A characteristic spectrum of Amaranthus retroflexus seeds, a construction method and application thereof, and a method for identifying adulterated Amaranthus retroflexus seeds in Celosia argentea L. seeds

Abstract

The present invention provides an amaranthus retroflexus L. seed characteristic fingerprint spectrum, a construction method and application thereof, and a method for identifying adulterated amaranthus retroflexus L. seeds in celosia argentea L. seeds, which relates to the technical field of traditional Chinese medicine detection. The present invention constructs an amaranthus retroflexus L. seed characteristic fingerprint spectrum, which has strong method specificity, good precision, good repeatability and good durability. By using the amaranthus retroflexus L. seed characteristic fingerprint spectrum constructed by the present invention to locate the HPLC chromatogram of the to-be-detected celosia argentea L. seeds, if no chromatographic peak with the same retention time appears in the chromatogram of the to-be-detected sample and the amaranthus retroflexus L. seed characteristic fingerprint spectrum, it indicates that the to-be-detected celosia argentea L. seeds do not contain amaranthus retroflexus L. seeds; if a chromatographic peak with the same retention time appears in the chromatogram of the to-be-detected sample and the amaranthus retroflexus L. seed characteristic fingerprint spectrum, and the ultraviolet spectrograms are consistent, it indicates that the to-be-detected celosia argentea L. seeds contain amaranthus retroflexus L. seeds. The method provided by the present invention solves the problem of adulteration of whether celosia argentea L. seeds are adulterated with amaranthus retroflexus L. seeds. This method is simple to operate, has simple steps, clear and reliable results, and is easy to judge, providing a more powerful guarantee for the authenticity identification and quality control of celosia argentea L. 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, there are reports on the high-performance liquid chromatography (HPLC) characteristic fingerprint of Celosiae Semen. The HPLC chromatographic conditions used are a Kromasil 100-5 C18 chromatographic column, with the mobile phase being acetonitrile-phosphoric acid solution (volume fraction 0.1%), 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%), the detection wavelength is 203 nm, the flow rate is 1 mL / min, and the column temperature is 30 °C; the detection results show that a total of 35 common peaks are marked in 10 batches of Celosiae Semen medicinal materials, and the similarity is 0.973 - 0.997. The ultraviolet absorption spectra of the main common peaks can be divided into 10 categories. However, the above detection method cannot detect whether Celosiae Semen is adulterated with Amaranthi retroflexi Semen. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a characteristic fingerprint of Amaranthi retroflexi Semen and its construction method and application, as well as a method for identifying adulterated Amaranthi retroflexi Semen in Celosiae Semen. The characteristic fingerprint of Amaranthi retroflexi Semen constructed by the present invention can accurately identify whether Celosiae Semen is adulterated with Amaranthi retroflexi Semen, providing a more powerful guarantee for the authenticity identification and quality control of Celosiae Semen.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for constructing a characteristic fingerprint of Amaranthi retroflexi Semen, comprising the following steps:

[0008] Extract Amaranthi retroflexi Semen with methanol to obtain a methanol extract;

[0009] Purify 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 275 nm, and the components with a retention time of 25.48 - 25.68 min are collected;

[0010] Dissolve the reference substance in a methanol aqueous solution to obtain a reference substance solution;

[0011] Perform high performance liquid chromatography (HPLC) detection on the reference substance solution to obtain the characteristic chromatogram of *Amaranthus retroflexus* seeds. The conditions for the HPLC detection include: the chromatographic column is a C18 chromatographic column; the mobile phase system includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution with the volume fraction of formic acid in the aqueous formic acid solution being 0.09 - 0.11%; the detection wavelength is 275 nm; the elution method is gradient elution, and the gradient elution program includes: from 0 to 15 min, the volume fraction of mobile phase A increases from 9.5 - 10.5% to 19.5 - 20.5%; from 15 to 33 min, the volume fraction of mobile phase A increases from 19.5 - 20.5% to 27.5 - 28.5%; from 33 to 38 min, the volume fraction of mobile phase A increases from 27.5 - 28.5% to 89.5 - 90.5%; from 38 to 44 min, the volume fraction of mobile phase A is 89.5 - 90.5%.

[0012] Preferably, the mass ratio of the *Amaranthus retroflexus* seeds to the volume of methanol is 1 kg: 5 - 15 L;

[0013] The extraction includes heating under reflux extraction, the number of extractions is 2 - 3 times, and the single extraction time is 1 - 3 h.

[0014] Preferably, the volume fraction of methanol in the methanol aqueous solution is 75 - 85%;

[0015] The concentration of the reference substance solution is 0.3 - 0.34 μg / mL.

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

[0017] The present invention also provides the characteristic chromatogram of *Amaranthus retroflexus* seeds obtained by the construction method described in the above technical solution.

[0018] The present invention also provides the application of the characteristic chromatogram of *Amaranthus retroflexus* seeds described in the above technical solution in the identification of *Celosia argentea* seeds.

[0019] The present invention also provides a method for identifying adulterated *Amaranthus retroflexus* seeds in *Celosia argentea* seeds, which includes the following steps:

[0020] Extract the test *Celosia argentea* seeds with a methanol aqueous solution, and the extract is the test sample solution;

[0021] Perform high performance liquid chromatography (HPLC) detection on the sample solution to be tested to obtain a chromatogram of the sample to be tested. The conditions for the HPLC detection include: the chromatographic column is a C18 chromatographic column; the mobile phase system includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution with a volume fraction of formic acid of 0.09 - 0.11%; the detection wavelength is 275 nm; the elution method is gradient elution, and the gradient elution program includes: from 0 to 15 minutes, the volume fraction of mobile phase A increases from 9.5 - 10.5% to 19.5 - 20.5%; from 15 to 33 minutes, the volume fraction of mobile phase A increases from 19.5 - 20.5% to 27.5 - 28.5%; from 33 to 38 minutes, the volume fraction of mobile phase A increases from 27.5 - 28.5% to 89.5 - 90.5%; from 38 to 44 minutes, the volume fraction of mobile phase A is 89.5 - 90.5%.

[0022] Compare the chromatogram of the sample to be tested with the characteristic chromatogram of *Amaranthus retroflexus* seeds described in the above technical solution. If there are no chromatographic peaks with the same retention time in the chromatogram of the sample to be tested and the characteristic chromatogram of *Amaranthus retroflexus* seeds, then the *Celosia argentea* seeds to be tested do not contain *Amaranthus retroflexus* seeds; if there are chromatographic peaks with the same retention time in the chromatogram of the sample to be tested and the characteristic chromatogram of *Amaranthus retroflexus* seeds, and the ultraviolet spectra Figure 1 are consistent, then the *Celosia argentea* seeds to be tested contain *Amaranthus retroflexus* seeds.

[0023] Preferably, the volume fraction of methanol in the aqueous methanol solution is 75 - 85%;

[0024] The ratio of the mass of the *Celosia argentea* seeds to be tested to the volume of the aqueous methanol solution is 1 g: 20 - 30 mL.

[0025] Preferably, the extraction includes heating under reflux extraction, and the extraction time is 55 - 65 minutes.

[0026] Preferably, the column temperature for the HPLC detection is 20 - 30 °C, the detector is a diode array detector, the flow rate of the mobile phase is 0.9 - 1.1 mL / min, and the injection volume is 5 - 15 μL.

[0027] The present invention constructs a characteristic chromatogram of *Amaranthus retroflexus* seeds. The construction method provided by the present invention has strong specificity, good precision, good repeatability, and good method durability.

[0028] Locate the HPLC chromatogram of the *Celosia argentea* seeds to be tested with the characteristic chromatogram of *Amaranthus retroflexus* seeds constructed by the present invention. If there are no chromatographic peaks with the same retention time in the chromatogram of the sample to be tested and the characteristic chromatogram of *Amaranthus retroflexus* seeds, then the *Celosia argentea* seeds to be tested do not contain *Amaranthus retroflexus* seeds; if there are chromatographic peaks with the same retention time in the chromatogram of the sample to be tested and the characteristic chromatogram of *Amaranthus retroflexus* seeds, and the ultraviolet spectra Figure 1If so, the tested Celosia seeds contain Amaranthus retroflexus seeds. The method provided by the present invention solves the problem of adulteration of whether Celosia seeds are adulterated with Amaranthus retroflexus seeds, filling the blank in the prior art that there is no detection method for whether Celosia seeds are adulterated with Amaranthus retroflexus seeds; this method is simple to operate, with simple steps, clear and reliable results, and easy to judge, providing a more powerful guarantee for the authenticity identification and quality control of Celosia seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the HPLC chromatogram of the reference substance;

[0030] Figure 2 is the ultraviolet absorption spectrum of the reference substance;

[0031] Figure 3 is the HPLC chromatogram of Amaranthus retroflexus seed 1;

[0032] Figure 4 is the HPLC chromatogram of Amaranthus retroflexus seed 2;

[0033] Figure 5 is the HPLC chromatogram of Amaranthus retroflexus seed 3;

[0034] Figure 6 is the HPLC chromatogram of Amaranthus retroflexus seed 4;

[0035] Figure 7 is the HPLC chromatogram of Amaranthus retroflexus seed 5;

[0036] Figure 8 is the HPLC chromatogram of Amaranthus retroflexus seed 6;

[0037] Figure 9 is the HPLC chromatogram of Amaranthus retroflexus seed 7;

[0038] Figure 10 is the HPLC chromatogram of Amaranthus retroflexus seed 8;

[0039] Figure 11 is the HPLC chromatogram of Amaranthus retroflexus seed 9;

[0040] Figure 12 is the HPLC chromatogram of Celosia seed 1;

[0041] Figure 13 is the HPLC chromatogram of Celosia seed 2;

[0042] Figure 14 is the HPLC chromatogram of Celosia seed 3;

[0043] Figure 15 is the HPLC chromatogram of Celosia seed 4;

[0044] Figure 16 is the HPLC chromatogram of Celosia seed 5;

[0045] Figure 17 It is the HPLC chromatogram of Celosia argentea L. 6;

[0046] Figure 18 It is the HPLC chromatogram of Celosia argentea L. 7;

[0047] Figure 19 It is the HPLC chromatogram of Celosia argentea L. 8;

[0048] Figure 20 It is the HPLC chromatogram of Celosia argentea L. 9;

[0049] Figure 21 It is the HPLC chromatogram for the detection limit investigation of the reference substance (i.e., the characteristic component);

[0050] Figure 22 It is the HPLC chromatogram of the reference substance solution under the acetonitrile - water mobile phase system;

[0051] Figure 23 It is the HPLC chromatogram of the reference substance solution under the acetonitrile - 0.1 v / v% phosphoric acid aqueous solution mobile phase system;

[0052] Figure 24 It is the HPLC chromatogram of the reference substance solution under the acetonitrile - 0.1 v / v% formic acid aqueous solution mobile phase system;

[0053] Figure 25 It is the HPLC chromatogram of the Amaranthus retroflexus L. sample solution under gradient elution program 1;

[0054] Figure 26 It is the HPLC chromatogram of the Amaranthus retroflexus L. sample solution under gradient elution program 2;

[0055] Figure 27 It is the HPLC chromatogram of the Amaranthus retroflexus L. sample solution under gradient elution program 3;

[0056] Figure 28 It is the HPLC chromatogram of the Amaranthus retroflexus L. sample solution detected by Agilent Eclipse Plus C18 chromatographic column;

[0057] Figure 29 It is the HPLC chromatogram of the Amaranthus retroflexus L. sample solution detected by Phenomenex luna C18 chromatographic column;

[0058] Figure 30 It is the HPLC chromatogram of the Amaranthus retroflexus L. sample solution detected by Lichrospher C18 chromatographic column;

[0059] Figure 31It is the HPLC chromatogram of the sample solution of Amaranthus retroflexus seeds detected by Waters high performance liquid chromatograph;

[0060] Figure 32 It is the HPLC chromatogram of the sample solution of Amaranthus retroflexus seeds detected by Agilent high performance liquid chromatograph;

[0061] Figure 33 It is the HPLC chromatogram of the sample solution of Amaranthus retroflexus seeds detected by Shimadzu LC high performance liquid chromatograph. Specific embodiments

[0062] The present invention provides a method for constructing a characteristic fingerprint of Amaranthus retroflexus seeds, comprising the following steps:

[0063] Using methanol to extract Amaranthus retroflexus seeds to obtain a methanol extract;

[0064] 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 275 nm, and the components with a retention time of 25.48-25.68 min are collected;

[0065] Dissolving the reference substance in a methanol aqueous solution to obtain a reference substance solution;

[0066] Performing high performance liquid chromatography detection on the reference substance solution to obtain a characteristic fingerprint of Amaranthus retroflexus seeds; 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 275 nm; the elution method is gradient elution, and the gradient elution program includes: 0-15 min, the volume fraction of mobile phase A increases from 9.5-10.5% to 19.5-20.5%, 20-28 min, the volume fraction of mobile phase A increases from 19.5-20.5% to 27.5-28.5%, 15-33 min, the volume fraction of mobile phase A increases from 27.5-28.5% to 89.5-90.5%, 38-44 min, and the volume fraction of mobile phase A is 89.5-90.5%.

[0067] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0068] The present invention uses methanol to extract Amaranthus retroflexus seeds to obtain a methanol extract.

[0069] In the present invention, the amaranth seeds are preferably used in the form of amaranth seed powder, and the particle size of the amaranth seed powder is preferably 250 - 350 µm. In the present invention, the mass - volume ratio of the amaranth 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.

[0070] In the present invention, the extraction preferably includes heating under reflux extraction; the number of extractions is preferably 2 - 3 times; the single - extraction time is 1 - 3 h, and in specific embodiments, it can be 1 h, 1.5 h, 2 h, 2.5 h, or 3 h.

[0071] After obtaining the methanol extract, the present invention purifies the methanol extract by semi - preparative high - performance liquid chromatography column to obtain the reference substance.

[0072] 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, and the volume fraction of acetonitrile in the acetonitrile aqueous solution is 19 - 21%, and in specific embodiments, it can be 19%, 19.5%, 20%, 20.5%, or 21%; the detection wavelength is 275 nm, and the components with a retention time of 25.48 - 25.68 min are collected. The present invention preferably collects the eluate with a retention time of 25.48 - 25.68 min, concentrates it under reduced pressure to dryness to obtain the reference substance.

[0073] The present invention dissolves the reference substance in a methanol aqueous solution to obtain a reference substance solution.

[0074] In the present invention, the volume fraction of methanol in the methanol aqueous solution is preferably 75 - 85%, and in specific embodiments, it can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%.

[0075] In the present invention, the concentration of the reference substance solution is preferably 0.30 - 0.35 μg / mL, and in specific embodiments, it can 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.

[0076] After obtaining the reference substance solution, the present invention performs high - performance liquid chromatography detection on the reference substance solution to obtain the characteristic chromatogram of amaranth seeds.

[0077] In the present invention, the conditions for high performance liquid chromatography detection include: the chromatographic column is a C18 chromatographic column, preferably including 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 specific embodiments, 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 mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is an aqueous formic acid solution, and the volume fraction of formic acid in the aqueous formic acid solution is 0.09 - 0.11%, and in specific embodiments, it can be 0.09%, 0.095%, 0.1%, 0.105% or 0.11%; the mobile phase flow rate is preferably 0.9 - 1.1 mL / min, and in specific embodiments, 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 specific embodiments, 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 method is gradient elution; the detection wavelength is 275 nm; the detector is preferably a diode array detector.

[0078] In the present invention, the gradient elution program includes:

[0079] 0 - 15 min, the volume fraction of mobile phase A increases from 9.5 - 10.5% (in specific embodiments, 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 specific embodiments, 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%);

[0080] 15 - 33 min, the volume fraction of mobile phase A increases from 19.5 - 20.5% (in specific embodiments, 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 specific embodiments, 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%);

[0081] From 33 to 38 minutes, the volume fraction of mobile phase A increases from 27.5 - 28.5% (which 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% in specific embodiments) to 89.5 - 90.5% (which 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% in specific embodiments).

[0082] From 38 to 44 minutes, the volume fraction of mobile phase A is 89.5 - 90.5%, which 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% in specific embodiments.

[0083] The present invention also provides the characteristic chromatogram of Amaranthus retroflexus seeds obtained by the construction method described in the above technical solution.

[0084] The present invention also provides the application of the characteristic chromatogram of Amaranthus retroflexus seeds described in the above technical solution in the identification of Celosia argentea L. seeds.

[0085] The present invention also provides a method for identifying adulterated Amaranthus retroflexus seeds in Celosia argentea L. seeds, comprising the following steps:

[0086] Extract the Celosia argentea L. seeds to be tested with an aqueous methanol solution, and the extract is the sample solution to be tested;

[0087] Perform high performance liquid chromatography (HPLC) detection on the sample solution to be tested to obtain the chromatogram of the sample to be tested; the conditions for the HPLC detection include: the chromatographic column is a C18 chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is an aqueous formic acid solution, and the volume fraction of formic acid in the aqueous formic acid solution is 0.09 - 0.11%; the detection wavelength is 275 nm; the elution method is gradient elution, and the gradient elution program includes: from 0 to 15 minutes, the volume fraction of mobile phase A increases from 9.5 - 10.5% to 19.5 - 20.5%, from 15 to 33 minutes, the volume fraction of mobile phase A increases from 19.5 - 20.5% to 27.5 - 28.5%, from 33 to 38 minutes, the volume fraction of mobile phase A increases from 27.5 - 28.5% to 89.5 - 90.5%, and from 38 to 44 minutes, the volume fraction of mobile phase A is 89.5 - 90.5%;

[0088] Compare the spectrum of the sample to be tested with the characteristic spectrum of *Amaranthus retroflexus* described in the above technical solution. If there are no chromatographic peaks with the same retention time in the spectrum of the sample to be tested and the characteristic spectrum of *Amaranthus retroflexus*, then the *Celosia argentea* to be tested does not contain *Amaranthus retroflexus*; if there are chromatographic peaks with the same retention time in the spectrum of the sample to be tested and the characteristic spectrum of *Amaranthus retroflexus*, and the ultraviolet spectra Figure 1 are consistent, then the *Celosia argentea* to be tested contains *Amaranthus retroflexus*.

[0089] In the present invention, the *Celosia argentea* to be tested is extracted with an aqueous methanol solution, and the extract is the sample solution to be tested.

[0090] In the present invention, the volume fraction of methanol in the aqueous methanol solution is preferably 75-85%, and in specific embodiments, it can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or 85%.

[0091] In the present invention, the *Celosia argentea* to be tested preferably includes *Celosia argentea* cut pieces or *Celosia argentea* powder to be tested; the *Celosia argentea* cut pieces are preferably pulverized before use; the particle size of the *Celosia argentea* powder to be tested is preferably 250-350 µm. In the present invention, the mass-volume ratio of the *Celosia argentea* to be tested to the aqueous methanol solution is preferably 1 g: 20-30 mL, and in specific embodiments, it can be 1 g: 20 mL, 1 g: 21 mL, 1 g: 22 mL, 1 g: 23 mL, 1 g: 24 mL, 1 g: 25 mL, 1 g: 26 mL, 1 g: 27 mL, 1 g: 28 mL, 1 g: 29 mL or 1 g: 30 mL.

[0092] In the present invention, the extraction preferably includes heating under reflux extraction, and the extraction time is preferably 55-65 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.

[0093] After completing the extraction, the present invention preferably further includes: cooling the extraction system to room temperature, weighing, making up the lost weight with an aqueous methanol solution, shaking well, filtering, and the filtrate is the sample solution to be tested. In the present invention, the aqueous methanol solution is preferably the same as the aqueous methanol solution used for the extraction.

[0094] After obtaining the sample solution to be tested, the present invention performs high performance liquid chromatography detection on the sample solution to be tested to obtain a spectrum of the sample to be tested. In the present invention, the conditions for the high performance liquid chromatography detection are the same as those used in the construction process of the characteristic spectrum of *Amaranthus retroflexus*, and will not be elaborated here.

[0095] 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 there are no chromatographic peaks with the same retention time in the spectrum of the sample to be tested and the characteristic spectrum of *Amaranthus retroflexus*, then the *Celosia argentea* to be tested does not contain *Amaranthus retroflexus*; if there are chromatographic peaks with the same retention time in the spectrum of the sample to be tested and the characteristic spectrum of *Amaranthus retroflexus*, and the ultraviolet spectra Figure 1 are consistent, then the *Celosia argentea* to be tested contains *Amaranthus retroflexus*.

[0096] To further illustrate the present invention, the characteristic spectrum of *Amaranthus retroflexus*, its construction method and application, and the identification method of adulterated *Amaranthus retroflexus* in *Celosia argentea* will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0097] In the following examples, the instruments used are: METTLER TOLEDO electronic balance; Waters 2695 high performance liquid chromatograph (PDA detector, Empower workstation);

[0098] Preparation method of reference substance: Add 80 v / v% methanol aqueous solution to the powder of *Amaranthus retroflexus*, heat under reflux for 2 h, filter to obtain filtrate and residue. Add 80 v / v% methanol aqueous solution to the residue and repeat the above heat reflux extraction step, filter twice, and combine the filtrates obtained from the two extractions to obtain the extract; Concentrate the extract and then purify it by semi-preparative HPLC column. The chromatographic column is octadecylsilane-bonded silica gel, the mobile phase is 20 v / v% acetonitrile-water, the detection wavelength is 275 nm, collect the eluate with a retention time of 25.48 - 25.68 min, and concentrate it under reduced pressure to dryness to obtain the reference substance. The solid-liquid ratio of the *Amaranthus retroflexus* powder to the 80 v / v% methanol aqueous solution used for single extraction is 1 kg:10 L.

[0099] Reagents: Acetonitrile and formic acid are chromatographically pure, water is MILLIPORE purified water, and methanol is analytically pure.

[0100] The sources of the *Celosia argentea* decoction pieces and *Amaranthus retroflexus* decoction pieces are shown in Table 1.

[0101] Table 1 Sources of multiple batches of *Celosia argentea* and *Amaranthus retroflexus* decoction pieces

[0102]

[0103] Example 1

[0104] Preparation of reference substance solution: Weigh the reference substance precisely, dissolve it with 80 v / v% methanol aqueous solution to obtain a reference substance solution with a concentration of 0.32 μg / mL.

[0105] Perform HPLC detection on the reference substance solution to obtain the reference substance spectrum (characteristic spectrum of *Amaranthus retroflexus*).

[0106] HPLC detection conditions: The chromatographic column was Agilent Eclipse Plus C18 (250×4.6 mm, 5 μm); the column temperature was 25 °C; a diode array detector was used, and the detection wavelength was 275 nm; mobile phase A was acetonitrile, mobile phase B was 0.1 v / v% HCOOH aqueous solution, the flow rate of the mobile phase was 1.0 mL / min, the injection volume was 10 μL; the elution method was gradient elution, and the gradient elution program is shown in Table 2.

[0107] Table 2 Gradient elution program

[0108]

[0109] Figure 1 As for the HPLC chromatogram of the reference substance, it can be seen that the characteristic peak of Amaranthus retroflexus seeds (i.e., the reference substance chromatographic peak) is at the retention time of 25.526 min.

[0110] Figure 2 As for the ultraviolet absorption spectrum of the reference substance, it can be seen that the maximum absorption wavelength is 275 nm.

[0111] Example 2

[0112] Preparation of the Celosia argentea L. sample solution: Weigh accurately 1 g of the Celosia argentea L. powder to be tested, place it in a stoppered conical flask, add 25 mL of 80 v / v% methanol aqueous solution, weigh it, reflux it in a water bath for 1 h, let it cool to room temperature, weigh it again, make up the lost weight with 80 v / v% methanol aqueous solution, shake well, filter, and the filtrate is the Celosia argentea L. sample solution.

[0113] Preparation of the Amaranthus retroflexus seeds sample solution: Weigh accurately 1 g of the Amaranthus retroflexus seeds powder, place it in a stoppered conical flask, add 25 mL of 80 v / v% methanol aqueous solution, weigh it, reflux it in a water bath for 1 h, let it cool to room temperature, weigh it again, make up the lost weight with 80 v / v% methanol aqueous solution, shake well, filter, and the filtrate is the Amaranthus retroflexus seeds sample solution.

[0114] Detect the Celosia argentea L. sample solution under the HPLC conditions of Example 1 to obtain the Celosia argentea L. sample chromatogram. Detect the Amaranthus retroflexus seeds sample solution under the HPLC conditions of Example 1 to obtain the Amaranthus retroflexus seeds sample chromatogram.

[0115] Compare the Amaranthus retroflexus seeds characteristic chromatogram, the Celosia argentea L. sample chromatogram and the Amaranthus retroflexus seeds sample chromatogram obtained in Example 1. If there are no chromatographic peaks with the same retention time in the Celosia argentea L. sample chromatogram and the Amaranthus retroflexus seeds characteristic chromatogram, then Celosia argentea L. does not contain Amaranthus retroflexus seeds; if there are chromatographic peaks with the same retention time in the Celosia argentea L. sample chromatogram, the Amaranthus retroflexus seeds characteristic chromatogram and the Amaranthus retroflexus seeds sample chromatogram, and the ultraviolet spectra Figure 1 are consistent, then Celosia argentea L. contains Amaranthus retroflexus seeds.

[0116] The present invention detects the collected experimental materials such as Celosiae Semen and Amaranthi Retroflexi Semen, verifies the established method, and checks whether there is adulteration of Amaranthi Retroflexi Semen in Celosiae Semen with the identification method established by the present invention. Multiple batches of Celosiae Semen and Amaranthi Retroflexi Semen decoction pieces collected are shown in Table 1.

[0117] Figures 3 to 11 They are the HPLC chromatograms of Amaranthi Retroflexi Semen 1 - 9 in sequence. By comparing Figure 1 and Figures 3 to 11 it can be seen that chromatographic peaks with the same retention time as the reference substance appear in the chromatograms of Amaranthi Retroflexi Semen samples.

[0118] Figures 12 to 20 They are the HPLC chromatograms of Celosiae Semen 1 - 9 in sequence. By comparing Figure 1 and Figures 12 to 20 it can be seen that chromatographic peaks with the same retention time as the reference substance and in the solution of Amaranthi Retroflexi Semen samples do not appear in the chromatograms of Celosiae Semen samples, that is, characteristic peaks are not detected. This shows that Celosiae Semen 1 - 9 does not contain Amaranthi Retroflexi Semen.

[0119] This 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 great role in standardizing Celosiae Semen decoction pieces.

[0120] Example 3

[0121] Optimization of HPLC Chromatographic Conditions

[0122] (1) Selection of Mobile Phase

[0123] The reference substance solution prepared in Example 1 is detected by HPLC. The difference in the HPLC detection conditions from those in Example 1 is only that the mobile phase systems are 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 respectively. The HPLC chromatograms of the reference substance solution are shown in Figures 22 to 24 in sequence. It can be seen that the chromatographic peak shape in the acetonitrile - water mobile phase system is poor. Both the acetonitrile - 0.1 v / v% phosphoric acid aqueous solution mobile phase system and the acetonitrile - 0.1 v / v% formic acid aqueous solution mobile phase system can better achieve the identification of characteristic peaks. However, the baseline of the acetonitrile - 0.1 v / v% phosphoric acid aqueous solution mobile phase system is not stable, and salts are likely to precipitate during long - term analysis, damaging the instrument. Therefore, the acetonitrile - 0.1 v / v% formic acid aqueous solution is finally selected as the mobile phase system.

[0124] (2) Investigation of Elution Program

[0125] The HPLC detection was carried out on the sample solution of Amaranthus retroflexus prepared in Example 2. The difference in the HPLC detection conditions from those in Example 1 was only 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 are shown successively in Figures 25 to 27 , and it can be seen that the reference peak elutes at about 25 min with less interference, which is beneficial for detection. Therefore, the gradient elution program shown in Table 2 was finally selected.

[0126] Table 3 Gradient elution program 1

[0127]

[0128] Table 4 Gradient elution program 2

[0129]

[0130] Example 4

[0131] Methodology investigation

[0132] The HPLC detection conditions were the same as those in Example 1.

[0133] (1) Specificity investigation

[0134] Seeds of Celosia argentea L. and Amaranthus retroflexus were taken, and the sample solutions were prepared according to Example 2 and detected under the chromatographic conditions of Example 1. The results showed that the characteristic component was not contained in Celosia argentea L., while it was contained in Amaranthus retroflexus.

[0135] (2) Detection limit

[0136] Preparation of reference solution: The reference substance was accurately weighed, dissolved and made up to volume with 80 v / v% methanol aqueous solution to obtain a solution with a concentration of 84.4 μg / mL as the reference stock solution.

[0137] 1 mL of the reference stock solution was accurately measured and diluted 25-fold, 125-fold and 250-fold respectively with 80 v / v% methanol aqueous solution. Each diluted solution was injected into the liquid chromatograph, and the signal-to-noise ratio was calculated. S / N = 3 was taken as the detection limit. The results are shown in Table 5 and Figure 21 , and it can be seen that the method provided by the present invention has a low detection limit and high method sensitivity.

[0138] Table 5 Sample detection limit

[0139]

[0140] (3) Precision

[0141] The reference solution prepared in Example 1 was continuously injected 6 times, the peak areas were measured, the RSD% was calculated, and the precision was investigated. The results are shown in Table 6. It can be seen that the method provided by the present invention has good precision.

[0142] Table 6 Results of precision test

[0143]

[0144] (4) Repeatability experiment

[0145] Six samples of Amaranthus retroflexus L. seeds solution were prepared according to the method of Example 2 and detected by HPLC. The results are shown in Table 7. It can be seen that the method provided by the present invention has good repeatability.

[0146] Table 7 Results of repeatability test

[0147]

[0148] (5) Stability investigation

[0149] The Amaranthus retroflexus L. seeds solution prepared in Example 2 was stored at room temperature, and samples were taken at 0 h, 2 h, 4 h, 8 h, 12 h, 18 h, and 24 h for HPLC detection. The results are shown in Table 8. It can be seen that the sample solution is stable for 24 h under room temperature conditions.

[0150] Table 8 Results of stability test

[0151]

[0152] (6) