A quality control method for Amaranthus australis

Abstract

The invention discloses a quality control method for Amaranthus tricolor, comprising the following steps: (1) preparing a test solution; using Amaranthus tricolor as a control medicinal material, preparing a control medicinal material reference solution; using gallic acid, corilagin, and ellagic acid as reference substances, respectively preparing reference substance reference solutions; (2) respectively drawing the reference medicinal material reference solution, the reference substance reference solution, and the test solution for ultra-high performance liquid chromatography analysis, constructing a corresponding characteristic spectrum, selecting 6 characteristic peaks in the characteristic spectrum of the test solution corresponding to the retention time in the characteristic chromatogram of the control medicinal material solution as common characteristic peaks, constructing a characteristic spectrum of Amaranthus tricolor, wherein characteristic peak 1 corresponds to the gallic acid reference substance peak, characteristic peak 4 corresponds to the corilagin reference substance peak, and characteristic peak 6 corresponds to the ellagic acid reference substance peak; (3) performing qualitative analysis on the test solution through the characteristic spectrum obtained in step (2). The method of the invention has good reproducibility and stability and reliable recovery rate.

Description

Technical Field

[0001] The invention relates to a drug quality control method, in particular to a comprehensive quality control method for Amaranthus chinensis. Background Art

[0002] Acalypha australis is the dried aerial part of Acalypha australis L., a plant of the Euphorbiaceae family (Jiangsu Provincial Food and Drug Administration. Jiangsu Province Traditional Chinese Medicine Preparation Specifications (Volume 2) [S]. Jiangsu Science and Technology Press. 2020: 372). Acalypha australis contains organic acids (gallic acid, caffeic acid, protocatechuic acid, etc.), flavonoids, terpenes, tannins and other compounds (Chen Yilong, Lina, Yao Yuanyuan, et al. Rapid analysis of chemical components of Acalypha australis based on UHPLC-Q-TOF-MS / MS technology [J]. Chinese Medicinal Materials, 2018, 41(6): 1352-1358). It tastes bitter and astringent, and is cool in nature; it enters the heart, lung, large intestine, and small intestine meridians. It has anti-inflammatory, antioxidant, anti-cancer, antibacterial, and hemostatic pharmacological activities; it is used to clear away heat and detoxify, promote dampness, astringe, and stop diarrhea, and can be used to treat enteritis, dysentery, hematemesis, epistaxis, hematochezia, hematuria, metrorrhagia, etc. (Jiangsu Provincial Food and Drug Administration. Jiangsu Province Traditional Chinese Medicine Preparation Specifications (Volume 2) [S]. Jiangsu Science and Technology Press. 2020: 372).

[0003] Modern pharmacological effects and clinical application studies have shown that Acalypha alopecuroidea has anti-inflammatory (Deng Li, Li Fengqian, Zou Hao, et al. Effective components of Acalypha alopecuroidea against ulcerative colitis [J]. Chinese Patent Medicine, 2007, 29 (7): 969-971), antioxidant (RAJKUMAR V, GGUHA'RASHOK KUMAR. Therapeutic potentia of Acalypha fruticosa [J]. Food and Toxicology, 2010, 48 (6): 1709-1713), anticancer (MADLENER, SVACINOVA, KITNER, et al. Invitro anti-inflammatory and anticancer activities of extracts of Acalypha alopecuroidea (Euphorbiaceae) [J]. Int J Oncol, 2009, 35 (4): 881-911), antibacterial (KRISHNARAJ C, JAPAN EG, MOHAN N, et al. al.Synthesis of silver nanoparticles usingAcalypha indica leaf extractsand its antibacterial activity against waterborne pathogens[J].Colloids and Surfaces B:Biointerfaces,2010,76(1):50-56), hemostasis(Wang Jinyuan, Yang Yangxian, et al. Clinical study on the treatment of uterine bleeding with Acalypha indica[J].Modern Chinese Medicine,1997,10(1):9-10) and other aspects have good therapeutic effects.

[0004] Chinese herbal formula granules are made from single-ingredient Chinese herbal pieces through water extraction, concentration, drying, and granulation. They represent a new form of herbal medicine reform. Compared to traditional decoctions, they are more portable and simple to use, meeting the demands of modern, fast-paced work and life. In recent years, Chinese herbal formula granules have experienced rapid development, with the release of national standards for formula granules for multiple Chinese herbal varieties. These standards have significantly contributed to the standardization of their production and the modernization of traditional Chinese medicine. Amaranthus tricolor formula granules are made from Amaranthus tricolor through modern extraction, concentration, drying, and granulation processes. They retain the clinical efficacy of Amaranthus tricolor while ensuring portability.

[0005] There are some reports on the quality control methods of Amaranthus chinensis. In the 2020 edition of "Jiangsu Province Traditional Chinese Medicine Preparation Specifications" (Volume 2), properties, powder microscopic identification, and thin layer identification are used as the standards for controlling the quality of Amaranthus chinensis (Jiangsu Food and Drug Administration. Jiangsu Province Traditional Chinese Medicine Preparation Specifications (Volume 2) [S]. Jiangsu Science and Technology Press. 2020: 372-374). Xu Yuexin et al. studied the thin layer of Amaranthus chinensis (Xu Yuexin, Zhang Xuefeng, Hu Shengjun. Establishment of quality standards for Amaranthus chinensis preparations [J]. Pharmacy and Clinical Research, 2019, 27(4): 264-266). Deng Xinyu et al. established a UPLC method for the determination of corilagin in Amaranthus chinensis (Deng Xinyu, Deng Shiren, Xia Linbo, et al. Simultaneous determination of corilagin in Amaranthus chinensis by UPLC [J]. Guangzhou Chemical Industry, 2021, 49(15): 119, 127). Ma Qifeng et al. established a UPLC method for the simultaneous determination of gallic acid, protocatechuic acid, rutin and methyl gallate in Amaranthus chinensis (Ma Qifeng, Liu Wei, Xia Linbo, et al. Simultaneous determination of four components in Amaranthus chinensis by UPLC [J]. Pharmaceutical Research, 2021, 40(4): 221-223). Amaranthus tricolor is not currently included in the 2020 edition of the Chinese Pharmacopoeia. Although some literature has conducted partial research on the quality control of Amaranthus tricolor medicinal materials, no comprehensive, effective and systematic quality evaluation methods and standards have been established. For example, there has been no analysis and research on the characteristic spectrum of Amaranthus tricolor; in terms of content determination, they all focus on organic acid compounds such as gallic acid, and no quantitative analysis and research on other active ingredients in Amaranthus tricolor has been conducted. There are still many deficiencies in its effectiveness evaluation means and the establishment of overall quality control methods. In addition, existing literature shows that the quality control research of Amaranthus tricolor is mainly concentrated on medicinal materials and decoction pieces, and no research on the quality control of Amaranthus tricolor formula granules has been retrieved. Therefore, the patent of this invention uses a liquid phase detection method to comprehensively control the overall profile and active ingredients of Amaranthus tricolor samples, realizing simultaneous qualitative and quantitative control to ensure the effectiveness of product quality. Summary of the Invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a quality control method for Amaranthus tricolor with good reproducibility and stability, reliable recovery, low detection cost and high detection efficiency.

