HPLC characteristic chromatogram construction method of acalypha australis medicinal material, decoction pieces, standard decoction and formula granules thereof

Abstract

The invention provides an HPLC characteristic chromatogram construction method for acalypha australis medicinal materials, decoction pieces, standard decoction and formula granules thereof, which comprises the following steps: A) extracting an acalypha australis raw material by adopting a solvent to obtain a to-be-detected solution; b) determining the liquid to be detected by adopting a high performance liquid chromatography to obtain an HPLC characteristic chromatogram of the acalypha australis raw material; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; a mobile phase A is methanol, a mobile phase B is a 0.1% phosphoric acid solution, and gradient elution is carried out. According to the method, a high performance liquid chromatography is adopted, the mobile phase is selected for gradient elution, gallic acid and ellagic acid are used as reference substances, the HPLC characteristic chromatogram of the copperleaf herb medicinal material, decoction pieces, standard decoction and formula granules thereof is established, repeatability and precision are good, the method is stable and reliable, and the method is suitable for industrial production. The quality of acalypha australis medicinal materials, decoction pieces, standard decoction and formula granules thereof can be controlled.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for constructing an HPLC characteristic spectrum of a Amaranthus tricolor medicinal material, a decoction piece, a standard decoction and formula particles thereof. Background Art

[0002] Acalypha australis is the dried aerial part of Acalypha australis L., a plant of the Euphorbiaceae family. It is harvested in summer and autumn, impurities are removed, and it is dried in the sun. It has the effects of clearing away heat and detoxifying, promoting dampness, astringing and stopping bleeding, and is often used for enteritis, dysentery, hematemesis, epistaxis, etc.

[0003] In order to ensure the uniformity and stability of the quality of Amaranthus tricolor medicinal materials, standard decoctions of medicinal pieces and their formula granules, a new characteristic spectrum method was established in this paper to control their quality. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic spectra of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula granules. The HPLC characteristic spectra method of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula granules constructed by the present invention is stable and reliable, and can control the quality of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula granules.

[0005] The present invention provides a method for constructing HPLC characteristic spectrum of Amaranthus tricolor medicinal materials, decoction pieces, standard decoction and formula particles thereof, comprising:

[0006] A) extracting the raw material of Amaranthus tricolor with a solvent to obtain a solution to be tested;

[0007] B) the test solution is measured by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the Amaranthus tricolor raw material;

[0008] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is a 0.1% phosphoric acid solution, and the elution is performed in a gradient manner;

[0009] The gradient elution is specifically:

[0010] 0-7min, phase A: 12%→30%, phase B: 88%→70%;

[0011] 7-14 min, phase A: 30%→38%, phase B: 88%-62%;

[0012] 14-19 min, phase A: 38%-53%, phase B: 62%-47%;

[0013] 19-25min, phase A: 53%-69%, phase B: 47%-31%.

[0014] The invention provides a method for constructing HPLC characteristic spectrum of Amaranthus tricolor medicinal material, decoction pieces, standard decoction and its formula particles. Firstly, Amaranthus tricolor raw material is taken and solvent extraction is performed to obtain a test solution. The solvent is preferably 50% methanol.

[0015] According to the present invention, the Amaranthus tricolor raw materials are Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and formula granules thereof.

[0016] In the present invention, the preparation of the test solution of Amaranthus tricolor medicinal material / cooked slices is specifically as follows: after boiling the Amaranthus tricolor raw material with water, filtering, dissolving the residue with 50% methanol, ultrasonicating, cooling, shaking, filtering, and obtaining the product. The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 30 to 40 minutes; preferably, the extraction is 30 minutes; the ratio of the mass g of the Amaranthus tricolor raw material to the volume mL of water is 1:50.

[0017] In the present invention, the preparation of the test solution of the standard decoction / formulated granules of Amaranthus tricolor is specifically as follows:

[0018] The Amaranthus truncatus raw material is ultrasonically extracted with 50% methanol, cooled, shaken, filtered, and the filtrate is obtained; the ultrasonic power is 600W, the frequency is 40kHz; the extraction time is 30 to 40min; the ratio of the mass g of the Amaranthus truncatus raw material to the volume mL of 50% methanol is 0.1:25.

[0019] The present invention adopts the above-mentioned extraction solvent, and the chromatographic peak information volume is large, the extraction efficiency is high, and the effect is good; at the same time, when the amount of the extraction solvent added is the above-mentioned ratio, the peak shape and separation degree of each chromatographic peak are good, and the peak size is appropriate.

[0020] The raw material of Amaranthus chinensis described in the present invention is Amaranthus chinensis medicinal material, Amaranthus chinensis decoction pieces or standard decoction, formula granules. The present invention does not limit it, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0021] The present invention also includes preparing reference substances and reference medicinal material reference solutions:

[0022] Take gallic acid reference substance and ellagic acid reference substance respectively, add water and use 50% methanol to dissolve them to obtain reference substance solutions;

[0023] The concentration of the gallic acid reference substance is 100 μg / mL; the concentration of the ellagic acid reference substance is 100 μg / mL;

[0024] Take the Amaranthus tricolor reference medicinal material, add water to boil, filter, dissolve the residue with 50% methanol, sonicate, cool, filter, and obtain a reference medicinal material solution;

[0025] The reference substance and reference medicinal material reference substance solutions are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference substance, respectively; and the components of the HPLC characteristic spectra of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively measured based on the chromatograms of the reference substance and the reference medicinal material reference substance.

[0026] The mobile phase A of the present invention is methanol, and the mobile phase B is 0.1% phosphoric acid solution.

[0027] The inventors found that the above mobile phase has a stable chromatogram baseline, more chromatographic peaks and a good peak shape compared with other mobile phases.

[0028] The gradient elution of the present invention is preferably specifically:

[0029] 0-7min, phase A: 12%→30%, phase B: 88%→70%;

[0030] 7-14 min, phase A: 30%→38%, phase B: 88%-62%;

[0031] 14-19 min, phase A: 38%-53%, phase B: 62%-47%;

[0032] 19-25min, phase A: 53%-69%, phase B: 47%-31%.

[0033] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0034] C18 column, specification: 5μm, 4.6×250mm; column temperature: 40℃.

[0035] The mobile phase flow rate is preferably 1 ml / min.

[0036] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 1 ml / min, which was the most preferred solution.

[0037] The detection wavelength of the present invention is preferably 260 nm.

[0038] The inventors found that at 260 nm, the chromatographic information was rich, each component had good absorption, the response value was moderate, the peak separation was good, and the baseline was stable.

[0039] The injection volume of the present invention is preferably 10 μL.

[0040] The beneficial effect of the present invention is that the similarity of HPLC characteristic spectra of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula granules is evaluated by using a Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula granules consisting of 6 characteristic peaks is obtained, wherein Peak 1 (S): gallic acid; Peak 2: protocatechuic acid; Peak 3: corilagin; Peak 6: ellagic acid.

[0041] In the characteristic spectrum of the standard decoction of Amaranthus tricolor, gallic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), and 2.58 (peak 5).

[0042] Quality judgment standard: Take samples of Amaranthus oleraceus medicinal materials, decoction pieces, standard decoctions and their formula granules, and operate according to the same method as above to obtain the characteristic spectra of Amaranthus oleraceus medicinal materials, decoction pieces, standard decoctions and their formula granules. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) of the National Pharmacopoeia Committee to analyze the standard characteristic spectra and sample characteristic spectra of Amaranthus oleraceus medicinal materials, decoction pieces, standard decoctions and their formula granules, and the similarity is greater than 0.90.

[0043] The method provided by the present invention can effectively monitor the quality of different batches of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and formula granules thereof, so as to stabilize the quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensively monitoring the quality of products.

[0044] The characteristic spectrum of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and formula granules thereof established by the present invention takes gallic acid and ellagic acid as references, focuses on the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the products, and the method is scientific and reliable.

[0045] The newly developed characteristic spectrum method of the present invention can detect components with greater polarity in Amaranthus tricolor and its standard decoction. The test sample preparation method is simple and easy to operate, and relatively more characteristic peaks can be identified. Accurate and reliable characteristic spectrum detection can be performed on Amaranthus tricolor and its preparations. The authenticity, quality consistency and stability of Amaranthus tricolor and its preparations can be effectively detected and controlled. It provides a basis for effectively controlling and more comprehensively evaluating the quality of Amaranthus tricolor standard decoction. It ensures the uniformity and stability of the quality of Amaranthus tricolor and its standard decoction.

