Construction method and application of ginseng yiyuan granules HPLC fingerprint

Abstract

The present application relates to a kind of ginseng Yi Yuan granules fingerprint detection method.By this method, the quality of ginseng Yi Yuan granules can be effectively characterized, which is beneficial to the overall monitoring of product quality stability, consistency, controllability.The ginseng Yi Yuan granules fingerprint constructed by the present application has 21 common peaks, and contains effective components belonging to six medicinal materials such as white peony root, white atractylodes, licorice, astragalus, caulis Bambusae and ginseng, effectively characterizing the quality of ginseng Yi Yuan granules, which is beneficial to the overall monitoring of product quality.The present application has the characteristics of convenience, speed, stability, high precision and good reproducibility, and can more comprehensively, objectively and scientifically evaluate the quality of ginseng Yi Yuan granules, thereby ensuring the safety and effectiveness of clinical medication.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a high performance liquid chromatography fingerprint of Renshen Yiyuan granules for controlling the quality of Renshen Yiyuan granules. BACKGROUND

[0002] In recent years, the overall incidence of malignant tumors continues to rise, and has become a major disease threatening human health. It is reported that more than 90% of chemotherapeutic drugs can cause myelosuppression, mainly manifested as different degrees of decrease in peripheral white blood cells, platelets, red blood cells and hemoglobin, which can cause infection, bleeding and anemia, leading to interruption of chemotherapy, reducing the quality of life of patients, and even endangering the lives of patients. At present, the main drugs for treating myelosuppression in Western medicine are colony stimulating factor, recombinant human thrombopoietin and glucocorticoids, etc., but have many adverse reactions. Studies have shown that although colony stimulating factor can rapidly increase neutrophils, it needs to be repeatedly used because it cannot promote the proliferation of hematopoietic stem cells, and has the risk of aggravating the immunosuppressive microenvironment of tumors. Myelosuppression after chemotherapy of malignant tumors belongs to the category of "blood deficiency", "asthenia" and "blood syndrome" in traditional Chinese medicine. The treatment principle of traditional Chinese medicine single herb and classical prescription for myelosuppression is tonifying qi and nourishing blood. In recent years, clinical research and reports have shown that the combination of traditional Chinese medicine and radiotherapy and chemotherapy drugs can reduce toxicity and increase efficacy, and can enable patients to complete radiotherapy and chemotherapy on time and in adequate amount, and improve prognosis.

[0003] Renshen Yiyuan granules have the effects of tonifying qi and nourishing blood, and supporting healthy energy and root. It is used for treating myelosuppression and low immune function caused by postoperative or post-chemotherapy of malignant tumors. It is composed of ginseng, astragalus, white peony root, atractylodes, caulis Broussonetiae and licorice. The ginseng and astragalus are used as monarch drugs, which tonify qi and nourish yin, tonify qi and replenish qi, raise yang and consolidate the exterior; the atractylodes tonifies qi and assists ginseng and astragalus to tonify qi and replenish the spleen; the white peony root nourishes blood and soothes the liver and ying, and is a ministerial drug; the caulis Broussonetiae activates blood and tonifies blood, and is used with other drugs to tonify without stagnation, and is a ministerial drug; and the licorice is used to tonify qi and harmonize the middle, and harmonize various drugs. The combination of various drugs achieves the effects of tonifying qi and nourishing blood, and supporting healthy energy and root.

[0004] Before the completion of the present application, there is no report on the method for establishing a fingerprint by taking the chemical components contained in Renshen Yiyuan granules as an index. SUMMARY

[0005] One of the purposes of the present application is to provide a fingerprint detection method of Renshen Yiyuan granules and a fingerprint thereof. The fingerprint constructed by the method can comprehensively reflect the overall chemical components of Renshen Yiyuan granules, and provides an effective means for the overall quality control and evaluation of Renshen Yiyuan granules, aiming to solve the defect that there is no literature report on the fingerprint of Renshen Yiyuan granules in the prior art, and effectively control the quality of Renshen Yiyuan granules.

