Characteristic chromatogram of amaranthus retroflexus seeds, construction method and application of characteristic chromatogram, and identification method of adulterated amaranthus retroflexus seeds in feather cockscomb seeds

Abstract

The invention provides a characteristic spectrum of amaranthus retroflexus seeds, a construction method and application of the characteristic spectrum and a method for identifying adulterated amaranthus retroflexus seeds in feather cockscomb seeds, and relates to the technical field of traditional Chinese medicine detection. According to the method, the characteristic chromatogram of the amaranthus retroflexus seeds is constructed, and the method is high in specificity, good in precision, good in repeatability and relatively good in durability. The HPLC chromatogram of the feather cockscomb seeds to be detected is positioned by using the characteristic chromatogram of the amaranthus retroflexus seeds constructed by the method, and if the spectrum of the sample to be detected and the characteristic chromatogram of the amaranthus retroflexus seeds do not have chromatographic peaks with the same retention time, the feather cockscomb seeds to be detected do not contain the amaranthus retroflexus seeds; if the spectrogram of the sample to be detected and the characteristic chromatogram of the amaranthus retroflexus have chromatographic peaks with the same retention time and the ultraviolet spectrograms are consistent, the feather cockscomb seeds to be detected contain the amaranthus retroflexus seeds. The method provided by the invention solves the adulteration problem of whether the feather cockscomb seeds are adulterated with amaranthus retroflexus seeds, is simple and convenient to operate, simple in step, clear and reliable in result and easy to judge, and provides more powerful guarantee for authenticity identification and quality control of the feather cockscomb seeds.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a characteristic spectrum of Amaranthus retroflexus seeds and a construction method and application thereof, and a method for identifying adulterated Amaranthus retroflexus seeds in Amaranthus chinensis seeds. Background Art

[0002] Cyanobium officinale is a plant of the genus Cyanobium in the Amaranthaceae family. Celosia argentea L.), also known as oxtail flower seeds, cassia, dogtail seeds, etc., were first recorded in Shennong's Herbal Classic and listed as a lower grade. They have been recorded in pharmacopoeias of all dynasties. Amaranthus seeds are bitter and slightly cold, belonging to the liver meridian. They have the effects of clearing the liver and purging fire, improving eyesight and removing cataracts. They are used for liver heat, red eyes, cataracts, blurred vision, and liver fire dizziness. Modern pharmacological studies have shown that Amaranthus seeds have the effects of protecting the liver, anti-tumor, lowering blood sugar, and treating cataracts. They can especially effectively prevent and treat chemical liver damage and have a high application value. Amaranthus seeds contain a variety of components, mainly saponins, cyclic peptides, fatty acids, amino acids, and minerals.

[0003] In addition to the Celosiae Semen listed in the standard, the Celosiae Semen on the market is easy to be confused with similar seeds because of its small seeds and unclear appearance. Its counterfeit products mainly come from Amaranthaceae, Convolvulaceae, Phytolaccae, etc., such as Amaranthus retroflexus ( Amaranthus retroflexus L.), Amaranth seeds ( Amaranthus tricolor L.), cockscomb seeds ( Celosia cristata L.), Amaranthus multiflorus ( Amaranthus paniculatus L.), Amaranthus spinulosus ( Amaranthus spinosus L.), Amaranthus rugosa ( Amaranthus viridis L.), Cuscuta australis ( Cuscuta chinensis Lam.), Phytolacca seeds ( Phytolacca acinosa Roxb.), Phytolacca longituba seeds ( Phytolacca Americana L.), bitter wormwood seeds ( Corydalis bungeana Turcz., etc. Among them, the most common counterfeits are Amaranthus retroflexus and Celosia cristata. Amaranthus retroflexus is Amaranthus retroflexus of the Amaranthaceae family ( Amaranthus retroflexus L.), which are often sold and used as Amaranthus seeds; Cockscomb seeds are from the same genus Cockscomb ( Celosia cristata L.) Dried seeds, which are slightly larger than Amaranthus seeds and are easily recognizable by their appearance.