[0007] Technical solution: The quality control method of Amaranthus chinensis of the present invention comprises the following steps:

[0008] (1) Prepare the test solution; use Amaranthus tricolor as the control medicinal material to prepare the reference solution of the control medicinal material; use gallic acid, corilagin, and ellagic acid as the reference substance, and prepare the reference solution of the reference substance respectively;

[0009] (2) The control medicinal material reference solution, the reference substance reference solution and the test solution were respectively taken for ultra-performance liquid chromatography analysis to construct corresponding characteristic spectra, and six characteristic peaks corresponding to the retention times in the characteristic chromatogram of the test solution and the control medicinal material solution were selected as common characteristic peaks to construct the characteristic spectra of Amaranthus chinensis, wherein characteristic peak 1 corresponds to the peak of the gallic acid reference substance, characteristic peak 4 corresponds to the peak of the corilagin reference substance, and characteristic peak 6 corresponds to the peak of the ellagic acid reference substance;

[0010] (3) Perform qualitative analysis on the test solution using the characteristic spectrum obtained in step (2).

[0011] Furthermore, in step (1), the extractant for preparing the reference substance solution and / or the test solution is water, methanol or a methanol aqueous solution with a volume concentration of 30% to 70%.

[0012] Furthermore, in preparing the reference substance solution, the amount of extractant used per gram of Amaranthus chinensis is 15ml-160ml, the extraction time is 15-60 minutes, and the extraction method is heating reflux.

[0013] Furthermore, in step (1), the concentration of the gallic acid reference substance solution is 20-100 μg / ml; the concentration of the corilagin reference substance solution is 20-100 μg / ml; and the concentration of the ellagic acid reference substance solution is 4-400 μg / ml.

[0014] Furthermore, the conditions for the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; acetonitrile is used as mobile phase A, and 0.05% to 0.2% by volume phosphoric acid solution is used as mobile phase B, and gradient elution is adopted: 0 to 1 min, 5% A, 1 to 3 min, 5→10% A, 3 to 9 min, 10→12% A, 9 to 16 min, 12→18% A, 16 to 20 min, 18→35% A, 20 to 21 min, 35→5% A, 21 to 26 min, 5% A.

[0015] Furthermore, the detection wavelength is 230-300 nm; the flow rate is 0.25 ml-0.35 ml per minute; the column temperature is 35° C.-40° C.; and an ultraviolet detector is used.

[0016] Furthermore, the injection volume of the test solution, the control medicinal material reference solution and the control substance reference solution is 1 μl to 4 μl.

[0017] Furthermore, the characteristic spectrum of Amaranthus tricolor constructed in step (2) is based on the characteristic peak corresponding to the corilagin reference substance as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated; the relative retention time should be within ±10% of the specified value, and the specified value is: characteristic 0.81, peak 3: 0.91, peak 5: 1.21.

[0018] Furthermore, the test sample Amaranthus chinensis is Amaranthus chinensis medicinal material, Amaranthus chinensis decoction pieces, Amaranthus chinensis preparation intermediate or Amaranthus chinensis preparation finished product.

[0019] Furthermore, when the test sample is a finished product of Amaranthus chinensis preparation, the preparation method is: take 0.2g to 0.6g of Amaranthus chinensis formula granules, grind them into powder, add water, 30% to 70% methanol or methanol for extraction, and take the filtrate to obtain the test sample solution.

[0020] Furthermore, the preparation method of the control medicinal material reference solution is: take 0.6g~2.0g of Amaranthus tricolor control medicinal material, add water, methanol, 30%~70% methanol or 50% ethanol to extract, and take the filtrate as the control medicinal material reference solution; the preparation method of the reference substance solution is: take appropriate amounts of gallic acid reference substance, corilagin reference substance, and ellagic acid reference substance, add methanol aqueous solution to dissolve, and use as the reference substance solution.

[0021] Furthermore, step (3) also includes a quantitative analysis step: using the content of ellagic acid as a content indicator component, and simultaneously detecting the content of ellagic acid in the test solution.

[0022] Furthermore, when the test sample is a finished product of Amaranthus tricolor preparation, the ellagic acid content should be above 0.5 mg / g; when the test sample is Amaranthus tricolor medicinal material, the ellagic acid content should be above 0.03%, then the test sample is an effective Amaranthus tricolor medicinal material.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: (1) The present invention establishes for the first time a characteristic spectrum that meets the requirements of the overall characteristics of Amaranthus tricolor. The characteristic spectrum contains 6 characteristic peaks and identifies the characteristic peaks of gallic acid, corilagin and ellagic acid. Using the control medicinal materials as reference materials can better reflect the consistency of the formula granules and the medicinal materials; (2) The quality control method established by the present invention can simultaneously perform qualitative and quantitative detection on Amaranthus tricolor, which is beneficial to the overall quality control of Amaranthus tricolor formula granules and takes into account the effectiveness and overall quality evaluation of traditional Chinese medicine; (3) This method uses ellagic acid as the content index of Amaranthus tricolor to analyze other phenolic acid compounds in Amaranthus tricolor. The results of the analysis showed that ellagic acid in Amaranthus tricolor has an antioxidant effect and protects biological molecules from damage caused by nitrite oxidation, which can fully characterize the detoxification effect of Amaranthus tricolor. The ellagic acid content is high and the composition is relatively stable, which provides a technical control method for the quality of Amaranthus tricolor raw materials, intermediates and formula granules, especially for the process control and product quality stability of Amaranthus tricolor formula granules, and provides a strong guarantee for its industrialization and quality control. (4) The method of the present invention is simple to operate, has good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency, and provides an important guarantee for standardizing the quality inspection of Amaranthus tricolor and its related traditional Chinese medicine preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the characteristic spectrum of Amaranthus tricolore granules, Peak 1: Gallic acid; Peak 4 (S): Corilagin; Peak 6: Ellagic acid;

[0025] Figure 2 This is the characteristic spectrum of the intermediate of Amaranthus tricolor, Peak 1: Gallic acid; Peak 4 (S): Corilagin; Peak 6: Ellagic acid;

[0026] Figure 3 This is the characteristic spectrum of Amaranthus tricolor medicinal material / decoction piece, Peak 1: Gallic acid; Peak 4 (S): Corilagin; Peak 6: Ellagic acid;

[0027] Figure 4 The chromatograms are for reference substance, reference medicinal material, and Amaranthus chinensis formula granules;

[0028] Figure 5 UPLC chromatograms of Amaranthus tricolor granules at different detection wavelengths;

[0029] Figure 6 This is the full wavelength scan of ellagic acid;

[0030] Figure 7 UPLC diagrams for different extraction solvents;

[0031] Figure 8 UPLC diagrams of different extraction methods;

[0032] Figure 9 UPLC diagrams for different extraction times;

[0033] Figure 10 UPLC diagrams for different extraction volumes;

[0034] Figure 11 For chromatographic column investigation;

[0035] Figure 12 For column temperature investigation;

[0036] Figure 13 For flow rate investigation;

[0037] Figure 14 is the linear relationship graph of ellagic acid reference substance;

[0038] Figure 15 It is a specific test;

[0039] Figure 16 It is a holistic test;

[0040] Figure 17 Overlay of characteristic spectra of three batches of formula particles;

[0041] Figure 18 Overlay of characteristic maps of ten batches of medicinal materials. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0043] The instruments and reagents used in the following examples are:

[0044] Agilent high-performance liquid chromatograph (1290 Infinity, Agilent); Waters high-performance liquid chromatograph (Waters H-Class, Waters); one-ten-thousandth balance (AP224W, Mettler-Toledo); one-millionth balance (XBR36, Mettler-Toledo); CNC ultrasonic cleaner (2700TH, Shanghai Anpu Laboratory Technology Co., Ltd.); electric constant temperature water bath (J-HH-6A, Shanghai Shengwei Electronic Technology Co., Ltd.); ultrapure water system (Comfort1, Sartorius, Germany); phosphoric acid (chromatographic grade, Shanghai MacLean Biochemical Technology Co., Ltd.); acetonitrile (chromatographic grade, CNW); water was ultrapure water; all other reagents were of analytical grade.

[0045] Gallic acid reference substance, corilagin reference substance, and ellagic acid reference substance were all purchased from the China Food and Drug Inspection Institute with batch numbers 110831-201906, 111623-200302, and 111959-201903, respectively. The purities were 91.5%, 100%, and 88.8%, respectively, and no treatment was required before use.

[0046] The control medicinal material Amaranthus chinensis was purchased from Shanghai Hongyong Biotechnology Co., Ltd. with batch number 410070-202105.

[0047] Amaranthus tricolor formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0048] Example 1 Establishment of the characteristic spectrum of Amaranthus chinensis

[0049] This embodiment provides a method for detecting the quality control of Amaranthus chinensis, wherein the Amaranthus chinensis sample to be tested can be Amaranthus chinensis medicinal materials, Amaranthus chinensis decoction pieces, Amaranthus chinensis preparation intermediates or Amaranthus chinensis preparation finished products. Taking Amaranthus chinensis formula granules as an example, the quality control method (characteristic spectrum and content determination) thereof includes the following steps:

[0050] (1) Select Amaranthus tricolor granule samples (Jiangyin Tianjiang Pharmaceutical Co., Ltd., batch numbers 21050349, 21050359, and 21050369) and prepare test solution; use Amaranthus tricolor as a control medicinal material to prepare a reference solution of the control medicinal material; use gallic acid, corilagin, and ellagic acid as reference substances to prepare reference solution of the reference substance respectively;

[0051] Preparation of control medicinal material reference solution: Take 1.0 g of Amaranthus tricolor control medicinal material, place it in a stoppered conical flask, add 25 ml of water, heat and reflux for 45 minutes, cool, centrifuge (speed is 7000 r / min) for 5 minutes, take the supernatant, evaporate to dryness, dissolve it in 50% methanol, transfer it to a 25 mL volumetric flask, add 50% methanol to the scale, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0052] Preparation of reference substance solution: Take appropriate amount of gallic acid reference substance, corilagin reference substance, and ellagic acid reference substance, weigh accurately, and add 50% methanol aqueous solution to make a solution containing 100 μg of gallic acid, 100 μg of corilagin, and 50 μg of ellagic acid per 1 ml, which is used as the reference substance solution.

[0053] Preparation of test solution: Take about 0.3g of Amaranthus chinensis granules, accurately weigh, place in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper, weigh, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0054] (2) The control medicinal material reference solution, the reference substance reference solution, and the test solution were respectively aspirated for ultra-high performance liquid chromatography analysis to construct the corresponding characteristic spectrum. The chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.1% volume fraction phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate 0.3 ml per minute; column temperature 35 ° C; detection wavelength 300 nm. The injection volume was 2 μl. Detection was performed using an Agilent high performance liquid chromatograph.

[0055]

[0056] The spectrum of one of the batches obtained is as follows Figure 4 As shown, the chromatogram of the test solution (Amaranthus tricolor formula granules) showed 6 characteristic peaks, and the retention times corresponded to the 6 characteristic peaks in the chromatogram of the control medicinal material reference, among which Peak 1, Peak 4, and Peak 6 were consistent with the peak retention times of the gallic acid reference, corilagin reference, and ellagic acid reference, respectively; the corresponding peak of the corilagin reference was taken as the S peak, and the relative retention times of the other characteristic peaks and the S peak were calculated; the relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.81 (peak 2), 0.91 (peak 3), and 1.21 (peak 5).

[0057] (3) Using the characteristic spectrum obtained in step (2), the qualitative analysis of the sample to be tested is performed; using ellagic acid as the content determination index, the quantitative analysis of the sample to be tested is performed. Figure 6 shown.

[0058] Finally, the spectra of the three batches of Amaranthus chinensis formula granules were fitted into the control characteristic spectrum, such as Figure 1 shown.

[0059] Repeat the above test steps, replacing the test samples with Amaranthus tricolor preparation intermediates (batch numbers 210443311, 210443321, and 210443331) and Amaranthus tricolor medicinal materials (purchased from Jiangyin Tianjiang Pharmaceuticals, YC2103325, YC2103326, and YC2103327 in Xuzhou, Jiangsu Province, YC2103508, YC2103509, and YC2103510 in Yulin, Guangxi Zhuang Autonomous Region, YC2103619, YC2103620, and YC2103621 in Baoji, Shaanxi Province, and YC2104232 in Nanyang, Henan Province). The control characteristic spectrum of the Amaranthus tricolor preparation intermediates after fitting is shown in the figure below. Figure 2 As shown in the figure, the reference characteristic spectrum of Amaranthus tricolor is as follows: Figure 3 As shown (herbal medicines can be simply cut into pieces, so one atlas can be shared).

[0060] The quality control method provided in this example is a liquid-phase method that simultaneously determines the characteristic spectrum and ellagic acid content. This establishes a rapid, comprehensive, and highly specific quality control method for Amaranthus chinensis granules. This dual-aspect quality control approach, focusing on both the characteristic spectrum and content determination, provides stronger support for the industrialization of Amaranthus chinensis granules.

[0061] Example 2

[0062] Three batches of Amaranthus chinensis granules (Jiangyin Tianjiang Pharmaceutical Co., Ltd., batch numbers 21050349, 21050359, and 21050369) and 10 batches of Amaranthus chinensis medicinal materials (purchased by Jiangyin Tianjiang Pharmaceutical, YC2103325, YC2103326, and YC2103327 in Xuzhou, Jiangsu Province, YC2103508, YC2103509, and YC2103510 in Yulin, Guangxi Zhuang Autonomous Region, YC2103619, YC2103620, and YC2103621 in Baoji, Shaanxi Province, and YC2104232 in Nanyang, Henan Province) were used as Amaranthus chinensis test samples, and characteristic spectra and content determination were performed according to the following steps:

[0063] (1) Selecting Amaranthus tricolor formulated granules or medicinal materials and preparing a test solution; using Amaranthus tricolor as a control medicinal material, preparing a control medicinal material reference solution; using gallic acid, corilagin, and ellagic acid as reference substances, respectively preparing reference substance solutions;

[0064] Preparation of control medicinal material reference solution: Take 1 g of Amaranthus tricolor control medicinal material, place it in a stoppered conical flask, add 25 mL of water, heat and reflux for 45 minutes, cool, centrifuge (speed is 7000 r / min) for 5 minutes, take the supernatant, evaporate to dryness, add 50% methanol to dissolve the residue, and make up to the volume in a 25 mL volumetric flask, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0065] Preparation of reference substance solution: Take appropriate amount of gallic acid reference substance, corilagin reference substance, and ellagic acid reference substance, accurately weigh them, and add 50% methanol to make a solution containing 100 μg of gallic acid, 100 μg of corilagin, and 50 μg of ellagic acid per 1 ml, which is used as the reference substance solution.

[0066] Preparation of the test solution of Amaranthus formula granules: Take about 0.3g of Amaranthus formula granules, accurately weigh, place in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper, weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0067] Preparation of Amaranthus chinensis test solution (characteristic spectrum): Accurately weigh approximately 1 g of Amaranthus chinensis powder (passed through a No. 3 sieve) and place in a stoppered conical flask. Accurately add 25 mL of water and reflux for 45 minutes. Cool and centrifuge at 7000 rpm for 5 minutes. Remove the supernatant and evaporate to dryness. Dissolve the residue in 50% methanol and dilute to a 25 mL volumetric flask. Shake well, filter, and collect the filtrate.

[0068] Preparation of the test solution of Amaranthus tricolor medicinal material (content determination): Take about 1g of the powder of this product (passed through No. 3 sieve), accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 50% ethanol, stopper it, weigh it, heat and reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0069] (2) The test solution and the reference solution prepared in step (1) are subjected to ultra-high performance liquid chromatography to obtain corresponding ultra-high performance liquid chromatograms; 2 μl of each of the reference solution and the test solution are accurately aspirated and injected into the liquid chromatograph for determination. The chromatographic conditions and parameters are the same as those in Example 1.

[0070] The test results of the test solution are shown in the table below and the spectrum is shown in Figure 17 (Overlay of characteristic spectra of three batches of formula particles), Figure 18 (Overlay of characteristic maps of ten batches of medicinal materials).

[0071] Table 1 Determination results of Amaranthus chinensis formula granule samples (relative retention time)

[0072]

[0073] Table 2 Determination results of 10 batches of Amaranthus chinensis medicinal material samples (relative retention time)

[0074]

[0075] (3) The ultra-high performance liquid chromatograms obtained in step (2) were compared with the formulated particles ( Figure 1 ), medicinal materials / cooked pieces( Figure 3 ) to conduct qualitative analysis of the samples;

[0076] The quantitative analysis of the samples was carried out using ellagic acid as the content determination indicator.

[0077] The content of ellagic acid in the samples was determined. The experimental results are shown in Tables 3 and 4.

[0078] Table 3 Determination of ellagic acid content in Amaranthus chinensis formula granules

[0079]

[0080] Table 4 Determination of ellagic acid content in Amaranthus chinensis

[0081]

[0082]

[0083] From the above test results, it can be seen that from a qualitative analysis, the characteristic spectra of the three batches of Amaranthus chinensis granules and the ten batches of Amaranthus chinensis medicinal materials all meet the requirements of the characteristic spectra in step (2). From a quantitative analysis, the ellagic acid content of the three batches of Amaranthus chinensis granules and the ten batches of Amaranthus chinensis medicinal materials is stable, ensuring the effectiveness of the Amaranthus chinensis granules and the medicinal materials.

[0084] Example 3 Establishment of the Characteristic Spectrum Method of Amaranthus chinensis Formula Granules and Its Methodological Research

[0085] This example is used to confirm the optimal scheme for establishing the characteristic spectrum in the quality control method of Amaranthus tricolor samples, specifically including the comparison of wavelength selection and sample solution preparation schemes, selecting the optimal detection wavelength and sample preparation scheme; and examining the optimal scheme for specificity, integrity, precision, intermediate precision, stability, repeatability, and durability.

[0086] 1. Determination of detection wavelength

[0087] Take an appropriate amount of Amaranthus chinensis granules, grind them into powder, take about 0.3g, place in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper tightly, weigh the weight, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate. Collect the chromatogram of the sample at 230nm, 300nm and 380nm, as shown Figure 5 shown.

[0088] The results showed that the chromatographic information of Amaranthus tricolor granules detected at 300nm was richer, and 300nm detection wavelength was finally selected as the detection wavelength.

[0089] 2. Determination of the test solution preparation method

[0090] The test samples used in this embodiment are all Amaranthus chinensis formula granules.

[0091] 2.1 Investigation of different extraction solvents

[0092] This experiment investigated the effects of different extraction solvents on the characteristic spectra of Amaranthus tricolor formula granules. Water, 30% methanol, 50% methanol, 70% methanol, methanol and 50% ethanol were selected as extraction solvents. The peak area / sample weight and chromatograms of the six chromatographic peaks were used to compare the effects of different extraction solvents on the characteristic spectra of Amaranthus tricolor formula granules.

[0093] Take an appropriate amount of this product, grind it into powder, take about 0.3g, and divide it into 5 parallel parts. Weigh it accurately and place it in a stoppered conical flask. Add 25ml of water, 30% methanol, 50% methanol, 70% methanol or methanol accurately. Stopper it tightly and weigh it. Ultrasonicate it (power 250W, frequency 40kHz) for 30 minutes respectively. Let it cool and weigh it again. Make up the lost weight with the corresponding solvent. Shake it well and filter it. Take the filtrate to obtain the product. Sample injection and detection are carried out according to the chromatographic conditions of Example 1. The results are shown in Table 5. Figure 7 .

[0094] Table 5 Comparison of extraction efficiency of different extraction solvents (peak area / sample weight)

[0095]

[0096]

[0097] It can be seen from the above table that when 70% methanol is used as the extraction solvent, the extraction efficiency is higher, so 70% methanol is selected as the extraction solvent.

[0098] 2.2 Investigation of different extraction methods

[0099] This experiment investigated the effects of different extraction methods on the characteristic spectra of Amaranthus tricolor formula granules. Three extraction methods, ultrasonic treatment, shaking extraction, and heating reflux, were selected. The peak area / sample weight and chromatograms of the six chromatographic peaks were used to compare the effects of different extraction methods on the characteristic spectra of Amaranthus tricolor formula granules.

[0100] Take an appropriate amount of this product, grind it into powder, take about 0.3g, and divide it into 3 parallel parts, accurately weigh it, put it into a stoppered conical flask, accurately add 25ml of 70% methanol each, seal it tightly, weigh it, and treat it ultrasonically (power 250W, frequency 40kHz), shake it to extract, heat it under reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it. The chromatographic conditions of Example 1 were used for sample injection and detection. The results are shown in Table 6. Figure 8 .

[0101] Table 6 Comparison of extraction efficiency of different extraction methods (peak area / sample weight)

[0102]

[0103] It can be seen from the above table that the extraction efficiency is the highest when using the heating reflux method. At the same time, considering that the content is higher under the heating reflux conditions, the heating reflux method is selected as the extraction method.

[0104] 2.3 Investigation of different extraction times

[0105] This experiment investigated the effects of four different extraction times of 15 minutes, 30 minutes, 45 minutes and 60 minutes on the characteristic spectrum of Amaranthus tricolor formula granules. The effects of different extraction times on the characteristic spectrum of Amaranthus tricolor formula granules were compared through the peak area / sample weight and chromatograms of the six chromatographic peaks.

[0106] Take an appropriate amount of this product, grind it into powder, take about 0.3g, divide it into 4 parallel parts, put it into a stoppered conical flask, add 25ml of 70% methanol, reflux for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, let it cool and weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it. The sample was injected and tested according to the chromatographic conditions of Example 1. The results are shown in Table 7. Figure 9 .

[0107] Table 7 Comparison of extraction efficiency at different extraction times (peak area / sample weight)

[0108]

[0109] It can be seen from the above table that when the extraction time is 30 minutes, the extraction is basically sufficient. In order to save time, the extraction time is selected to be 30 minutes.

[0110] 2.4 Investigation of different extraction volumes

[0111] This experiment investigated the effects of three different extraction solvent volumes of 15ml, 25ml and 50ml on the characteristic spectrum of Amaranthus chinensis formula granules. The effects of different extraction volumes on the characteristic spectrum of Amaranthus chinensis formula granules were compared through the peak area of ​​6 chromatographic peaks / sample weight × extraction volume / 25 and chromatograms.

[0112] Take an appropriate amount of this product, grind it into powder, take about 0.3g, and divide it into three parallel parts, accurately weigh them, put them into stoppered conical flasks, add 15ml, 25ml and 50ml of 70% methanol respectively, stopper them, weigh them, heat and reflux for 30 minutes, let them cool, weigh them again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate. The chromatographic conditions of Example 1 were used for sample injection and detection. The results are shown in Table 8. Figure 10 .

[0113] Table 8 Comparison of extraction efficiency of different extraction volumes (peak area / sample weight*extraction volume / 25)

[0114]

[0115] It can be seen from the above table that when the extraction volume is 25 ml, the extraction efficiency is higher, and the extraction volume is finally determined to be 25 ml.

[0116] 2.5 Determination of the test solution preparation method

[0117] According to the above research results, the preparation method of the test solution of the characteristic spectrum of the Amaranthus chinensis formula granules is determined as follows:

[0118] Take an appropriate amount of the sample to be tested, grind it into powder, take about 0.3g, weigh it accurately, place it in a stoppered conical flask, add 25ml of 70% methanol, heat and reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0119] 3. Feature Map Methodology Research

[0120] 3.1 Specificity Investigation

[0121] The excipients added to the Amaranth formula granules are maltodextrin, silicon dioxide and magnesium stearate. This experiment investigated the effect of negative samples of iron-deficient Amaranth on the characteristic spectrum of the Amaranth formula granules.

[0122] Take the negative sample of iron-deficient Amaranthus (the extract of the formula granules without Amaranthus), and prepare the negative sample solution according to the test solution preparation method in 2.5. Take the Amaranthus formula granules and prepare the test solution according to 2.5. Take the negative sample solution (blank solvent) and the test solution and inject 2μl of them according to the chromatographic conditions of Example 1 for detection. The results are shown in Figure 15 .

[0123] The experimental results showed that the solvent and excipients had no interference on the determination of the characteristic spectrum of Amaranthus chinensis formula granules, and the method had strong specificity.

[0124] 3.2 Overall Investigation

[0125] Take the Amaranthus formula granules and prepare the test solution according to 2.5. Under the chromatographic conditions of Example 1, keep the elution gradient when the acetonitrile ratio is the highest, double the elution time, and record the chromatogram. The results are shown below. Figure 16 .

[0126] The experimental results show that no obvious chromatographic peaks are eluted after the original gradient elution, indicating that the chromatographic conditions basically meet the principle of maximum information content.

[0127] 3.3 Repeatability study

[0128] Take an appropriate amount of Amaranthus chinensis granules, grind them into powder, take about 0.3 g, accurately weigh, and prepare 6 parallel portions. Prepare the test solution according to the test solution preparation method in 2.5, and analyze the sample according to the chromatographic conditions of Example 1 to examine the consistency of the relative retention time of the characteristic peaks. The results are shown in Table 9.

[0129] Table 9 Repeatability test (relative retention time)

[0130]

[0131] The results showed that the RSDs of the relative retention times of the characteristic peaks were all less than 1%, indicating a good repeatability.

[0132] 3.4 Precision investigation

[0133] Take an appropriate amount of Amaranthus chinensis granules, grind them into powder, take about 0.3 g, and prepare a test solution according to the test solution preparation method in 2.5. According to the chromatographic conditions of Example 1, inject 6 times continuously, 2 μl each time, and examine the consistency of the relative retention time of the characteristic peaks. The results are shown in Table 10.

[0134] Table 10 Precision test results (relative retention time)

[0135]

[0136] The results showed that the RSDs of the relative retention times of the characteristic peaks were all less than 1%, indicating good precision.

[0137] 3.5 Stability investigation

[0138] Take an appropriate amount of Amaranthus chinensis granules, grind them into powder, take about 0.3 g, weigh accurately, and prepare a test solution according to the test solution preparation method in 2.5. According to the chromatographic conditions of Example 1, 2 μl of the sample was injected at 0, 2, 4, 8, 12, and 24 hours after the preparation of the test solution to examine the consistency of the relative retention time of the characteristic peaks. The results are shown in Table 11.

[0139] Table 11 Stability study (relative retention time)

[0140]

[0141]

[0142] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the test solution had good stability within 24 hours.

[0143] 3.6 Intermediate precision study

[0144] Three test solutions of Amaranthus tricolor granules were prepared by two experimenters A and B according to the test solution preparation method in 2.5. 2 μl of the solution was injected at different times and on different instruments according to the chromatographic conditions of Example 1 to examine the consistency of the relative retention times of the characteristic peaks. The results are shown in Table 12.

[0145] Table 12 Intermediate precision investigation (relative retention time)

[0146]

[0147] The results showed that the RSD of the relative retention time of each characteristic peak was less than 3%, and the intermediate precision was good.

[0148] 3.7 Durability Assessment

[0149] (1) Investigation of different chromatographic columns

[0150] The same batch of samples was taken and the test solution was prepared according to the preparation method of the test solution in 2.5. According to the chromatographic conditions of Example 1, three different chromatographic columns, ZORBAX SB-C18 RRHD (Agilent, 2.1 mm × 100 mm, 1.8 μm); ACQUITY UPLC BE H C18 (Waters, 2.1 mm × 100 mm, 1.7 μm); and Kinetex XB-C18 (phenomenex, 2.1 mm × 100 mm, 1.7 μm), were used to investigate their separation effects. The results are shown in the figure.

[0151] Table 13 Column investigation (relative retention time)

[0152]

[0153] The results showed that the Amaranthus tricolor granules were well separated on three different brands of chromatographic columns. A ZORBAX SB-C18 RRHD (Agilent, 2.1 mm × 100 mm, 1.8 μm) column was used in subsequent studies.

[0154] (2) Investigation of different column temperatures

[0155] Take the same batch number sample and prepare the test solution according to the preparation method of the test solution in 2.5. According to the chromatographic conditions of Example 1, use ZORBAX SB-C18 RRHD (Agilent, 2.1mm×100mm, 1.8μm) as the chromatographic column to investigate the sample separation effect at column temperatures of 30℃, 35℃, and 40℃. Figure 12 , Table 14.

[0156] Table 14 Column temperature investigation (relative retention time)

[0157]

[0158] The results showed that small column temperature changes within the range of 30℃ to 40℃ could meet the system suitability requirements, and a column temperature of 35℃ was used in subsequent studies.

[0159] (3) Investigation of different flow rates

[0160] Take the same batch number sample and prepare the test solution according to the preparation method of the test solution in 2.5. According to the chromatographic conditions of Example 1, a ZORBAX SB-C18 RRHD (Agilent, 2.1mm×100mm, 1.8μm) column was used to investigate the separation effect of the sample at flow rates of 0.25ml / min, 0.30ml / min, and 0.35ml / min. The results are shown in FIG. Figure 13 , Table 15.

[0161] Table 15 Flow rate investigation (relative retention time)

[0162]

[0163] The results showed that small flow rate changes in the range of 0.25ml / min to 0.35ml / min could meet the system suitability requirements, and a flow rate of 0.30ml / min was used in subsequent studies.

[0164] Example 4 Establishment of the content determination method of Amaranthus chinensis formula granules and its methodological research

[0165] 1. Determination of detection wavelength

[0166] In this experiment, the ellagic acid reference solution was scanned at full wavelength and its UV absorption graph was recorded. Figure 6 .

[0167] The results showed that ellagic acid had a strong absorption at around 250nm. Combined with the characteristic chromatographic conditions, 300nm was finally selected as the detection wavelength.

[0168] 2 Preparation of test solution

[0169] 2.1 Investigation of different extraction solvents

[0170] Take an appropriate amount of the formula granules, grind them finely, take about 0.3g, and divide them into 5 parallel groups, 2 portions per group, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of water, 30% methanol, 50% methanol, 70% methanol or methanol, respectively, stopper them, weigh them, ultrasonically treat them (power 250W, frequency 40kHz) for 30 minutes, let them cool, weigh them again, make up the lost weight with the corresponding solvent, shake them well, filter them, and take the filtrate. Accurately take 2μl of each test solution, inject them into the liquid chromatograph, and determine the content of ellagic acid according to the chromatographic conditions of Example 1. The results are shown in Table 1. Figure 7 , Table 16.

[0171] Table 16 Comparison of different extraction solvents

[0172]

[0173] The results showed that water, 30% methanol, 50% methanol, 70% methanol and methanol were used as extraction solvents respectively. The ellagic acid content extracted when 70% methanol was used as the extraction solvent was relatively high, so 70% methanol was selected as the extraction solvent.

[0174] 2.2 Investigation of different extraction methods

[0175] Take an appropriate amount of the formula granules, grind them finely, take about 0.3g, and divide them into 3 parallel groups, 2 portions each, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of 70% methanol, seal it tightly, weigh the weight, perform ultrasonic treatment (power 250W, frequency 40kHz), heat reflux and shake extraction for 30 minutes, let it cool, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate. Accurately take 2μl of each test solution, inject it into the liquid chromatograph, and determine the content of ellagic acid according to the chromatographic conditions of Example 1. The results are shown in Figure 2. Figure 8 , Table 17.

[0176] Table 17 Comparison of different extraction methods

[0177]

[0178]

[0179] The results show that the content of ellagic acid is the highest when the extraction method is heated and refluxed, so the extraction method is selected as heated and refluxed.

[0180] 2.3 Investigation of different extraction times

[0181] Take an appropriate amount of the formula granules, grind them finely, take about 0.3g, and divide them into 4 parallel groups, 2 portions each, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of 70% methanol, seal it tightly, weigh the weight, heat and reflux for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, let it cool, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate. Accurately take 2μl of each test solution, inject it into the liquid chromatograph, and determine the content of ellagic acid according to the chromatographic conditions of Example 1. The results are shown in Table 1. Figure 9 , Table 18.

[0182] Table 18 Comparison of different extraction times

[0183]

[0184] The results show that the ellagic acid content is relatively high when the extraction time is 30 minutes, so the extraction time is selected as 30 minutes.

[0185] 2.4 Investigation of different extraction volumes

[0186] Take an appropriate amount of the formula granules, grind them finely, take about 0.3g, and divide them into 3 parallel groups, 2 portions each, accurately weigh them, place them in stoppered conical flasks, accurately add 15ml, 25ml, and 50ml of 70% methanol respectively, stopper them tightly, weigh them, heat and reflux for 30 minutes, let them cool, weigh them again, make up the lost weight with 70% methanol, shake them well, filter them, and take the filtrate. Accurately take 2μl of each test solution, inject them into the liquid chromatograph, and determine the content of ellagic acid according to the chromatographic conditions of Example 1. The results are shown in Table 1. Figure 10 , Table 19.

[0187] Table 19 Comparison of different extraction volumes

[0188]

[0189] The results show that complete extraction can be achieved when the amount of extraction solvent is 25 ml, and the highest ellagic acid content is measured, so the amount of solvent added is selected to be 25 ml.

[0190] 2.5 Determination of the test solution preparation method

[0191] According to the above research results, the final method for preparing the sample for the determination of the content of Amaranthus serrata particles was determined as follows:

[0192] Take an appropriate amount of formula granules, grind them into powder, take about 0.3g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper tightly, weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0193] 3 Methodological validation

[0194] 3.1 Linearity

[0195] Accurately pipette 0.1 ml, 0.25 ml, 0.5 ml, 1.0 ml, and 5.0 ml of the ellagic acid reference stock solution (concentration of 405.1944 μg / ml) into 10 ml volumetric flasks, add methanol to the mark, shake well, and prepare reference solutions containing 4.0519 μg, 10.1299 μg, 20.2597 μg, 40.5194 μg, and 202.5972 μg of ellagic acid per ml, respectively. Accurately pipette the above reference solution and stock solution, inject 2 μl in sequence, and measure according to the chromatographic conditions of Example 1; plot a standard curve with the peak area integral value as the ordinate and the injection amount (μg) as the abscissa to obtain the regression equation for ellagic acid. Figure 14 , Tables 20 and 21.

[0196] Table 20 Regression equation

[0197]

[0198] Table 21 Relationship between injection volume and peak area of ​​ellagic acid reference substance

[0199]

[0200] The results showed that the injection amount of ellagic acid was in the range of 0.00810-0.81039 μg, and there was a good linear relationship between the peak area value.

[0201] 3.2 Precision test

[0202] 3.2.1 Instrument precision test

[0203] Take the Amaranthus tricolor granules and prepare a test solution according to the test solution preparation method in 2.5. Accurately pipette 2 μl and inject it into the liquid chromatograph. Determine according to the chromatographic conditions of Example 1. Continuously inject 6 times, record the measured value of the ellagic acid peak area, and calculate its RSD. The results are shown in Table 22 below.

[0204] Table 22 Instrument precision test

[0205]

[0206] The results showed that the RSD of the ellagic acid peak area was 0.17%, indicating that the instrument precision test was good.

[0207] 3.2.2 Repeatability test

[0208] Take an appropriate amount of Amaranthus chinensis granules, grind them into powder, take about 0.3 g, accurately weigh, and prepare 6 parallel portions. Prepare test solutions according to the test solution preparation method in 2.5, inject 2 μl of each, and determine the ellagic acid peak area value according to the chromatographic conditions of Example 1. Calculate its content and RSD. The results are shown in Table 23.

[0209] Table 23 Repeatability test results of samples

[0210]

[0211]

[0212] The results showed that the RSD of ellagic acid content was 0.81%, indicating that the repeatability of the test was good.

[0213] 3.2.3 Intermediate precision test

[0214] Take the Amaranthus tricolor formula granules, and two experimenters A and B prepare three test solutions according to the test solution preparation method in 2.5. 2 μl of each solution is injected at different times and on different instruments. The ellagic acid peak area value is determined according to the chromatographic conditions of Example 1, and its content and RSD are calculated. The results are shown in Table 24.

[0215] Table 24 Intermediate precision test

[0216]

[0217] The results showed that the RSD of ellagic acid content was 4.03%, and the intermediate precision test was good.

[0218] 3.3 Accuracy test

[0219] Take 0.15g of a sample with a known content (ellagic acid content: 3.16mg / g), a total of 9 parts, accurately weighed, and added three parts each of 50%, 100%, and 150% of the ellagic acid contained in 0.15g of the sample. Prepare the sample recovery solution according to the test solution preparation method in 2.5. According to the chromatographic conditions of Example 1, 2μl of the sample was injected respectively. The recovery rate and RSD were calculated using the following formula:

[0220] Table 25.

[0221]

[0222] Table 25 Accuracy results

[0223]

[0224]

[0225] The results showed that the recovery rate of ellagic acid content was between 95.28% and 100.95%, and the accuracy test was good.

[0226] 3.4 Durability test

[0227] 3.4.1 Stability test

[0228] Take the Amaranthus tricolor formula granules and prepare the test solution according to the preparation method of the test solution in 2.5. Inject 2 μl of the sample at 0, 2, 4, 8, 12, 18, and 24 hours after the preparation of the test solution. Determine the peak area value according to the chromatographic conditions of Example 1 and calculate its RSD. The results are shown in Table 26.

[0229] Table 26 Stability test results

[0230]

[0231] The results showed that the RSD of the ellagic acid peak area was 0.58%, indicating that the sample solution had good stability within 24 hours.

[0232] 3.4.2 Investigation of different flow rates

[0233] Take the Amaranthus chinensis formula granules and prepare the test solution according to the preparation method of the test solution in 2.5. According to the chromatographic conditions of Example 1, the ellagic acid content at three flow rates of 0.25ml / min, 0.30ml / min, and 0.35ml / min was investigated. The results are shown in FIG. Figure 13 , Table 27.

[0234] Table 27 Investigation of different flow rates

[0235]

[0236]

[0237] The results show that when the flow rate is in the range of 0.25ml / min to 0.35ml / min, small changes in the flow rate have no significant effect on the content determination of the sample, and the durability is good.

[0238] 3.4.3 Investigation of different column temperatures

[0239] Take the Amaranthus chinensis formula granules, prepare the test solution according to the preparation method of the test solution in 2.5, and investigate the ellagic acid content at three temperatures of 30°C, 35°C, and 40°C according to the chromatographic conditions of Example 1. Figure 12 , Table 28.

[0240] Table 28 Investigation of different column temperatures

[0241]

[0242] The results showed that when the column temperature was within the range of 30℃~40℃, small changes in column temperature had no significant effect on the content determination of the sample, and the column had good durability.

[0243] 3.4.4 Investigation of different chromatographic columns

[0244] Take the same batch number sample and prepare the test solution according to the preparation method of the test solution in 2.5. According to the chromatographic conditions of Example 1, three different chromatographic columns, ZORBAX SB-C18 RRHD (Agilent, 2.1mm×100mm, 1.8μm); ACQUITY UPLCBEH C18 (Waters, 2.1mm×100mm, 1.7μm); Kinetex XB-C18 (phenomenex, 2.1mm×100mm, 1.7μm), were used to measure the content of ellagic acid. The results are shown in Figure 2. Figure 11 , Table 29.

[0245] Table 29 Comparison of different chromatographic columns

[0246]

[0247] The results showed that the three different types of chromatographic columns had good separation of target components, similar ellagic acid contents, and good durability.

Claims

1. A method for controlling the quality of Amaranthus chinensis, characterized in that: The following steps are involved: (1) Prepare the test solution; use Amaranthus tricolor as the control medicinal material and prepare the control medicinal material reference solution; Gallic acid, corilagin, and ellagic acid were used as reference substances to prepare reference substance solutions; The extractants for preparing reference substance solutions and test sample solutions are water, methanol, or a methanol-water solution with a volume concentration of 30% to 70%; The test sample is Amaranthus chinensis medicinal material, Amaranthus chinensis decoction pieces or Amaranthus chinensis formula granules; (2) The control medicinal material reference solution, the reference substance reference solution and the test solution were respectively taken for ultra-high performance liquid chromatography analysis to construct the corresponding characteristic spectrum. The six characteristic peaks in the characteristic spectrum of the test solution corresponding to the retention time in the characteristic chromatogram of the control medicinal material solution were selected as common characteristic peaks to construct the characteristic spectrum of Amaranthus trichoderma, wherein characteristic peak 1 corresponds to the peak of the gallic acid reference substance, characteristic peak 4 corresponds to the peak of the corilagin reference substance, and characteristic peak 6 corresponds to the peak of the ellagic acid reference substance; The conditions for the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel with a particle size of 1.7 or 1.8 μm; acetonitrile is used as mobile phase A, and 0.05% to 0.2% by volume phosphoric acid solution is used as mobile phase B, and gradient elution is adopted: 0-1 min, 5% A, 1-3 min, 5→10% A, 3-9 min, 10→12% A, 9-16 min, 12→18% A, 16-20 min, 18→35% A, 20-21 min, 35→5% A, 21-26 min, 5% A; The detection wavelength was 300 nm, using an ultraviolet detector; (3) Perform qualitative analysis on the test solution using the characteristic spectrum obtained in step (2).

2. The quality control method of Amaranthus chinensis according to claim 1, characterized in that: In preparing the reference substance solution, the amount of extractant used per gram of Amaranthus chinensis is 15ml~160ml, the extraction time is 15~60 minutes, and the extraction method is heating reflux.

3. The quality control method of Amaranthus chinensis according to claim 1, characterized in that: In step (1), the concentration of the gallic acid reference substance solution is 20-100 μg / ml; the concentration of the corilagin reference substance solution is 20-100 μg / ml; and the concentration of the ellagic acid reference substance solution is 4-400 μg / ml.

4. The quality control method of Amaranthus chinensis according to claim 1, characterized in that: The flow rate is 0.25ml~0.35ml per minute; the column temperature is 35℃~40℃.

5. The quality control method of Amaranthus chinensis according to claim 1, characterized in that: In the characteristic spectrum of Amaranthus tricolor constructed in step (2), the characteristic peak corresponding to the corilagin reference substance is taken as the S peak, and the relative retention time of the other characteristic peaks and the S peak is calculated; the relative retention time should be within ±10% of the specified value, and the specified value is: Peak 2: 0.81, Peak 3: 0.91, Peak 5: 1.

21.

6. The quality control method of Amaranthus chinensis according to claim 1, characterized in that: Step (3) also includes a quantitative analysis step: using the content of ellagic acid as a content indicator component, and simultaneously detecting the content of ellagic acid in the test solution.

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