[0006] The second object of the present application is to comprehensively reflect the current situation of each component in the Renshen Yiyuan Granules by component identification and single herb attribution of common peaks of different batches of drugs, thereby providing a reference for the quality of the Renshen Yiyuan Granules.

[0007] The present application adopts the following technical scheme: a fingerprint detection method of Renshen Yiyuan Granules, which adopts high performance liquid chromatography to determine the fingerprint.

[0008] 80% methanol is used to extract the Renshen Yiyuan Granules, and the extract is used as the test solution; the protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid and atractylodes lactone III control samples are dissolved in methanol to serve as the control solution; the control solution and the test solution are subjected to high performance liquid chromatography analysis, and the chromatographic peaks of the control solution are used as the reference to obtain the fingerprint of the Renshen Yiyuan Granules.

[0009] Optimally, the weight of the Renshen Yiyuan Granules is 0.2-2 g, and the amount of 80% methanol is 20-200 mL.

[0010] Optimally, the extraction is performed under ultrasonic conditions, the power of the ultrasonic is 200-300 W, and the frequency is 30-50 kHz; the extraction time is 20-40 min.

[0011] Preferably, when the high performance liquid chromatography analysis is performed, the mobile phase A is acetonitrile, and the mobile phase B is 0.02-0.2% phosphoric acid aqueous solution; the flow rate of the mobile phase system is 0.7-1.2 mL / min; the gradient elution mode is adopted, and the proportion of the mobile phases A and B changes as follows during the gradient elution process: 0-10 min, 3% A; 10-15 min, 3-10% A; 15-35 min, 10-15% A; 35-65 min, 15-30% A; 65-90 min, 30-40% A; 90-105 min, 40-80% A.

[0012] Preferably, when the high performance liquid chromatography analysis is performed, octadecylsilane-bonded silica gel is used as the filler, the column specification is 250*4.60 mm, the column temperature is 20-40℃, the detection wavelength is 210-360 nm, and the injection volume is 5-15 μL.

[0013] Preferably, when the high performance liquid chromatography analysis is performed, the theoretical plate number calculated according to the paeoniflorin peak is not less than 5000.

[0014] Preferably, the ginseng Yi Yuan granule fingerprint contains 21 common peaks, wherein peaks 4, 5, 8, 10, 12, 18, 19 and 21 are respectively indicated as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid and atractylodes lactone III.

[0015] Preferably, the 21 common peaks respectively belong to six medicinal materials of radix paeoniae alba, atractylodes, glycyrrhiza, astragalus, caudate vitex and ginseng; wherein peak 1 belongs to ginseng and astragalus, peak 2 belongs to radix paeoniae alba and atractylodes, peak 3 belongs to radix paeoniae alba and caudate vitex, peak 4 belongs to caudate vitex, peak 5 belongs to caudate vitex, peak 6 belongs to glycyrrhiza, peak 7 belongs to radix paeoniae alba, peak 8 belongs to radix paeoniae alba, peak 9 belongs to glycyrrhiza and astragalus, peak 10 belongs to glycyrrhiza, peak 11 belongs to radix paeoniae alba, peak 12 belongs to astragalus, peak 13 belongs to ginseng, peak 14 belongs to radix paeoniae alba, peak 15 belongs to glycyrrhiza, astragalus and caudate vitex, peak 16 belongs to glycyrrhiza, peak 17 belongs to radix paeoniae alba, peak 18 belongs to caudate vitex, peak 19 belongs to glycyrrhiza, peak 20 belongs to glycyrrhiza, and peak 21 belongs to atractylodes.

[0016] The present application provides a ginseng Yi Yuan granule fingerprint obtained by the construction method of the ginseng Yi Yuan granule HPLC fingerprint according to the technical scheme.

[0017] The present application provides an application of the ginseng Yi Yuan granule fingerprint in quality control of ginseng Yi Yuan granule.

[0018] The beneficial effects of the present application are embodied in:

[0019] 1. The present application establishes HPLC fingerprints of 10 batches of ginseng Yi Yuan granules, the similarity is greater than 0.90, 21 common peaks are marked, 7 Chinese medicines are covered, and the chemical information contained in ginseng Yi Yuan granules can be more comprehensively reflected, and the quality thereof can be better characterized.

[0020] 2. Since the fingerprint is not for determining the accurate content of a certain component, but for fully reflecting the information of chemical components, the present application selects to determine at a wavelength of 230 nm, the peaks are more, the reflected information is more complete, the absorption value of each peak is good, and the baseline is smooth.

[0021] 3. The high performance liquid chromatography fingerprint method established by the present application realizes quality control of ginseng Yi Yuan granules for the first time, is not for identifying a single compound or medicinal material, overcomes the problems of single and one-sidedness of the existing quality control methods, can more effectively guide feeding, strictly regulate production operation, and ensure the safety and effectiveness of clinical medication. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 HPLC chromatogram of mixed reference solution

[0023] Figure 2 Common pattern of ginseng Yi Yuan granules

[0024] Figure 3 Superimposed figure of 10 batches of ginseng Yi Yuan granules fingerprint

[0025] Figure 4 Sample and each medicinal material chromatogram comparison attribution figure

[0026] Figure 5 Ginseng Yi Yuan granules spectrum under 360nm detection wavelength condition

[0027] Figure 6 Ginseng Yi Yuan granules spectrum under 320nm detection wavelength condition

[0028] Figure 7 Ginseng Yi Yuan granules spectrum under 280nm detection wavelength condition

[0029] Figure 8 Ginseng Yi Yuan granules spectrum under 254nm detection wavelength condition

[0030] Figure 9 Ginseng Yi Yuan granules spectrum under 230nm detection wavelength condition

[0031] Figure 10 Ginseng Yi Yuan granules spectrum under 210nm detection wavelength condition

[0032] Figure 11203nm detection wavelength condition ginseng Yi Yuan granules spectrum DETAILED DESCRIPTION

[0033] The ginseng Yi Yuan granules in the application are prepared from 160g of ginseng, 480g of astragalus, 240g of atractylodes, 240g of white peony root, 240g of caulis spatholobi and 120g of honey-fried licorice. The specific preparation method is as follows: the ginseng and astragalus are decocted twice with 10 times the amount of water, 3 hours for the first time and 2 hours for the second time, the decoction is combined, left to stand, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.30-1.35 (60°C), which is ready for use; the remaining four kinds of atractylodes and the like are decocted twice with 8 times the amount of water, 2 hours each time, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.10-1.15 (60°C), 2 times the amount of ethanol is added, stirred, left to stand for 12-24 hours, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.30-1.35 (60°C), which is combined with the above-mentioned thick paste, dried, pulverized, sieved, an appropriate amount of dextrin-sucrose (1:4) is added, mixed uniformly, granulated with 90% ethanol, dried, and made into 1000g, which is obtained.

[0034] The application provides a method for constructing HPLC fingerprint of ginseng Yi Yuan granules, comprising the following steps:

[0035] a. The ginseng Yi Yuan granules are extracted with 80% methanol, and the obtained ginseng Yi Yuan granule extract is used as the test sample solution;

[0036] b. The methanol solutions of protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid, and atractylodes lactone III are used as the control sample solutions;

[0037] c. The control sample solutions and the test sample solution are subjected to high performance liquid chromatography analysis, and the chromatographic peaks of the control sample solutions are used as the reference to obtain the ginseng Yi Yuan granule fingerprint.

[0038] In the present application, the ginseng Yi Yuan granules are extracted with 80% methanol, and the obtained ginseng Yi Yuan granule extract is used as the test sample solution. Preferably, the ginseng Yi Yuan granules are finely ground before extraction. The weight of the ginseng Yi Yuan granules is 0.2-2 g, and the amount of 80% methanol is 20-200 mL. More preferably, the weight of the ginseng Yi Yuan granules is 0.5 g, and the amount of 80% methanol is 50 mL. The power of the ultrasonic treatment is 200-300 W, and the frequency is 30-50 kHz. More preferably, the power is 250 W, and the frequency is 40 kHz. The extraction time is 20-40 min, and more preferably, 30 min.