[0046] The present invention provides a method for constructing an HPLC characteristic spectrum of amaranth medicinal material, a decoction piece, a standard decoction and a formula granule thereof, comprising: A) extracting the raw material of amaranth with a solvent to obtain a test liquid; B) determining the test liquid with a high performance liquid chromatography method to obtain an HPLC characteristic spectrum of the raw material of amaranth; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is a 0.1% phosphoric acid solution, and the gradient elution is performed. The present invention adopts high performance liquid chromatography, selects the above mobile phase for gradient elution, and uses gallic acid and ellagic acid as reference substances to establish an HPLC characteristic spectrum of amaranth medicinal material, a decoction piece, a standard decoction and a formula granule thereof, which has good repeatability and precision, and the method is stable and reliable, and the quality of the amaranth medicinal material, a decoction piece, a standard decoction and a formula granule thereof can be controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Mobile phase selection; Figure 2 Chromatograms of Amaranthus tricolori in different wavelengths;

[0048] Figure 3 Column temperature investigation;

[0049] Figure 4 Flow rate investigation;

[0050] Figure 5 Delayed inspection;

[0051] Figure 6 Investigation of extraction methods;

[0052] Figure 7 Investigation of extraction solvents; Figure 8 Extraction time investigation;

[0053] Fig. 9 Investigation of solvent addition amount;

[0054] Fig.10 Chromatographic peak identification;

[0055] Fig.11 Spectrum of Gallic Acid - Medicinal Herbs;

[0056] Fig.12 Spectrum of Gallic Acid - Reference;

[0057] Fig.13 Protocatechuic acid spectrum - medicinal materials;

[0058] Fig.14 Protocatechuic acid spectrum - reference;

[0059] Fig.15 Corilagin Spectrum - Medicinal Herbs;

[0060] Fig.16 Spectrum of Corilagin - Reference;

[0061] Fig.17 Ellagic acid spectrum - medicinal materials;

[0062] Fig.18 Ellagic acid spectrum - reference;

[0063] Fig.19 Different instrument surveys.

[0064] Fig. 20 Investigation of different chromatographic columns.

[0065] Fig.21 Amaranthus tricolor medicinal material characteristic spectrum;

[0066] Fig. 22 Control feature map;

[0067] Fig.23 Characteristic spectrum of Amaranthus tricolor decoction pieces;

[0068] Fig.24 Comparative characteristic spectrum of Amaranthus tricolor decoction pieces;

[0069] Fig.25 Mobile phase selection.

[0070] Fig.26 Chromatograms of Amaranthus tricolor soup at different wavelengths;

[0071] Fig. 27 Column temperature investigation

[0072] Fig.28 Flow rate inspection.

[0073] Fig.29 Delayed inspection.

[0074] Fig.30 Investigation of extraction methods;

[0075] Fig.31 Investigation of extraction solvents.

[0076] Fig.32 Extraction time investigation

[0077] Fig.33 Investigation of solvent addition amount.

[0078] Fig.34 Chromatographic peak identification;

[0079] Fig.35 Gallic acid - standard decoction;

[0080] Fig.36 Gallic acid-reference substance;

[0081] Fig.37 Protocatechuic acid - standard decoction;

[0082] Fig.38 Protocatechuic acid-reference substance;

[0083] Fig.39 Corilagin - standard decoction;

[0084] Fig.40 Corilagin - control;

[0085] Fig.41 Ellagic acid - standard decoction;

[0086] Fig.42 Ellagic acid-reference substance;

[0087] Fig.43 Different instrument surveys.

[0088] Fig.44 Investigation of different chromatographic columns.

[0089] Fig.45 Characteristic spectrum of standard decoction of Amaranthus tricolor

[0090] Fig.46 Control feature map;

[0091] Fig.47 Mobile phase selection.

[0092] Fig.48 Chromatograms of Amaranthus tricolori formula granules at different wavelengths;

[0093] Fig.49 Column temperature investigation

[0094] Fig.50 Flow rate inspection.

[0095] Fig.51 Delayed inspection.

[0096] Fig.52 Investigation of extraction methods.

[0097] Fig.53 Investigation of extraction solvents;

[0098] Fig.54 Extraction time investigation;

[0099] Fig.55 Investigation of solvent addition amount;

[0100] Fig.56 Chromatographic peak identification;

[0101] Fig.57 Gallic acid-formulated granules;

[0102] Fig.58 Gallic acid-reference substance;

[0103] Fig.59 Protocatechuic acid-formulated granules;

[0104] Fig.60 Protocatechuic acid-reference substance;

[0105] Fig.61 Corilagin - Formula Granules;

[0106] Fig.62 Corilagin - control;

[0107] Fig.63 Ellagic acid-formulated granules;

[0108] Fig.64 Ellagic acid-reference substance;

[0109] Fig.65 Different instrument surveys.

[0110] Fig.66 Investigation of different chromatographic columns.

[0111] Fig.67 Characteristic spectrum of Amaranthus tricolor formula granules.

[0112] Fig.68 Control feature map;

[0113] Fig.69 This is the chromatogram of the medicinal material particles in Comparative Example 1.

[0114] Fig.70 This is the result diagram of comparative example 2.

[0115] Fig.71 This is the result diagram of Comparative Example 3. DETAILED DESCRIPTION

[0116] The present invention provides a method for constructing HPLC characteristic spectra of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula particles. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0117] To further illustrate the present invention, the following describes in detail a method for constructing an HPLC characteristic spectrum of a Amaranthus tricolor medicinal material, a decoction piece, a standard decoction and a formula granule thereof provided by the present invention in combination with an embodiment.

[0118] Example 1 HPLC characteristic spectrum of Amaranthus tricolor medicinal materials and decoction pieces

[0119] 2.2.1.1 Materials, reagents and instruments

[0120] 2.2.1.1.1 Instruments and materials

[0121] Ultra-high performance liquid chromatograph: Shimadzu LC-20AD high performance liquid chromatograph (instrument 1), Agilent 1260 high performance liquid chromatograph (instrument 2), Thermo Fisher Vanquish Core high performance liquid chromatograph (instrument 3);

[0122] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0123] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0124] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0125] Chromatographic columns: ZORBAX Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 1), Luna C18 (2) 100A, 4.6 mm × 250 mm, 5 μm (chromatographic column 2), Agilent 5TC-C18 (2) 4.6 mm × 250 mm, 5 μm (chromatographic column 3).

[0126] 2.2.1.1.2 Reagents and test drugs

[0127] Methanol and phosphoric acid were of chromatographic grade; water was ultrapure water, and other reagents were of analytical grade;

[0128] Gallic acid reference substance (China Food and Drug Inspection Institute, batch number: 110831-201906, purity: 91.5%);

[0129] Corilagin reference substance (China Food and Drug Control Institutes, batch number: 111623-200302, purity: 100%);

[0130] Ellagic acid reference substance (China Food and Drug Inspection Institute, batch number: 111959-201903, purity: 88.8%);

[0131] Protocatechuic acid reference substance (China Food and Drug Inspection Institute, batch number: 110809-202207, purity: 97.5%);

[0132] Amaranthus tricolor control medicinal material (China Food and Drug Inspection Institute, batch number: 121655-201502);

[0133] Batch numbers of Amaranthus tricolor medicinal materials: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16.

[0134] Batch numbers of Amaranthus tricolor slices: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16.

[0135] 2.2.1.2 Characteristic spectrum determination method

[0136] The chromatographic conditions and system suitability test determined the conditions for the medicinal material of Amaranthus tricolor based on the characteristic chromatographic conditions of the standard decoction of Amaranthus tricolor: octadecylsilane bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); methanol is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, gradient elution is performed according to the provisions in the following table, and the flow rate is 1.0 ml per minute; the column temperature is 40°C; the detection wavelength is 260m, and the number of theoretical plates calculated based on the gallic acid peak should be not less than 5000.

[0137]

[0138] Preparation of reference solution Take 1g of Amaranthus tricolor reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 25ml of 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of gallic acid and ellagic acid reference substances, accurately weigh, add 50% methanol to make a solution containing 100μg per 1ml, as the reference substance solution.

[0139] Preparation of the test solution: Take 1.0 g of Amaranthus tricolor powder (passed through No. 3 sieve), add 50 mL of water, boil for 30 minutes, filter, evaporate to dryness, add 25 ml of 50% methanol to the residue, seal, and treat with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0140] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0141] 2.2.1.3 Chromatographic conditions and system suitability test

[0142] 2.2.1.3.1 Mobile phase selection

[0143] Based on the experimental conditions proposed above, methanol was used as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Figure 1 . Figure 1 Selection of mobile phase: The results showed that the chromatogram baseline was relatively stable under the conditions of methanol-0.1% phosphoric acid solution gradient elution, so methanol-0.1% phosphoric acid solution gradient elution was used as the mobile phase for the characteristic spectrum determination method of Amaranthus trichosanthis.

