A characteristic spectrum detection method for Euphorbia rubrum, Euphorbia rubrum and their preparations

Abstract

The present invention provides a method for detecting characteristic patterns of Euphorbia rubrum, Euphorbia rubrum with vinegar, and their preparations, comprising the following steps: S1) extracting a raw material of a test sample using a solvent to obtain a test liquid; the raw material of the test sample is Euphorbia rubrum or Euphorbia rubrum with vinegar; S2) subjecting the test liquid to high-performance liquid chromatography to obtain an HPLC characteristic pattern of the test sample. The method for detecting characteristic patterns of Euphorbia rubrum, Euphorbia rubrum with vinegar, and their preparations provided by the present invention can be used for quality testing of Euphorbia rubrum medicinal materials, decoction pieces, standard decoctions, and granules, or Euphorbia rubrum with vinegar decoction pieces, standard decoctions, and granules, providing reliable results and providing a new method for quality control of Euphorbia rubrum and Euphorbia rubrum with vinegar.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis methods, in particular to a method for detecting characteristic spectra of Euphorbia rubrum, Euphorbia rubrum and their preparations. Background Art

[0002] Euphorbia valerianoides is the dried tuberous root of the Rubiaceae plant Knoxia valerianoides Thorel et Pitard, which is included in the 2020 edition of the "Chinese Pharmacopoeia". It has the effects of purging water and fluid, reducing swelling and dispersing nodules. It is used for symptoms such as edema, chest and abdominal effusion, phlegm accumulation, qi reversal, cough and asthma, difficulty in defecation and urination, carbuncles, sores, and tuberculosis phlegm nodules.

[0003] Vinegar-red euphorbia is a processed product of red euphorbia using the vinegar-fried method outlined in the 2015 edition of the "Sichuan Province Traditional Chinese Medicine Preparation Specifications." Distinguishing and identifying red euphorbia from vinegar-red euphorbia currently involves characterization, identification, inspection, and content determination, but a comprehensive testing method for both red and vinegar-red euphorbia is lacking.

[0004] Based on this, a method for identifying Euphorbia rubrum, Euphorbia rubrum and their preparations was established, which is of great significance for the formulation of quality standards. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a characteristic spectrum detection method for red euphorbia, vinegar red euphorbia and their preparations. The detection method can be used for quality detection of red euphorbia medicinal materials, decoction pieces, standard decoctions, formula granules and vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions, and formula granules. The results are reliable, providing a new method for quality control of red euphorbia and vinegar red euphorbia.

[0006] In view of this, the present application provides a method for detecting characteristic patterns of Euphorbia rubrum, Euphorbia rubrum and their preparations, comprising the following steps:

[0007] S1) extracting the raw material of the test product with a solvent to obtain a test solution; the raw material of the test product is a red euphorbia raw material or a vinegar red euphorbia raw material;

[0008] S2) measuring the test solution by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the test product;

[0009] The chromatographic conditions of the high performance liquid chromatography method are as follows: a C18 chromatographic column; mobile phase A is methanol; mobile phase B is 0.2% phosphoric acid solution; and gradient elution.

[0010] Preferably, the method further comprises preparing a reference substance solution 1, a reference substance solution 2, and a reference medicinal material reference solution:

[0011] Dissolve scutellariae alcohol in methanol to obtain reference substance solution 1;

[0012] Dissolve 3-hydroxybajiquinone in methanol to obtain reference substance solution 2;

[0013] The red euphorbia medicinal material was decocted with water, the filtered filtrate was evaporated to dryness, and then extracted with methanol, and the control medicinal material reference solution was obtained after ultrasonication;

[0014] The reference substance solution 1, the reference substance solution 2 and the reference medicinal material reference solution were respectively determined by high performance liquid chromatography to obtain chromatograms of cyperus alcohol and chromatograms of 3-hydroxybajiquinone;

[0015] The components of the HPLC characteristic spectra of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules were qualitatively determined based on the chromatograms of cyperiol and 3-hydroxybajiquinone.

[0016] The components of the HPLC characteristic spectra of Euphorbia acetosporum medicinal materials, decoction pieces, standard decoction and its formula granules were qualitatively determined based on the chromatograms of tiglol and 3-hydroxybajiquinone.

[0017] Preferably, the raw materials of the test product are Euphorbia rubra medicinal materials, decoction pieces, standard decoctions and formula granules thereof, or Euphorbia rubra medicinal materials, decoction pieces, standard decoctions and formula granules thereof.

[0018] Preferably, step S1) is specifically:

[0019] The preparation of the test solution of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules is as follows:

[0020] Adding methanol to the medicinal materials, decoction pieces, standard decoction or its formula granules of Euphorbia rubrum and performing ultrasonic extraction;

[0021] The preparation of the test solution of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules is as follows:

[0022] Add the medicinal materials, decoction pieces, standard decoction or its formula granules of Euphorbia pulex to methanol and perform ultrasonic extraction.

[0023] Preferably, the ultrasonic extraction power is 600W, the frequency is 40kHz, and the ultrasonic extraction time is 20 to 40 minutes;

[0024] The ratio of the test sample raw material to the solvent is (0.5-1) g: (10-30) ml.

[0025] Preferably, the gradient elution is specifically:

[0026] 0-12 min, phase A: 10-40%, phase B: 90-60%;

[0027] 12-15 min, phase A: 40-43%, phase B: 60-57%;

[0028] 15-24 min, phase A: 43-45%, phase B: 57-55%;

[0029] 24-28 min, phase A: 45-55%, phase B: 55-45%;

[0030] 28-36 min, phase A: 55-65%, phase B: 45-35%;

[0031] 36~42min, phase A: 65~73%, phase B: 35~27%;

[0032] 42-43 min, phase A: 73-90%, phase B: 27-10%;

[0033] 43-50min, phase A: 90%, phase B: 10%.

[0034] Preferably, the chromatographic column has a column length of 150 mm, an inner diameter of 2.1 mm, a particle size of 2.7 μm, and a column temperature of 30°C.

[0035] Preferably, the flow rate of the mobile phase is 0.25 ml / min, the detection wavelength is 280 nm, and the injection volume is 1 μL.

[0036] Preferably, the similarity of the HPLC characteristic spectra of the red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules is evaluated by using a Chinese medicine chromatographic fingerprint similarity evaluation system, and a HPLC standard characteristic spectra of the red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules consisting of 12 characteristic peaks is obtained, wherein peak 8 (S): scutellaria alcohol peak, peak 11: 3-hydroxybajiquinone peak;

[0037] The similarity of the HPLC characteristic spectra of Euphorbia oxyphylla, its decoction pieces, standard decoction and its formula granules was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system, and the HPLC standard characteristic spectra of Euphorbia oxyphylla, its decoction pieces, standard decoction and its formula granules were obtained, which consisted of 12 characteristic peaks, including peak 8 (S): scutellaria alcohol peak, and peak 11: 3-hydroxybajiquinone peak.

[0038] Preferably, in the characteristic spectra of the red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules, the relative retention time of each characteristic peak and the S peak is calculated with thorn alcohol as the reference peak S, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 8), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 12), 1.61 (peak 12) ); In the characteristic spectra of the vinegar-red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules thereof, with thorn alcohol as the reference peak S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 8), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 12), and 1.61 (peak 12).

[0039] The present invention provides a method for detecting characteristic patterns of euphorbia rubrum, euphorbia rubrum with vinegar, and their preparations. First, a test solution of euphorbia rubrum raw material or euphorbia rubrum with vinegar raw material is prepared, and then the test solution is tested using high-performance liquid chromatography to obtain an HPLC of the test product. The present invention adopts high-performance liquid chromatography, selects methanol-0.2% phosphoric acid solution as the mobile phase for gradient elution, and uses cypermethrin as a reference substance to establish HPLC characteristic patterns of euphorbia rubrum medicinal materials, decoction pieces, standard decoctions and formulated granules thereof, and euphorbia rubrum with vinegar medicinal materials, decoction pieces, standard decoctions and formulated granules thereof. The method has good repeatability and precision, is stable, and is reliable. The method can be used to control the quality of euphorbia rubrum medicinal materials, decoction pieces, standard decoctions and formulated granules thereof, and euphorbia rubrum with vinegar medicinal materials, decoction pieces, standard decoctions and formulated granules thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The chromatogram of Euphorbia rubrum medicinal material was used as the basis for mobile phase selection;

[0041] Figure 2 This is the chromatogram of Euphorbia rubrum medicinal material at different wavelengths;

[0042] Figure 3 The chromatograms of Euphorbia rubrum medicinal materials under different column temperature conditions;

[0043] Figure 4 The chromatograms of Euphorbia rubrum medicinal materials at different flow rates are shown;

[0044] Figure 5 This is the chromatogram of the delayed investigation of the medicinal material of red euphorbia;

[0045] Figure 6This is the chromatogram of Euphorbia rubrum under different extraction solvents;

[0046] Figure 7 This is the chromatogram of Euphorbia rubrum at different extraction times;

[0047] Figure 8 The chromatograms of Euphorbia rubrum medicinal materials with different solvent addition amounts are shown;

[0048] Figure 9 This is the chromatogram of Euphorbia rubrum under different extraction methods;

[0049] Figure 10 Chromatogram for peak identification of Euphorbia rubrum medicinal material;

[0050] Figure 11 This is the spectrum of thorn alcohol from Euphorbia rubrum;

[0051] Figure 12 This is the spectrum of scutellariae alcohol in the reference substance;

[0052] Figure 13 This is the spectrum of 3-hydroxybajiquinone of Euphorbia rubrum medicinal material;

[0053] Figure 14 This is the spectrum of 3-hydroxybamoquinone in the reference substance;

[0054] Figure 15 This is the chromatogram of Euphorbia rubrum under different instruments;

[0055] Figure 16 The chromatograms of Euphorbia rubrum under different chromatographic columns are shown;

[0056] Figure 17 Characteristic maps of 21 batches of Euphorbia rubrum medicinal materials;

[0057] Figure 18 It is a comparison characteristic spectrum of the characteristic spectrum of Euphorbia rubrum medicinal material;

[0058] Figure 19 Characteristic maps of 21 batches of Euphorbia rubrum slices;

[0059] Figure 20 It is the reference characteristic spectrum of the characteristic spectrum of Euphorbia rubrum decoction pieces;

[0060] Figure 21 This is the characteristic spectrum of Euphorbia acetophylla after the mobile phase is selected;

[0061] Figure 22 This is the chromatogram of the vinegar-red Euphorbia slices at different wavelengths;

[0062] Figure 23 The chromatograms of Euphorbia rubra slices at different column temperatures are shown;

[0063] Figure 24 The chromatograms of Euphorbia rubra slices at different flow rates are shown;

[0064] Figure 25 This is the chromatogram for the delayed investigation of the vinegar-red Euphorbia slices;

[0065] Figure 26 The chromatograms of Euphorbia rubra slices extracted with different solvents;

[0066] Figure 27 The chromatograms of Euphorbia rubra slices at different extraction times;

[0067] Figure 28 The chromatograms of Euphorbia rubra slices with different solvent addition amounts are shown;

[0068] Figure 29 The chromatograms of Euphorbia acetochrome under different extraction methods are shown;

[0069] Figure 30 This is the chromatographic peak identification diagram of the vinegar-red euphorbia slices;

[0070] Figure 31 This is the spectrum of cyperus alcohol in vinegar-red euphorbia slices;

[0071] Figure 32 This is the spectrum of the reference substance, schisandra alcohol;

[0072] Figure 33 This is the spectrum of 3-hydroxybajiquinone of the vinegar-red euphorbia slices;

[0073] Figure 34 This is the spectrum of 3-hydroxybamoquinone, a reference substance;

[0074] Figure 35 This is the chromatogram of vinegar-red euphorbia slices under different instruments;

[0075] Figure 36 The chromatograms of Euphorbia rubra slices under different types of chromatographic columns are shown;

[0076] Figure 37 This is the chromatogram of different batches of vinegar-red Euphorbia slices;

[0077] Figure 38 It is the reference characteristic spectrum of the characteristic spectrum of Euphorbia vulgaris decoction pieces;

[0078] Figure 39 This is the chromatogram of the standard decoction of Euphorbia rubrum after the mobile phase was determined;

[0079] Figure 40 This is the wavelength selection diagram of the chromatogram of the standard decoction of Euphorbia rubrum;

[0080] Figure 41 The chromatograms of the standard decoction of Euphorbia rubrum at different column temperatures are shown;

[0081] Figure 42 The chromatograms of the standard decoction of Euphorbia rubrum at different flow rates are shown;

[0082] Figure 43 This is the chromatogram of the delayed investigation of the standard decoction of Euphorbia rubrum;

[0083] Figure 44 The chromatograms of the standard decoction of Euphorbia rubrum under different extraction solvents are shown;

[0084] Figure 45 The chromatograms of the standard decoction of Euphorbia rubrum extracted by different extraction methods are shown below;

[0085] Figure 46 The chromatograms of the standard decoction of Euphorbia rubrum at different extraction times are shown;

[0086] Figure 47 The chromatograms of the standard decoction of Euphorbia rubrum with different solvent addition amounts are shown;

[0087] Figure 48 This is the chromatogram for peak identification of the standard decoction of Euphorbia rubrum;

[0088] Figure 49 This is the spectrum of Euphorbia rubrum standard decoction Huciol;

[0089] Figure 50 This is the spectrum of the reference substance, schisandra alcohol;

[0090] Figure 51 This is the spectrum of 3-hydroxybajiquinone of the standard decoction of red euphorbia;

[0091] Figure 52 This is the spectrum of the reference substance 3-hydroxybamoquinone;

[0092] Figure 53 The chromatograms of the durability of Euphorbia rubrum standard decoction on different instruments are shown;

[0093] Figure 54 The chromatograms of different types of chromatographic columns for standard decoction of Euphorbia rubrum are shown below;

[0094] Figure 55 This is the characteristic spectrum of the standard decoction of Euphorbia rubrum;

[0095] Figure 56 It is a comparison chart of the characteristic chart of the standard decoction of Euphorbia rubrum;

[0096] Figure 57 This is the chromatogram of the standard decoction of Euphorbia acetophylla after the mobile phase was determined;

[0097] Figure 58 The chromatograms of the standard decoction of Euphorbia rubra at different wavelengths are shown;

[0098] Figure 59 The chromatograms of Euphorbia acetophylla standard decoction at different column temperatures are shown;

[0099] Figure 60 The chromatograms of Euphorbia acetophylla standard decoction at different flow rates are shown;

[0100] Figure 61 This is the chromatogram for the delayed investigation of the standard decoction of Euphorbia rubra;

[0101] Figure 62 The chromatograms of Euphorbia acetophylla standard decoction under different extraction solvents are shown;

[0102] Figure 63 The chromatograms of the standard decoction of Euphorbia acetophylla at different extraction times are shown;

[0103] Figure 64 The chromatograms of the standard decoction of Euphorbia acetophylla at different extraction times are shown;

[0104] Figure 65 The chromatograms of Euphorbia acetophylla standard decoction with different solvent addition amounts are shown;

[0105] Figure 66 This is the chromatogram for peak identification of the standard decoction of Euphorbia rubra;

[0106] Figure 67 This is the spectrum of Euphorbia cyrtonema in the standard decoction of Euphorbia cyrtonema;

[0107] Figure 68 This is the spectrum of the reference substance, schisandra alcohol;

[0108] Figure 69 This is the spectrum of 3-hydroxybajiquinone in the standard decoction of Euphorbia vulgaris;

[0109] Figure 70 This is the spectrum of the reference substance 3-hydroxybamoquinone;

[0110] Figure 71 Chromatograms for the durability investigation of different instruments for the standard decoction of Euphorbia rubrum;

[0111] Figure 72 This is the chromatogram for the durability test of Euphorbia rubra standard decoction on different types of chromatographic columns;

[0112] Figure 73 Characteristic spectra of different batches of standard decoction of Euphorbia rubrum;

