (3) preparing negative sample solutions of each missing single herb: weigh three kinds of dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata respectively according to the prescription ratio of Reyanning Granules to obtain the prescription raw materials, add water 8 times the weight of the prescription raw materials and reflux for 2 h, then filter, concentrate and dry to obtain a dry extract;

[0074] 0.5 g of the ground dry extract is placed in a conical flask, 25 mL of 50-100% methanol solution is added, and ultrasonic treatment is performed at a power of 100 W and a frequency of 40 kHz for 20-60 min. After cooling, the amount lost is supplemented with 50-100% methanol solution, and the solution is filtered through a membrane, and the subsequent filtrate is taken to obtain negative sample solutions of each single drug deficiency, including negative sample solutions of dandelion deficiency, negative sample solutions of polygonum cuspidatum deficiency, negative sample solutions of Scutellaria barbata deficiency, and negative sample solutions of Patrinia scabra deficiency;

[0075] (4) Using high performance liquid chromatography, 10 μL of the test solution, mixed reference solution, and each single-drug-deficient negative sample solution were injected for testing; the chromatographic conditions were the same as those in Experimental Example 1; the characteristic spectra of each single-drug-deficient negative sample solution were as follows: Figure 2 to Figure 5 As shown;

[0076] (5) HPLC characteristic spectra of the test solution, mixed reference solution and negative samples lacking a single herb were constructed, and the HPLC characteristic peak comparison spectra of the Reyanning Granules sample were obtained by comparative analysis, such as Figure 6 As shown in the figure, the characteristic peaks of Reyanning Granules were assigned, and after identification by reference substances, a total of 9 chromatographic peaks were identified, namely, caffeic acid No. 5, chlorogenic acid No. 7, polydatin No. 8, baicalin No. 9, chicoric acid No. 11, resveratrol No. 15, quercetin No. 16, luteolin No. 17, and apigenin No. 18. Peaks 4 and 12 were assigned to Taraxacum mongolicum, peaks 8 (polydatin), 13, and 15 (resveratrol) were assigned to Polygonum cuspidatum, peaks 9 (baicalin), 10, 14, and 18 (apigenin) were assigned to Scutellaria barbata, and peak 6 was assigned to Patrinia scabra.

[0077] Test Example 3

[0078] The 18 common peak areas of 15 batches of Reyanning Granule samples obtained in Experimental Example 1 were imported into IBM SPSS Statistics 27 software, and cluster analysis (CA) and principal component analysis (PCA) were performed using the intergroup linkage method. The results are shown in Figure 7 and Figure 8 As shown; the 18 common peak areas of 15 batches of Reyanning Granule samples obtained in Experimental Example 1 were imported into SIMCA software for principal component analysis. The result is shown in FIG. Fig. 9 shown.

[0079] Depend on Figure 7It can be seen that when the square Euclidean distance is 5, 15 batches of samples can be divided into 2 categories: S1-5, S7, S9, and S15 are one category, and S6, S8, and S10-14 are another category, indicating that there are differences between the samples, which may be related to the supply of medicinal materials, the origin of medicinal materials, or the source of samples.

[0080] Principal component analysis was performed to improve data analysis efficiency by reducing data dimension. The results are shown in Figure 8 , based on the condition of eigenvalue>1, 6 principal components were selected, and the cumulative variance contribution rate was 90.391%. In the scree map, the slopes of these 6 principal components were large, and the slopes of the subsequent components were gentle, indicating that these 6 principal components can reflect the main characteristics of the original data and achieve the purpose of dimensionality reduction.

[0081] like Fig. 9 As shown in the figure, the characteristic distribution among samples can be intuitively reflected, and they can be divided into two categories, and the classification results are consistent with the cluster analysis results: S1-5, S7, S9, and S15 are one category, and S6, S8, and S10-14 are another category.

[0082] Test Example 4

[0083] The test solution S10 in Test Example 1 was taken and placed at room temperature for 0, 2, 4, 8, 12, and 24 hours, respectively, and then detected according to the chromatographic conditions in Test Example 1, and the peak areas were recorded.

[0084] The results showed that the RSD values ​​of the peak areas of caffeic acid, chlorogenic acid, polydatin, baicalin, chicoric acid, resveratrol, quercetin, luteolin and apigenin were 1.77%, 1.25%, 1.66%, 2.13%, 1.51%, 1.07%, 1.32%, 2.05% and 1.67%, respectively, indicating that the test solution had good stability within 24 hours.

[0085] Test Example 5

[0086] Take the test solution S10 in Experimental Example 1, prepare 6 test solutions in parallel, and then detect according to the chromatographic conditions in Experimental Example 1.

[0087] The results showed that the RSD values ​​of caffeic acid, chlorogenic acid, polydatin, baicalin, chicoric acid, resveratrol, quercetin, luteolin and apigenin were 1.37%, 1.43%, 1.52%, 1.13%, 1.61%, 1.18%, 1.24%, 1.09% and 1.57%, respectively, indicating that the method had good repeatability.

[0088] Test Example 6

[0089] Weigh 6 portions of Reyanning granules with known contents, each portion is about 0.25 g, and add corresponding reference substances to each portion according to 100% of the contents of 9 components of caffeic acid, chlorogenic acid, polydatin, scutellarin, chicoric acid, resveratrol, quercetin, luteolin, and apigenin in the sample. Prepare the test solution according to step (2) in Test Example 1, detect and calculate the recovery rate according to the chromatographic conditions in Test Example 1, and the results are shown in Table 1, indicating that the recovery rate of this method is good.

[0090] Table 1 Results of sample recovery test

[0091] Element Average recovery rate (%) RSD(%) Caffeic acid 99.89 0.97 Chlorogenic acid 100.30 0.63 Polydatin 101.25 1.86 Scutellarin 100.78 1.20 Chicoric acid 100.01 1.30 Resveratrol 100.71 1.51 Quercetin 100.19 1.74 Luteolin 100.58 1.48 Apigenin 100.14 1.72

[0092] Test Example 7

[0093] (1) Preparation of mixed reference solution: same as step (1) in Experimental Example 1;

[0094] (2) Preparation of test solution: same as step (2) in Experimental Example 1;

[0095] (3) The test solutions S1 to S15 are subjected to high performance liquid chromatography detection, a characteristic spectrum is constructed, and the chromatographic peaks of the characteristic spectrum are calibrated to obtain characteristic peaks; the chromatographic conditions are the same as those in Experimental Example 1;

[0096] (4) Performing content determination processing on the characteristic peaks to obtain content determination results; as shown in Table 2.

[0097] Table 2 Results of determination of 9 components

[0098]

[0099] According to Table 2, the inventors analyzed that the most active ingredients in dandelion are phenolic compounds, including flavonoids and phenolic acid compounds. Modern pharmacological studies have shown that phenolic compounds have antioxidant, hypoglycemic, anti-tumor, anti-inflammatory and other effects. Polygonum cuspidatum has anti-inflammatory and antiviral activities, among which flavonoids, phenylpropanoids and diphenylethylene components play a role. Scutellaria barbata has multiple pharmacological activities, including anti-tumor, anti-viral, antioxidant, etc., and the main active substances are flavonoids. Patrinia odoratum has the effects of clearing heat and dampness, reducing swelling and pus discharge, etc. Its chemical composition is complex, mainly flavonoids and organic acids, including quercetin, chlorogenic acid, etc. Therefore, according to the pharmacodynamic characteristics of the chemical components and their roles in the pharmacodynamics of traditional Chinese medicine, the present invention selects apigenin, polydatin, baicalin, caffeic acid, chlorogenic acid, quercetin, resveratrol, chicoric acid and luteolin as content determination indicators of Reyanning granules, and the test sample of Reyanning granules is easily determined by high performance liquid chromatography to obtain characteristic peaks and content determination results of the above components, and the quality of Reyanning granules is evaluated, thereby providing a scientific basis for the quality control of Reyanning granules, being able to more accurately and comprehensively monitor the quality of Reyanning granules, and the method is simple and has good repeatability, thus providing a reference for subsequent clinical application and research.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein by equivalents. If these modifications and variations fall within the scope of the claims of the present invention and their equivalent technologies, they should be considered to be within the protection scope of the present invention.

Claims

1. A quality evaluation method for Reyanning granules based on characteristic spectrum, characterized in that: The following steps are involved: Performing high performance liquid chromatography detection on a test solution including Reyanning granules, constructing a characteristic spectrum, and calibrating the chromatographic peak of the characteristic spectrum to obtain a characteristic peak; Performing content determination processing on the characteristic peak to obtain content determination results; According to the types and content determination results of characteristic peaks, the quality evaluation results of the Reyanning granules are obtained.