[0144] 2.2.1.3.2 Wavelength selection

[0145] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution at wavelengths of 220nm, 240nm, 260nm, 280nm, 300nm, and 320nm were extracted respectively. Figure 2 . Figure 2 Chromatograms of Amaranthus tricolor at different wavelengths (from top to bottom: 220nm, 240nm, 280nm, 300nm, 320nm, 260nm). The results show that when the detection wavelength is 260nm, the chromatographic peak information is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 260nm.

[0146] 2.2.1.3.3 Column temperature investigation

[0147] Based on the experimental conditions proposed above, the column temperatures of 35°C, 40°C, and 45°C were investigated. Figure 3 .

[0148] Figure 3 Column temperature investigation: The results show that when the column temperature is 40℃, the chromatogram peak shape is more symmetrical, the separation is better, and the peaks are more complete. Therefore, the column temperature is determined to be 40℃.

[0149] 2.2.1.3.4 Flow rate investigation

[0150] Based on the experimental conditions proposed above, the flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min were investigated. Figure 4 . Figure 4 Flow rate investigation: The results show that when the flow rate is 1.0 mL / min, the chromatogram peak shape is better and the separation is moderate. Therefore, the flow rate is determined to be 1.0 mL / min.

[0151] 2.2.1.3.5 Delay Investigation

[0152] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Figure 5 . Figure 5 Delayed inspection.

[0153] The results showed that the sample had almost no chromatographic peak after 25 minutes, so the sample detection time was set at 25 minutes.

[0154] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of Amaranthus tricolor were determined as follows: methanol was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed as specified in the following table, the flow rate was 1.0 ml per minute; the column temperature was 40°C; the detection wavelength was 260m, and the number of theoretical plates calculated based on the gallic acid peak should be no less than 5000.

[0155]

[0156] 2.2.1.4 Preparation of test solution

[0157] 2.2.1.4.1 Investigation of extraction methods

[0158] Take 1g of Amaranthus tricolor powder (passed through No. 3 sieve, batch number: YC-16), put it in a stoppered conical flask, add 25mL of 50% methanol and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes and add 50mL of water, boil for 30 minutes, let cool, filter, evaporate the filtrate, add 25mL of 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain. Figure 6 . Figure 6 The extraction method was investigated; the results showed that when the test sample was first decocted and then ultrasonically extracted, the peaks of the decoction were better separated, so the extraction method of the test sample was determined to be decocting first and then ultrasonically extracted.

[0159] 2.2.1.4.2 Investigation of extraction solvent

[0160] Take 1g of Amaranthus tricolor powder (passed through No. 3 sieve, batch number: YC-16), put it in a stoppered conical flask, add 50mL of water, boil for 30 minutes, filter, evaporate to dryness, add 25ml of 30% methanol, 70% methanol, methanol, 50% ethanol, ethanol, water, 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Figure 7 . Figure 7 Investigation of extraction solvent; the results showed that when the extraction solvent was 50% methanol, the peak shapes of the characteristic peaks were good and the separation was moderate. The extraction solvent was temporarily set to 50% methanol.

[0161] 2.2.1.4.3 Extraction time investigation

[0162] Take 1g of Amaranthus tricolor powder (passed through No. 3 sieve, batch number: YC-16), put it in a stoppered conical flask, add 50mL of water, boil for 30 minutes, filter, evaporate to dryness, add 25ml of 50% methanol to the residue, and treat the sample with ultrasound (power 600W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively, cool, shake well, filter, and take the filtrate to obtain. Figure 8 . Figure 8 The results of the extraction time showed that the peak shape and separation of the chromatogram were better when the extraction time was 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0163] 2.2.1.4.4 Investigation of solvent addition amount

[0164] Take 1g of Amaranthus tricolor powder (passed through No. 3 sieve, batch number: YC-16), put it in a stoppered conical flask, add 50mL of water, boil for 30 minutes, filter, evaporate to dryness, add 10ml, 25ml and 50ml of 50% methanol to the residue, seal it, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain. Fig. 9 . Fig. 9 The amount of solvent added was investigated; the results showed that when the amount of extraction solvent added was 25 ml, the peak shapes and separation of each chromatographic peak were better, so 25 ml of solvent was selected.

[0165] In summary, the preparation method of the test solution of the characteristic spectrum of Amaranthus tricolor medicinal material is determined as follows: take 1 g of Amaranthus tricolor medicinal material powder (passed through No. 3 sieve), put it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 50% methanol to the residue, seal it, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain.

[0166] 2.2.1.5 Methodological investigation

[0167] 2.2.1.5.1 Chromatographic peak identification

[0168] Preparation of test solution: According to the experimental conditions determined above, prepare the test solution of Amaranthus tricolor medicinal material.

[0169] Preparation of reference solution: Take 1 g of Amaranthus tricolor reference medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 25 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0170] Take appropriate amount of gallic acid reference substance, protocatechuic acid reference substance, corilagin reference substance and ellagic acid reference substance, weigh accurately, add 50% methanol to make a solution containing 100 μg per 1 ml, which serves as the reference substance solution.

[0171] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of iron-deficient Amaranthus medicinal material.

[0172] Locate the characteristic spectrum peaks of Amaranthus tricolor. Figure 10-18 . Fig.10 Chromatographic peak identification; Fig.11 Spectrum of Gallic Acid - Medicinal Herbs; Fig.12 Spectrum of Gallic Acid - Reference; Fig.13 Protocatechuic acid spectrum - medicinal materials; Fig.14 Protocatechuic acid spectrum - reference; Fig.15 Corilagin Spectrum - Medicinal Herbs; Fig.16 Spectrum of Corilagin - Reference; Fig.17 Ellagic acid spectrum - medicinal materials; Fig.18 Spectrum of ellagic acid - reference.

[0173] The results showed that peak 1 was gallic acid, peak 2 was protocatechuic acid, peak 3 was corilagin, and peak 6 was ellagic acid.

[0174] 2.2.1.5.2 Precision test

[0175] Take the test solution of Amaranthus tricolor (batch number: YC-16) and inject it 6 times continuously according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 1.

[0176] Table 1 Precision investigation - retention time

[0177]

[0178] The results showed that the RSD of the retention time of each characteristic peak was 0.04%-0.14%, and the precision of the instrument was good.

[0179] 2.2.1.5.3 Repeatability study

[0180] Six samples of Amaranthus tricolor (batch number: YC-16) were prepared and tested according to the proposed experimental method. See Table 2.

[0181] Table 2 Repeatability study - relative retention time

[0182]

[0183] The results showed that the RSD of the relative retention time of the six samples was 0.08% to 0.24%, indicating that the method had good repeatability.

[0184] 2.2.1.5.4 Intermediate precision study

[0185] 2.2.1.5.4.1 Investigation with different instruments

[0186] Based on the above experimental conditions, the medicinal materials of Amaranthus tricolor (batch number: YC-16) were weighed to prepare the test solution, which was measured on Shimadzu LC-20AD HPLC (Instrument 1), Agilent 1260 HPLC (Instrument 2), and Thermo Fisher Vanquish Core HPLC (Instrument 3). The relative retention time of each characteristic peak was calculated. Fig.19 , see Table 3. Fig.19 Different instrument surveys.

[0187] Table 3 Instrument durability investigation - relative retention time

[0188]

[0189] The results showed that when the three instruments were used to detect the test samples, the RSD of the relative retention time of each characteristic peak was 1.73%-5.17%, indicating that the instruments had good durability.

[0190] 2.2.1.5.4.2 Investigation by different personnel and time

[0191] Based on the above experimental conditions, different personnel (A, B) weighed two portions of Amaranthus tricolor (batch number: YC-16) at different times (T1, T2) to prepare the test sample and conduct the determination. The relative retention time of each characteristic peak was calculated. See Table 4.

[0192] Table 4 Personnel and time inspection-relative retention time

[0193]

[0194] The results showed that when different people measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.03% to 0.07%, and the method had good stability.

[0195] 2.2.1.5.5 Durability test

[0196] 2.2.1.5.5.1 Study on the durability of chromatographic columns

[0197] Based on the experimental conditions proposed above, ZORBAX Eclipse XDB-C18, 4.6mm×250mm, 5μm (chromatographic column 1), Luna C18(2)100A, 4.6mm×250mm, 5μm (chromatographic column 2), Agilent 5TC-C18(2)4.6mm×250mm, 5μm (chromatographic column 3) were used for analysis and investigation, and the relative retention time of each characteristic peak was calculated. The results are shown in Fig. 20 , Table 5. Fig. 20 Investigation of different chromatographic columns.