[0113] Figure 74 It is a comparison chart of the characteristic chart of the standard decoction of Euphorbia rubra;

[0114] Figure 75 This is the chromatogram of the Euphorbia rubra granules after the mobile phase was determined;

[0115] Figure 76 This is the chromatogram of the red euphorbia formula granules at different wavelengths;

[0116] Figure 77 This is the chromatogram of the red euphorbia granules at different column temperatures;

[0117] Figure 78 The chromatograms of the Euphorbia rubra granules at different flow rates are shown;

[0118] Figure 79 This is the chromatographic spectrum of the delayed investigation of the red euphorbia formula granules;

[0119] Figure 80 The chromatograms of Euphorbia rubra granules extracted with different solvents are shown;

[0120] Figure 81 The chromatograms of the granules of Euphorbia rubra obtained by different extraction methods are shown;

[0121] Figure 82 The chromatograms of the Euphorbia rubra granules extracted at different times are shown;

[0122] Figure 83 The chromatograms of the Euphorbia rubra granules with different solvent addition amounts are shown;

[0123] Figure 84 This is the chromatogram for peak identification of the Hongdaji Formula Granules;

[0124] Figure 85 This is the spectrum of cyperus alcohol in the Euphorbia rubra granules;

[0125] Figure 86 This is the spectrum of the reference substance Huciol in the photo;

[0126] Figure 87 This is the spectrum of 3-hydroxybajiquinone in the red euphorbia formula granules;

[0127] Figure 88 This is the spectrum of 3-hydroxybamoquinone, a reference material for the photograph;

[0128] Figure 89 This is the durability chromatogram of the Euphorbia rubra granules under different instruments;

[0129] Figure 90 The chromatograms of the Euphorbia rubra formula granules on different types of chromatographic columns are shown below;

[0130] Figure 91 Characteristic spectra of different batches of Hongdaji granules;

[0131] Figure 92 It is the control characteristic spectrum of the characteristic spectrum of Hongdaji formula granules;

[0132] Figure 93This is the chromatogram of the Euphorbia acetophylla formula granules after the mobile phase was determined;

[0133] Figure 94 The chromatograms of the granules of Euphorbia acetophora at different wavelengths are shown;

[0134] Figure 95 This is the chromatogram of the vinegar-red euphorbia granules at different column temperatures;

[0135] Figure 96 The chromatograms of Euphorbia acetophylla granules at different flow rates are shown;

[0136] Figure 97 This is the chromatogram for the delayed investigation of the vinegar-red euphorbia granules;

[0137] Figure 98 This is the chromatogram of different extraction solvents of Euphorbia glutinosa granules;

[0138] Figure 99 This is the chromatogram of different extraction methods of Vinegar Red Euphorbia Granules;

[0139] Figure 100 This is the chromatogram of different extraction times of the vinegar red euphorbia granules;

[0140] Figure 101 This is the chromatogram of different solvent addition amounts of Euphorbia acetochrome;

[0141] Figure 102 This is the chromatogram for peak identification of the granules of Euphorbia glutinosus;

[0142] Figure 103 This is the spectrum of cyperus alcohol in the Euphorbia glutinosa granules;

[0143] Figure 104 This is the spectrum of the reference substance, schisandra alcohol;

[0144] Figure 105 This is the spectrum of 3-hydroxybajiquinone in the vinegar-red euphorbia granules;

[0145] Figure 106 This is the spectrum of the reference substance 3-hydroxybamoquinone;

[0146] Figure 107 This is the durability chromatogram of the vinegar-red euphorbia granules under different instruments;

[0147] Figure 108 The chromatograms of different chromatographic columns for the granules of Euphorbia glutinosa are shown;

[0148] Figure 109 This is the characteristic spectrum verification diagram of different batches of Vinegar-Red Euphorbia Granules;

[0149] Figure 110It is a comparison chart of the characteristic chart of the vinegar-red euphorbia formula granules. DETAILED DESCRIPTION

[0150] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0151] In view of the need for establishing a method for identifying red euphorbia, vinegar red euphorbia and their preparations in the prior art, the present application provides a method for detecting a characteristic spectrum of red euphorbia, vinegar red euphorbia and their preparations, which can respectively establish a characteristic spectrum identification method for red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules, as well as vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules. The method has high stability and good repeatability, and can effectively and comprehensively reflect the status of red euphorbia, vinegar red euphorbia and their preparations, providing a basis for the formulation of their subsequent quality standards. Specifically, the embodiment of the present invention discloses a method for detecting a characteristic spectrum of red euphorbia, vinegar red euphorbia and their preparations, comprising the following steps:

[0152] S1) extracting the raw material of the test product with a solvent to obtain a test solution; the raw material of the test product is a red euphorbia raw material or a vinegar red euphorbia raw material;

[0153] S2) measuring the test solution by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the test product;

[0154] The chromatographic conditions of the high performance liquid chromatography method are as follows: a C18 chromatographic column; mobile phase A is methanol; mobile phase B is 0.2% phosphoric acid solution; and gradient elution.

[0155] In the process of establishing the characteristic spectrum of red euphorbia, vinegar red euphorbia and its preparations in the present application, first, the raw material of the product to be tested is extracted with a solvent to obtain a test liquid, and the raw material to be tested is a red euphorbia raw material or a vinegar red euphorbia raw material. Specifically, the red euphorbia raw material is a red euphorbia medicinal material, a decoction piece, a standard decoction or a formula granule, and the vinegar red euphorbia raw material is a vinegar red euphorbia medicinal material, a decoction piece, a standard decoction or a formula granule. In the process of preparing the test liquid, the red euphorbia medicinal material, a decoction piece, a standard decoction or a formula granule are respectively extracted with a solvent to obtain a red euphorbia medicinal material test liquid, a red euphorbia decoction piece test liquid, a red euphorbia standard decoction test liquid or a red euphorbia formula granule test liquid; the vinegar red euphorbia medicinal material, a decoction piece, a standard decoction or a formula granule are respectively extracted with a solvent to obtain a vinegar red euphorbia medicinal material test liquid, a vinegar red euphorbia decoction piece test liquid, a vinegar red euphorbia standard decoction test liquid or a vinegar red euphorbia formula granule test liquid. In the present application, the solvent is selected from methanol, and the extraction method is ultrasonic extraction. The ultrasonic power of the ultrasonic extraction is 600W, the frequency is 40kHz, and the time of the ultrasonic extraction is 20 to 40 minutes; the ratio of the raw material to be tested and the solvent is (0.5 to 1) g: (10 to 30) ml; specifically, the time of the ultrasonic extraction is 30 minutes, and the ratio of the raw material to be tested and the solvent is 0.5 g: 20 ml.

[0156] According to the present invention, the preparation of the test solution of red euphorbia medicinal materials, decoction pieces, standard decoctions or formula granules is specifically as follows: red euphorbia medicinal materials, decoction pieces, standard decoctions or formula granules are added to methanol and ultrasonically extracted; the ultrasonic power of the ultrasonic extraction is 600W, the frequency is 40kHz, and the extraction time is 30min; the ratio of the red euphorbia medicinal materials, decoction pieces, standard decoctions or formula granules and methanol is 0.5g:20ml.

[0157] According to the present invention, the preparation of the test solution of the vinegar-red euphorbia medicinal materials, decoction pieces, standard decoctions or formula granules is specifically as follows: the vinegar-red euphorbia medicinal materials, decoction pieces, standard decoctions or formula granules are added to methanol and ultrasonically extracted; the ultrasonic power of the ultrasonic extraction is 600W, the frequency is 40kHz, and the extraction time is 30min; the ratio of the vinegar-red euphorbia medicinal materials, decoction pieces, standard decoctions or formula granules and methanol is 0.5g:20ml.

[0158] The present invention adopts the selection of the above-mentioned extraction solvent to ensure that the chromatographic peak shape is good and the separation is moderate. The extraction time is selected to be 30 minutes, the chromatogram peak shape separation is good, and the extraction is complete.

[0159] The present invention also includes preparing reference substance solution 1 and reference substance solution 2:

[0160] The red euphorbia medicinal material was decocted with water, the filtered filtrate was evaporated to dryness, and then extracted with methanol, and the control medicinal material reference solution was obtained after ultrasonication;

[0161] The reference substance solution 1, the reference substance solution 2 and the reference medicinal material reference solution were respectively determined by high performance liquid chromatography to obtain a chromatogram of cyperus alcohol and a chromatogram of 3-hydroxybajiquinone;

[0162] The components of the HPLC characteristic spectra of Euphorbia acetosporum medicinal materials, decoction pieces, standard decoction and its formula granules were qualitatively determined based on the chromatograms of tiglol and 3-hydroxybajiquinone.

[0163] The reference substances, euphorbia pulex and 3-hydroxybajiquinone solution, are determined by high performance liquid chromatography to obtain chromatograms of the reference substances, and the components of the HPLC characteristic spectra of the red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules are qualitatively determined based on the chromatograms of the reference substances, or the components of the HPLC characteristic spectra of the vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules are qualitatively determined.

[0164] The present application then uses high performance liquid chromatography to determine the liquid to be tested. The chromatographic conditions of the high performance liquid chromatography are: mobile phase A is methanol, mobile phase B is 0.2% phosphoric acid solution, and gradient elution is performed. Specifically, the gradient elution is as follows:

[0165] 0-12 min, phase A: 10-40%, phase B: 90-60%;

[0166] 12-15 min, phase A: 40-43%, phase B: 60-57%;

[0167] 15-24 min, phase A: 43-45%, phase B: 57-55%;

[0168] 24-28 min, phase A: 45-55%, phase B: 55-45%;

[0169] 28-36 min, phase A: 55-65%, phase B: 45-35%;

[0170] 36~42min, phase A: 65~73%, phase B: 35~27%;

[0171] 42-43 min, phase A: 73-90%, phase B: 27-10%;

[0172] 43-50min, phase A: 90%, phase B: 10%.

[0173] The present invention has good baseline separation under the above gradient elution, good separation of each peak and a stable baseline.

[0174] The chromatographic column used for gradient elution in this application is a C18 column with a column length of 150 mm, an inner diameter of 2.1 mm, a particle size of 2.7 μm, and a column temperature of 30°C.

[0175] Under the above-mentioned 30° C. condition, the chromatographic column of the present invention has symmetrical chromatographic peaks, good separation, and relatively complete peak extraction.

[0176] In this application, the flow rate of the mobile phase is 0.25 ml / min. Through experiments, it was found that the chromatographic peak shape is better and the separation degree is moderate at a flow rate of 0.25 ml / min.

[0177] The detection wavelength of this application is preferably 280nm; through experiments, it is found that the chromatographic peak information volume is larger at a wavelength of 280nm, and the chromatogram baseline is more stable.

[0178] The injection volume in this application is 1 μl.

[0179] After the relevant parameter conditions of the above-mentioned high-performance liquid chromatography are determined, the red euphorbia medicinal material solution is determined by high-performance liquid chromatography, or the red euphorbia decoction piece solution is determined by high-performance liquid chromatography, or the red euphorbia standard decoction solution is determined by high-performance liquid chromatography, or the red euphorbia formula granule solution is determined by high-performance liquid chromatography, or the vinegar red euphorbia medicinal material solution is determined by high-performance liquid chromatography, or the vinegar red euphorbia decoction piece solution is determined by high-performance liquid chromatography, or the vinegar red euphorbia standard decoction solution is determined by high-performance liquid chromatography, or the vinegar red euphorbia formula granule solution is determined by high-performance liquid chromatography.

[0180] The characteristic spectrum detection method provided by the present invention uses fingerprint spectrum to control the substance group of red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules or the substance group of vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules under a liquid chromatography condition, and uses thorn alcohol to locate the fingerprint spectrum; it can greatly reduce the cost of detection and realize qualitative detection.

[0181] The HPLC characteristic spectrum similarity of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules was evaluated by using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectrum of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules was obtained, which consisted of 12 characteristic peaks, including peak 8 (S): scutellarin peak, peak 11: 3-hydroxybajiquinone peak;

[0182] At the same time, the Chinese medicine chromatographic fingerprint similarity evaluation system was used to evaluate the similarity of the HPLC characteristic spectra of the vinegar-red Euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules, and the HPLC standard characteristic spectra of the vinegar-red Euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules were obtained, which consisted of 12 characteristic peaks, including peak 8 (S): scutellaria alcohol peak, and peak 11: 3-hydroxybajiquinone peak.

[0183] In the characteristic spectra of the red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules thereof, the relative retention time of each characteristic peak and the S peak is calculated with tiglol as the reference peak S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 8), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 12), and 1.61 (peak 12);

[0184] In the characteristic spectra of the vinegar-red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules thereof, tiglol is used as the reference peak S peak to calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 8), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 12), and 1.61 (peak 12).

[0185] Quality judgment standard: Take samples of red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules, and operate according to the same method as above to obtain characteristic spectra of red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules. Use the 2012 edition of the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" of the State Pharmacopoeia Committee to analyze the standard characteristic spectra and sample characteristic spectra of red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules, and the similarity is extremely high. Similarly, take samples of vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules, and operate according to the same method as above to obtain characteristic spectra of vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules. Use the 2012 edition of the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" of the State Pharmacopoeia Committee to analyze the standard characteristic spectra and sample characteristic spectra of vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions and their formula granules, and the similarity is extremely high.

[0186] The method provided by the present invention can effectively monitor the quality of different batches of red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules thereof, so that the quality is stable. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensive monitoring of product quality. At the same time, the method provided by the present invention can effectively monitor the quality of different batches of vinegar red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules thereof, so that the quality is stable. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensive monitoring of product quality.

[0187] The characteristic maps of Euphorbia rubrum medicinal materials, decoction pieces, standard decoctions and their formula granules or vinegar Euphorbia rubrum medicinal materials, decoction pieces, standard decoctions and their formula granules established by the present invention use trichol as a reference, focus 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.

[0188] The present invention provides a characteristic spectrum detection method for Euphorbia rubrum, Euphorbia rubrum and their preparations, comprising the following steps: S1) extracting a test sample raw material with a solvent to obtain a test liquid; the test sample raw material is Euphorbia rubrum raw material or Euphorbia rubrum raw material; S2) measuring the test liquid with high performance liquid chromatography to obtain an HPLC characteristic spectrum of the test sample; the newly developed characteristic spectrum method of the present invention is simple and easy to operate for sample preparation, identifies a large number of characteristic peaks, and accurately and reliably detects Euphorbia rubrum, Euphorbia rubrum and their preparations; and effectively controls the authenticity, quality consistency and stability of Euphorbia rubrum, Euphorbia rubrum and their preparations, providing a basis for comprehensive quality evaluation of Euphorbia rubrum, Euphorbia rubrum and their preparations.

[0189] In order to further understand the present invention, the red euphorbia, vinegar red euphorbia and their preparation characteristic spectrum detection method provided by the present invention are described in detail below in conjunction with the examples. The protection scope of the present invention is not limited by the following examples.

[0190] Example 1 Detection of characteristic patterns of Euphorbia rubrum medicinal materials

[0191] The 2020 edition of the "Chinese Pharmacopoeia" stipulates that the dried tubers of the red euphorbia Knoxiavalerianoides Thorel et Pitard of the Rubiaceae family are collected; the fibrous roots are removed, washed, slightly blanched in boiling water, and dried.