[0004] At present, the HPLC characteristic spectrum of C. amaranthus has been reported. The HPLC chromatographic conditions used are Kromasil 100-5 C18 column, acetonitrile-phosphoric acid solution (volume fraction of 0.1%) as the mobile phase, gradient elution (0-10 min, A is 5%→10%, 10-40 min, A is 10%→28%, 40-45 min, A is 28%→30%, 45-55 min, A is 30%→50%, 55-60 min, A is 50%→80%), detection wavelength is 203 nm, flow rate is 1 mL / min, column temperature is 30 °C; the test results show that 10 batches of C. amaranthus herbal medicine materials have 35 common peaks marked, with a similarity of 0.973-0.997, and the UV absorption spectra of the main common peaks can be divided into 10 categories. However, the above detection method cannot detect whether C. amaranthus herbal medicine is adulterated with Amaranthus retroflexus seeds. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a characteristic spectrum of Amaranthus retroflexus seeds and its construction method and application, and a method for identifying adulterated Amaranthus retroflexus seeds in Amaranthus chinensis seeds. The characteristic spectrum of Amaranthus retroflexus seeds constructed by the present invention can accurately identify whether Amaranthus retroflexus seeds are adulterated with Amaranthus retroflexus seeds, providing a more powerful guarantee for the authenticity identification and quality control of Amaranthus chinensis seeds.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for constructing a characteristic map of Amaranthus retroflexus, comprising the following steps: Using methanol to extract Amaranthus retroflexus seeds to obtain a methanol extract; Purifying the methanol extract by semi-preparative high performance liquid chromatography column to obtain a reference substance; the conditions for the semi-preparative high performance liquid chromatography column purification include: the chromatographic column is a C18 chromatographic column, the mobile phase is an acetonitrile aqueous solution, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 19-21%, the detection wavelength is 275nm, and the components with a retention time of 25.48-25.68min are collected; Dissolving the reference substance in a methanol aqueous solution to obtain a reference substance solution; The reference solution is subjected to high performance liquid chromatography to obtain a characteristic spectrum of Amaranthus retroflexus seeds; the conditions for the high performance liquid chromatography detection include: a C18 chromatographic column; a mobile phase system includes a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.09-0.11%; the detection wavelength is 275 nm; the elution mode is gradient elution, and the gradient elution program includes: 0-15 min, the volume fraction of the mobile phase A increased from 9.5-10.5% to 19.5-20.5%, 15-33 min, the volume fraction of the mobile phase A increased from 19.5-20.5% to 27.5-28.5%, 33-38 min, the volume fraction of the mobile phase A increased from 27.5-28.5% to 89.5-90.5%, 38-44 min, the volume fraction of the mobile phase A was 89.5-90.5%.

[0007] Preferably, the ratio of the mass of Amaranthus retroflexus seeds to the volume of methanol is 1 kg: 5-15 L; The extraction includes heating reflux extraction, the number of extractions is 2 to 3 times, and the single extraction time is 1 to 3 hours.

[0008] Preferably, the volume fraction of methanol in the methanol aqueous solution is 75-85%; The concentration of the reference solution is 0.3-0.34 μg / mL.

[0009] Preferably, the column temperature of the HPLC detection is 20-30° C., the detector is a diode array detector, the mobile phase flow rate is 0.9-1.1 mL / min, and the injection volume is 5-15 μL.

[0010] The present invention also provides a characteristic spectrum of Amaranthus retroflexus obtained by the construction method described in the above technical solution.

[0011] The present invention also provides the application of the characteristic spectrum of Amaranthus retroflexus seeds described in the above technical solution in the identification of Amaranthus chinensis seeds.

[0012] The present invention also provides a method for identifying adulterated Amaranthus retroflexus seeds in Amaranthus chinensis seeds, comprising the following steps: The Cercidiphyllum amarum seeds to be tested are extracted with methanol aqueous solution, and the extract is the sample solution to be tested; The sample liquid to be tested is subjected to high performance liquid chromatography detection to obtain a sample spectrum to be tested; the conditions for the high performance liquid chromatography detection include: a chromatographic column is a C18 chromatographic column; a mobile phase system includes a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.09-0.11%; the detection wavelength is 275nm; the elution mode is gradient elution, and the gradient elution program includes: 0-15min, the volume fraction of the mobile phase A increases from 9.5-10.5% to 19.5-20.5%, 15-33min, the volume fraction of the mobile phase A increases from 19.5-20.5% to 27.5-28.5%, 33-38min, the volume fraction of the mobile phase A increases from 27.5-28.5% to 89.5-90.5%, and 38-44min, the volume fraction of the mobile phase A is 89.5-90.5%.

[0013] The spectrum of the sample to be tested is compared with the characteristic spectrum of Amaranthus retroflexus described in the above technical solution. If the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus do not have chromatographic peaks with the same retention time, the Amaranthus retroflexus seeds to be tested do not contain Amaranthus retroflexus seeds; if the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus have chromatographic peaks with the same retention time, and the ultraviolet spectrum Figure 1 If the test result is positive, then the Amaranthus retroflexus seeds are contained in the tested Amaranthus retroflexus seeds.

[0014] Preferably, the volume fraction of methanol in the methanol aqueous solution is 75-85%; The ratio of the mass of the Celosia chinensis seeds to be tested to the volume of the methanol aqueous solution is 1 g: 20-30 mL.

[0015] Preferably, the extraction comprises heating reflux extraction, and the extraction time is 55 to 65 minutes.

[0016] Preferably, the column temperature of the HPLC detection is 20-30° C., the detector is a diode array detector, the mobile phase flow rate is 0.9-1.1 mL / min, and the injection volume is 5-15 μL.

[0017] The present invention constructs a characteristic map of Amaranthus retroflexus. The construction method provided by the present invention has strong specificity, good precision, good repeatability and good durability.