[0198] Table 5 Column durability investigation - relative retention time

[0199]

[0200] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 1.91% and 5.87% when the three chromatographic columns were used to detect the samples, indicating that the chromatographic columns had good durability.

[0201] 2.2.1.5.5.2 Stability test

[0202] Based on the above experimental conditions, take the same test solution and measure it at 0h, 2h, 4h, 8h, 16h, and 24h. Calculate the retention time of each characteristic peak 6. See Table 6.

[0203] Table 6 Stability Study - Retention Time

[0204]

[0205] The results showed that the RSD of the characteristic peak retention time was between 0.14% and 0.26%, and the sample solution was stable within 24 hours.

[0206] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 6 characteristic peaks were included in the subsequent investigation.

[0207] 2.2.1.6 Verification of the characteristic spectrum of Amaranthus tricolor

[0208] The characteristic spectrum of 16 batches of Amaranthus australis was analyzed and the relative retention time was calculated. The results are shown in Fig.21 , Table 7. Fig.21 Characteristic spectrum of Amaranthus tricolor medicinal material; Peak 1 (S): gallic acid; Peak 2: protocatechuic acid; Peak 3: corilagin; Peak 6: ellagic acid (S1-S16 are: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16 respectively).

[0209] Table 7 Relative retention time of characteristic spectrum of Amaranthus tricolor

[0210]

[0211] According to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peak is relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative peak area RSD of the characteristic peaks of medicinal materials was too different to specify the relative peak area, so it was not included in the quality standard text.

[0212] The relative retention times of the 16 batches of Amaranthus tricolor medicinal materials were all within the range of ±10% of the mean relative retention time of the 3 batches of Amaranthus tricolor granules, so the mean relative retention time of the 3 batches of Amaranthus tricolor granules was used as the prescribed value of Amaranthus tricolor medicinal materials. See Table 8.

[0213] Table 8 Relative retention time of Amaranthus chinensis formula granules

[0214]

[0215]

[0216] Final regulations: There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1 and peak 6 should correspond to the retention time of the peaks of the reference material of gallic acid and ellagic acid, respectively. The peak corresponding to the gallic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), 2.58 (peak 5).

[0217] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of Amaranthus chinensis medicinal materials, and a reference characteristic spectrum of Amaranthus chinensis medicinal materials was established. Fig. 22 . Fig. 22 Reference characteristic spectrum; Peak 1 is gallic acid, Peak 2 is protocatechuic acid, Peak 3 is corilagin, Peak 6 is ellagic acid (ZORBAX Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 1)).

[0218] 2.2.1.7 Verification of characteristic spectrum of Amaranthus australis decoction pieces

[0219] According to the characteristic spectrum determination method, chromatographic conditions and system suitability in 2.2.1.2, 16 batches of test sample solutions of Amaranthus australis slices were prepared, and characteristic spectrum analysis was performed to calculate the relative retention time and relative peak area ratio. Fig.23 , Table 9. Fig.23Characteristic spectrum of Amaranthus tricolor slices; Peak 1 (S): Gallic acid; Peak 2: Protocatechuic acid; Peak 3: Corilagin; Peak 6: Ellagic acid; (S1-S16 are: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16)

[0220] Table 9 Relative retention time of characteristic spectra of decoction pieces

[0221]

[0222] According to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peaks are relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative peak area RSD of the characteristic peaks of the decoction pieces was too different to specify the relative peak area, so it was not included in the main text of the quality standard.

[0223] The relative retention times of the 16 batches of Amaranthus chinensis slices were all within the range of ±10% of the mean relative retention time of the 3 batches of Amaranthus chinensis granules. Therefore, the mean relative retention time of the 3 batches of Amaranthus chinensis granules was used as the prescribed value for Amaranthus chinensis slices. See Table 10.

[0224] Table 10 Relative retention time of Amaranthus chinensis formula granules

[0225]

[0226] Final regulations: There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1 and peak 6 should correspond to the retention time of the peaks of the reference material of gallic acid and ellagic acid, respectively. The peak corresponding to the gallic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), 2.58 (peak 5).

[0227] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of Amaranthus herba slices, and a reference characteristic spectrum of Amaranthus herba slices was established. Fig.24 . Fig.24 Reference spectrum; Peak 1 (S): Gallic acid; Peak 2: Protocatechuic acid; Peak 3: Corilagin; Peak 6: Ellagic acid (ZORBAX Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 1))

[0228] 2.2.1.8 Determination of characteristic spectra of Amaranthus tricolor medicinal materials and decoction pieces

[0229] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table, flow rate: 1.0 ml per minute; column temperature: 40°C; detection wavelength: 260 m, theoretical plate number calculated based on gallic acid peak should be not less than 5000.

[0230]

[0231] Preparation of reference solution Take 1g of Amaranthus tricolor reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 25ml of 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of gallic acid reference substance, accurately weigh it, add 50% methanol to make a solution containing 100μg per 1ml, as the reference substance solution.

[0232] Preparation of the test solution: Take 1.0 g of Amaranthus tricolor powder (passed through No. 3 sieve), add 50 mL of water, boil for 30 minutes, filter, evaporate to dryness, add 25 ml of 50% methanol to the residue, seal, and treat with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0233] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0234] Final regulations: The test sample characteristic spectrum should show 6 characteristic peaks, and should correspond to the retention time of the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1 and peak 6 should correspond to the retention time of the reference peaks of gallic acid and ellagic acid, respectively. The peak corresponding to the gallic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), 2.58 (peak 5). Fig.24 Comparative characteristic spectrum of Amaranthus tricolor decoction pieces; Peak 1 (S): Gallic acid; Peak 2: Protocatechuic acid; Peak 3: Corilagin; Peak 6: Ellagic acid.

[0235] Example 2 HPLC characteristic spectrum of Amaranthus tricolor standard decoction

[0236] 2.2.2.1 Experimental instruments and materials

[0237] Batch numbers of standard decoction of Amaranthus tricolor: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16.

[0238] The rest is the same as in Example 1.

[0239] 2.2.2.2 Characteristic spectrum determination method

[0240] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table, flow rate: 1.0 ml per minute; column temperature: 40°C; detection wavelength: 260 m, theoretical plate number calculated based on gallic acid peak should be not less than 5000.

[0241]

[0242] Preparation of reference solution Take 1g of Amaranthus tricolor reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 25ml of 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of gallic acid and ellagic acid reference substances, accurately weigh, add 50% methanol to make a solution containing 100μg of each per 1ml, as the reference substance solution.

[0243] Preparation of the test solution: Take 0.1 g of standard decoction of Amaranthus tricolor, place it in a stoppered conical flask, add 25 ml of 50% methanol, stopper it, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the test solution.

[0244] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0245] 2.2.2.3 Chromatographic conditions and system suitability test

[0246] 2.2.2.3.1 Mobile phase selection

[0247] Based on the experimental conditions proposed above, the separation effect of using methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B was investigated. Fig.25 . Fig.25 Mobile phase selection.

[0248] The results showed that the chromatogram baseline was relatively stable under the condition of methanol-0.1% phosphoric acid solution gradient elution. Therefore, methanol-0.1% phosphoric acid solution gradient elution was used as the mobile phase for the determination of the characteristic spectrum of the standard decoction of Amaranthus tricolor.

[0249] 2.2.2.3.2 Wavelength selection

[0250] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution at wavelengths of 220nm, 240nm, 260nm, 280nm, 300nm, and 320nm were extracted respectively. Fig.26 . Fig.26 Chromatograms of Amaranthus tricolori soup at different wavelengths; (from top to bottom: 220nm, 240nm, 280nm, 300nm, 320nm, 260nm).

[0251] The results showed that when the detection wavelength was 260 nm, the chromatographic peak had more information and the chromatogram baseline was more stable, so the detection wavelength was determined to be 260 nm.

[0252] 2.2.2.3.3 Column temperature investigation

[0253] Based on the experimental conditions proposed above, the column temperatures of 35℃, 40℃ and 45℃ were investigated respectively. Fig.28 , Tables 11-12. Fig. 27 Column temperature inspection.

[0254] Table 11 Column temperature investigation-retention time

[0255]

[0256] Table 12 Column temperature investigation-relative retention time

[0257]

[0258] The results show that when the column temperature is 40℃, the chromatogram peak shape is more symmetrical, the separation is better, and the peaks are more complete. Therefore, the column temperature is determined to be 40℃.