[0039] In the present application, the ginseng Yi Yuan granules are extracted with 80% methanol, and the obtained ginseng Yi Yuan granule extract is used as the test sample solution. Preferably, the ginseng Yi Yuan granules are finely ground before extraction. The weight of the ginseng Yi Yuan granules is 0.2-2 g, and the amount of 80% methanol is 20-200 mL. More preferably, the weight of the ginseng Yi Yuan granules is 0.5 g, and the amount of 80% methanol is 50 mL. The power of the ultrasonic treatment is 200-300 W, and the frequency is 30-50 kHz. More preferably, the power is 250 W, and the frequency is 40 kHz. The extraction time is 20-40 min, and more preferably, 30 min.

[0040] In the present application, the ginseng Yi Yuan granules are extracted with 80% methanol, and the obtained ginseng Yi Yuan granule extract is used as the test sample solution. Preferably, the ginseng Yi Yuan granules are finely ground before extraction. The weight of the ginseng Yi Yuan granules is 0.2-2 g, and the amount of 80% methanol is 20-200 mL. More preferably, the weight of the ginseng Yi Yuan granules is 0.5 g, and the amount of 80% methanol is 50 mL. The power of the ultrasonic treatment is 200-300 W, and the frequency is 30-50 kHz. More preferably, the power is 250 W, and the frequency is 40 kHz. The extraction time is 20-40 min, and more preferably, 30 min.

[0041] After preparing the control sample solution and the sample solution, the present application carries out high performance liquid chromatography analysis on the control sample solution and the sample solution respectively, and obtains the fingerprint of ginseng Yiyuan granules based on the chromatographic peak of the control sample solution. In the present application, when carrying out the high performance liquid chromatography analysis, the mobile phase A is acetonitrile, the mobile phase B is 0.02-0.2% phosphoric acid aqueous solution, and the phosphoric acid aqueous solution is further preferably 0.1%; the flow rate of the mobile phase system is 0.7-1.2 mL / min, and is further preferably 1.0 mL / min; the gradient elution mode is adopted, and during the gradient elution process, the ratio of the mobile phase A and B changes as follows: 0-10 min, 3% A; 10-15 min, 3-10% A; 15-35 min, 10-15% A; 35-65 min, 15-30% A; 65-90 min, 30-40% A; 90-105 min, 40-80% A.

[0042] In the present application, when carrying out the high performance liquid chromatography analysis, the filler in the chromatographic column is preferably octadecylsilane bonded silica gel, the chromatographic column is preferably Alphasil C18 column, the specification is 250x4.60 mm; the column temperature is 20-40°C, and is more preferably 30°C; the detection wavelength is preferably 210-360 nm, and is more preferably 230 nm; and the injection volume is preferably 5-15 μL, and is more preferably 10 μL.

[0043] In the present application, when carrying out the high performance liquid chromatography analysis, the theoretical plate number calculated based on the peak of paeoniflorin is preferably not less than 5000, and is more preferably 6000-8000.

[0044] After the control sample solution and the test sample solution are respectively subjected to high performance liquid chromatography analysis, in the present application, after the control sample solution is subjected to high performance liquid chromatography analysis, a control sample chromatogram is obtained; after the test sample solution is subjected to high performance liquid chromatography analysis, a sample chromatogram is obtained; preferably, the sample chromatogram is introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis to obtain a ginseng Yi Yuan granule fingerprint. In the present application, the traditional Chinese medicine chromatographic fingerprint similarity evaluation system is preferably a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012A version; preferably, the similarity analysis is performed by using a median method, the time window width is preferably set to 0.1 min, and 21 common peaks are calibrated. In the examples of the present application, 10 batches of ginseng Yi Yuan granules are subjected to high performance liquid chromatography analysis, and the obtained sample chromatograms are introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis, and the similarity of each batch of ginseng Yi Yuan granules is greater than 0.90. Preferably, the sample chromatogram is compared with the control sample chromatogram, and the chromatographic peaks at the same retention time as each control sample are identified, and the results show that the 4th, 5th, 8th, 10th, 12th, 18th, 19th and 21st chromatographic peaks in the 21 common peaks are identified as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid and atractylodes lactone III. The present application attributes the 21 common peaks to six medicinal materials, including radix paeoniae alba, atractylodes, licorice, astragalus, caudate vitex and ginseng; wherein the 1st peak is attributed to ginseng and astragalus, the 2nd peak is attributed to radix paeoniae alba and atractylodes, the 3rd peak is attributed to radix paeoniae alba and caudate vitex, the 4th peak is attributed to caudate vitex, the 5th peak is attributed to caudate vitex, the 6th peak is attributed to licorice, the 7th peak is attributed to radix paeoniae alba, the 8th peak is attributed to radix paeoniae alba, the 9th peak is attributed to licorice and astragalus, the 10th peak is attributed to licorice, the 11th peak is attributed to radix paeoniae alba, the 12th peak is attributed to astragalus, the 13th peak is attributed to ginseng, the 14th peak is attributed to radix paeoniae alba, the 15th peak is attributed to licorice, astragalus and caudate vitex, the 16th peak is attributed to licorice, the 17th peak is attributed to radix paeoniae alba, the 18th peak is attributed to caudate vitex, the 19th peak is attributed to licorice, the 20th peak is attributed to licorice, and the 21st peak is attributed to atractylodes.

[0045] The present application provides a ginseng Yi Yuan granule fingerprint obtained by the method for constructing a ginseng Yi Yuan granule fingerprint according to the technical scheme.

[0046] The present application provides an application of the ginseng Yi Yuan granule fingerprint according to the technical scheme in quality control of ginseng Yi Yuan granules

[0047] In order to facilitate the understanding of the technical solutions of the present application, the establishment of the HPLC fingerprint of the Ginseng Yi Yuan granules of the present application and the fingerprint thereof are further described below in combination with specific test examples. The examples are only a part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0048] Example 1: Detection of different batches of ginseng Yi Yuan granules fingerprint

[0049] 1. Instruments and reagents

[0050] 1.1 Agilent 1220 high performance liquid chromatograph (Agilent, USA); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); BSA 124S electronic balance (Sartorius); QUINTIX 35-1CN (Sartorius).

[0051] 1.2 Wahaha purified water, acetonitrile (chromatographically pure), and other reagents are analytically pure. Protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, ammonium glycyrrhizinate, and atractylodes lactone III control samples (batch numbers are 110809-202207, 110810-202210, 110736-202145, 111610-202209, 111530-201914, 111704-202104, 110731-202122, and 111978-201501, respectively, with purities of 97.5%, 99.9%, 94.6%, 95.2%, 95.2%, 98.8%, 94.4%, and 99.9%, respectively, all purchased from China Institute for Food and Drug Control). Ginseng Yi Yuan granules (batch numbers: 220901, 220902, 220903, 221101, 221102, 221103, 230101, 230102, 230103, and 230104).

[0052] 2. Fingerprint determination

[0053] 2.1 Chromatographic conditions: octadecylsilane-bonded silica gel as the filler, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution, flow rate 1.0 ml / min, column temperature 25°C, detection wavelength 230 nm, and the gradient elution program of the mobile phase as follows: 0-10 min, 3% A; 10-15 min, 3-10% A; 15-35 min, 10-15% A; 35-65 min, 15-30% A; 65-90 min, 30-40% A; 90-105 min, 40-80% A.