[0192] In order to ensure the reliability of the established quality standards for Euphorbia rubrum, a total of 21 batches of Euphorbia rubrum medicinal materials were collected. The specific origin information is shown in Table 1:

[0193] Table 1 Summary of origin information of Euphorbia rubrum medicinal materials

[0194]

[0195]

[0196] 1.1 Instruments and Materials

[0197] High performance liquid chromatograph: three different models of ultra-high performance liquid chromatographs from Shimadzu, Agilent, and Thermo Fisher Scientific;

[0198] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0199] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0200] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0201] Chromatographic columns: Column 1: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm; Column 2: Agilent ZORBAX SB-C18 2.1×150mm 1.8μm; Column 3: Waters CORTECST3 2.1×150mm 1.6μm;

[0202] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0203] 3-Hydroxybamoquinone reference substance (China Food and Drug Inspection Institute, batch number: 111962-202102, purity: 97.2%);

[0204] Huciol reference substance (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH033801, purity: 99.76%);

[0205] Red euphorbia medicinal material batch number: 010200-2307001, XXLS202307649, XXLS202307650, XXLS202307651, XXLS202307652, XXLS202307653, XXLS202307654, XXLS202307655, XXLS202307656, XXLS202307657, XX LS202307658, XXLS202307659, XXLS202307660, XXLS202307661, XXLS202307662, XXLS2023 07663, XXLS202307664, XXLS202307665, XXLS202307666, XXLS202307667, XXLS202307668.

[0206] 1.2 Feature Spectrum Detection Method

[0207] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 2; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm; theoretical plate number calculated based on the 3-hydroxyephrinone peak should be no less than 3000;

[0208] Table 2 Initial relevant parameter data of gradient elution of red euphorbia medicinal materials

[0209] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0210] Preparation of reference solution

[0211] Take 1 g of Euphorbia rubrum control medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the control medicinal material reference solution;

[0212] Take appropriate amount of cyperus alcohol and 3-hydroxybajiquinone reference substances, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions;

[0213] Preparation of test solution: Take about 0.5 g of the product powder (passed through a No. 4 sieve), add 20 ml of methanol, plug tightly, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Let cool, shake well, filter, and take the filtrate to obtain the product;

[0214] Determination method: Accurately aspirate 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. 1.3 Chromatographic conditions and system suitability test

[0215] 1.3.1 Mobile phase selection

[0216] Methanol was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B for gradient elution. The results were as follows: Figure 1 As shown; 1.3.2 Wavelength selection

[0217] Based on the experimental conditions proposed above, the test solution was scanned in the full band using a diode array detector, and the chromatograms of the test solution at wavelengths of 220nm, 260nm, 270nm, 280nm, 300nm, and 320nm were extracted respectively. The results are as follows: Figure 2 As shown, Figure 2 The chromatograms of Euphorbia rubrum medicinal materials at different wavelengths are shown in the figure. The wavelengths from top to bottom are 220nm, 260nm, 270nm, 300nm, 320nm, and 280nm. Figure 2 It can be seen that when the detection wavelength is 280 nm, the chromatographic peak information volume is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 280 nm.

[0218] 1.3.3 Column temperature investigation

[0219] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. The results are as follows: Figure 3 As shown, Figure 3 The chromatograms of red euphorbia medicinal materials under different column temperature conditions are shown in the figure. It can be seen from the figure that when the column temperature is 30℃, the chromatogram peak shape is more symmetrical, the separation is better, and the peaks are more complete, so the column temperature is temporarily set at 30℃.

[0220] 1.3.4 Flow rate investigation

[0221] Based on the experimental conditions proposed above, the flow rates of 0.2ml / min, 0.25ml / min and 0.3ml / min were investigated respectively. The results are as follows: Figure 4 As shown, Figure 4 The chromatograms of Euphorbia rubrum medicinal materials at different flow rates are shown in the figure. It can be seen from the figure that when the flow rate is 0.25ml / min, the chromatogram peak shape is better and the separation degree is moderate, so the flow rate is temporarily set at 0.25ml / min.

[0222] 1.3.5 Delay Investigation

[0223] Based on the experimental conditions proposed above, a delay test was conducted, and the results are as follows: Figure 5 As shown, Figure 5 This is the chromatogram of the delayed detection of red euphorbia medicinal material. It can be seen from the figure that the sample has basically no chromatographic peak after 50 minutes, so the sample detection time is set to 50 minutes.

[0224] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of Euphorbia rubrum medicinal material were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 150 mm, inner diameter was 2.1 mm, and particle size was 2.7 μm); methanol was used as mobile phase A, and 0.2% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the regulations in the following table; the flow rate was 0.25 ml per minute; the column temperature was 30°C; the detection wavelength was 280 nm; and the number of theoretical plates calculated based on the trichol peak should be no less than 3000.

[0225] Table 3 Gradient elution related parameter data of red euphorbia medicinal materials

[0226] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27

[0227] 1.4 Preparation and inspection of test samples

[0228] 1.4.1 Extraction solvent investigation

[0229] Take 0.5g of this product powder (passed through No. 4 sieve, batch number: 010200-2307001) and place it in a stoppered conical flask. Add 20ml of 30% methanol, 50% methanol, 70% methanol, methanol, water, and 50% ethanol respectively. Then ultrasonicate (power 600W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate. The result is as follows: Figure 6 As shown, Figure 6 The chromatograms of Euphorbia rubrum under different extraction solvents are shown in the figure. It can be seen from the figure that when the extraction solvent is methanol, the peak shapes of the characteristic peaks are good and the separation is moderate. The extraction solvent is temporarily determined to be methanol.

[0230] 1.4.2 Extraction time investigation

[0231] Take 0.5g of the powder of this product (passed through a No. 4 sieve, batch number: 010200-2307001), place it in a stoppered conical flask, add 20ml of methanol, and ultrasonically extract the test sample for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and take the filtrate; the results are shown in Table 7. Figure 7 The chromatograms of Euphorbia rubrum medicinal materials at different extraction times are shown in the figure. It can be seen from the figure that when the extraction time is 30 minutes, the chromatogram peak shape and separation are better, and the extraction is complete; therefore, the extraction time is determined to be 30 minutes.

[0232] 1.4.3 Investigation of solvent addition amount

[0233] Take 0.5g of this product powder (passed through No. 4 sieve, batch number: 010200-2307001) and place it in a stoppered conical flask. Add 10ml, 20ml and 50ml of methanol respectively, stopper it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Shake well, filter and take the filtrate. The result is as follows: Figure 8 As shown, Figure 8 The chromatograms of Euphorbia rubrum medicinal materials with different solvent addition amounts are shown in the figure. It can be seen from the figure that the chromatogram effects are basically the same under the conditions of different extraction solvent addition amounts. In order to ensure sufficient extraction, the solvent addition amount is determined to be 20 ml.

[0234] 1.4.4 Investigation of extraction methods

[0235] Take 1.0 g of this product powder (passed through a No. 4 sieve, batch number: 010200-2307001), place it in a stoppered conical flask, add 50 ml of water and boil for 30 minutes, filter, evaporate to dryness, add 20 ml of methanol to the residue, stopper tightly, and sonicate for 30 minutes (power 600 W, frequency 40 kHz), let cool, shake well, filter, and take the filtrate to obtain;

[0236] Take another 0.5g of powder (passed through No. 4 sieve, batch number: 010200-2307001), place it in a stoppered conical flask, add 20ml of methanol, stopper it, reflux and ultrasonicate for 30 minutes (power 600W, frequency 40kHz), cool it, shake it well, filter it, and take the filtrate to obtain the result. Figure 9 As shown, Figure 9 The chromatograms of red euphorbia medicinal materials under different extraction methods can be seen from the figure. When the test sample is first decocted and then ultrasonically extracted, there are fewer peaks. There is little difference in peaks between directly adding solvent for ultrasonic treatment and treatment, and the peak shapes are better separated. Therefore, the extraction method of the test sample is determined to be ultrasonic.

[0237] 1.4.5 Determine the test sample preparation method

[0238] The preparation method of the test solution of the red euphorbia medicinal material atlas is as follows: take about 0.5g of the powder of the product (passed through a No. 4 sieve), add 20ml of methanol, plug it tightly, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Then cool it, shake it evenly, filter it, and take the filtrate to obtain the product.

[0239] 1.5 Methodological Review

[0240] 1.5.1 Chromatographic peak identification

[0241] Preparation of test solution According to the experimental conditions proposed above, the test solution of Euphorbia rubrum medicinal material was prepared.

[0242] Preparation of reference solution: Take appropriate amount of cyperus alcohol and 3-hydroxybamoquinone reference substances, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions.

[0243] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking Euphorbia rubrum medicinal materials was prepared.

[0244] Locate the characteristic spectrum peaks of Euphorbia rubrum, such as Figures 10-14 As shown, Figure 10 This is the chromatogram for peak identification of Euphorbia rubrum. Figure 11 This is the spectrum of Euphorbia rubrum. Figure 12 This is the spectrum of cyperus alcohol in the reference substance. Figure 13 This is the spectrum of 3-hydroxybajiquinone of red euphorbia medicinal material. Figure 14 The spectrum of 3-hydroxybajiquinone in the reference substance is shown in the figure. As can be seen from the figure, peak 8 is thorn alcohol and peak 11 is 3-hydroxybajiquinone. The retention time and spectrum of the reference substance can be compared with the retention time and spectrum of the target peak in the red euphorbia medicinal material. Figure 1 There is one-to-one correspondence and no interference from the negative solution, so the method has good specificity.

[0245] In summary, the 12 characteristic peaks in the medicinal materials were included in the subsequent investigation and methodological research was carried out.

[0246] 1.5.2 Precision investigation

[0247] Take the test solution of Euphorbia rubrum (Batch No.: 010200-2307001) and inject 6 times in a row according to the proposed experimental method, each time with 1 μl, and calculate the retention time of each characteristic peak. The results are shown in Table 4;

[0248] Table 4 Precision Investigation-Retention Time Data Table

[0249]

[0250] The results showed that the RSD of the retention time of each characteristic peak was 0.09%-0.26%, indicating that the method has good precision.

[0251] 1.5.3 Repeatability Study

[0252] Six samples of the test solution of Euphorbia rubrum (Batch No. 010200-2307001) were prepared and tested according to the proposed experimental method. The results are shown in Table 5.

[0253] Table 5 Repeatability study - relative retention time ratio data table

[0254]

[0255] The results showed that the RSD of the relative retention time of the six samples was 0.09% to 0.46%, indicating that the method had good repeatability.

[0256] 1.5.4 Intermediate precision study

[0257] Based on the experimental conditions proposed above, two portions of Euphorbia rubrum (batch number: 010200-2307001) were accurately weighed to prepare test solutions. The solutions were then analyzed on three different ultra-high performance liquid chromatographs (Shimadzu, Agilent, and Thermo Fisher). The results were as follows: Figure 15 As shown in Table 6; Figure 15 This is the chromatogram of Euphorbia rubrum under different instruments;

[0258] Table 6 Intermediate precision-relative retention time ratio

[0259]

[0260] The results showed that when the three instruments were used to detect the test samples, the RSD range of the relative retention time of each characteristic peak was 3.32% to 44.96%, indicating that different instruments had a great influence on the retention time of the characteristic method.

[0261] 1.5.5 Inspection by different personnel and time

[0262] Based on the experimental conditions proposed above, two portions of Euphorbia rubrum (batch number: 010200-2307001) were accurately weighed by different personnel (A and B) at different times (T1 and T2) to prepare the test samples and perform the assay. The results are shown in Table 7.

[0263] Table 7 Personnel and time inspection-relative retention time

[0264]

[0265]

[0266] The results showed that when different people measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks were 0.09% to 0.46%, indicating that the method was stable.

[0267] 1.5.6 Durability inspection

[0268] Based on the experimental conditions proposed above, different types of chromatographic columns were investigated; the results are as follows Figure 16 , as shown in Table 8, Figure 16 The chromatograms of Euphorbia rubrum under different chromatographic columns are shown;

[0269] Table 8 Durability Study - Relative Retention Time Ratio Data

[0270]

[0271] The results showed that when the samples were detected using the above chromatographic column, the RSD of the relative retention time of the characteristic peaks was between 1.36% and 2.69%, indicating that the chromatographic column had good durability.

[0272] 1.5.7 Stability Study

[0273] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. The results are shown in Table 9.

[0274] Table 9 Stability Study-Relative Retention Time Data

[0275]

[0276] The results showed that the RSDs of the characteristic peak retention times were between 0.26% and 0.69%, and the sample solutions were stable within 24 hours.

[0277] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above 12 characteristic peaks were included in the subsequent investigation.

[0278] 1.6 Determination of characteristic peaks and establishment of reference spectrum

[0279] Using this method, characteristic spectrum analysis was performed on 21 batches of samples and relative retention time ratios were calculated. The results are as follows: Figure 17 As shown in Table 10, Figure 17 Characteristic spectra of 21 batches of Euphorbia rubrum medicinal materials; in the figure, S1-S21 are: 010200-2307001, XXLS202307649, XXLS202307650, XXLS202307651, XXLS202307652, XXLS202307653, XXLS202307654, XXLS202307655, XXLS202307656, XXLS2020 2307657, XXLS202307658, XXLS202307659, XXLS202307660, XXLS202307661, XXLS202307662, XXL S202307663, XXLS202307664, XXLS202307665, XXLS202307666, XXLS202307667, XXLS202307668.

[0280] Table 10 Relative retention time data of 21 batches of Euphorbia rubrum medicinal materials

[0281] Number batch number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8(S) Peak 9 Peak 10 Peak 11 Peak 12 1 010200-2307001 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.33 1.39 1.53 1.61 2 XXLS202307649 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.33 1.37 1.53 1.61 3 XXLS202307650 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.33 1.37 1.53 1.61 4 XXLS202307651 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 5 XXLS202307652 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 6 XXLS202307653 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 7 XXLS202307654 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 8 XXLS202307655 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 9 XXLS202307656 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 10 XXLS202307657 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 11 XXLS202307658 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 12 XXLS202307659 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 13 XXLS202307660 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.60 14 XXLS202307661 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.61 15 XXLS202307662 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.61 16 XXLS202307663 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.33 1.37 1.52 1.61 17 XXLS202307664 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.33 1.37 1.52 1.61 18 XXLS202307665 0.12 0.34 0.38 0.40 0.46 0.48 0.60 1.00 1.33 1.37 1.52 1.61 19 XXLS202307666 0.12 0.34 0.38 0.40 0.46 0.49 0.60 1.00 1.33 1.37 1.53 1.61 20 XXLS202307667 0.12 0.34 0.38 0.40 0.46 0.49 0.60 1.00 1.33 1.39 1.53 1.61 21 XXLS202307668 0.12 0.34 0.38 0.40 0.46 0.49 0.60 1.00 1.33 1.39 1.53 1.61 average value 0.12 0.34 0.38 0.40 0.45 0.48 0.60 1.00 1.32 1.37 1.52 1.61 RSD% 0.20 0.08 0.10 0.10 0.12 0.11 0.15 0.00 0.21 0.49 0.16 0.21 90％ 0.11 0.31 0.34 0.36 0.41 0.44 0.54 0.90 1.19 1.24 1.37 1.44 110％ 0.13 0.38 0.42 0.44 0.50 0.53 0.66 1.10 1.46 1.51 1.67 1.77

[0282] Based on the principle of stable relative retention times and the fact that peaks can be detected in all batches of samples at relatively high levels, a total of 12 peaks with good reproducibility were selected as characteristic peaks. The results showed that the RSD of the relative retention times of the 12 characteristic peaks in 21 batches of Euphorbia rubrum medicinal materials was less than 1.0%.

[0283] Final regulations: The test sample's characteristic spectrum should show 12 characteristic peaks, of which Peak 8 should correspond to the retention time of the corresponding reference peak of the control. The peak corresponding to the scutellariae reference peak 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: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 7), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 11), and 1.61 (peak 12).

[0284] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 21 batches of red euphorbia medicinal materials, and a reference characteristic spectrum of red euphorbia medicinal materials was established, such as Figure 18 shown.

[0285] Example 2

[0286] The process of establishing the HPLC characteristic spectrum of the vinegar red euphorbia medicinal material is the same as that of the red euphorbia medicinal material.