2. The quality evaluation method of Reyanning Granules based on characteristic spectrum according to claim 1, characterized in that: The method of obtaining the quality evaluation result of the Reyanning Granules according to the type and content determination results of the characteristic peaks includes: The types of characteristic peaks include chromatographic peaks of apigenin, polydatin, baicalin, caffeic acid, chlorogenic acid, quercetin, resveratrol, chicoric acid and luteolin.

3. The quality evaluation method of Reyanning granules based on characteristic spectrum according to claim 2 is characterized in that: The method of obtaining the quality evaluation result of the Reyanning Granules according to the type and content determination results of the characteristic peaks includes: The content of apigenin, polydatin, baicalin, caffeic acid, chlorogenic acid, quercetin, resveratrol, chicoric acid and luteolin was 0.15-0.17 mg·g -1 , 4.62~5.11 mg·g -1 , 0.88~1.11 mg·g -1 , 0.17~0.21 mg·g -1 , 0.42~0.50 mg·g -1 , 0.27~0.39 mg·g -1 , 0.42~0.50 mg·g -1 , 3.30~3.92 mg·g -1 , 0.25~0.30 mg·g -1 .

4. The quality evaluation method of Reyanning granules based on characteristic spectrum according to claim 3 is characterized in that: The content was determined by external standard method.

5. The quality evaluation method of Reyanning granules based on characteristic spectrum according to claim 4 is characterized in that: A reference solution is also required in the high performance liquid chromatography detection process. The preparation method of the reference solution comprises the following steps: Take polydatin, baicalin, chicoric acid, apigenin, caffeic acid, chlorogenic acid, quercetin, resveratrol, and luteolin reference substances, dissolve them in methanol, shake well, and prepare them to have mass concentrations of 400.90, 49.58, 156.86, 8.56, 12.56, 34.04, 12.00, 48.32, and 16.81 μg mL -1 of the reference solution.

6. The quality evaluation method of Reyanning Granules based on characteristic spectrum according to any one of claims 1 to 5, characterized in that: The method for preparing the test solution comprises the following steps: (1) According to the prescription ratio of Reyanning Granules, dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata were weighed respectively to obtain the prescription raw materials, and 3 to 5 times the weight of water of the prescription raw materials were added to reflux for 2 to 2.5 hours, and then 4 to 6 times the weight of water of the prescription raw materials were added to reflux for 1 to 1.5 hours, the decoctions were combined, and then filtered, concentrated and dried to obtain a dry extract; (2) Take 0.5 g (± 2%) of the ground dry extract and place it in a conical flask. Add 20-30 mL of 50-100% methanol solution and perform ultrasonic treatment at a power of 100 W and a frequency of 40 kHz for 20-60 min. After cooling, make up the loss with 50-100% methanol solution, pass through a membrane, and take the filtrate to obtain the test solution.

7. The quality evaluation method of Reyanning granules based on characteristic spectrum according to claim 6 is characterized in that: The mass concentration of the methanol solution is 100%; and / or, The ultrasonic treatment time is 40 to 45 minutes.

8. The quality evaluation method of Reyanning granules based on characteristic spectrum according to claim 6, characterized in that: High performance liquid chromatography (HPLC) was used for the detection and treatment. The chromatographic column was ShimNex CS C 18 (4.6×250nm, 5μm) chromatographic column.

9. The quality evaluation method of Reyanning granules based on characteristic spectrum according to claim 6, characterized in that: Mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution; the gradient elution program was: 0-40min: Liquidity A increases from 10% to 30% at a constant rate, and liquidity B decreases from 87% to 70% at a constant rate; 40-50min: Liquidity A increases from 30% to 35% at a constant rate, and liquidity B decreases from 70% to 65% at a constant rate; 50~60min: Liquidity A increases uniformly from 35% to 40%, and liquidity B decreases uniformly from 65% to 60%.

10. The quality evaluation method of Reyanning granules based on characteristic spectrum according to claim 6, characterized in that: During the high performance liquid chromatography detection process, the column temperature is 30°C; and / or, The detection wavelength is 330 nm; and / or, The test sample flow rate is 1.0 ml / min; and / or, The injection volume was 10 μL.

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