[0018] The HPLC chromatogram of the tested Amaranthus retroflexus fruit is positioned by using the Amaranthus retroflexus fruit characteristic spectrum constructed by the present invention. If the spectrum of the tested sample and the Amaranthus retroflexus fruit characteristic spectrum do not have a chromatographic peak with the same retention time, the Amaranthus retroflexus fruit is not contained in the tested Amaranthus retroflexus fruit; if the spectrum of the tested sample and the Amaranthus retroflexus fruit characteristic spectrum have a chromatographic peak with the same retention time, and the ultraviolet spectrum Figure 1If the test result is the same, the Amaranthus retroflexus seeds are contained in the Amaranthus retroflexus seeds. The method provided by the present invention solves the problem of whether the Amaranthus retroflexus seeds are adulterated with Amaranthus retroflexus seeds, and fills the gap in the prior art in the lack of a method for detecting whether the Amaranthus retroflexus seeds are adulterated with Amaranthus retroflexus seeds; the method is easy to operate, the steps are simple, the results are clear and reliable, and it is easy to judge, which provides a more powerful guarantee for the authenticity identification and quality control of Amaranthus retroflexus seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the HPLC chromatogram of the reference substance; Figure 2 is the UV absorption spectrum of the reference substance; Figure 3 is the HPLC chromatogram of Amaranthus retroflexus seeds 1; Figure 4 is the HPLC chromatogram of Amaranthus retroflexus 2; Figure 5 is the HPLC chromatogram of Amaranthus retroflexus 3; Figure 6 is the HPLC chromatogram of Amaranthus retroflexus seeds 4; Figure 7 is the HPLC chromatogram of Amaranthus retroflexus 5; Figure 8 is the HPLC chromatogram of Amaranthus retroflexus 6; Fig. 9 is the HPLC chromatogram of Amaranthus retroflexus 7; Fig.10 is the HPLC chromatogram of Amaranthus retroflexus 8; Fig.11 is the HPLC chromatogram of Amaranthus retroflexus 9; Fig.12 is the HPLC chromatogram of Cercidiphyllum Nobilis 1; Fig.13 is the HPLC chromatogram of Cnidium officinale seeds 2; Fig.14 is the HPLC chromatogram of Cnidium officinale seeds 3; Fig.15 is the HPLC chromatogram of Cnidium officinale seeds 4; Fig.16 is the HPLC chromatogram of Cnidium officinale seeds 5; Fig.17 is the HPLC chromatogram of Cnidium officinale seeds 6; Fig.18 is the HPLC chromatogram of Cnidium officinale seeds 7; Fig.19 is the HPLC chromatogram of Cnidium officinale Seed 8; Fig. 20 is the HPLC chromatogram of Cnidium officinale seeds 9; Fig.21To investigate the HPLC chromatogram for the detection limit of the reference substance (i.e., characteristic component); Fig. 22 It is the HPLC chromatogram of the reference substance solution in the acetonitrile-water mobile phase system; Fig.23 It is the HPLC chromatogram of the reference substance solution in the mobile phase system of acetonitrile-0.1 v / v% phosphoric acid aqueous solution; Fig.24 It is the HPLC chromatogram of the reference substance solution in the mobile phase system of acetonitrile-0.1 v / v% formic acid aqueous solution; Fig.25 HPLC chromatogram of Amaranthus retroflexus sample solution under gradient elution program 1; Fig.26 It is the HPLC chromatogram of the Amaranthus retroflexus sample solution under the gradient elution program 2; Fig. 27 It is the HPLC chromatogram of the Amaranthus retroflexus sample solution under the gradient elution program 3; Fig.28 This is the HPLC chromatogram of the Amaranthus retroflexus sample solution detected by Agilent Eclipse Plus C18 column; Fig.29 This is the HPLC chromatogram of the Amaranthus retroflexus sample solution detected by Phenomenex luna C18 column; Fig.30 This is the HPLC chromatogram of the Amaranthus retroflexus sample solution detected by Lichrospher C18 column; Fig.31 This is the HPLC chromatogram of the Amaranthus retroflexus sample solution detected by Waters high performance liquid chromatograph; Fig.32 The HPLC chromatogram of the Amaranthus retroflexus sample solution detected by Agilent high performance liquid chromatograph; Fig.33 This is the HPLC chromatogram of the Amaranthus retroflexus sample solution detected by Shimadzu LC high performance liquid chromatograph. DETAILED DESCRIPTION

[0020] The present invention provides a method for constructing a characteristic map of Amaranthus retroflexus, comprising the following steps: Using methanol to extract Amaranthus retroflexus seeds to obtain a methanol extract; Purifying the methanol extract by semi-preparative high performance liquid chromatography column to obtain a reference substance; the conditions for the semi-preparative high performance liquid chromatography column purification include: the chromatographic column is a C18 chromatographic column, the mobile phase is an acetonitrile aqueous solution, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 19-21%, the detection wavelength is 275nm, and the components with a retention time of 25.48-25.68min are collected; Dissolving the reference substance in a methanol aqueous solution to obtain a reference substance solution; The reference solution is subjected to high performance liquid chromatography to obtain a characteristic spectrum of Amaranthus retroflexus seeds; the conditions for the high performance liquid chromatography detection include: a C18 chromatographic column; a mobile phase system includes a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.09-0.11%; the detection wavelength is 275 nm; the elution mode is gradient elution, and the gradient elution program includes: 0-15 min, the volume fraction of the mobile phase A increased from 9.5-10.5% to 19.5-20.5%, 20-28 min, the volume fraction of the mobile phase A increased from 19.5-20.5% to 27.5-28.5%, 15-33 min, the volume fraction of the mobile phase A increased from 27.5-28.5% to 89.5-90.5%, 38-44 min, the volume fraction of the mobile phase A was 89.5-90.5%.