[0259] 2.2.2.3.4 Flow rate investigation

[0260] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Fig.28 , Tables 13-14. Fig.28 Flow rate inspection.

[0261] Table 13 Flow rate investigation-retention time

[0262]

[0263] Table 14 Flow rate investigation - relative retention time

[0264]

[0265] The results show that when the flow rate is 1.0 ml / min, the chromatogram peak shape is better and the separation is moderate. Therefore, the flow rate is determined to be 1.0 ml / min.

[0266] 2.2.2.3.5 Delay Investigation

[0267] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Fig.29 . Fig.29 Delayed inspection.

[0268] The results showed that the sample had almost no chromatographic peak after 25 minutes, so the sample detection time was set at 25 minutes.

[0269] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of the standard decoction of Amaranthus tricolor were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, and particle size was 5 μm); methanol was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the regulations in the following table, with a flow rate of 1.0 ml per minute; the column temperature was 40°C; the detection wavelength was 260 m, and the number of theoretical plates calculated based on the gallic acid peak should be no less than 5000.

[0270]

[0271] 2.2.2.4 Preparation of test solution

[0272] 2.2.2.4.1 Investigation of extraction methods

[0273] Take 0.1g of standard decoction of Amaranthus tricolor (batch number: BT-16), place it in a stoppered conical flask, add 25ml of 50% methanol, perform reflux and ultrasonic extraction respectively, the extraction time is 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Fig.30 . Fig.30 The results showed that the results of ultrasonic extraction and reflux extraction were consistent. Ultrasonic extraction was more convenient, so the extraction method was determined to be ultrasonic extraction.

[0274] 2.2.2.4.2 Investigation of extraction solvent

[0275] Take 0.1 g of standard decoction of Amaranthus tricolor (batch number: BT-16), place it in a stoppered conical flask, add 25 ml of 30% methanol, 70% methanol, methanol, 50% ethanol, ethanol, water, and 50% methanol, respectively, and treat with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain the product. Fig.31 . Fig.31 Investigation of extraction solvents.

[0276] The results showed that when 50% methanol was used as the extraction solvent, the chromatographic peak information was larger and the separation effect was better, so the extraction solvent for the test sample was determined to be 50% methanol.

[0277] 2.2.2.4.3 Extraction time investigation

[0278] Take 0.1g of standard decoction of Amaranthus tricolor (batch number: BT-16), place it in a stoppered conical flask, add 25ml of 50% methanol, and treat the sample with ultrasound (power 600W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively, cool, shake well, filter, and take the filtrate to obtain the product. Fig.32 . Fig.32 Extraction time investigation

[0279] The results showed that sufficient extraction was achieved when the extraction time was 30 minutes. Therefore, the extraction time of the test sample was determined to be 30 minutes.

[0280] 2.2.2.4.4 Investigation of solvent addition amount

[0281] Take 0.1g of standard decoction of Amaranthus tricolor (batch number: BT-16), place it in a stoppered conical flask, add 10ml, 25ml, and 50ml of 50% methanol respectively, stopper it, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, shake it well, filter it, and take the filtrate to obtain the product. Fig.33 . Fig.33 Investigation of solvent addition amount.

[0282] The results show that when the amount of solvent added is 25 ml, the peak area of ​​the characteristic spectrum is moderate. Therefore, the amount of solvent added for the test sample is determined to be 25 ml.

[0283] In summary, the preparation method of the test solution of the characteristic spectrum of the standard decoction of Amaranthus chinensis is determined as follows: take 0.1 g of the standard decoction of Amaranthus chinensis, place it in a stoppered conical flask, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and obtain the filtrate.

[0284] 2.2.2.5 Methodological investigation

[0285] 2.2.2.5.1 Chromatographic peak identification

[0286] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Amaranthus tricolor decoction.

[0287] Preparation of reference solution: Take 1 g of Amaranthus tricolor reference medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate to dryness, add 25 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution.

[0288] Also, accurately weigh appropriate amounts of gallic acid reference substance, protocatechuic acid reference substance, corilagin reference substance, and ellagic acid reference substance, add 50% methanol to make a solution containing 100 μg per 1 ml, which is used as the reference substance solution.

[0289] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of iron-deficient Amaranth standard decoction.

[0290] Position the characteristic peaks of the standard decoction of Amaranthus tricolor. Figure 34-42 . Fig.34 Chromatographic peak identification; Fig.35 Gallic acid - standard decoction; Fig.36 Gallic acid-reference substance; Fig.37 Protocatechuic acid - standard decoction; Fig.38 Protocatechuic acid-reference substance; Fig.39 Corilagin - standard decoction; Fig.40 Corilagin - control; Fig.41 Ellagic acid - standard decoction; Fig.42 Ellagic acid - reference.

[0291] As can be seen from the figure, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is corilagin, and peak 6 is ellagic acid. In the following methodological investigation, the six characteristic peaks in the sample were investigated.

[0292] 2.2.2.5.2 Precision test

[0293] Take the test solution of Amaranthus tricolor standard decoction (batch number: BT-16), inject 6 times continuously according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 15.

[0294] Table 15 Precision investigation - retention time

[0295]

[0296]

[0297] The results showed that the RSD of the retention time of each characteristic peak was 0.01%-0.03%, indicating that the instrument had good precision.

[0298] 2.2.2.5.3 Repeatability study

[0299] Accurately weigh 6 portions of Amaranthus tricolor standard decoction (batch number: BT-16), prepare and measure according to the proposed experimental method, and calculate the relative retention time of each characteristic peak. See Table 16.

[0300] Table 16 Repeatability study - relative retention time

[0301]

[0302] The results showed that the RSD of the relative retention time of the six samples was 0.01%-0.03%, indicating that the method had good repeatability.

[0303] 2.2.2.5.4 Intermediate precision study

[0304] 2.2.2.5.4.1 Investigation with different instruments

[0305] Based on the experimental conditions proposed above, the standard decoction of Amaranthus tricolor (batch number: BT-16) was weighed to prepare the test solution, which was measured on Shimadzu LC-20AD HPLC (Instrument 1), Agilent 1260 HPLC (Instrument 2), and Thermo Fisher Vanquish Core HPLC (Instrument 3). The relative retention time of each characteristic peak was calculated. Fig.43 , Table 17. Fig.43 Different instrument surveys.

[0306] Table 17 Instrument durability study - relative retention time

[0307]

[0308] The results showed that when the three instruments were used to detect the test products, the RSD of the relative retention time of each characteristic peak was 0.75% to 2.42%, indicating that the instruments had good durability.

[0309] 2.2.2.5.4.2 Investigation by different personnel and time

[0310] Based on the experimental conditions proposed above, different personnel (A, B) weighed the standard decoction of Amaranthus tricolor (batch number: BT-16) at different times (T1, T2), prepared the test sample, carried out the determination, and calculated the relative retention time of each characteristic peak. See Table 18.

[0311] Table 18 Personnel and time inspection-relative retention time

[0312]

[0313] The results showed that when different people measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.02%-0.05%, and the method had good stability.

[0314] 2.2.2.5.5 Durability test

[0315] 2.2.2.5.5.1 Study on the durability of chromatographic columns

[0316] Based on the experimental conditions proposed above, ZORBAX Eclipse XDB-C18, 4.6mm×250mm, 5μm (chromatographic column 1), Luna C18(2)100A, 4.6mm×250mm, 5μm (chromatographic column 2), Agilent 5TC-C18(2)4.6mm×250mm, 5μm (chromatographic column 3) were used for analysis and investigation, and the relative retention time of each characteristic peak was calculated. The results are shown in Fig.44 , Table 19. Fig.44 Investigation of different chromatographic columns.

[0317] Table 19 Column durability study - relative retention time

[0318]

[0319] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.05% and 1.37% when the three chromatographic columns were used to detect the samples, indicating that the chromatographic columns had good durability.

[0320] 2.2.2.5.5.2 Stability study

[0321] Based on the above experimental conditions, take the same test solution and measure it at 0h, 2h, 4h, 8h, 16h, and 24h. Calculate the retention time of each characteristic peak. See Table 20.

[0322] Table 20 Stability Study-Retention Time

[0323]

[0324] The results showed that the RSD of the characteristic peak retention time was between 0.06% and 0.14%, and the sample solution was stable within 24 hours.

[0325] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 6 characteristic peaks were included in the subsequent investigation.

[0326] 2.2.2.6 Determination of characteristic peaks and establishment of reference spectrum

[0327] 2.2.2.6.1 Verification results of 16 batches of Amaranthus chinensis standard decoction

[0328] Using this method, 16 batches of samples were analyzed for characteristic spectra and relative retention times were calculated. Fig.45 , Table 21. Fig.45 Characteristic spectrum of standard decoction of Amaranthus tricolor (S1-S16 are: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16).

[0329] Table 21 Relative retention time of standard decoction of Amaranthus tricolor

[0330]

[0331]

[0332] Based on the principle of stable relative retention time and the fact that each batch of samples can be detected and the peak is relatively high, a total of 6 peaks with good durability were selected as characteristic peaks. According to the results of the methodological investigation and the verification results of 16 batches of standard soup, the theoretical plate number calculated based on the gallic acid peak should be no less than 5000.

[0333] 2.2.2.6.2 Establishment of relative retention time limit values

[0334] The summary of the methodology investigation items and verification results is shown in Table 22:

[0335] Table 22 Summary of RSD% results of each item of the methodology - relative retention time / retention time

[0336]

[0337] The relative retention time of each characteristic peak is stable and within the range of ±10% of the average value, so the relative retention time of each peak is temporarily set at ±10%.

[0338] The relative retention times of the 16 batches of Amaranthus chinensis standard decoctions were all within the range of ±10% of the mean relative retention time of the 3 batches of Amaranthus chinensis formula granules, so the mean relative retention time of the 3 batches of Amaranthus chinensis formula granules was used as the specified value of the Amaranthus chinensis standard decoction. See Table 23.

[0339] Table 23 Relative retention time of Amaranthus chinensis formula granules

[0340]

[0341] Final regulations: The test sample characteristic spectrum should show 6 characteristic peaks, and should correspond to the retention time of the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1 and peak 6 should correspond to the retention time of the reference peaks of gallic acid and ellagic acid, respectively. The peak corresponding to the gallic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), 2.58 (peak 5).

[0342] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of standard decoctions of Amaranthus chinensis, and a comparison spectrum of the characteristic spectrum of the standard decoction of Amaranthus chinensis was established. Fig.46 . Fig.46 Reference characteristic spectrum; Peak 1 (S): gallic acid; Peak 2: protocatechuic acid; Peak 3: corilagin; Peak 6: ellagic acid; (ZORBAX Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 1)).

[0343] 2.2.2.7 Determination of characteristic spectrum of standard decoction of Amaranthus tricolor

[0344] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table, flow rate: 1.0 ml per minute; column temperature: 40°C; detection wavelength: 260 m, theoretical plate number calculated based on gallic acid peak should be not less than 5000.

[0345]

[0346]

[0347] Preparation of reference solution Take 1g of Amaranthus tricolor reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 25ml of 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of gallic acid and ellagic acid reference substances, accurately weigh, add 50% methanol to make a solution containing 100μg of each per 1ml, as the reference substance solution.

[0348] Preparation of the test solution: Take 0.1 g of standard decoction of Amaranthus tricolor, place it in a stoppered conical flask, add 25 ml of 50% methanol, stopper it, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the test solution.

[0349] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0350] There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1 and peak 6 should correspond to the retention time of the reference peaks of gallic acid and ellagic acid, respectively. The peak corresponding to the peak of the gallic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), and 2.58 (peak 5). Figure 2 Fig.46 .

[0351] Example 3 HPLC characteristic spectrum of Amaranthus tricolor formula granules

[0352] 2.2.3.1 Materials, reagents and instruments

[0353] Amaranthus tricolor formula granules batch numbers: KL-01, KL-02, KL-03.

[0354] The rest is the same as in Example 1.

[0355] 2.2.3.2 Characteristic spectrum determination method

[0356] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table, flow rate: 1.0 ml per minute; column temperature: 40°C; detection wavelength: 260 nm, theoretical plate number calculated based on gallic acid peak should be not less than 5000.

[0357]

[0358] Preparation of reference solution Take 1g of Amaranthus tricolor reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 25ml of 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of gallic acid and ellagic acid reference substances, accurately weigh, add 50% methanol to make a solution containing 100μg of each per 1ml, as the reference substance solution.

[0359] Preparation of the test solution: Take 0.1 g of Amaranthus tricolor granules, place in a stoppered conical flask, add 25 ml of 50% methanol, stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0360] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0361] 2.2.3.3 Chromatographic conditions and system suitability test

[0362] 2.2.3.3.1 Mobile phase selection

[0363] Based on the experimental conditions proposed above, the separation effect of using methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B was investigated. Fig.47 . Fig.47 Mobile phase selection.

[0364] The results showed that the chromatogram baseline was relatively stable under the condition of methanol-0.1% phosphoric acid solution gradient elution. Therefore, methanol-0.1% phosphoric acid solution gradient elution was used as the mobile phase for the characteristic spectrum determination method of Amaranthus trichosanthis formula granules.

[0365] 2.2.3.3.2 Wavelength selection

[0366] Based on the experimental conditions proposed above, the diode array detector was used to scan the test solution in the full band, and the chromatograms of the test solution at wavelengths of 220nm, 240nm, 260nm, 280nm, 300nm, and 320nm were extracted respectively. Fig.48 . Fig.48 Chromatograms of Amaranthus tricolor formula granules at different wavelengths (from top to bottom: 220nm, 240nm, 280nm, 300nm, 320nm, 260nm). The results show that when the detection wavelength is 260nm, the chromatographic peak information is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 260nm.

[0367] 2.2.3.3.3 Column temperature investigation

[0368] Based on the experimental conditions proposed above, the column temperatures of 35℃, 40℃ and 45℃ were investigated respectively. Fig.50 . See Tables 24-25. Fig.49 Column temperature inspection.

[0369] Table 24 Column temperature investigation-retention time

[0370]

[0371]

[0372] Table 25 Column temperature investigation-relative retention time

[0373]

[0374] The results showed that when the column temperature was 40℃, the chromatogram peak shape was more symmetrical and the separation was better, so the column temperature was determined to be 40℃.

[0375] 2.2.3.3.4 Flow rate investigation

[0376] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Fig.50 , Tables 26-27. Fig.50 Flow rate inspection.

[0377] Table 26 Flow rate investigation-retention time

[0378]

[0379] Table 27 Flow rate investigation - relative retention time

[0380]

[0381] The results show that when the flow rate is 1.0 ml / min, the chromatogram peak shape is better and the separation is moderate. Therefore, the flow rate is determined to be 1.0 ml / min.

[0382] 2.2.3.3.5 Delay Investigation

[0383] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Fig.52 . Fig.51 Delayed inspection.

[0384] The results showed that the sample had almost no chromatographic peak after 25 minutes, so the sample detection time was set at 25 minutes.

[0385] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of Amaranthus tricolor formula granules were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, and particle size was 5 μm); methanol was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the regulations in the following table, with a flow rate of 1.0 ml per minute; the column temperature was 40°C; the detection wavelength was 260 nm, and the number of theoretical plates calculated based on the gallic acid peak should be no less than 5000.

[0386]

[0387] 2.2.3.4 Preparation of test solution

[0388] 2.2.3.4.1 Investigation of extraction methods

[0389] Take 0.1g of Amaranthus chinensis granules (batch number: KL-01), grind them into powder, put them into a stoppered conical flask, add 25ml of 50% methanol, perform reflux and ultrasonic extraction respectively, the extraction time is 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Fig.52 . Fig.52 Investigation of extraction methods.

[0390] The results showed that the ultrasonic extraction and reflux extraction of the test sample had the same effect. Because the ultrasonic extraction operation is simpler, the ultrasonic extraction method was determined to be the extraction method of the test sample.

[0391] 2.2.3.4.2 Investigation of extraction solvent

[0392] Take 0.1g of Amaranthus oleifera granules (batch number: KL-01), grind them into powder, put them into a stoppered conical flask, add 25ml of 30% methanol, 70% methanol, methanol, 50% ethanol, ethanol, water, 50% methanol, respectively, and treat with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain the product. Fig.53 . Fig.53 Investigation of extraction solvents;

[0393] The results showed that when 50% methanol was used as the extraction solvent, the chromatographic peak information was larger and the separation effect was better, so the extraction solvent for the test sample was determined to be 50% methanol.