[0054] 2.2 Preparation of test solution: Take the Renshen Yiyuan granules, grind them finely, and accurately weigh 0.5 g into a conical flask with a stopper. Accurately add 50 mL of 80% methanol, weigh it, and ultrasonically treat it (power 40 W, frequency 250 kHz) for 30 minutes. Let it cool, re-weigh it, make up the weight loss with 80% methanol, shake it well, filter it, and take the filtrate, which is the test solution.

[0055] 2.3 Preparation of control medicinal materials and control extracts: According to the prescription ratio and preparation method, accurately weigh the control extracts or control medicinal materials, and prepare them according to the preparation method of the test solution.

[0056] 2.4 Preparation of control solution: Accurately weigh the appropriate amount of protocatechuic acid, protocatechualdehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, ammonium glycyrrhizinate, and atractylodes lactone III control substances, accurately weigh them, and place them in a 10 mL volumetric flask with methanol as the solvent. Prepare the control stock solution with a mass concentration of 0.08034, 0.06149, 0.9810, 0.9672, 0.0421, 0.05088, 0.0436, and 0.06214 mg / mL, respectively. Then accurately pipette 0.1, 0.1, 0.5, 0.2, 0.1, 0.1, 0.1, and 0.1 mL of the control stock solution into the same 10 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and prepare the mixed control solution with a mass concentration of 0.008034, 0.006149, 0.04905, 0.01934, 0.00421, 0.005088, 0.00436, and 0.006214 mg / mL, respectively. Filter it through a 0.45 μm microporous filter, and take the filtrate as the mixed control solution.

[0057] 2.4 Determination: Accurately pipette 10 μL of the control solution and the test solution, respectively, into the high performance liquid chromatograph, and record the chromatogram within 105 minutes, as shown in Figure 1 、 Figure 2 .

[0058] Example 2: 10 batches of ginseng Yi Yuan granules fingerprint analysis

[0059] 1. Fingerprint similarity analysis

[0060] Ten batches of Renshen Yiyuan granules were prepared according to the preparation method of the test sample, and the chromatographic conditions were analyzed. The fingerprint of Renshen Yiyuan granules was recorded. The similarity of the fingerprint of the composition of 10 batches of Renshen Yiyuan granules was analyzed by the Similarity Evaluation System of Traditional Chinese Medicine Chromatographic Fingerprint 2012A version issued by the National Pharmacopoeia Committee. S1 was set as the reference spectrum. The median method was used, and the time window width was set to 0.1 min. A total of 21 common peaks were calibrated. The similarity of 10 batches of Renshen Yiyuan granules was greater than 0.90. The similarity evaluation results are shown in Table 1. The relative retention times of the 21 common peaks were basically consistent, while the relative peak areas were quite different, as shown in Table 2. The fingerprint of 10 batches of Renshen Yiyuan granules is shown in Figure 3 ( Figure 3 S1-S10 correspond to batch numbers 220901, 220902, 220903, 221101, 221102, 221103, 230101, 230102, 230103, and 230104, respectively.

[0061] Table 1 Similarity evaluation results of 10 batches of Renshen Yiyuan granule samples

[0062]

[0063] Table 2 Standard fingerprint data

[0064]

[0065]

[0066] 2. Common peak identification: The chromatogram measured by the control sample was compared with the sample fingerprint. The chromatographic peak at the same retention time as each control sample was identified. The control chromatogram is shown in Figure 1 、 2 (4, 5, 8, 10, 12, 18, 19, and 21 chromatographic peaks were identified as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid, and atractylodes lactone III, respectively).