[0021] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0022] The invention utilizes methanol to extract Amaranthus retroflexus seeds to obtain a methanol extract.

[0023] In the present invention, the Amaranthus retroflexus seeds are preferably used in the form of Amaranthus retroflexus seeds powder, and the particle size of the Amaranthus retroflexus seeds powder is preferably 250-350 μm. In the present invention, the mass volume ratio of the Amaranthus retroflexus seeds to methanol is preferably 1 kg: 5-15 L, and in specific embodiments, it can be 1 kg: 5 L, 1 kg: 6 L, 1 kg: 7 L, 1 kg: 8 L, 1 kg: 9 L, 1 kg: 10 L, 1 kg: 11 L, 1 kg: 12 L, 1 kg: 13 L, 1 kg: 14 L or 1 kg: 15 L.

[0024] In the present invention, the extraction preferably includes heating reflux extraction; the number of extractions is preferably 2 to 3 times; the single extraction time is 1 to 3 hours, and in specific embodiments it can be 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours.

[0025] After obtaining the methanol extract, the present invention purifies the methanol extract using a semi-preparative high performance liquid chromatography column to obtain a reference substance.

[0026] In the present invention, the conditions for semi-preparative high performance liquid chromatography column purification include: the chromatographic column is a C18 chromatographic column, the mobile phase is an acetonitrile aqueous solution, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 19-21%, and in a specific embodiment, it can be 19%, 19.5%, 20%, 20.5% or 21%; the detection wavelength is 275nm, and the components with a retention time of 25.48-25.68min are collected. The present invention preferably collects the eluent with a retention time of 25.48-25.68min, and concentrates it to dryness under reduced pressure to obtain a reference substance.

[0027] The present invention dissolves the reference substance in a methanol aqueous solution to obtain a reference substance solution.

[0028] In the present invention, the volume fraction of methanol in the methanol aqueous solution is preferably 75-85%, and in specific embodiments may be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or 85%.

[0029] In the present invention, the concentration of the reference solution is preferably 0.30-0.35 μg / mL, and in specific embodiments may be 0.30 μg / mL, 0.31 μg / mL, 0.32 μg / mL, 0.33 μg / mL, 0.34 μg / mL or 0.35 μg / mL.

[0030] After obtaining the reference substance solution, the present invention performs high performance liquid chromatography detection on the reference substance solution to obtain a characteristic spectrum of Amaranthus retroflexus seeds.

[0031] In the present invention, the conditions for the high performance liquid chromatography detection include: the chromatographic column is a C18 chromatographic column, preferably an Agilent Eclipse Plus C18 chromatographic column, a Phenomenex luna C18 chromatographic column or a Lichrospher C18 chromatographic column; the column temperature is preferably 20-30°C, and in a specific embodiment, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C; the mobile phase system includes a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, the volume fraction of formic acid in the formic acid aqueous solution is 0.09-0.11%, and in a specific embodiment, it can be 0.09%, 0.095%, 0.1%, 0.105% or 0.11%; the mobile phase flow rate is preferably The speed is preferably 0.9-1.1 mL / min, and in a specific embodiment it can be 0.90 mL / min, 0.95 mL / min, 1.0 mL / min, 1.05 mL / min or 1.1 mL / min; the injection volume is preferably 5-15 μL, and in a specific embodiment it can be 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL or 15 μL; the elution mode is gradient elution; the detection wavelength is 275 nm; the detector is preferably a diode array detector.

[0032] In the present invention, the gradient elution procedure comprises: 0-15min, the volume fraction of the mobile phase A increases from 9.5-10.5% (in a specific embodiment, it can be 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10.0%, 10.1%, 10.2%, 10.3%, 10.4% or 10.5%) to 19.5-20.5% (in a specific embodiment, it can be 19.5%, 19.6%, 19.7%, 19.8%, 19.9%, 20.0%, 20.1%, 20.2%, 20.3%, 20.4% or 20.5%); 15-33 min, the volume fraction of the mobile phase A increases from 19.5-20.5% (in a specific embodiment, it can be 19.5%, 19.6%, 19.7%, 19.8%, 19.9%, 20.0%, 20.1%, 20.2%, 20.3%, 20.4% or 20.5%) to 27.5-28.5% (in a specific embodiment, it can be 27.5%, 27.6%, 27.7%, 27.8%, 27.9%, 28.0%, 28.1%, 28.2%, 28.3%, 28.4% or 28.5%); 33-38 min, the volume fraction of the mobile phase A increases from 27.5-28.5% (in a specific embodiment, it can be 27.5%, 27.6%, 27.7%, 27.8%, 27.9%, 28.0%, 28.1%, 28.2%, 28.3%, 28.4% or 28.5%) to 89.5-90.5% (in a specific embodiment, it can be 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90.0%, 90.1%, 90.2%, 90.3%, 90.4% or 90.5%); 38-44 min, the volume fraction of the mobile phase A is 89.5-90.5%, and in specific embodiments can be 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90.0%, 90.1%, 90.2%, 90.3%, 90.4% or 90.5%.

[0033] The present invention also provides a characteristic spectrum of Amaranthus retroflexus obtained by the construction method described in the above technical solution.

[0034] The present invention also provides the application of the characteristic spectrum of Amaranthus retroflexus seeds described in the above technical solution in the identification of Amaranthus chinensis seeds.

[0035] The present invention also provides a method for identifying adulterated Amaranthus retroflexus seeds in Amaranthus chinensis seeds, comprising the following steps: The Cercidiphyllum amarum seeds to be tested are extracted with methanol aqueous solution, and the extract is the sample solution to be tested; The sample liquid to be tested is subjected to high performance liquid chromatography detection to obtain a sample spectrum to be tested; the conditions of the high performance liquid chromatography detection include: the chromatographic column is a C18 chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.09-0.11%; the detection wavelength is 275nm; the elution mode is gradient elution, and the gradient elution program includes: 0-15min, the volume fraction of the mobile phase A increases from 9.5-10.5% to 19.5-20.5%, 15-33min, the volume fraction of the mobile phase A increases from 19.5-20.5% to 27.5-28.5%, 33-38min, the volume fraction of the mobile phase A increases from 27.5-28.5% to 89.5-90.5%, 38-44min, the volume fraction of the mobile phase A is 89.5-90.5%; The spectrum of the sample to be tested is compared with the characteristic spectrum of Amaranthus retroflexus described in the above technical solution. If the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus do not have chromatographic peaks with the same retention time, the Amaranthus retroflexus seeds to be tested do not contain Amaranthus retroflexus seeds; if the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus have chromatographic peaks with the same retention time, and the ultraviolet spectrum Figure 1 If the test result is positive, then the Amaranthus retroflexus seeds are contained in the tested Amaranthus retroflexus seeds.

[0036] The present invention utilizes methanol aqueous solution to extract the Cercis amaranth seeds to be tested, and the extract is the sample solution to be tested.

[0037] In the present invention, the volume fraction of methanol in the methanol aqueous solution is preferably 75-85%, and in specific embodiments may be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or 85%.

[0038] In the present invention, the C. amaranth seeds to be tested preferably include C. amaranth seeds decoction pieces or C. amaranth seeds powder to be tested; the C. amaranth seeds decoction pieces are preferably crushed before use; the particle size of the C. amaranth seeds powder to be tested is preferably 250-350µm. In the present invention, the mass volume ratio of the C. amaranth seeds to be tested to the methanol aqueous solution is preferably 1g:20-30mL, and in specific embodiments can be 1g:20mL, 1g:21mL, 1g:22mL, 1g:23mL, 1g:24mL, 1g:25mL, 1g:26mL, 1g:27mL, 1g:28mL, 1g:29mL or 1g:30mL.

[0039] In the present invention, the extraction preferably includes heating reflux extraction, and the extraction time is preferably 55 to 65 min, and in specific embodiments it can be 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min or 65 min.

[0040] After the extraction is completed, the present invention preferably further comprises: cooling the extraction system to room temperature, weighing, supplementing the lost weight with methanol aqueous solution, shaking, filtering, and the filtrate is the sample solution to be tested. In the present invention, the methanol aqueous solution is preferably the same as the methanol aqueous solution used in the extraction.

[0041] After obtaining the sample liquid to be tested, the present invention performs high performance liquid chromatography detection on the sample liquid to obtain a sample spectrum to be tested. In the present invention, the conditions of the high performance liquid chromatography detection are the same as those of the high performance liquid chromatography detection used in the construction process of the characteristic spectrum of Amaranthus retroflexus seeds, and will not be repeated here.

[0042] After obtaining the spectrum of the sample to be tested, the present invention compares the spectrum of the sample to be tested with the characteristic spectrum of Amaranthus retroflexus described in the above technical solution. If the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus do not have chromatographic peaks with the same retention time, the Amaranthus retroflexus seeds to be tested do not contain Amaranthus retroflexus seeds; if the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus have chromatographic peaks with the same retention time, and the ultraviolet spectrum Figure 1 If the test result is positive, then the Amaranthus retroflexus seeds are contained in the tested Amaranthus retroflexus seeds.

[0043] To further illustrate the present invention, the characteristic spectrum of Amaranthus retroflexus seeds, its construction method and application, and the identification method of adulterated Amaranthus retroflexus seeds in Amaranthus chinensis seeds are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] In the following examples, the instruments used were: METTLER TOLEDO electronic balance; Waters 2695 high performance liquid chromatograph (PDA detector, Empower workstation); Preparation method of reference substance: add 80v / v% methanol aqueous solution to the powder of Amaranthus retroflexus, heat and reflux for extraction for 2h, filter to obtain filtrate and residue, add 80v / v% methanol aqueous solution to the residue and repeat the above heating and reflux extraction steps, filter twice, combine the filtrate obtained by two extractions to obtain an extract; concentrate the extract and purify it with a semi-preparative HPLC column, the chromatographic column is octadecylsilane bonded silica gel, the mobile phase is 20v / v% acetonitrile-water, the detection wavelength is 275nm, collect the eluent with a retention time of 25.48~25.68min, and concentrate to dryness under reduced pressure to obtain a reference substance. The solid-liquid ratio of Amaranthus retroflexus powder to the 80v / v% methanol aqueous solution used for single extraction is 1kg:10L.

[0045] Reagents: acetonitrile and formic acid were of chromatographic grade, water was MILLIPORE purified water, and methanol was of analytical grade.

[0046] The sources of the decoction pieces of Amaranthus antiqua and Amaranthus retroflexus are shown in Table 1.

[0047] Table 1 Sources of multiple batches of Amaranthus chinensis and Amaranthus retroflexus slices

[0048] Example 1 Preparation of reference solution: Accurately weigh the reference substance and dissolve it in 80 v / v% methanol aqueous solution to obtain a reference solution with a concentration of 0.32 μg / mL.

[0049] The reference substance solution is subjected to HPLC detection to obtain a reference substance spectrum (Amaranthus retroflexus characteristic spectrum).

[0050] HPLC detection conditions: the chromatographic column was Agilent Eclipse Plus C18 (250×4.6mm, 5μm); the column temperature was 25°C; a diode array detector was used with a detection wavelength of 275nm; mobile phase A was acetonitrile, mobile phase B was 0.1v / v% HCOOH aqueous solution, the mobile phase flow rate was 1.0mL / min, and the injection volume was 10μL; the elution method was gradient elution, and the gradient elution program is shown in Table 2.

[0051] Table 2 Gradient elution program

[0052] Figure 1 This is the HPLC chromatogram of the reference substance. It can be seen that the characteristic peak of Amaranthus retroflexus (i.e. the chromatographic peak of the reference substance) is at a retention time of 25.526 min.

[0053] Figure 2 This is the UV absorption spectrum of the reference substance, and it can be seen that the maximum absorption wavelength is 275nm.

[0054] Example 2 Preparation of C. amaranthus seed sample solution: Accurately weigh 1 g of C. amaranthus seed powder to be tested, place it in a stoppered conical flask, add 25 mL of 80 v / v% methanol aqueous solution, weigh the weight, heat under reflux in a water bath for 1 h, return to room temperature, weigh again, make up the lost weight with 80 v / v% methanol aqueous solution, shake well, filter, and the filtrate is the C. amaranthus seed sample solution.

[0055] Preparation of Amaranthus retroflexus sample solution: Accurately weigh 1 g of Amaranthus retroflexus powder, place it in a stoppered conical flask, add 25 mL of 80 v / v% methanol aqueous solution, weigh the weight, heat under reflux in a water bath for 1 h, return to room temperature, weigh again, make up the lost weight with 80 v / v% methanol aqueous solution, shake well, filter, and the filtrate is the Amaranthus retroflexus sample solution.

[0056] The C. amaranthus sample solution was tested according to the HPLC conditions of Example 1 to obtain a C. amaranthus sample spectrum. The Amaranthus retroflexus sample solution was tested according to the HPLC conditions of Example 1 to obtain a Amaranthus retroflexus sample spectrum.

[0057] The characteristic spectrum of Amaranthus retroflexus obtained in Example 1, the spectrum of the sample of Amaranthus retroflexus and the spectrum of the sample of Amaranthus retroflexus are compared. If the spectrum of the sample of Amaranthus retroflexus and the characteristic spectrum of Amaranthus retroflexus do not have chromatographic peaks with the same retention time, then the Amaranthus retroflexus does not contain Amaranthus retroflexus; if the spectrum of the sample of Amaranthus retroflexus and the characteristic spectrum of Amaranthus retroflexus and the spectrum of the sample of Amaranthus retroflexus appear chromatographic peaks with the same retention time, and the ultraviolet spectrum Figure 1 If it is caused by the Amaranthus seeds, then the Amaranthus retroflexus seeds are contained in the Amaranthus retroflexus seeds.

[0058] The present invention verifies the established method by testing the collected experimental materials such as Amaranthus retroflexus seeds and Amaranthus retroflexus seeds, and uses the identification method established by the present invention to check whether Amaranthus retroflexus seeds are mixed with Amaranthus retroflexus seeds. The collected batches of Amaranthus retroflexus seeds and Amaranthus retroflexus seeds are shown in Table 1.

[0059] Figure 3~Figure 11 The HPLC chromatograms of Amaranthus retroflexus seeds 1 to 9 are shown in order. Figure 1 and Figure 3~Figure 11 It can be seen that the chromatographic peaks with the same retention time as the reference substance appeared in the spectra of the Amaranthus retroflexus samples.

[0060] Figure 12~Figure 20 The HPLC chromatograms of Cercidiphyllum seeds 1 to 9 are shown in order. Figure 1 and Figure 12~Figure 20 It can be seen that the chromatographic peaks with the same retention time as those in the reference substance and Amaranthus retroflexus sample solutions did not appear in the spectra of the Amaranthus chinensis samples, that is, no characteristic peaks were detected, indicating that Amaranthus retroflexus seeds were not contained in Amaranthus retroflexus seeds 1 to 9.