[0394] 2.2.3.4.3 Extraction time investigation

[0395] Take 0.1g of Amaranthus tricolor granules (batch number: KL-01), grind them into powder, put them into a stoppered conical flask, add 25ml of 50% methanol, and treat the sample with ultrasound (power 600W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively, cool, shake well, filter, and take the filtrate to obtain the product. Fig.54 . Fig.54 The results of the extraction time showed that the peak shape and separation of the chromatogram were better when the extraction time was 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0396] 2.2.3.4.4 Investigation of solvent addition amount

[0397] Take 0.1g of Amaranthus chinensis granules (batch number: KL-01), grind them into powder, put them into a stoppered conical flask, add 10ml, 25ml, and 50ml of 50% methanol respectively, stopper them tightly, and treat them with ultrasound (power 600W, frequency 40kHz) for 30 minutes, shake them well, filter them, and take the filtrate to obtain the product. Fig.55 . Fig.55 The results showed that when the amount of solvent added was 25 ml, the peak area of ​​the characteristic spectrum was moderate. Therefore, the amount of solvent added for the test sample was determined to be 25 ml.

[0398] In summary, the preparation method of the test solution of the characteristic spectrum of the Amaranthus tricolor formula granules is determined as follows: take 0.1 g of the Amaranthus tricolor formula granules, grind them into powder, put them in a stoppered conical flask, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and obtain the filtrate.

[0399] 2.2.3.5 Methodological investigation

[0400] 2.2.3.5.1 Chromatographic peak identification

[0401] Preparation of test solution: According to the experimental conditions determined above, prepare the test solution of Amaranthus tricolor formula granules.

[0402] Preparation of reference solution: Take 1 g of Amaranthus tricolor reference medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate to dryness, add 25 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution.

[0403] Separately, weigh an appropriate amount of gallic acid reference substance, protocatechuic acid reference substance, corilagin reference substance, and ellagic acid reference substance, add 50% methanol to prepare a solution containing 100 μg per 1 ml, which serves as the reference substance solution.

[0404] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of iron-deficient Amaranth formula granules.

[0405] Locate the characteristic spectrum peaks of Amaranthus tricolor formula granules. Figure 56-64 . Fig.56 Chromatographic peak identification; Fig.57 Gallic acid-formulated granules; Fig.58 Gallic acid-reference substance; Fig.59 Protocatechuic acid-formulated granules; Fig.60 Protocatechuic acid-reference substance; Fig.61 Corilagin - Formula Granules; Fig.62 Corilagin - control; Fig.63 Ellagic acid-formulated granules; Fig.64 Ellagic acid - reference.

[0406] As can be seen from the figure, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is corilagin, and peak 6 is ellagic acid.

[0407] 2.2.3.5.2 Precision test

[0408] Take the test solution of Amaranthus tricolor formula granules (batch number: KL-01), inject 6 times continuously according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 28.

[0409] Table 28 Precision Investigation-Retention Time

[0410]

[0411] The results showed that the RSD range of the relative retention time of each characteristic peak of the sample was 0.03%-0.07%, indicating that the method had good injection precision.

[0412] 2.2.3.5.3 Repeatability study

[0413] Take 6 portions of Amaranthus tricolor formula granules (batch number: KL-01), prepare and measure according to the proposed experimental method, and calculate the relative retention time of each characteristic peak. See Table 29.

[0414] Table 29 Repeatability Study - Relative Retention Time

[0415]

[0416]

[0417] The results showed that the RSD range of the relative retention time of the six samples was 0.02%-0.04%, indicating that the method had good repeatability.

[0418] 2.2.3.5.4 Intermediate precision study

[0419] 2.2.3.5.4.1 Investigation with different instruments

[0420] Based on the experimental conditions proposed above, 3 portions of Amaranthus tricolor granules (batch number: KL-01) were weighed to prepare test solutions, which were measured on Shimadzu LC-20AD HPLC (Instrument 1), Agilent 1260 HPLC (Instrument 2), and Thermo Fisher Vanquish Core HPLC (Instrument 3). The relative retention time of each characteristic peak was calculated. Fig.65 , Table 30. Fig.65 Different instrument surveys.

[0421] Table 30 Instrument durability study - relative retention time

[0422]

[0423] The results showed that when the three instruments were used to detect the test samples, the RSD of the relative retention time of each characteristic peak was 0.93%-3.95%, indicating that the instrument precision of this method was good.

[0424] 2.2.3.5.4.2 Investigation by different personnel and time

[0425] Based on the experimental conditions proposed above, different personnel (A, B) weighed Amaranthus tricolor formula granules (batch number: KL-01) at different times (T1, T2) to prepare test samples and conduct the determination. The relative retention time of each characteristic peak was calculated. See Table 31.

[0426] Table 31 Personnel and time inspection - relative retention time

[0427]

[0428] The results showed that when different people measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.02% to 0.04%, indicating that the intermediate precision of this method was good.

[0429] 2.2.3.5.5 Durability test

[0430] 2.2.3.5.5.1 Column durability test

[0431] Based on the experimental conditions proposed above, ZORBAX Eclipse XDB-C18, 4.6mm×250mm, 5μm (chromatographic column 1), Luna C18(2)100A, 4.6mm×250mm, 5μm (chromatographic column 2), Agilent 5TC-C18(2)4.6mm×250mm, 5μm (chromatographic column 3) were used for analysis and investigation, and the relative retention time of each characteristic peak was calculated. The results are shown in Fig.66 , Tables 32 and 33. Fig.66 Investigation of different chromatographic columns.

[0432] Table 32 Chromatographic column durability investigation - relative retention time

[0433]

[0434] The results showed that the RSD of the relative retention time of the characteristic peaks was between 1.84% and 5.43% when the samples were detected by the three chromatographic columns mentioned above, indicating that the chromatographic columns of this method had good durability.

[0435] 2.2.3.5.5.2 Stability study

[0436] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 4h, 8h, 16h, and 24h. Calculate the retention time of each characteristic peak. See Table 33.

[0437] Table 33 Stability Study-Retention Time

[0438]

[0439] The results showed that the RSD of the characteristic peak retention time was between 0.14% and 0.29%, indicating that the sample solution was stable within 24 hours.

[0440] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 6 characteristic peaks were included in the subsequent investigation.

[0441] 2.2.3.6 Determination of characteristic peaks and establishment of reference spectrum

[0442] 2.2.3.6.1 Verification results of three batches of Amaranthus chinensis formula granules

[0443] Using this method, characteristic spectra were analyzed on three batches of samples and relative retention times were calculated. Fig.67 , Table 34. Fig.67 Characteristic spectrum of Amaranthus tricolor formula granules.

[0444] Table 34 Relative retention time of Amaranthus chinensis formula granules

[0445]

[0446] Based on the principle of stable relative retention time and the fact that each batch of samples can be detected and the peak is relatively high, a total of 6 peaks with good durability were selected as characteristic peaks, namely Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, and Peak 6. According to the results of the methodological investigation and the verification results of 3 batches of particles, the theoretical plate number calculated based on the gallic acid peak should be no less than 5000.

[0447] 2.2.3.6.2 Establishment of relative retention time limit values

[0448] The methodology investigation items and verification results are summarized in Table 35:

[0449] Table 35 Summary of RSD% results of each item of methodology - relative retention time / retention time

[0450]

[0451] The relative retention time of each characteristic peak is stable and within the range of ±10% of the average value, so the specified value range of the relative retention time of each peak is temporarily set at ±10%. The relative peak areas of each batch vary too much to be specified, so the relative peak areas are not included in the text.

[0452] Final regulations: There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1 and peak 6 correspond to the retention time of the reference peak. The peak corresponding to the peak of the gallic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), 2.58 (peak 5).

[0453] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize three batches of Amaranthus chinensis formula granules, and a comparison spectrum of the characteristic spectrum of Amaranthus chinensis formula granules was established. Fig.68 . Fig.68 Reference characteristic spectrum; Peak 1 (S): gallic acid; Peak 2: protocatechuic acid; Peak 3: corilagin; Peak 6: ellagic acid (ZORBAX Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 1)).

[0454] 2.2.3.7 Determination of characteristic spectrum of Amaranthus tricolor formula granules

[0455] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table, flow rate: 1.0 ml per minute; column temperature: 40°C; detection wavelength: 260 nm, theoretical plate number calculated based on gallic acid peak should be not less than 5000.

[0456]

[0457] Preparation of reference solution Take 1g of Amaranthus tricolor reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate, add 25ml of 50% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of gallic acid and ellagic acid reference substances, accurately weigh, add 50% methanol to make a solution containing 100μg of each per 1ml, as the reference substance solution.

[0458] Preparation of the test solution: Take 0.1 g of Amaranthus tricolor granules, place in a stoppered conical flask, add 25 ml of 50% methanol, stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0459] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0460] There should be 6 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1 and peak 6 correspond to the retention time of the reference peak. The peak corresponding to the peak of the gallic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), 2.58 (peak 5). Figure 2 Fig.68 .

[0461] Comparative Example 1

[0462] Chromatographic conditions and system suitability test Chromatographic column: Agilent TC-C18 (4.6 mm × 150 mm, 5 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table, flow rate of 1.0 ml per minute; column temperature of 30 ° C; detection wavelength of 270 nm.

[0463]

[0464] Preparation of reference solution: Take an appropriate amount of gallic acid and ellagic acid reference substances, weigh them accurately, and add 50% methanol to prepare a solution containing 0.2 mg per 1 ml; take an appropriate amount of protocatechuic acid reference substance, weigh them accurately, and add 60% ethanol to prepare a solution containing 0.09 mg per milliliter; take an appropriate amount of corilagin reference substance, weigh them accurately, dissolve them in methanol to prepare a reference solution containing 0.24 mg per milliliter.

[0465] Preparation of test solution: Medicinal materials and granules Take 0.5 g of the powder of this product, place it in a round-bottom flask, add 50 mL of 4 mol / L hydrochloric acid, weigh it, heat and reflux for 4 h, cool it, weigh it again, make up the lost weight with 4 mol·L hydrochloric acid, shake it well, filter it, and take the filtrate to obtain the product.

[0466] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0467] Results Fig.69 , Fig.69 This is the chromatogram of the medicinal material particles in Comparative Example 1.

[0468] According to the above results, it can be seen that the method of Comparative Example 1 cannot simultaneously detect the four index components of gallic acid, protocatechuic acid, corilagin and ellagic acid, and the sample preparation method is complicated.

[0469] Comparative Example 2

[0470] Chromatographic conditions and system suitability test: Chromatographic column: Elite ODS C18 (4.6 mm×250 mm, 5 μm); methanol: 0.1% phosphoric acid solution (25:75) as the mobile phase, the flow rate is 1.0 ml per minute; the column temperature is 25°C; the detection wavelength is 260 nm.

[0471] Preparation of reference solution: Take an appropriate amount of gallic acid and ellagic acid reference substances, weigh them accurately, and add 50% methanol to prepare a solution containing 0.2 mg per 1 ml; take an appropriate amount of protocatechuic acid reference substance, weigh them accurately, and add 60% ethanol to prepare a solution containing 0.09 mg per milliliter; take an appropriate amount of corilagin reference substance, weigh them accurately, dissolve them in methanol to prepare a reference solution containing 0.24 mg per milliliter.

[0472] Preparation of test solution: Medicinal materials and granules Take 1.0 g of the powder of this product, place it in a stoppered conical flask, accurately add 50 ml of 60% ethanol, weigh it, reflux and extract it for 1.5 h, weigh it to make up the weight, filter it, and take the filtrate as the test solution.

[0473] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0474] The results are as follows Fig.70 As shown, Fig.70 This is the result diagram of Comparative Example 2. According to the above results, it can be seen that the method of Comparative Example 2 cannot simultaneously detect the four indicator components of gallic acid, protocatechuic acid, corilagin and ellagic acid.

[0475] Comparative Example 3

[0476] Chromatographic conditions and system suitability test Chromatographic column: Poroshell 120 SB-C18 (3.0 mm×150 mm, 2.7 μm); 0.1% formic acid as mobile phase A, 0.1% formic acid-acetonitrile as mobile phase B, gradient elution as specified in the table below, flow rate of 0.6 ml per minute; column temperature of 40°C; detection wavelength of 274 nm.

[0477]

[0478] Preparation of reference solution: Take an appropriate amount of gallic acid and ellagic acid reference substances, weigh them accurately, and add 50% methanol to prepare a solution containing 0.2 mg per 1 ml; take an appropriate amount of protocatechuic acid reference substance, weigh them accurately, and add 60% ethanol to prepare a solution containing 0.09 mg per milliliter; take an appropriate amount of corilagin reference substance, weigh them accurately, dissolve them in methanol to prepare a reference solution containing 0.24 mg per milliliter.

[0479] Preparation of test solution: Medicinal materials and granules Take 1.0 g of the powder of this product, place it in a stoppered conical flask, accurately add 20 mL of methanol, weigh it, soak it in cold water for 1 hour, extract it with ultrasound for 50 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0480] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0481] The results are as follows Fig.71 As shown, Fig.71 This is the result diagram of comparative example 3. According to the above results, the method of comparative document 3 cannot simultaneously detect the four indicator components of gallic acid, protocatechuic acid, corilagin and ellagic acid, and the peak shape and separation are poor.

[0482] In summary, the methods of reference documents 1, 2, and 3 cannot simultaneously detect the four index components in the sample, and the sample preparation method is complicated. The mobile phase system and sample preparation method used in this project are simple and easy to obtain, which can well achieve the separation of chromatographic peaks in Amaranthus tricolor (see Fig.10 , Fig.34 and Fig.56 ).

[0483] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic spectrum of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising: A) extracting the raw material of Amaranthus tricolor with a solvent to obtain a solution to be tested; B) the test solution is measured by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the Amaranthus tricolor raw material; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is a 0.1% phosphoric acid solution, and the elution is performed in a gradient manner; The gradient elution is specifically: 0-7min, phase A: 12%→30%, phase B: 88%→70%; 7-14 min, phase A: 30%→38%, phase B: 88%-62%; 14-19 min, phase A: 38%-53%, phase B: 62%-47%; 19-25min, phase A: 53%-69%, phase B: 47%-31%.

2. The method according to claim 1, characterized in that It also includes the preparation of reference substances and reference medicinal material solutions: Take gallic acid reference substance and ellagic acid reference substance respectively, add water and use 50% methanol to dissolve them to obtain reference substance solutions; The concentration of the gallic acid reference substance is 100 μg / mL; the concentration of the ellagic acid reference substance is 100 μg / mL; Take the Amaranthus tricolor reference medicinal material, add water to boil, filter, dissolve the residue with 50% methanol, sonicate, cool, filter, and obtain a reference medicinal material solution; The reference substance and reference medicinal material reference substance solutions are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference substance, respectively; and the components of the HPLC characteristic spectra of Amaranthus tricolor medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively measured based on the chromatograms of the reference substance and the reference medicinal material reference substance.

3. The method according to claim 1, characterized in that: The chromatographic column is a C18 column with specifications of 5 μm and 4.6×250 mm; the column temperature is 40° C.

4. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 1 mL / min; the detection wavelength is 260 nm; the theoretical plate number calculated based on the gallic acid peak should not be less than 5000, and the injection volume is 10 μL.

5. The method according to claim 1, characterized in that: The similarity of HPLC characteristic spectra of Amaranthus tricolor, its decoction pieces, standard decoction and its formula granules was evaluated by using the similarity evaluation system of Chinese medicine chromatographic fingerprint. The HPLC standard characteristic spectra of Amaranthus tricolor, its decoction pieces, standard decoction and its formula granules were obtained, which consisted of 6 characteristic peaks, including Peak 1 (S): gallic acid; Peak 2: protocatechuic acid; Peak 3: corilagin; Peak 6: ellagic acid.

6. The method according to claim 1, characterized in that In the characteristic spectrum of the standard decoction of Amaranthus tricolor, gallic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 1.51 (peak 2), 2.09 (peak 3), 2.17 (peak 4), and 2.58 (peak 5).

7. The method according to claim 1, characterized in that The Amaranthus chinensis raw materials of step A) are Amaranthus chinensis medicinal materials, decoction pieces, standard decoctions and formula granules thereof.

8. The method according to claim 7, characterized in that The preparation of the test solution of Amaranthus tricolor medicinal material / cooked slices is specifically as follows: after boiling the Amaranthus tricolor raw material with water, filtering, evaporating to dryness, dissolving the residue with 50% methanol, ultrasonicating, cooling, shaking, filtering, and obtaining the solution.

9. The method according to claim 8, characterized in that The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 30 to 40 minutes; The ratio of the mass g of the Amaranthus tricolor raw material to the volume mL of water is 1:

50.

10. The method according to claim 7, characterized in that The preparation of the test solution of the standard decoction / formulated granules of Amaranthus tricolor is as follows: The Amaranthus tricolor raw material is ultrasonically extracted with 50% methanol, cooled, shaken, filtered, and the filtrate is obtained; the ultrasonic power is 600W, the frequency is 40kHz; the extraction time is 30 to 40min; the ratio of the mass g of the Amaranthus tricolor raw material to the volume mL of 50% methanol is 0.1:25.

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