[0067] 3. Medicinal material attribution: The chromatographic peak attribution was performed according to the standard fingerprint conditions, as shown in Figure 4(S1 is the sample, and S2-S7 are six medicinal materials: white peony root, white atractylodes rhizome, licorice root, astragalus root, chicken blood vine, and ginseng, respectively). The 21 common peaks are respectively attributed to six medicinal herbs: white peony root, atractylodes macrocephala, licorice root, astragalus root, chicken blood vine, and ginseng. Among them, peak 1 belongs to ginseng and astragalus root; peak 2 belongs to white peony root and atractylodes macrocephala; peak 3 belongs to white peony root and chicken blood vine; peak 4 belongs to chicken blood vine; peak 5 belongs to chicken blood vine; peak 6 belongs to licorice root; peak 7 belongs to white peony root; peak 8 belongs to white peony root; peak 9 belongs to licorice root and astragalus root; peak 10 belongs to licorice root; peak 11 belongs to white peony root; peak 12 belongs to astragalus root; peak 13 belongs to ginseng; peak 14 belongs to white peony root; peak 15 belongs to licorice root, astragalus root, and chicken blood vine; peak 16 belongs to licorice root; peak 17 belongs to white peony root; peak 18 belongs to chicken blood vine; peak 19 belongs to licorice root; peak 20 belongs to licorice root; and peak 21 belongs to atractylodes macrocephala.

[0068] Example 3, wavelength selection

[0069] 220,901 batches of samples were tested, and the overall effect of the chromatograms at different wavelengths (360, 320, 280, 250, 230, 210, and 203) was examined. Other chromatographic conditions were the same as in Example 1. Figure 5 , 6 7, 8, 9, 10, 11. The measured chromatograms show that at a wavelength of 360 nm, the number of chromatographic peaks is small, and all peaks cannot fully reflect the characteristics of each medicinal material. Figure 5 At a wavelength of 320 nm, the number of chromatographic peaks is small, and all peaks cannot fully reflect the characteristics of each medicinal material. Figure 6 At a wavelength of 280 nm, the number of chromatographic peaks is small, and all peaks cannot fully reflect the characteristics of each medicinal material. Figure 7 At a wavelength of 254 nm, the number of chromatographic peaks is small, and all peaks cannot fully reflect the characteristics of each medicinal material. Figure 8 At a wavelength of 230 nm, there are more chromatographic peaks, and each peak can comprehensively reflect the characteristics of each medicinal material. The chromatogram baseline is relatively flat, and the peak shape is better. Therefore, this is the optimal condition. Figure 9 At a wavelength of 210 nm, the chromatogram baseline is uneven, and the chromatographic peaks fluctuate greatly. Figure 10 At a wavelength of 203 nm, the chromatogram baseline was not in the standard position. Figure 11 Based on comprehensive selection, fingerprint spectra with a detection wavelength of 230nm best reflect the composition of the formulation and have the best peak shape. Therefore, 230nm is the preferred detection wavelength.

[0070] Example 4, methodology investigation

[0071] 220901 batches of samples were tested, and the chromatographic conditions and preparation methods of the test samples were the same as in Example 1.

[0072] (1) Precision test

[0073] Take ginseng Yi Yuan particles, preparation of test solution, 6 times continuously sampling, record 21 main chromatographic peak retention time and peak area. With paeoniflorin as reference peak, relative peak area RSD <3.0% (n=6), relative retention time RSD <1.0% (n=6), to prove the instrument precision is good. The experimental results are shown in Table 3.

[0074] (2) Stability test

[0075] Take ginseng Yi Yuan particles, preparation of test solution, respectively, at 0, 2, 4, 8, 12, 24h test, record 21 main chromatographic peak retention time and peak area. With paeoniflorin as reference peak, relative peak area RSD <3.0% (n=6), relative retention time RSD <1.0% (n=6), to prove the sample within 24h stability is good. The experimental results are shown in Table 3.

[0076] (3) Reproducibility test

[0077] Take ginseng Yi Yuan particles, preparation of test solution 6, test, record 21 main chromatographic peak retention time and peak area. With paeoniflorin as reference peak, relative peak area RSD <3.0% (n=6), relative retention time RSD <1.0% (n=6), to prove the reproducibility is good. The experimental results are shown in Table 3.

[0078] Table 3 results of methodology test

[0079]

[0080]

Claims

1. A method for constructing a HPLC fingerprint of ginseng Yi Yuan granules, characterized by comprising the following steps: It comprises the following steps: ​ a. Extracting the ginseng Yi Yuan granules with 80% methanol, and taking the extract as the test sample solution; b. Taking the protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid, and atractylodes lactone III control samples, dissolving them in methanol as the control sample solution; c. Carrying out high performance liquid chromatography analysis on the control sample solution and the test sample solution, taking the chromatographic peak of the control sample solution as the benchmark to obtain the ginseng Yi Yuan granule fingerprint; When carrying out the high performance liquid chromatography analysis, the mobile phase A is acetonitrile, and the mobile phase B is 0.02-0.2% phosphoric acid aqueous solution; the flow rate of the mobile phase system is 0.7-1.2 mL / min; the gradient elution mode is adopted, and the proportion of the mobile phase A and B changes as follows during the gradient elution process: 0-10 min, 3% A; 10-15 min, 3-10% A; 15-35 min, 10-15% A; 35-65 min, 15-30% A; 65-90 min, 30-40% A; 90-105 min, 40-80% A; When carrying out the high performance liquid chromatography analysis, octadecylsilane-bonded silica gel is used as the filler, the column specification is 250x4.60 mm, 5 μm, the column temperature is 20-40 °C, the detection wavelength is 230 nm, and the injection volume is 5-15 μL; The ginseng Yi Yuan granules are prepared from 160 g of ginseng, 480 g of astragalus, 240 g of atractylodes, 240 g of white peony root, 240 g of caulis Bambusae, and 120 g of fried licorice.

2. The method for constructing the HPLC fingerprint of ginseng Yi Yuan granules according to claim 1, characterized in that, The weight of the ginseng Yi Yuan granules is 0.2-2 g, and the dosage of 80% methanol is 20-200 mL.

3. The method for constructing the HPLC fingerprint of ginseng Yi Yuan granules according to claim 2, characterized in that, The extraction is carried out under ultrasonic conditions, the power of the ultrasonic is 200-300 W, and the frequency is 30-50 kHz; the extraction time is 20-40 min.

4. The method for constructing the HPLC fingerprint of ginseng Yi Yuan granules according to claim 1, characterized in that, When carrying out the high performance liquid chromatography analysis, the theoretical plate number calculated according to the paeoniflorin peak is not less than 5000.

5. The method for constructing the HPLC fingerprint of Renshen Yiyuan Granules according to any one of claims 1-4, characterized in that, The ginseng Yi Yuan granule fingerprint contains 21 common peaks, wherein the 4th, 5th, 8th, 10th, 12th, 18th, 19th, and 21st chromatographic peaks are respectively identified as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid, and atractylodes lactone III.

6. The method for constructing the HPLC fingerprint of ginseng Yi Yuan granules according to claim 5, characterized in that, The 21 common peaks respectively belong to the six medicinal materials of white peony root, atractylodes, licorice, astragalus, caulis Bambusae, and ginseng; wherein the 1st peak belongs to ginseng and astragalus, the 2nd peak belongs to white peony root and atractylodes, the 3rd peak belongs to white peony root and caulis Bambusae, the 4th peak belongs to caulis Bambusae, the 5th peak belongs to caulis Bambusae, the 6th peak belongs to licorice, the 7th peak belongs to white peony root, the 8th peak belongs to white peony root, the 9th peak belongs to licorice and astragalus, the 10th peak belongs to licorice, the 11th peak belongs to white peony root, the 12th peak belongs to astragalus, the 13th peak belongs to ginseng, the 14th peak belongs to white peony root, the 15th peak belongs to licorice, astragalus, and caulis Bambusae, the 16th peak belongs to licorice, the 17th peak belongs to white peony root, the 18th peak belongs to caulis Bambusae, the 19th peak belongs to licorice, the 20th peak belongs to licorice, and the 21st peak belongs to atractylodes.

7. The use of the construction method of the HPLC fingerprint of the ginseng Yiyuan granules according to any one of claims 1-6 in the quality control of the ginseng Yiyuan granules.

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