[0061] It shows that the method established by the present invention is accurate and reliable, and the identification method provided by the present invention will play a huge role in standardizing the decoction pieces of Cnidium monnieri.

[0062] Example 3 Optimization of HPLC chromatographic conditions (1) Selection of mobile phase The reference solution prepared in Example 1 was subjected to HPLC detection. The HPLC detection conditions used were different from those in Example 1 only in that the mobile phase systems were acetonitrile-water mobile phase system, acetonitrile-0.1 v / v% phosphoric acid aqueous solution mobile phase system, and acetonitrile-0.1 v / v% formic acid aqueous solution mobile phase system. The HPLC chromatograms of the reference solution are shown in order. Figure 22~Figure 24 . It can be seen that the peak shape of the chromatographic peak of the acetonitrile-water mobile phase system is poor. The acetonitrile-0.1v / v% phosphoric acid aqueous solution mobile phase system and the acetonitrile-0.1v / v% formic acid aqueous solution mobile phase system can both achieve good identification of characteristic peaks. However, the baseline of the acetonitrile-0.1v / v% phosphoric acid aqueous solution mobile phase system is not stable, and salt is easily precipitated during long-term analysis, which damages the instrument. Therefore, acetonitrile-0.1v / v% formic acid aqueous solution was finally selected as the mobile phase system.

[0063] (2) Elution procedure investigation The Amaranthus retroflexus sample solution prepared in Example 2 was subjected to HPLC detection. The HPLC detection conditions used were different from those in Example 1 in that the gradient elution program 1 shown in Table 3, the gradient elution program 2 shown in Table 4, and the gradient elution program 3 shown in Table 2 were used respectively. The HPLC chromatograms of the Amaranthus retroflexus sample solution were shown in Table 3 and Table 4. Figure 25~Figure 27 It can be seen that the reference sample peak at around 25 minutes has less interference and is conducive to detection, so the gradient elution program shown in Table 2 was finally selected.

[0064] Table 3 Gradient elution program 1

[0065] Table 4 Gradient elution program 2

[0066] Example 4 Methodological review The HPLC detection conditions are the same as those in Example 1.

[0067] (1) Specificity investigation Take samples of Amaranthus chinensis seeds and Amaranthus retroflexus seeds, prepare sample solutions according to Example 2, and perform detection according to the chromatographic conditions of Example 1. The results show that Amaranthus chinensis seeds do not contain the characteristic component, while Amaranthus retroflexus seeds contain the characteristic component.

[0068] (2) Detection limit Preparation of reference substance solution: accurately weigh the reference substance, add 80v / v% methanol aqueous solution to dissolve and make up to volume to obtain a solution with a concentration of 84.4μg / mL to obtain the reference substance stock solution.

[0069] Accurately measure 1 mL of the mother solution of the reference substance, add 80 v / v% methanol aqueous solution to dilute 25 times, 125 times and 250 times respectively, inject each dilution into the liquid chromatograph, calculate the signal-to-noise ratio, and S / N=3 is the detection limit. The results are shown in Table 5 and Fig.21 It can be seen that the method provided by the present invention has a low detection limit and high sensitivity.

[0070] Table 5 Sample detection limits

[0071] (3) Precision The reference solution prepared in Example 1 was injected 6 times continuously, the peak area was measured, and the RSD% was calculated to examine the precision. The results are shown in Table 6. It can be seen that the method provided by the present invention has good precision.

[0072] Table 6 Precision test results

[0073] (4) Repeatability experiment According to the method of Example 2, 6 Amaranthus retroflexus sample solutions were prepared and subjected to HPLC detection. The results are shown in Table 7. It can be seen that the method provided by the present invention has good repeatability.

[0074] Table 7 Repeatability test results

[0075] (5) Stability test The Amaranthus retroflexus sample solution prepared in Example 2 was stored at room temperature, and samples were taken for HPLC detection at 0 h, 2 h, 4 h, 8 h, 12 h, 18 h, and 24 h. The results are shown in Table 8. It can be seen that the sample solution is stable when stored at room temperature for 24 h.

[0076] Table 8 Stability test results

[0077] (6) Durability test Chromatographic column model investigation: The Amaranthus retroflexus sample solution prepared in Example 2 was subjected to HPLC detection according to the method of Example 1. The only difference from Example 1 was that the three types of chromatographic columns shown in Table 9 were used. The results are shown in Table 9. Fig.28 (Agilent Eclipse Plus C18 column), Fig.29 (Phenomenex luna C18 column) and Fig.30 (Lichrospher C18 column), the results showed that this method has good durability on the column.

[0078] High performance liquid chromatograph model investigation: The Amaranthus retroflexus sample solution prepared in Example 2 was subjected to HPLC detection according to the method of Example 1. The only difference from Example 1 is that different detection instruments shown in Table 10 were used. The results are shown in Table 10. Fig.31 (Waters high performance liquid chromatograph), Fig.32 (Agilent HPLC) and Fig.33 (Shimadzu LC high performance liquid chromatograph), verified by UV spectroscopy, can achieve good separation, and the spectral characteristics of the sample chromatographic peaks are basically consistent with those of the reference, indicating that the method provided by the present invention has good durability.

[0079] Table 9 Durability study of different types of chromatographic columns

[0080] Table 10 Durability study of high performance liquid chromatographs of different brands

[0081] In summary, in order to effectively solve the problem of adulteration of Amaranthus seeds, the present invention, on the basis of systematic analysis of the chemical components of Amaranthus seeds and Amaranthus retroflexus seeds, uses high performance liquid chromatography to establish chromatographic fingerprints of Amaranthus seeds and mixed and counterfeit Amaranthus retroflexus seeds, searches for and discovers characteristic components in Amaranthus retroflexus seeds that are different from Amaranthus seeds, that is, differential components, and establishes a method for constructing a characteristic map of Amaranthus retroflexus seeds and a method for identifying adulterated Amaranthus retroflexus seeds in Amaranthus seeds.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic map of Amaranthus retroflexus, comprising the following steps: Using methanol to extract Amaranthus retroflexus seeds to obtain a methanol extract; Purifying the methanol extract using a semi-preparative high performance liquid chromatography column to obtain a reference substance; The semi-preparative high performance liquid chromatography column purification conditions include: the chromatographic column is a C18 chromatographic column, the mobile phase is an acetonitrile aqueous solution, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 19-21%, the detection wavelength is 275nm, and the components with a retention time of 25.48-25.68min are collected; Dissolving the reference substance in a methanol aqueous solution to obtain a reference substance solution; The reference solution is subjected to high performance liquid chromatography detection to obtain a characteristic spectrum of Amaranthus retroflexus seeds; the conditions of the high performance liquid chromatography detection include: a chromatographic column is a C18 chromatographic column; a mobile phase system includes a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.09-0.11%; the elution mode is gradient elution, and the gradient elution program includes: 0-15min, the volume fraction of the mobile phase A increases from 9.5-10.5% to 19.5-20.5%, 15-33min, the volume fraction of the mobile phase A increases from 19.5-20.5% to 27.5-28.5%, 33-38min, the volume fraction of the mobile phase A increases from 27.5-28.5% to 89.5-90.5%, and 38-44min, the volume fraction of the mobile phase A is 89.5-90.5%.

2. The construction method according to claim 1, characterized in that: The ratio of the mass of Amaranthus retroflexus seeds to the volume of methanol is 1 kg: 5-15 L; The extraction includes heating reflux extraction, the number of extractions is 2 to 3 times, and the single extraction time is 1 to 3 hours.

3. The construction method according to claim 1, characterized in that: The volume fraction of methanol in the methanol aqueous solution is 75-85%; The concentration of the reference solution is 0.30-0.35 μg / mL.

4. The construction method according to claim 1, characterized in that: The column temperature of the high performance liquid chromatography detection is 20-30° C., the detector is a diode array detector, the mobile phase flow rate is 0.9-1.1 mL / min, and the injection volume is 5-15 μL.

5. The characteristic spectrum of Amaranthus retroflexus obtained by the construction method according to any one of claims 1 to 4.

6. Use of the characteristic spectrum of Amaranthus retroflexus seeds as claimed in claim 5 in the identification of Amaranthus chinensis seeds.

7. A method for identifying adulteration of Amaranthus retroflexus seeds in Amaranthus chinensis seeds, comprising the following steps: The Cercidiphyllum amarum seeds to be tested are extracted with methanol aqueous solution, and the extract is the sample solution to be tested; The sample liquid to be tested is subjected to high performance liquid chromatography to obtain a spectrum of the sample to be tested; The conditions for the high performance liquid chromatography detection include: the chromatographic column is a C18 chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.09-0.11%; the detection wavelength is 275nm; the elution method is gradient elution, and the gradient elution program includes: 0-15min, the volume fraction of the mobile phase A increases from 9.5-10.5% to 19.5-20.5%, 15-33min, the volume fraction of the mobile phase A increases from 19.5-20.5% to 27.5-28.5%, 33-38min, the volume fraction of the mobile phase A increases from 27.5-28.5% to 89.5-90.5%, and 38-44min, the volume fraction of the mobile phase A is 89.5-90.5%. Compare the spectrum of the sample to be tested with the characteristic spectrum of Amaranthus retroflexus described in claim 5. If the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus do not have chromatographic peaks with the same retention time, the Amaranthus retroflexus seeds to be tested do not contain Amaranthus retroflexus seeds; if the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus seeds have chromatographic peaks with the same retention time, and the ultraviolet spectra are consistent, the Amaranthus retroflexus seeds to be tested contain Amaranthus retroflexus seeds.

8. The identification method according to claim 7, characterized in that: The volume fraction of methanol in the methanol aqueous solution is 75-85%; The ratio of the mass of the Celosia chinensis seeds to be tested to the volume of the methanol aqueous solution is 1 g: 20-30 mL.

9. The identification method according to claim 7 or 8, characterized in that: The extraction includes heating reflux extraction, and the extraction time is 55 to 65 minutes.

10. The identification method according to claim 7, characterized in that: The column temperature of the high performance liquid chromatography detection is 20-30° C., the detector is a diode array detector, the mobile phase flow rate is 0.9-1.1 mL / min, and the injection volume is 5-15 μL.

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