[0287] Example 3 Detection of characteristic patterns of Euphorbia rubrum slices

[0288] 3.1 Experimental instruments and materials

[0289] High performance liquid chromatograph: three different models of ultra-high performance liquid chromatographs from Shimadzu, Agilent, and Thermo Fisher Scientific;

[0290] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0291] Ultrapure water machine: tissue type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0292] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0293] Chromatographic columns: Column 1: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm; Column 2: Agilent ZORBAX SB-C18 2.1×150mm 1.8μm; Column 3: Waters CORTECST3 2.1×150mm 1.6μm;

[0294] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0295] 3-Hydroxybamoquinone reference substance (China Food and Drug Inspection Institute, batch number: 111962-202102, purity: 97.2%);

[0296] Huciol reference substance (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH033801, purity: 99.76%);

[0297] Batch numbers of Euphorbia rubrum slices: HDJ-230701, HDJ-230702, HDJ-230703, HDJ-230704, HDJ-230705, HDJ-230706, HDJ-230707, HDJ-230708, HDJ-230709, HDJ-230710, HDJ-230711, HDJ-230712, HDJ-230713, HDJ-230714, HDJ-230715, HDJ-230716, HDJ-230717, HDJ-230718, HDJ-230719, HDJ-230720, HDJ-230721.

[0298] 3.2 Chromatographic conditions

[0299] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, and gradient elution as specified in the table below; the flow rate was 0.25 ml / min; the column temperature was 30°C; the detection wavelength was 280 nm; the number of theoretical plates calculated based on the trichol peak should be no less than 3000; the gradient elution parameters are shown in Table 11.

[0300] Table 11 Initial data table of gradient elution of Euphorbia rubrum slices

[0301] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0302] Preparation of reference solution

[0303] Take 1 g of Euphorbia rubrum control medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the control medicinal material reference solution;

[0304] Take appropriate amount of cyperus alcohol and 3-hydroxybajiquinone reference substances, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions;

[0305] Preparation of test solution: Take about 0.5 g of the product powder (passed through a No. 4 sieve), add 20 ml of methanol, plug tightly, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Let cool, shake well, filter, and take the filtrate to obtain the product;

[0306] Determination method: Accurately aspirate 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0307] 21 batches of Euphorbia rubrum slices were tested, and the results were as follows: Figure 19 As shown in Table 12, Figure 19 Characteristic maps of 21 batches of Euphorbia rubrum slices;

[0308] Table 12 Relative retention time of characteristic spectra of Euphorbia rubrum slices

[0309]

[0310]

[0311] Based on the principle of stable relative retention time and the fact that peaks can be detected in all batches of samples at relatively high levels, 12 peaks with good repeatability were selected as characteristic peaks. The results showed that the RSD of the relative retention times of the 12 characteristic peaks in 21 batches of Euphorbia rubrum slices was less than 1.0%.

[0312] Final regulations: The test sample's characteristic spectrum should show 12 characteristic peaks, of which Peak 8 should correspond to the retention time of the corresponding reference peak of the control. The peak corresponding to the scutellariae reference peak 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: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 7), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 11), and 1.61 (peak 12).

[0313] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 21 batches of Euphorbia rubrum slices, and a comparison map of the characteristic maps of Euphorbia rubrum slices was established. Figure 20 shown.

[0314] Example 4 Establishment of characteristic spectrum of Euphorbia rubra slices

[0315] 4.1 Materials, reagents, and instruments

[0316] High performance liquid chromatograph: three different models of ultra-high performance liquid chromatographs from Shimadzu, Agilent, and Thermo Fisher Scientific;

[0317] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0318] Ultrapure water machine: tissue type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0319] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0320] Chromatographic columns: Column 1: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm; Column 2: Agilent ZORBAX SB-C18 2.1×150mm 1.8μm; Column 3: Waters CORTECST3 2.1×150mm 1.6μm;

[0321] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0322] 3-Hydroxybamoquinone reference substance (China Food and Drug Inspection Institute, batch number: 111962-202102, purity: 97.2%);

[0323] Huciol reference substance (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH033801, purity: 99.76%);

[0324] Batch numbers of vinegar-red euphorbia slices: CHDJ-230701, CHDJ-230702, CHDJ-230703, CHDJ-230704, CHDJ-230705, CHDJ-230706, CHDJ-230707, CHDJ-230708, CHDJ-230709, SNT-2307 10, CHDJ-230711, CHDJ-230712, CHDJ-230713, CHDJ-230714, CHDJ-230715, CHDJ-230716, CHDJ-230717, CHDJ-230718, CHDJ-230719, CHDJ-230720, CHDJ-230721.

[0325] 4.2 Chromatographic conditions

[0326] Chromatographic conditions and system suitability tests were conducted using octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, and gradient elution as specified in the table below; the flow rate was 0.25 ml / min; the column temperature was 30°C; the detection wavelength was 280 nm; the number of theoretical plates calculated based on the 3-hydroxyephrinone peak should be no less than 3000; the gradient elution parameters are shown in Table 13.

[0327] Table 13 Initial data table of gradient elution of Euphorbia rubrum slices

[0328] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0329] Preparation of reference solution: Take 1 g of Euphorbia rubrum reference medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the reference medicinal material solution;

[0330] Preparation of reference solution Take appropriate amount of cyperus alcohol and 3-hydroxybajiquinone reference substances, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions;

[0331] Preparation of test solution: Take about 0.5 g of the product powder (passed through a No. 4 sieve), add 20 ml of methanol, plug tightly, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Let cool, shake well, filter, and take the filtrate to obtain the product;

[0332] Determination method: Accurately aspirate 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. 4.3 Chromatographic conditions and system suitability test

[0333] 4.3.1 Mobile phase selection

[0334] Methanol was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B for gradient elution. The results were as follows: Figure 21 As shown, Figure 21 This is the characteristic spectrum of Euphorbia acetophylla after the mobile phase is selected.

[0335] 4.3.2 Wavelength Selection

[0336] Based on the experimental conditions proposed above, the test solution was scanned in the full band using a diode array detector, and the chromatograms of the test solution at wavelengths of 220nm, 260nm, 270nm, 280nm, 300nm, and 320nm were extracted respectively. The results are as follows: Figure 22 As shown, Figure 22 These are the chromatograms of vinegar-red euphorbia slices at different wavelengths. From top to bottom in the figure, they are the chromatograms of vinegar-red euphorbia slices at 220nm, 260nm, 270nm, 300nm, 320nm, and 280nm. It can be seen from the figure that when the detection wavelength is 280nm, the amount of chromatographic peak information is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 280nm.

[0337] 4.3.3 Column temperature investigation

[0338] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. The results are as follows: Figure 23 As shown, Figure 23The chromatograms of the vinegar-red euphorbia slices at different column temperatures are shown in the figure. It can be seen that when the column temperature is 30℃, the chromatogram peak shape is more symmetrical, the separation is better, and the peaks are more complete; therefore, the column temperature is temporarily set at 30℃.

[0339] 4.3.4 Flow rate investigation

[0340] Based on the experimental conditions proposed above, the flow rates of 0.2ml / min, 0.25ml / min and 0.3ml / min were investigated respectively. The results are as follows: Figure 24 As shown, Figure 24 The chromatograms of the vinegar-red euphorbia slices at different flow rates are shown in the figure. It can be seen that when the flow rate is 0.25ml / min, the chromatogram peak shape is better and the separation degree is moderate; therefore, the flow rate is temporarily set at 0.25ml / min.

[0341] 4.3.5 Delay Investigation

[0342] Based on the experimental conditions proposed above, a delay test was conducted, and the results are as follows: Figure 25 As shown, Figure 25 This is the chromatogram of the delayed investigation of the vinegar-red Euphorbia decoction slices. It can be seen from the figure that the sample has basically no chromatographic peak after 50 minutes, so the sample detection time is set to 50 minutes.

[0343] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of Euphorbia rubra slices were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the requirements of Table 14; the flow rate was 0.25 ml per minute; the column temperature was 30°C; the detection wavelength was 280 nm; and the number of theoretical plates calculated based on the trichol peak should be no less than 3000.

[0344] Table 14 Gradient elution parameters of Euphorbia rubra slices

[0345] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27

[0346] 4.4 Preparation of test solution

[0347] 4.4.1 Extraction solvent investigation

[0348] Take 0.5g of this product powder (passed through No. 4 sieve, batch number: CHDJ-230701) and place it in a stoppered conical flask. Add 20ml of 30% methanol, 50% methanol, 70% methanol, methanol, water, and 50% ethanol respectively. Ultrasonicate (power 600W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate. The result is as follows: Figure 26 As shown, Figure 26The chromatograms of Euphorbia pulex slices with different extraction solvents are shown in the figure. As can be seen from the figure, when the extraction solvent is methanol, the peak shapes of the characteristic peaks are good and the separation is moderate. The extraction solvent is tentatively set to methanol.

[0349] 4.4.2 Extraction time investigation

[0350] Take 0.5g of the powder of this product (passed through No. 4 sieve, batch number: CHDJ-230701), place it in a stoppered conical flask, add 20ml of methanol, and ultrasonically extract the sample for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and take the filtrate; the results are as follows: Figure 27 As shown, Figure 27 The chromatograms of the Euphorbia rapa slices at different extraction times are shown in the figure. As can be seen from the figure, when the extraction time is 30 minutes, the chromatogram peak shape and separation are better, and the extraction is complete; therefore, the extraction time is determined to be 30 minutes.

[0351] 4.4.3 Investigation of solvent addition amount

[0352] Take 0.5g of this product powder (passed through No. 4 sieve, batch number: CHDJ-230701) and place it in a stoppered conical flask. Add 10ml, 20ml and 50ml of methanol respectively, stopper it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Shake well, filter and take the filtrate. The result is as follows: Figure 28 As shown, Figure 28 The chromatograms of Euphorbia rubra slices with different solvent addition amounts are shown in the figure. It can be seen from the figure that when the extraction solvent addition amount is 20 ml, the peak shape and separation of each chromatographic peak are better; therefore, 20 ml of solvent is selected.

[0353] 4.4.4 Investigation of extraction methods

[0354] Take 1.0 g of this product powder (passed through a No. 4 sieve, batch number: CHDJ-230701), place it in a stoppered conical flask, add 50 ml of water and boil for 30 minutes, filter, evaporate to dryness, add 20 ml of methanol to the residue, stopper tightly, and ultrasonicate for 30 minutes (power 600 W, frequency 40 kHz), let cool, shake well, filter, and take the filtrate to obtain;

[0355] Take another 0.5g of powder (passed through No. 4 sieve, batch number: 010200-2307001), place it in a stoppered conical flask, add 20ml of methanol, stopper it, reflux and ultrasonicate for 30 minutes (power 600W, frequency 40kHz), cool it, shake it well, filter it, and take the filtrate to obtain the result. Figure 29 As shown, Figure 29 The chromatograms of Euphorbia acetochrome under different extraction methods are shown in the figure. It can be seen from the figure that when the sample is first decocted and then ultrasonically extracted, there are fewer peaks. There is little difference in the peaks between directly adding solvent for ultrasonic treatment and treatment, and the peak shapes are better separated. Therefore, the extraction method of the sample is determined to be ultrasonic.

[0356] In summary, the preparation method of the test solution of the Euphorbia rubrum medicinal material atlas is determined as follows: take about 0.5 g of the product powder (passed through a No. 4 sieve), add 20 ml of methanol, plug it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Then cool it, shake it evenly, filter it, and take the filtrate to obtain the product.

[0357] 4.5 Methodological Review

[0358] 4.5.1 Chromatographic peak identification

[0359] Preparation of test solution According to the experimental conditions proposed above, prepare the test solution of Euphorbia rubrum slices;

[0360] Preparation of reference solution Take appropriate amount of cyperus alcohol and 3-hydroxybamoquinone reference substances, weigh accurately, add methanol to make solutions containing 130μg and 30μg per ml respectively, as reference substance solutions;

[0361] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Euphorbia rubrum slices lacking vinegar;

[0362] Position the characteristic spectrum peaks of Euphorbia rubra slices; Figures 30-34 , Figure 30 This is the chromatographic peak identification diagram of the vinegar-red euphorbia slices. Figure 31 This is the spectrum of cyperus alcohol in vinegar-red euphorbia slices; Figure 32 The spectrum of the reference substance Huciol is shown in Figure 2. Figure 33 This is the spectrum of 3-hydroxybajiquinone of the vinegar-red euphorbia slices; Figure 34 This is the spectrum of 3-hydroxybajiquinone, a reference substance. As can be seen from the figure, peak 8 is trichol and peak 11 is 3-hydroxybajiquinone.

[0363] 4.5.2 Precision test

[0364] Take the test solution of Euphorbia pulex (lot number: CHDJ-230701) and inject 6 times continuously according to the proposed experimental method, 1 μl each time, and calculate the retention time of each characteristic peak, as shown in Table 15;

[0365] Table 15 Precision Investigation-Retention Time Data Table

[0366]

[0367]

[0368] The results showed that the RSD of each characteristic peak retention time was 0.06%-0.26%, indicating that the method has good precision.

[0369] 4.5.3 Repeatability Study

[0370] Six portions of the test solution of Euphorbia rubrum (Batch No.: CHDJ-230701) were prepared and tested according to the proposed experimental method. The results are shown in Table 16.

[0371] Table 16 Repeatability Study-Relative Retention Time Data

[0372]

[0373] The results showed that the RSD of the relative retention time of the six samples was 0.18% to 0.52%, indicating that the method had good repeatability.

[0374] 4.6 Intermediate precision study

[0375] 4.6.1 Investigation of different instruments

[0376] Based on the experimental conditions proposed above, two portions of Euphorbia rubrum (batch number: CHDJ-230701) were accurately weighed to prepare test solutions, which were then analyzed on three different models of ultra-high performance liquid chromatographs: Shimadzu, Agilent, and Thermo Fisher. The results are shown in Tables 17 and 17. Figure 35 As shown, Figure 35 This is the chromatogram of vinegar-red euphorbia slices under different instruments;

[0377] Table 17 Instrument durability investigation - relative retention time ratio data table

[0378]

[0379] The results showed that when the three instruments were used to detect the test samples, the RSD range of the relative retention time of each characteristic peak was 0.05%-3.07%; the durability of the instruments was good.

[0380] 4.6.2 Inspection by different personnel and time

[0381] Based on the experimental conditions proposed above, two portions of Euphorbia rubrum (Batch No.: CHDJ-230701) were accurately weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples and perform the assay. The results are shown in Figure 18.

[0382] Table 18 Personnel and time inspection-relative retention time data table

[0383]

[0384]

[0385] The results showed that when different people measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks were 0.18% to 0.52%, indicating that the method was stable.

[0386] 4.6.3 Durability assessment

[0387] Based on the experimental conditions proposed above, different types of chromatographic columns were investigated; the results are as follows Figure 36 As shown in Table 19, Figure 36 The chromatograms of Euphorbia rubra slices under different types of chromatographic columns are shown;

[0388] Table 19 Column durability investigation - relative retention time data

[0389]

[0390] The results showed that when the above chromatographic column was used to detect samples, the RSD of the relative retention time of the characteristic peaks was between 1.00% and 3.44%, indicating that the chromatographic column had good durability.

[0391] 4.6.4 Stability investigation

[0392] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. The results are shown in Table 20.

[0393] Table 20 Stability Study-Retention Time Data

[0394]

[0395] The results showed that the RSDs of the characteristic peak retention times were between 0.31% and 0.94%, and the sample solutions were stable within 24 hours.

[0396] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above 12 characteristic peaks were included in the subsequent investigation.

[0397] 4.7 Verification of the Characteristic Spectrum of Vinegar-red Euphorbia decoction pieces

[0398] 21 batches of Euphorbia rubra slices were tested and the relative retention time was calculated. Figure 37 As shown in Table 21, Figure 37The chromatograms of different batches of vinegar-red Euphorbia slices are shown in Figures S1-S21: CHDJ-230701, CHDJ-230702, CHDJ-230703, CHDJ-230704, CHDJ-230705, CHDJ-230706, CHDJ-230707, CHDJ-230708, CHDJ-230709, and SNT-2307 10. CHDJ-230711, CHDJ-230712, CHDJ-230713, CHDJ-230714, CHDJ-230715, CHDJ -230716, CHDJ-230717, CHDJ-230718, CHDJ-230719, CHDJ-230720, CHDJ-230721;

[0399] Table 21 Relative retention time data of characteristic spectra of Euphorbia vulgaris slices

[0400]

[0401] Based on the principle of stable relative retention times and the fact that peaks can be detected in all batches of samples at relatively high levels, a total of 12 peaks with good reproducibility were selected as characteristic peaks. The results showed that the RSD of the relative retention times of the 12 characteristic peaks in 21 batches of Euphorbia rubrum slices was less than 1.0%.

[0402] Final regulations: The test sample's characteristic spectrum should show 12 characteristic peaks, of which Peak 8 should correspond to the retention time of the corresponding reference peak of the control. The peak corresponding to the scutellariae reference peak 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: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.45 (peak 5), 0.48 (peak 6), 0.60 (peak 7), 1.31 (peak 9), 1.36 (peak 10), 1.51 (peak 11), and 1.59 (peak 12).

[0403] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 21 batches of Euphorbia rubrum medicinal materials, and the reference characteristic spectrum of the vinegar Euphorbia rubrum decoction pieces was established. Figure 38 As shown, Figure 38 Peak 8 (S): cyperiol; Peak 11: 3-hydroxybamoquinone; Chromatographic column: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm.

[0404] Example 5 Characteristic Spectrum Detection of Standard Decoction of Euphorbia rubrum

[0405] 5.1 Experimental instruments and materials

[0406] Ultra-high performance liquid chromatograph: three different models of ultra-high performance liquid chromatographs from Shimadzu, Agilent, and Thermo Fisher Scientific;

[0407] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0408] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0409] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0410] Chromatographic columns: Column 1: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm; Column 2: Agilent ZORBAX SB-C18 2.1×150mm 1.8μm; Column 3: Waters CORTECST3 2.1×150mm 1.6μm;

[0411] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0412] 3-Hydroxybamoquinone reference substance (China Food and Drug Inspection Institute, batch number: 111962-202102, purity: 97.2%);

[0413] Huciol reference substance (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH033801, purity: 99.76%);

[0414] Euphorbia rubrum control medicinal material: (China Food and Drug Inspection Institute, batch number: 121102-201603);

[0415] Euphorbia rubrum standard decoction freeze-dried powder (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: (HDJ-BT-230701, HDJ-BT-230702, HDJ-BT-230703, HDJ-BT-230704, HDJ-BT-230705, HDJ-BT-230706, HDJ-BT-230707, HDJ-BT-230708, HDJ-BT-230709, HD ... T-230710, HDJ-BT-230711, HDJ-BT-230712, HDJ-BT-230713, HDJ-BT-230714, HDJ-BT-230715, HDJ -BT-230716, HDJ-BT-230717, HDJ-BT-230718, HDJ-BT-230719, HDJ-BT-230720, HDJ-BT-230721).

[0416] 5.2 Chromatographic conditions

[0417] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, and gradient elution as specified in Table 22 below; the flow rate was 0.25 ml / min; the column temperature was 30°C; the detection wavelength was 280 nm; and the number of theoretical plates calculated based on the 3-hydroxyephrinone peak was not less than 3000.

[0418] Table 22 Initial data of gradient elution of Euphorbia rubrum standard decoction

[0419] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0420] 5.3 Preparation of reference solution

[0421] Take 1 g of Euphorbia rubrum control medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the control medicinal material reference solution;

[0422] Take appropriate amount of cyperus alcohol and 3-hydroxybajiquinone reference substances, weigh them accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions.

[0423] 5.4 Preparation of test solution

[0424] Take 0.5 g of this product, place it in a stoppered conical flask, add 20 ml of methanol, stopper it tightly, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0425] 5.5 Determination method

[0426] Accurately pipette 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0427] 5.5.1 Chromatographic conditions and system suitability test

[0428] 5.5.1.1 Mobile Phase Selection

[0429] Methanol was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B for gradient elution. The results were as follows: Figure 39 As shown, Figure 39 This is the chromatogram of the standard decoction of Euphorbia rubrum.

[0430] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 220 nm, 260 nm, 270 nm, 280 nm, 300 nm, and 320 nm were extracted respectively. Figure 40 As shown, Figure 40 This is the wavelength selection diagram of the chromatogram of the standard decoction of red euphorbia. From top to bottom, the wavelengths are 220nm, 260nm, 270nm, 300nm, 320nm, and 280nm. It can be seen from the figure that when the detection wavelength is 280nm, the chromatographic peak information volume is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 280nm.

[0431] 5.5.1.2 Column temperature investigation

[0432] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively; as shown in Table 23, Figure 41 As shown, Figure 41 The chromatograms of the standard decoction of Euphorbia rubrum at different column temperatures are shown in Figure 2.

[0433] Table 23 Column temperature-relative retention time data table

[0434]

[0435] The results of column temperature investigation showed that when the column temperature was 30℃, the chromatogram peaks were symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of the red euphorbia standard decoction.

[0436] 5.5.1.3 Flow rate investigation

[0437] Based on the experimental conditions proposed above, the flow rates of 0.20ml / min, 0.25ml / min, and 0.3ml / min were investigated, and the results are shown in Table 24. Figure 42 As shown, Figure 42 The chromatograms of the standard decoction of Euphorbia rubrum at different flow rates are shown;

[0438] Table 24 Flow rate-relative retention time data table

[0439]

[0440] The results showed that when the flow rate was 0.25 ml / min, the chromatogram peak shape was better and the separation was moderate, so the flow rate was determined to be 0.25 ml / min.

[0441] 5.5.1.4 Delay Investigation

[0442] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 100 minutes. The results are as follows: Figure 43 As shown, Figure 43 This is the chromatogram of the delayed investigation of the standard decoction of Euphorbia rubrum. It can be seen from the figure that the sample has basically no chromatographic peak after 50 minutes, so the sample detection time is set to 50 minutes.

[0443] In summary, the characteristic spectrum chromatographic conditions and system suitability test of the standard decoction of Euphorbia rubrum are determined as follows:

[0444] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, and gradient elution as specified in Table 25 below; the flow rate was 0.25 ml / min; the column temperature was 30°C; the detection wavelength was 280 nm; and the number of theoretical plates calculated based on the 3-hydroxyephrinone peak was not less than 3000.

[0445] Table 25 Gradient elution data of red euphorbia standard decoction

[0446] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0447] 5.6 Preparation of test solution

[0448] 5.6.1 Extraction solvent investigation

[0449] Take 0.5g of this product (batch number HDJ-BT-230701) and place it in a stoppered conical flask. Add 20ml of 30% methanol, 50% methanol, 70% methanol, methanol, water, and 50% ethanol respectively. Ultrasonicate (power 600W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate. The result is as follows: Figure 44 As shown, Figure 44 The chromatograms of the standard decoction of Euphorbia rubrum under different extraction solvents are shown in the figure. As can be seen from the figure, the chromatographic peak information volume is large when methanol is used as the extraction solvent, so the extraction solvent of the test sample is determined to be methanol.

[0450] 5.6.2 Investigation of extraction methods

[0451] Take 0.5g of this product (batch number HDJ-BT-230701), place it in a stoppered conical flask, add 20ml of methanol, and conduct reflux and ultrasonic (power 600W, frequency 40kHz) inspection respectively. The extraction time is 30 minutes, let it cool, shake well, filter, and take the filtrate. The results are as follows: Figure 45 As shown, Figure 45 The chromatograms of the standard decoction of Euphorbia rubrum under different extraction methods are shown in the figure. It can be seen from the figure that the effects are consistent when the test sample is subjected to ultrasonic extraction and reflux extraction respectively; because ultrasonic extraction operation is simpler, ultrasonic extraction is determined to be the extraction method for the test sample.

[0452] 5.6.3 Extraction time investigation

[0453] Take 0.5g of this product (batch number HDJ-BT-230701) and place it in a stoppered conical flask. Add 20ml of methanol and conduct ultrasonic extraction for 20 minutes, 30 minutes and 40 minutes respectively. Cool, shake well, filter and take the filtrate. The results are as follows: Figure 46 As shown, Figure 46 The chromatograms of the standard decoction of Euphorbia pulex at different extraction times are shown in the figure. As can be seen from the figure, sufficient extraction can be achieved when the extraction time is 30 minutes; therefore, the extraction time of the test sample is determined to be 30 minutes.

[0454] 5.6.4 Investigation of solvent addition amount

[0455] Take 0.5g of this product (batch number HDJ-BT-230701) and place it in a stoppered conical flask. Add 10ml, 20ml and 50ml of methanol respectively, stopper it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate. The result is as follows: Figure 47 As shown, Figure 47 The chromatograms of the standard decoction of Euphorbia rubrum under different solvent addition amounts are shown in the figure. It can be seen from the figure that when the solvent addition amount is 20 ml, the chromatographic peak area of ​​the characteristic spectrum is moderate; therefore, the solvent addition amount of the test sample is determined to be 20 ml.

[0456] In summary, the preparation method of the test solution of the characteristic spectrum of the standard decoction of Euphorbia rubrum is determined as follows: take about 0.5 g of the product, place it in a stoppered conical flask, add 20 ml of methanol, stopper it, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, shake it evenly, filter it, and take the filtrate to obtain it.

[0457] 5.7 Methodological Review

[0458] 5.7.1 Chromatographic Peak Identification

[0459] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Euphorbia rubrum standard decoction.

[0460] Preparation of reference solution: Take appropriate amount of cyperus alcohol and 3-hydroxybamoquinone reference substances, accurately weigh them, and add methanol to make solutions containing 130μg and 30μg per 1ml respectively, which serve as reference substance solutions;

[0461] Preparation of negative solution: According to the experimental conditions proposed above, prepare the test solution of Euphorbia rubrum standard decoction;

[0462] The characteristic peaks of the red euphorbia standard decoction were located; the results are as follows Figures 48-52 As shown, Figure 48 This is the chromatogram for peak identification of the standard decoction of Euphorbia rubrum. Figure 49 This is the spectrum of Euphorbia rubrum standard decoction Huciol. Figure 50 This is the spectrum of the reference substance, Huciol. Figure 51 This is the spectrum of 3-hydroxybajiquinone in the standard decoction of red euphorbia. Figure 52 This is the spectrum of the reference substance 3-hydroxybajiquinone; it can be seen from the figure that peak 8 is trichol and peak 11 is 3-hydroxybajiquinone.

[0463] 5.7.2 Precision test

[0464] Take 0.5 g of red euphorbia standard decoction (batch number HDJ-BT-230701) to prepare the test solution. According to the proposed experimental method, inject 6 times continuously, each time with 1 μl, and calculate the retention time of each characteristic peak, as shown in Table 26;

[0465] Table 26 Precision Investigation-Retention Time Data Table

[0466]

[0467] The results showed that the RSD of the retention time of each characteristic peak was 0.26%-0.72%, and the precision of this method was good.

[0468] 5.7.3 Repeatability Study

[0469] Six portions of Euphorbia rubrum standard decoction (batch number HDJ-BT-230701) were prepared and tested according to the proposed experimental method. The results are shown in Table 27.

[0470] Table 27 Repeatability Study - Relative Retention Time Data

[0471]

[0472] The results showed that the RSD of the relative retention time of each characteristic peak was 0.13%-0.44%. The method has good reproducibility.

[0473] 5.7.4 Intermediate precision assessment

[0474] 5.7.4.1 Inspection of different instruments

[0475] Based on the experimental conditions proposed above, three portions of the standard decoction of Euphorbia rubrum (HDJ-BT-230701) were weighed to prepare the test solution. The solution was determined on three different types of ultra-high performance liquid chromatographs (Shimadzu, Agilent, and Thermo Fisher). The results are shown in Table 28. Figure 53 As shown, Figure 53 The chromatograms of the durability of Euphorbia rubrum standard decoction on different instruments are shown;

[0476] Table 28 Instrument durability investigation - relative retention time data table

[0477]

[0478] The results showed that when the three instruments were used to detect the test sample, the RSD of the relative retention time of each characteristic peak was less than 45.74%, indicating that different instruments had a great influence on the characteristic method of Euphorbia rubrum.

[0479] 5.7.4.2 Inspection by different personnel and time

[0480] Based on the experimental conditions proposed above, two aliquots of the standard decoction of Euphorbia rubrum (HDJ-BT-230701) were accurately weighed by different individuals (A and B) at different times (T1 and T2) to prepare the test samples for analysis. The results are shown in Table 29.

[0481] Table 29 Personnel and time inspection-relative retention time data table

[0482]

[0483] The results showed that the method was stable when different people measured the same sample at different times.

[0484] 5.7.4.3 Durability assessment

[0485] Based on the experimental conditions proposed above, different types of chromatographic columns were investigated. Figure 54 , as shown in Table 30, Figure 54 The chromatograms are of different types of chromatographic columns;

[0486] Table 30 Chromatographic column durability investigation - relative retention time data

[0487]

[0488] Depend on Figure 54It can be seen that the chromatographic peak separation of each column is good and the peaks are clear, and the chromatographic columns have good durability.

[0489] 5.7.4.4 Stability assessment

[0490] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. The results are shown in Table 31.

[0491] Table 31 Stability Study-Retention Time Data

[0492]

[0493] More stable inside.

[0494] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in all the above investigations, and the method is good. 5.8 Determination of characteristic peaks and establishment of reference spectrum

[0495] 5.8.1 Establishment of relative retention time limit values

[0496] The summary of the methodological investigation items and verification results is shown in Table 32;

[0497] Table 32 Summary of RSD% of each item results of the methodology - Retention time / relative retention time data table

[0498]

[0499]

[0500] In the column durability test, the RSD range of peaks 1 to 12 was 0.99%-3.01%, so the relative retention time of each peak was temporarily set at ±10%.

[0501] Final Stipulation: The relative retention time RSDs of each characteristic peak met the requirements in all the above assessments, and the method was considered good. The above 12 characteristic peaks were included in the subsequent assessment.

[0502] 5.8.2 Verification Results of 21 Batches of Euphorbia rubrum Standard Decoction

[0503] Using this method, characteristic spectrum analysis was performed on 21 batches of samples and relative retention times were calculated; the results are shown in Figure 2. Figure 55 , as shown in Table 33, Figure 55This is the characteristic spectrum of the standard decoction of red euphorbia. In the figure, peak 8 (S): cyperiol; peak 11: 3-hydroxybajiquinone. The batch numbers from bottom to top are: HDJ-BT-230701, HDJ-BT-230702, HDJ-BT-230703, HDJ-BT-230704, HDJ-BT-230705, HDJ-BT-230706, HDJ-BT-230707, HDJ-BT-230708, HDJ-BT-2307 09. HDJ-BT-230710, HDJ-BT-230711, HDJ-BT-230712, HDJ-BT-230713, HDJ-BT-230714, HDJ-BT-23071 5. HDJ-BT-230716, HDJ-BT-230717, HDJ-BT-230718, HDJ-BT-230719, HDJ-BT-230720, HDJ-BT-230721;

[0504] Table 33 Relative retention time data of 21 batches of Euphorbia rubrum standard decoction

[0505]

[0506] Based on the principle of stable relative retention time and that all batches of samples can be detected and the peaks are relatively high, a total of 12 peaks with good repeatability were selected as characteristic peaks. The above 12 characteristic peaks were included in subsequent investigations, among which peak 8 and peak 11 should correspond to the retention time of the corresponding reference substance peaks respectively, and the peak corresponding to schisandra alcohol is the S peak. The relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified value is: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.45 (peak 5), 0.48 (peak 6), 0.60 (peak 7), 1.31 (peak 9), 1.36 (peak 10), 1.51 (peak 11), and 1.59 (peak 12).

[0507] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 21 batches of red euphorbia standard decoctions, and a comparison map of the characteristic spectrum of the red euphorbia standard decoction was established. Figure 56 As shown, peak 8 (S) in the figure: scutellariae; peak 11: 3-hydroxybamoquinone.

[0508] Example 6 Establishment of Standard Atlas of Vinegar-Red Euphorbia Decoction

[0509] 6.1 Experimental instruments and materials

[0510] Ultra-high performance liquid chromatograph: three different models of ultra-high performance liquid chromatographs from Shimadzu, Agilent, and Thermo Fisher Scientific;

[0511] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0512] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0513] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0514] Column: Kromasil 100-5-C185 μm 250 × 4.6 2.1 × 150 mm 2.7 μm;

[0515] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0516] Huciol reference substance (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH033801, purity: 99.76%);

[0517] 3-Hydroxybamoquinone reference substance (China Food and Drug Inspection Institute, batch number: 111962-202102, purity: 97.2%);

[0518] Euphorbia cyrtonema standard decoction freeze-dried powder (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: (CHDJ-BT-230701, CHDJ-BT-230702, CHDJ-BT-230703, CHDJ-BT-230704, CHDJ-BT-230705, CHDJ-BT-230706, CHDJ-BT-230707, CHDJ-BT-230708, CHDJ-BT-230709, CHD ...1 T-230710, CHDJ-BT-230711, CHDJ-BT-230712, CHDJ-BT-230713, CHDJ-BT-230714, CHDJ-BT-230715, CHD J-BT-230716, CHDJ-BT-230717, CHDJ-BT-230718, CHDJ-BT-230719, CHDJ-BT-230720, CHDJ-BT-230721);

[0519] 6.2 Chromatographic conditions

[0520] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution according to the specifications in Table 34; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm; theoretical plate number calculated based on the 3-hydroxyephrinone peak should be no less than 3000;

[0521] Table 34 Gradient elution initial parameter data table of standard decoction of Euphorbia rubra

[0522]

[0523]

[0524] 6.3 Preparation of reference solution

[0525] Take 1 g of Euphorbia rubrum control medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the control medicinal material reference solution;

[0526] Take appropriate amount of cyperus alcohol reference substance and 3-hydroxybajiquinone reference substance, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions.

[0527] 6.4 Preparation of test solution

[0528] Take 0.5 g of this product, place it in a stoppered conical flask, add 20 ml of methanol, stopper it tightly, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0529] 6.5 Determination method

[0530] Accurately pipette 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0531] 6.6 Chromatographic conditions and system suitability test

[0532] 6.6.1 Mobile phase selection

[0533] Methanol was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B for gradient elution. Figure 57 As shown, Figure 57 This is the chromatogram of the standard decoction of Euphorbia pulex.

[0534] 6.6.2 Wavelength Selection

[0535] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 220 nm, 260 nm, 270 nm, 280 nm, 300 nm, and 320 nm were extracted respectively. Figure 58 As shown, Figure 58 The chromatograms of the standard decoction of Euphorbia rubra at different wavelengths are 220nm, 260nm, 270nm, 300nm, 320nm, and 380nm from top to bottom. The results show that when the detection wavelength is 280nm, the chromatographic peak information volume is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 280nm.

[0536] 6.6.3 Column temperature investigation

[0537] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively; the results are shown in Table 35, Figure 59 As shown, Figure 59 The chromatograms of Euphorbia acetophylla standard decoction at different column temperatures are shown;

[0538] Table 35 Column temperature-relative retention time data table

[0539]

[0540] The results of column temperature investigation showed that when the column temperature was 30℃, the chromatogram peaks were symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of the standard decoction of Euphorbia cyrtonema.

[0541] 6.6.4 Flow rate investigation

[0542] Based on the experimental conditions proposed above, the flow rates of 0.20ml / min, 0.25ml / min, and 0.3ml / min were investigated respectively; the results are shown in Table 36, Figure 60 As shown, Figure 60 The chromatograms of Euphorbia acetophylla standard decoction at different flow rates are shown;

[0543] Table 36 Flow rate-relative retention time data table

[0544]

[0545]

[0546] The results showed that when the flow rate was 0.25 ml / min, the chromatogram peak shape was better and the separation was moderate; therefore, the flow rate was determined to be 0.25 ml / min.

[0547] 6.6.5 Delay Investigation

[0548] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 100 min; the results are as follows Figure 61 As shown, Figure 61 This is the chromatogram of the delayed investigation of the standard decoction of Euphorbia rubra. The results show that there is basically no chromatographic peak after 50 minutes, so the sample detection time is set to 50 minutes.

[0549] In summary, the characteristic spectrum chromatographic conditions and system suitability test of the standard decoction of Euphorbia rubra were determined as follows:

[0550] Chromatographic conditions and system suitability tests were conducted using octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, using gradient elution as specified in Table 37 below; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm. The theoretical plate number, calculated based on the 3-hydroxyephrinone peak, should be no less than 3000.

[0551] Table 37 Gradient elution data of standard decoction of Euphorbia rubra

[0552] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0553] 6.6.6 Preparation of test solution

[0554] 6.6.6.1 Examination of extraction solvent

[0555] Take 0.5g of this product (batch number CHDJ-BT-230701) and place it in a stoppered conical flask. Add 20ml of 30% methanol, 50% methanol, 70% methanol, methanol, water, and 50% ethanol respectively. Ultrasonicate (power 600W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate. The result is as follows: Figure 62 As shown, Figure 62 The chromatograms of the standard decoction of Euphorbia acetophora under different extraction solvents are shown in the figure. As can be seen from the figure, the chromatographic peak information volume is large when methanol is used as the extraction solvent, so the extraction solvent of the test sample is determined to be methanol.

[0556] 6.6.6.2 Examination of extraction methods

[0557] Take 0.5g of this product (batch number CHDJ-BT-230701), place it in a stoppered conical flask, add 20ml of methanol, and examine the extraction method of the test sample when it is refluxed and ultrasonically. The extraction time is 30 minutes, cool, shake well, filter, and take the filtrate. The results are as follows: Figure 63 As shown, Figure 63The chromatograms of the standard decoction of Euphorbia acetophora at different extraction times are shown in the figure. As can be seen from the figure, the effects of ultrasonic extraction and reflux extraction are consistent. Since ultrasonic extraction is more convenient, ultrasonic extraction is determined to be the extraction method for the test sample.

[0558] 6.6.6.3 Extraction time investigation

[0559] Take 0.5g of this product (batch number CHDJ-BT-230701), place it in a stoppered conical flask, add 20ml of methanol, and observe the ultrasonic extraction time of the test sample at 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake well, filter, and take the filtrate; the results are as follows: Figure 64 As shown, Figure 64 The chromatograms of the standard decoction of Euphorbia acetophora at different extraction times are shown in Figure 1. As can be seen from the figure, sufficient extraction is achieved at an extraction time of 30 minutes. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0560] 6.6.6.4 Investigation of solvent addition amount

[0561] Take 0.5g of this product (batch number CHDJ-BT-230701) and place it in a stoppered conical flask. Add 10ml, 20ml and 50ml of methanol respectively. Seal the flask tightly and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake well, filter and take the filtrate. The result is as follows: Figure 65 As shown, Figure 65 The chromatograms of the standard decoction of Euphorbia cyrtonema under different solvent addition amounts are shown in the figure. It can be seen that when the solvent addition amount is 20 ml, the chromatographic peak area of ​​the characteristic spectrum is moderate; therefore, the solvent addition amount of the test sample is determined to be 20 ml.

[0562] In summary, the preparation method of the test solution of the characteristic spectrum of the standard decoction of Euphorbia rubra is determined as follows: take about 0.5 g of the product, place it in a stoppered conical flask, add 20 ml of methanol, stopper it, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain it.

[0563] 6.7 Methodological Review

[0564] 6.7.1 Chromatographic Peak Identification

[0565] Preparation of test solution: Prepare the test solution of Euphorbia acetophylla standard decoction according to the experimental conditions proposed above;

[0566] Preparation of reference solution: Take appropriate amount of cyperus alcohol reference substance and 3-hydroxybamoquinone reference substance, accurately weigh them, and add methanol to make solutions containing 130μg and 30μg per 1ml respectively, which are used as reference solution;

[0567] Preparation of negative solution: According to the experimental conditions proposed above, prepare the test solution of Euphorbia rubrum decoction lacking vinegar;

[0568] Position the characteristic spectrum peaks of the standard decoction of Euphorbia rubra, such as Figures 66-70 As shown, Figure 66 This is the chromatogram for peak identification of the standard decoction of Euphorbia rubra. Figure 67 This is the spectrum of Euphorbia cyrtonema in the standard decoction of Euphorbia cyrtonema. Figure 68 This is the spectrum of the reference substance, Huciol. Figure 69 This is the spectrum of 3-hydroxybajiquinone in the standard decoction of Euphorbia rubra. Figure 70 This is the spectrum of the reference substance 3-hydroxybajiquinone; it can be seen from the figure that peak 8 is trichol and peak 11 is 3-hydroxybajiquinone.

[0569] 6.7.2 Precision test

[0570] Take 0.5 g of the standard decoction of Euphorbia rubrum (CHDJ-BT-230701) to prepare the test solution. According to the proposed experimental method, inject 6 times continuously, 1 μl each time, and calculate the retention time of each characteristic peak, as shown in Table 38;

[0571] Table 38 Precision Investigation-Retention Time Data Table

[0572]

[0573] The results showed that the RSD of each characteristic peak retention time was 0.13%-0.41%, indicating that the method has good precision.

[0574] 6.7.3 Repeatability Study

[0575] Six portions of the standard decoction of Euphorbia rubrum (CHDJ-BT-230701) were prepared and assayed according to the proposed experimental method, as shown in Table 39.

[0576] Table 39 Repeatability Study - Relative Retention Time Data

[0577]

[0578]

[0579] The results showed that the RSD of the relative retention time of each characteristic peak was 0.27%-0.44%, indicating that the method has good reproducibility.

[0580] 6.7.4 Intermediate precision assessment

[0581] 6.7.4.1 Inspection of different instruments

[0582] Based on the experimental conditions proposed above, three portions of the standard decoction of Euphorbia rubrum (CHDJ-BT-230701) were weighed to prepare the test solution, which was then measured on three different ultra-high performance liquid chromatographs (Shimadzu, Agilent, and Thermo Fisher). The results are shown in Table 40. Figure 71 As shown, Figure 71 Chromatograms for the durability investigation of different instruments for the standard decoction of Euphorbia rubrum;

[0583] Table 40 Instrument durability investigation - relative retention time data table

[0584]

[0585] 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 less than 48.92%, indicating that different instruments had a great influence on the relative retention time of the characteristic method.

[0586] 6.7.4.2 Inspection by different personnel and time

[0587] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed the standard decoction of Euphorbia cyrtonema (CHDJ-BT-230701) at different times (T1, T2) to prepare the test samples for determination, as shown in Table 41.

[0588] Table 41 Personnel and time inspection-relative retention time data table

[0589]

[0590] The results showed that the method was stable when different people measured the same sample at different times.

[0591] 6.7.4.3 Durability assessment

[0592] Based on the experimental conditions proposed above, different types of chromatographic columns were investigated; the results are as follows Figure 72 , as shown in Table 42; Figure 72 To investigate the chromatograms for the durability of different types of chromatographic columns;

[0593] Table 42 Chromatographic column durability investigation - relative retention time data table

[0594]

[0595]

[0596] From the above, we can see that when different types of chromatographic columns were selected for investigation, the peak shapes and separation of each chromatographic peak were good, and the relative retention time RSD values ​​of each characteristic peak and S peak were in the range of 0.96% to 3.07%, indicating that the chromatographic column had good durability.

[0597] 6.7.4.4 Stability assessment

[0598] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 6h, 12h, and 24h, as shown in Table 43;

[0599] Table 43 Stability Study-Retention Time Data

[0600]

[0601] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.31% and 0.91%, and the sample solution was relatively stable within 24 hours.

[0602] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in all the above investigations, and the method is good. 6.8 Determination of characteristic peaks and establishment of reference spectrum

[0603] 6.8.1 Establishment of relative retention time limit values

[0604] The summary of the investigation items and validation results of the methodology is shown in Table 44;

[0605] Table 44 Summary of RSD% of each item results of the method - retention time / relative retention time data table

[0606]

[0607] In the column durability test, the RSD range of peaks 1 to 12 was 0.96%-3.07%, so the relative retention time of each peak was temporarily set at ±10%.

[0608] Final rule: The relative retention time RSDs of each characteristic peak met the requirements in all the above tests, and the method was considered good. The above 12 characteristic peaks were included in the subsequent investigation.

[0609] 6.8.2 Verification Results of 21 Batches of Vinegar-Red Euphorbia Standard Decoction

[0610] Using this method, characteristic spectrum analysis was performed on 21 batches of samples, and relative retention time ratios were calculated; Figure 73 , as shown in Table 45; Figure 73Characteristic spectra of different batches of standard decoction of Euphorbia acetosporum, including peak 8 (S): cyperiol; peak 11: 3-hydroxybajiquinone. The batch numbers from bottom to top are: CHDJ-BT-230701, CHDJ-BT-230702, CHDJ-BT-230703, CHDJ-BT-230704, CHDJ-BT-230705, CHDJ-BT-230706, CHDJ-BT-230707, CHDJ-BT-230708, CHDJ-BT-230709. 709. CHDJ-BT-230710, CHDJ-BT-230711, CHDJ-BT-230712, CHDJ-BT-230713, CHDJ-BT-230714, CHDJ-BT-23071 5. CHDJ-BT-230716, CHDJ-BT-230717, CHDJ-BT-230718, CHDJ-BT-230719, CHDJ-BT-230720, CHDJ-BT-230721;

[0611] Table 45 Relative retention time data of 21 batches of standard decoction of Euphorbia rubra

[0612]

[0613] Based on the principles of stable relative retention times and the ability to detect peaks at relatively high levels across all batches of samples, a total of 12 peaks with good reproducibility were selected as characteristic peaks. Peak 8 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the schisandra alcohol reference peak is the S peak. The relative retention times of the remaining characteristic peaks and the S peak were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.45 (peak 5), 0.48 (peak 6), 0.60 (peak 7), 1.31 (peak 9), 1.36 (peak 10), 1.51 (peak 11), and 1.59 (peak 12).

[0614] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 21 batches of standard decoctions of Euphorbia rubra. The comparison of the characteristic spectra of the standard decoctions of Euphorbia rubra was established. The results are as follows: Figure 74 As shown; Peak 8 (S) in the figure: cyperiol; Peak 11): 3-hydroxybajiquinone; Chromatographic column: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm2.7μm.

[0615] Example 7 Establishment of characteristic spectrum of Euphorbia rubra formula granules

[0616] 7.1 Experimental instruments and materials

[0617] Ultra-high performance liquid chromatograph: three different models of ultra-high performance liquid chromatographs from Shimadzu, Agilent, and Thermo Fisher Scientific;

[0618] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0619] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0620] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0621] Chromatographic columns: Column 1: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm; Column 2: Agilent ZORBAX SB-C18 2.1×150mm 1.8μm; Column 3: Waters CORTECST3 2.1×150mm 1.6μm;

[0622] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0623] 3-Hydroxybamoquinone reference substance (China Food and Drug Inspection Institute, batch number: 111962-202102, purity: 97.2%);

[0624] Huciol reference substance (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH033801, purity: 99.76%);

[0625] Euphorbia rubrum control medicinal material: (China Food and Drug Inspection Institute, batch number: 121102-201603);

[0626] Euphorbia rubra formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2309024, 2309025, 2309026).

[0627] 7.2 Chromatographic conditions

[0628] The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution according to the specifications in Table 46; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm. The number of theoretical plates calculated based on the trichol peak should be no less than 5000.

[0629] Table 46 Initial parameters of gradient elution of Euphorbia rubrum formula particles

[0630] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0631] 7.3 Preparation of reference solution

[0632] Take 1 g of Euphorbia rubrum control medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the control medicinal material reference solution;

[0633] Take appropriate amount of cyperus alcohol reference substance and 3-hydroxybajiquinone reference substance, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions.

[0634] 7.4 Preparation of test solution

[0635] Take 0.5g of this product, grind it into powder, add 20ml of methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0636] 7.5 Determination method

[0637] Accurately pipette 1 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography instrument, and measure to obtain the result.

[0638] 7.5.1 Chromatographic conditions and system suitability test

[0639] 7.5.1.1 Mobile Phase Selection

[0640] Methanol was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B for gradient elution. The results showed that the chromatogram baseline was relatively stable and there were many chromatographic peaks under the conditions of methanol-0.2% phosphoric acid solution gradient elution. Therefore, methanol-0.2% phosphoric acid solution gradient elution was used as the mobile phase for the characteristic spectrum determination method of Hong Da Ji formula granules. Figure 75 As shown, Figure 75 This is the chromatogram of the Euphorbia rubra formula granules.

[0641] 7.5.1.2 Wavelength Selection

[0642] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 220 nm, 260 nm, 270 nm, 280 nm, 300 nm, and 320 nm were extracted respectively. Figure 76 As shown, Figure 76The chromatograms of the red euphorbia formula granules at different wavelengths. In the figure, from top to bottom are the chromatograms of 220nm, 260nm, 270nm, 300nm, 320nm, and 280nm. The results show that when the detection wavelength is 280nm, the amount of chromatographic peak information is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 280nm.

[0643] 7.5.1.3 Column temperature investigation

[0644] Based on the experimental conditions proposed above, the column temperatures were investigated at 25℃, 30℃, and 35℃, and the results are shown in Table 47. Figure 77 As shown, Figure 77 This is the chromatogram of the red euphorbia granules at different column temperatures;

[0645] Table 47 Column temperature-relative retention time data table

[0646]

[0647] The results of column temperature investigation showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of Euphorbia rubra granules.

[0648] 7.5.1.4 Flow rate investigation

[0649] Based on the experimental conditions proposed above, the flow rates of 0.2ml / min, 0.25ml / min, and 0.3ml / min were investigated, and the results are shown in Table 48. Figure 78 As shown, Figure 78 The chromatograms of the Euphorbia rubra granules at different flow rates are shown;

[0650] Table 48 Flow rate-relative retention time data table

[0651]

[0652] The results showed that when the flow rate was 0.25 ml / min, the chromatogram peak shape was better and the separation was moderate; therefore, the flow rate was determined to be 0.25 ml / min.

[0653] 7.5.1.5 Delay Investigation

[0654] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 100 minutes. The results are as follows: Figure 79 As shown, Figure 79 The chromatographic spectrum of the delayed investigation of the red euphorbia formula granules showed that the sample had basically no chromatographic peak after 50 minutes, so the sample detection time was set at 50 minutes.

[0655] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of the Euphorbia pulex granules are determined as follows:

[0656] The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 49; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm; the number of theoretical plates calculated based on the trichol peak should be no less than 5000;

[0657] Table 49 Gradient elution related parameter data of red euphorbia formula granules

[0658] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0659] 7.6 Preparation of test solution

[0660] 7.6.1 Extraction solvent investigation

[0661] Take 0.5g of this product (batch number: 2309024), grind it into powder, put it into a stoppered conical flask, add 20ml of 30% methanol, 50% methanol, 70% methanol, methanol, water, 50% ethanol respectively, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate. The result is as follows: Figure 80 As shown, Figure 80 The chromatograms of the Euphorbia rubra formula granules under different extraction solvents are shown in the figure. It can be seen from the figure that the chromatographic peak information volume is large when methanol is used as the extraction solvent, so the extraction solvent of the test sample is determined to be methanol.

[0662] 7.6.2 Examination of extraction methods

[0663] Take 0.5g of this product (batch number: 2309024), grind it into powder, put it into a stoppered conical flask, add 20ml of methanol, and conduct reflux and ultrasonic (power 600W, frequency 40kHz) inspection respectively. The extraction time is 30 minutes, let it cool, shake it well, filter it, and take the filtrate. The results are as follows: Figure 81 As shown, Figure 81 The chromatograms of the Euphorbia rubra granules under different extraction methods show that the effects of ultrasonic extraction and reflux extraction are consistent. Since ultrasonic extraction is more convenient, ultrasonic extraction is determined to be the extraction method for the test sample.

[0664] 7.6.3 Extraction time investigation

[0665] Take 0.5g of this product (batch number: 2309024), grind it into powder, put it into a stoppered conical flask, add 20ml of methanol, and ultrasonically extract the sample for 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate to obtain the following: Figure 82 As shown, Figure 82 The chromatograms of the Euphorbia rubra formula granules at different extraction times show that sufficient extraction can be achieved when the extraction time is 30 minutes; therefore, the extraction time of the test sample is determined to be 30 minutes.

[0666] 7.6.4 Investigation of solvent addition amount

[0667] Take 0.5g of this product (batch number: 2309024), grind it into powder, put it into a stoppered conical flask, add 10ml, 20ml and 50ml of methanol respectively, stopper it, ultrasonicate it (power 600W, frequency 40kHz) for 30 minutes, shake it well, filter it, and take the filtrate to obtain the result. Figure 83 As shown, Figure 83 The chromatograms of the Euphorbia pulex granules with different solvent addition amounts are shown below. The results show that when the solvent addition amount is 20 ml, the characteristic chromatographic peak area is moderate; therefore, the solvent addition amount of the test sample is determined to be 20 ml. 7.6.5 Determine the preparation method of the test sample

[0668] Take 0.5g of this product, grind it into powder, put it into a stoppered conical flask, add 20ml of methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0669] 7.6.6 Characteristic Spectrum Method Determination

[0670] Determined by high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020 edition).

[0671] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 50; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm; the number of theoretical plates calculated based on the trichol peak should be no less than 5000;

[0672] Table 50 Gradient elution related parameter data of red euphorbia formula granules

[0673] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0674] Preparation of reference solution: Take 1 g of Euphorbia rubrum reference medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the reference medicinal material solution;

[0675] Take appropriate amount of cyperus alcohol reference substance and 3-hydroxybajiquinone reference substance, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions.

[0676] Preparation of the test solution: Take 0.5 g of the product, grind it into powder, add 20 ml of methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0677] Determination method: Accurately aspirate 1 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography instrument, and determine.

[0678] 7.7 Methodological Review

[0679] 7.7.1 Chromatographic Peak Identification

[0680] Preparation of test solution: Prepare the test solution of Euphorbia rubra granules according to the experimental conditions proposed above;

[0681] Preparation of reference solution: Take appropriate amount of cyperus alcohol and 3-hydroxybajiquinone reference substances, weigh accurately, add methanol to make solutions containing 130μg and 30μg per ml respectively, as reference substance solutions; Figures 84-88 As shown, Figure 84 This is the chromatogram for peak identification of the red euphorbia formula granules. Figure 85 This is the spectrum of euphorbia pulex in the granules of Euphorbia pulex. Figure 86 The spectrum of the reference substance cyperiol in the photo is shown below. Figure 87 This is the spectrum of 3-hydroxybajiquinone in red euphorbia formula granules. Figure 88 This is the spectrum of 3-hydroxybajiquinone, a reference material in the photo. As can be seen from the figure, peak 8 is trichol and peak 11 is 3-hydroxybajiquinone.

[0682] 7.7.2 Precision test

[0683] Take the test solution of the Euphorbia rubra granules and inject 1 μl of the sample 6 times continuously according to the proposed experimental method. Calculate the retention time of each characteristic peak, as shown in Table 51.

[0684] Table 51 Precision Investigation-Retention Time Data Table

[0685]

[0686] The results showed that the RSD of the retention time of each characteristic peak was 0.04%-0.11%; the method had good precision.

[0687] 7.7.3 Repeatability Study

[0688] Six portions of Euphorbia rubra granules were prepared and tested according to the proposed experimental method, as shown in Table 52.

[0689] Table 52 Repeatability Study-Relative Retention Time Data Table

[0690]

[0691] The results showed that the RSD of the relative retention time of each characteristic peak was 0.22%-0.56%; the method had good repeatability.

[0692] 7.7.4 Intermediate precision assessment

[0693] 7.7.4.1 Inspection of different instruments

[0694] Based on the experimental conditions proposed above, three portions of the Euphorbia rubra granules were accurately weighed to prepare the test solution, which was then measured on three different types of ultra-high performance liquid chromatographs, namely Shimadzu, Agilen, and Thermo Fisher. Figure 89 As shown, Figure 89 This is the durability chromatogram of the Euphorbia rubra granules under different instruments;

[0695] Table 53 Instrument durability investigation - relative retention time data table

[0696]

[0697] The results showed that when the three instruments were used to detect the test sample, the RSD range of the relative retention time of each characteristic peak was 3.51%-44.25%. The durability of Shimadzu was not good. It is recommended to use ultra-high performance liquid chromatographs such as Agilent or Thermo Fisher. 7.7.4.2 Inspection of different personnel and time

[0698] Based on the experimental conditions proposed above, two portions of Hong Da Ji formula granules (batch number: YJ04182307001-1) were weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples and perform the assays, as shown in Table 54.

[0699] Table 54 Personnel and time inspection-relative retention time data table

[0700]

[0701] The results showed that the method had good robustness when the same sample was measured by different personnel at different times.

[0702] 7.7.5 Durability assessment

[0703] 7.7.5.1 Column Durability Assessment

[0704] Based on the experimental conditions proposed above, different types of chromatographic columns were investigated; the results are as follows Figure 90 , as shown in Table 55, Figure 90 The chromatograms of the Euphorbia rubra formula granules on different types of chromatographic columns are shown below;

[0705] Table 55 Chromatographic column durability investigation - relative retention time data table

[0706]

[0707] Depend on Figure 90 It can be seen that the chromatographic peak separation of each column is good and the peaks are clear, and the chromatographic columns have good durability.

[0708] 7.7.6 Stability

[0709] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h, as shown in Table 56;

[0710] Table 56 Stability Study-Retention Time Data

[0711]

[0712] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.30% and 0.84%, and the sample solution was stable within 24 hours.

[0713] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above 12 characteristic peaks were included in the subsequent investigation.

[0714] 7.7.7 Determination of characteristic peaks and establishment of reference spectrum

[0715] 7.7.7.1 Establishment of relative retention time limit values

[0716] The summary of the methodological investigation items and verification results is shown in Table 57.

[0717] Table 57 Summary of RSD% of each item results of the method - retention time - relative retention time data table

[0718]

[0719] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0720] Final Rule: If the RSDs of the relative retention times of each characteristic peak meet the requirements in all of the above considerations, the method is considered good. The characteristic spectrum of the test sample should show 12 characteristic peaks, of which the peak corresponding to the cypermethrin reference is Peak S, and the peak corresponding to the 3-hydroxybamoquinone reference is Peak 11.

[0721] 7.7.7.2 Verification Results of Three Batches of Euphorbia rubra Granules

[0722] The proposed method was used to determine the characteristic spectra of three batches of samples of this product and calculate the relative retention time, such as Figure 91 , as shown in Table 58, Figure 91 The characteristic spectra of different batches of red euphorbia formula granules, in the figure, peak 8 (S): cyperus alcohol; peak 11: 3-hydroxybajiquinone;

[0723] Table 58 Relative retention time data of three batches of Euphorbia rubra granules

[0724]

[0725] Based on the principles of stable relative retention times and the ability to detect peaks at relatively high levels across all batches of samples, a total of 12 peaks with good reproducibility were selected as characteristic peaks. Peak 8 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the scutellariae reference peak is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 7), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 11), and 1.61 (peak 12).

[0726] Three batches of Hong Da Ji formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison map of the characteristic spectrum of Hong Da Ji formula granules was established. Figure 92 As shown, Figure 92 This is the characteristic spectrum of the red euphorbia formula granules. In the figure, peak 8 (S): cyperus alcohol; peak 11: 3-hydroxybajiquinone, chromatographic column: Agilent Infinitylab Poroshell120AQ-C18 chromatographic column 1 2.1×150mm 2.7μm.

[0727] Example 8 Establishment of Characteristic Spectrum of Vinegar-red Euphorbia Formula Granules

[0728] 8.1 Experimental instruments and materials

[0729] High performance liquid chromatograph: three different models of ultra-high performance liquid chromatographs from Shimadzu, Agilent, and Thermo Fisher Scientific;

[0730] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0731] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0732] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0733] Chromatographic columns: Column 1: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm; Column 2: Agilent ZORBAX SB-C18 2.1×150mm 1.8μm; Column 3: Waters CORTECST3 2.1×150mm 1.6μm;

[0734] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0735] Huciol reference substance (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH033801, purity: 99.76%);

[0736] 3-Hydroxybamoquinone reference substance (China Food and Drug Inspection Institute, batch number: 111962-202102, purity: 97.2%);

[0737] Vinegar-red Euphorbia formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2309027, 2309028, 2309029).

[0738] 8.2 Chromatographic conditions

[0739] The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution according to the specifications in Table 59; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm. The theoretical plate number calculated based on the trichol peak should be no less than 5000.

[0740] Table 59 Initial data table of gradient elution of vinegar red euphorbia formula particles

[0741] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0742] 8.3 Preparation of reference solution

[0743] Take 1 g of Euphorbia rubrum control medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the control medicinal material reference solution;

[0744] Take appropriate amount of cyperus alcohol reference substance and 3-hydroxybajiquinone reference substance, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions.

[0745] 8.4 Preparation of test solution

[0746] Take 0.5g of this product, grind it into powder, add 20ml of methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0747] 8.5 Determination method

[0748] Accurately pipette 1 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography instrument, and measure to obtain the result.

[0749] 8.6 Chromatographic Conditions and System Suitability Test

[0750] 8.6.1 Mobile Phase Selection

[0751] Methanol was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B for gradient elution. The results showed that the chromatogram baseline was relatively stable and there were many chromatographic peaks under the conditions of methanol-0.2% phosphoric acid solution gradient elution. Therefore, methanol-0.2% phosphoric acid solution gradient elution was used as the mobile phase for the characteristic spectrum determination method of red euphorbia formula granules. Figure 93 As shown, Figure 93 This is the chromatogram of the vinegar-red euphorbia formula granules.

[0752] 8.6.2 Wavelength Selection

[0753] Based on the experimental conditions proposed above, the test solution was scanned in the full band using a diode array detector, and the chromatograms of the test solution at wavelengths of 220nm, 260nm, 270nm, 280nm, 300nm, and 320nm were extracted respectively. The results are as follows: Figure 94 As shown, Figure 94 The chromatograms of the vinegar-red euphorbia formula granules at different wavelengths are 220nm, 260nm, 270nm, 300nm, 320nm, and 280nm from top to bottom. The results show that when the detection wavelength is 280nm, the chromatographic peak has a larger amount of information and the chromatogram baseline is more stable, so the detection wavelength is determined to be 280nm.

[0754] 8.6.3 Column temperature investigation

[0755] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated, as shown in Table 60. Figure 95 As shown; Figure 95 This is the chromatogram of the vinegar-red euphorbia granules at different column temperatures;

[0756] Table 60 Column temperature-relative retention time data table

[0757]

[0758] The results of column temperature investigation showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of the vinegar-red euphorbia formula granules.

[0759] 8.6.4 Flow rate investigation

[0760] Based on the experimental conditions proposed above, the flow rates of 0.2ml / min, 0.25ml / min, and 0.3ml / min were investigated respectively; see Table 61, Figure 96 As shown, Figure 96 The chromatograms of Euphorbia acetophylla granules at different flow rates are shown;

[0761] Table 61 Flow rate-relative retention time data table

[0762]

[0763] The results showed that when the flow rate was 0.25 ml / min, the chromatogram peak shape was better and the separation was moderate; therefore, the flow rate was determined to be 0.25 ml / min.

[0764] 8.6.5 Delay Investigation

[0765] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 100 minutes. The results are as follows: Figure 97 As shown; Figure 97 This is the chromatogram of the delayed investigation of the vinegar-red euphorbia formula granules. It can be seen from the figure that the sample has basically no chromatographic peak after 50 minutes, so the sample detection time is set to 50 minutes.

[0766] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of Euphorbia glutinosa granules were determined as follows:

[0767] The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution according to Table 62; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm. The theoretical plate number calculated based on the tricholol peak should be no less than 5000.

[0768] Table 62 Elution parameter data table of vinegar red euphorbia formula granules

[0769] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 2428 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0770] 8.7 Preparation of test solution

[0771] 8.7.1 Examination of extraction solvents

[0772] Take 0.5g of this product (batch number: 2309027), grind it into powder, put it into a stoppered conical flask, add 20ml of 30% methanol, 50% methanol, 70% methanol, methanol, water, 50% ethanol respectively, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate. The result is as follows: Figure 98 As shown, Figure 98 The chromatograms of different extraction solvents for the vinegar-red euphorbia granules are shown. The results show that the chromatographic peak has a large amount of information when methanol is used as the extraction solvent, so the extraction solvent for the test sample is determined to be methanol.

[0773] 8.7.2 Examination of extraction methods

[0774] Take 0.5g of this product (batch number: 2309027), grind it into powder, put it into a stoppered conical flask, add 20ml of methanol, and conduct reflux and ultrasonic (power 600W, frequency 40kHz) inspection respectively. The extraction time is 30 minutes, let it cool, shake well, filter, and take the filtrate. The results are as follows: Figure 99 As shown, Figure 99 The chromatograms of different extraction methods of the vinegar-red euphorbia granules show that the effects of ultrasonic extraction and reflux extraction are consistent. Since ultrasonic extraction is simpler, ultrasonic extraction is determined to be the extraction method for the test sample.

[0775] 8.7.3 Extraction time investigation

[0776] Take 0.5g of this product (batch number: 2309027), grind it into powder, put it into a stoppered conical flask, add 20ml of methanol, and extract the sample by ultrasonic (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate to obtain the following: Figure 100 As shown, Figure 100The chromatograms of different extraction times of the vinegar-red euphorbia granules are shown. The results show that sufficient extraction can be achieved when the extraction time is 30 minutes; therefore, the extraction time of the test sample is determined to be 30 minutes.

[0777] 8.7.4 Investigation of solvent addition amount

[0778] Take 0.5g of this product (batch number: 2309027), grind it into powder, put it into a stoppered conical flask, add 10ml, 20ml and 50ml of methanol respectively, stopper it, ultrasonicate it (power 600W, frequency 40kHz) for 30 minutes, shake it well, filter it, and take the filtrate. The result is as follows: Figure 101 As shown, Figure 101 The chromatograms of Euphorbia acetophora with different solvent addition amounts are shown in Figure 2. The results show that when the solvent addition amount is 20 ml, the characteristic chromatographic peak area is moderate. Therefore, the solvent addition amount for the test sample is determined to be 20 ml.

[0779] 8.7.5 Determine the test sample preparation method

[0780] Take 0.5g of this product, grind it into powder, put it into a stoppered conical flask, add 20ml of methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0781] 8.7.6 Characteristic Spectrum Method Determination

[0782] Determined by high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020 edition).

[0783] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 2.7 μm); methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution according to the requirements of Table 63; flow rate: 0.25 ml / min; column temperature: 30°C; detection wavelength: 280 nm; the number of theoretical plates calculated based on the trichol peak should be no less than 5000;

[0784] Table 63 Gradient elution parameters of vinegar red euphorbia formula granules

[0785] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 10→40 90→60 12～15 40→43 60→57 15～24 43→45 57→55 24～28 45→55 55→45 28～36 55→65 45→35 36～42 65→73 35→27 42～43 73→90 27→10 43～50 90 10

[0786] Preparation of reference solution: Take 1 g of Euphorbia rubrum reference medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate as the reference medicinal material solution;

[0787] Take appropriate amount of cyperus alcohol reference substance and 3-hydroxybajiquinone reference substance, weigh accurately, add methanol to make solutions containing 130μg and 30μg per 1ml respectively, as reference substance solutions.

[0788] Preparation of the test solution: Take 0.5 g of the product, grind it into powder, add 20 ml of methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0789] Determination method: Accurately aspirate 1 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography instrument, and determine.

[0790] 8.8 Methodological Review

[0791] 8.8.1 Chromatographic Peak Identification

[0792] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Euphorbia glutinosus granules;

[0793] Preparation of reference solution: Take appropriate amount of cyperus alcohol reference substance and 3-hydroxybamoquinone reference substance, accurately weigh them, and add methanol to make solutions containing 130μg and 30μg per 1ml respectively, which are used as reference solution;

[0794] Preparation of negative solution: According to the experimental conditions proposed above, prepare the test solution of Euphorbia glutinosa granules.

[0795] like Figures 102-106 As shown, Figure 102 This is the chromatogram for peak identification of the granules of Euphorbia glutinosus. Figure 103 This is the spectrum of cyperus alcohol in the Euphorbia glutinosa granules. Figure 104 This is the spectrum of the reference substance, Huciol. Figure 105 This is the spectrum of 3-hydroxybajiquinone in the vinegar-red euphorbia granules. Figure 106 This is the spectrum of the reference substance 3-hydroxybajiquinone; it can be seen from the figure that peak 8 is trichol and peak 11 is 3-hydroxybajiquinone.

[0796] 8.8.2 Precision test

[0797] Take the test solution of Euphorbia glutinosus granules and inject 1 μl of the sample 6 times continuously according to the proposed experimental method. Calculate the retention time of each characteristic peak as shown in Table 64.

[0798] Table 64 Precision Investigation-Retention Time Data Table

[0799]

[0800]

[0801] The results showed that the RSD of the retention time of each characteristic peak was 0.08% to 0.21%, and the method had good precision.

[0802] 8.8.3 Repeatability Study

[0803] Six portions of the vinegar-red euphorbia granules were prepared and tested according to the proposed experimental method, as shown in Table 65.

[0804] Table 65 Repeatability Study-Relative Retention Time Data Table

[0805]

[0806] The results showed that the RSD of the relative retention time of each characteristic peak was 0.12%-0.43%. The method has good reproducibility.

[0807] 8.8.4 Intermediate precision assessment

[0808] 8.8.4.1 Inspection of different instruments

[0809] Based on the experimental conditions proposed above, three portions of the vinegar-red euphorbia granules were accurately weighed to prepare test solutions, which were then measured on three different types of ultra-high performance liquid chromatographs, namely Shimadzu, Agilent, and Thermo Fisher, as shown in Table 66. Figure 107 As shown, Figure 107 This is the durability chromatogram of the vinegar-red euphorbia granules under different instruments;

[0810] Table 66 Instrument durability investigation - relative retention time data table

[0811]

[0812] The results showed that when the test samples were detected using the above three instruments, the RSD range of the relative retention time of each characteristic peak was 3.05%-44.56%. The durability of Shimadzu instruments was not good, and it was recommended to use ultra-high performance liquid chromatographs such as Agilent or Thermo Fisher.

[0813] 8.8.4.2 Inspection by different personnel and time

[0814] Based on the experimental conditions proposed above, two portions of Vinegar Red Euphorbia Granules (Batch No.: 2309027) were weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples for determination, as shown in Table 67.

[0815] Table 67 Personnel and time inspection-relative retention time data table

[0816]

[0817]

[0818] The results showed that the method was stable when different people measured the same sample at different times.

[0819] 8.8.4.3 Durability assessment

[0820] Based on the experimental conditions proposed above, different chromatographic columns were used: chromatographic column 1: Agilent Infinitylab Poroshell 120AQ-C18 2.1×150mm 2.7μm; chromatographic column 2: Agilent ZORBAX SB-C18 2.1×150mm 1.8μm; chromatographic column 3: Waters CORTECS T3 2.1×150mm 1.6μm. The results are as follows: Figure 108 , as shown in Table 68, Figure 108 The chromatograms of different chromatographic columns for the granules of Euphorbia glutinosa are shown;

[0821] Table 68 Chromatographic column durability investigation - relative retention time data table

[0822]

[0823] As can be seen from the figure, the chromatographic peak separation of each column is good and the peaks are clear, and the chromatographic columns have good durability.

[0824] 8.8.4.4 Stability

[0825] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 6h, 12h, and 24h, respectively; as shown in Table 69;

[0826] Table 69 Stability Study-Retention Time Data

[0827]

[0828] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.11% and 1.01%, and the sample solution was stable within 24 hours.

[0829] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above 12 characteristic peaks were included in the subsequent investigation.

[0830] 8.9 Determination of characteristic peaks and establishment of reference maps

[0831] 8.9.1 Establishment of Relative Retention Time Limits

[0832] The summary of the methodological investigation items and verification results is shown in Table 70;

[0833] Table 70 Summary of RSD% of each item results of the methodology - Retention time - Relative retention time data table

[0834]

[0835] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0836] Final Rule: If the RSDs of the relative retention times of each characteristic peak meet the requirements in all of the above considerations, the method is considered good. The characteristic spectrum of the test sample should show 12 characteristic peaks, of which the peak corresponding to the cypermethrin reference is Peak S, and the peak corresponding to the 3-hydroxybamoquinone reference is Peak 11.

[0837] 8.9.2 Verification Results of Three Batches of Vinegar-Red Euphorbia Granules

[0838] The proposed method was used to determine the characteristic spectra of three batches of samples of this product and calculate the relative retention time, such as Figure 109 , as shown in Table 71, Figure 109 This is a characteristic spectrum verification diagram of different batches of vinegar-red euphorbia formula granules; in the figure, peak 8 (S): cyperus alcohol; peak 11: 3-hydroxybajiquinone;

[0839] Table 71 Relative retention time data of three batches of vinegar-red euphorbia granules

[0840]

[0841] Based on the principles of stable relative retention times and the ability to detect peaks at relatively high levels across all batches of samples, a total of 12 peaks with good reproducibility were selected as characteristic peaks. Peak 8 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the schisandra alcohol reference peak is the S peak. The relative retention times of the remaining characteristic peaks and the S peak were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.45 (peak 5), 0.48 (peak 6), 0.60 (peak 7), 1.31 (peak 9), 1.36 (peak 10), 1.51 (peak 11), and 1.59 (peak 12).

[0842] Three batches of Vinegar-Red Euphorbia Formula Granules were synthesized using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), and a comparison of the characteristic spectra of Vinegar-Red Euphorbia Formula Granules was established. Figure 110 As shown, Figure 110 This is the characteristic spectrum of the vinegar-red euphorbia formula granules. In the figure, peak 8 (S): thorn alcohol; peak 11): 3-hydroxybajiquinone; chromatographic column: Infinitylab Poroshell120AQ-C18 2.1×150mm 2.7μm.

[0843] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0844] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting characteristic patterns of Euphorbia rubrum, Euphorbia rubrum and their preparations, comprising the following steps: S1) extracting the raw material of the test product with a solvent to obtain a test solution; the raw material of the test product is a red euphorbia raw material or a vinegar red euphorbia raw material; S2) measuring the test solution by high performance liquid chromatography to obtain a HPLC characteristic spectrum of the test product; The HPLC conditions are as follows: a C18 column; mobile phase A is methanol, mobile phase B is 0.2% phosphoric acid solution, gradient elution; the column length is 150 mm, the inner diameter is 2.1 mm, and the particle size is 2.7 μm; the detection wavelength of the mobile phase is 280 nm; Step S1) is specifically as follows: The preparation of the test solution of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules is as follows: Adding methanol to the medicinal materials, decoction pieces, standard decoction or its formula granules of Euphorbia rubrum and performing ultrasonic extraction; The preparation of the test solution of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules is as follows: Adding methanol to the medicinal materials, decoction pieces, standard decoction or its formula granules of Euphorbia pulex and performing ultrasonic extraction; The gradient elution is specifically as follows: 0-12 min, phase A: 10-40%, phase B: 90-60%; 12-15 min, phase A: 40-43%, phase B: 60-57%; 15-24 min, phase A: 43-45%, phase B: 57-55%; 24-28 min, phase A: 45-55%, phase B: 55-45%; 28-36 min, phase A: 55-65%, phase B: 45-35%; 36~42min, phase A: 65~73%, phase B: 35~27%; 42-43 min, phase A: 73-90%, phase B: 27-10%; 43~50min, phase A: 90%, phase B: 10%.

2. The characteristic spectrum detection method according to claim 1, characterized in that: The invention also includes preparing reference substance solution 1, reference substance solution 2 and reference medicinal material reference substance solution: Dissolve scutellariae alcohol in methanol to obtain reference substance solution 1; Dissolve 3-hydroxybajiquinone in methanol to obtain reference substance solution 2; The red euphorbia medicinal material was decocted with water, the filtered filtrate was evaporated to dryness, and then extracted with methanol, and the control medicinal material reference solution was obtained after ultrasonication; The reference substance solution 1, the reference substance solution 2 and the reference medicinal material reference solution were respectively determined by high performance liquid chromatography to obtain chromatograms of cyperus alcohol and chromatograms of 3-hydroxybajiquinone; The components of the HPLC characteristic spectra of Euphorbia rubrum medicinal materials, decoction pieces, standard decoction and its formula granules were qualitatively determined based on the chromatograms of cyperiol and 3-hydroxybajiquinone. The components of the HPLC characteristic spectra of Euphorbia acetosporum medicinal materials, decoction pieces, standard decoction and its formula granules were qualitatively determined based on the chromatograms of tiglol and 3-hydroxybajiquinone.

3. The characteristic spectrum detection method according to claim 1, characterized in that: The raw materials of the test products are Euphorbia rubrum medicinal materials, decoction pieces, standard decoctions and formula granules thereof, or Euphorbia rubrum medicinal materials, decoction pieces, standard decoctions and formula granules thereof.

4. The characteristic spectrum detection method according to claim 1, characterized in that: The ultrasonic extraction was performed with an ultrasonic power of 600 W and a frequency of 40 kHz, and the extraction time was 20 to 40 min. The ratio of the test sample raw material to the solvent is (0.5~1) g: (10~30) ml.

5. The characteristic spectrum detection method according to claim 1, wherein: The column temperature of the chromatographic column is 30°C.

6. The characteristic spectrum detection method according to claim 5, characterized in that: The flow rate of the mobile phase was 0.25 ml / min, and the injection volume was 1 μL.

7. The characteristic spectrum detection method according to claim 1, characterized in that: The similarity of the HPLC characteristic spectra of Euphorbia rubrum, its medicinal pieces, standard decoction and its formula granules was evaluated by using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectra of Euphorbia rubrum, its medicinal pieces, standard decoction and its formula granules were obtained, which consisted of 12 characteristic peaks, including peak 8: cyperus alcohol peak, peak 11: 3-hydroxybamoquinone peak; The similarity of the HPLC characteristic spectra of Euphorbia oxyphylla, its decoction pieces, standard decoction and its formula granules was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectra of Euphorbia oxyphylla, its decoction pieces, standard decoction and its formula granules were obtained, which consisted of 12 characteristic peaks, including peak 8: trichol peak and peak 11: 3-hydroxybajiquinone peak.

8. The method according to claim 7, characterized in that In the characteristic spectra of the Euphorbia rubrum medicinal materials, decoction pieces, standard decoctions and formula granules thereof, the relative retention time of each characteristic peak and the S peak was calculated with tiglol as the reference peak S, and the relative retention time was within ±10% of the specified value, and the specified values ​​were: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 8), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 12), and 1.61 (peak 12); In the characteristic spectra of the vinegar-red euphorbia medicinal materials, decoction pieces, standard decoctions and formula granules thereof, the relative retention time of each characteristic peak and the S peak was calculated with tiglol as the reference peak S, and the relative retention time was within ±10% of the specified value, and the specified values ​​were: 0.12 (peak 1), 0.34 (peak 2), 0.38 (peak 3), 0.40 (peak 4), 0.46 (peak 5), 0.49 (peak 6), 0.60 (peak 8), 1.33 (peak 9), 1.37 (peak 10), 1.53 (peak 12), and 1.61 (peak 12).

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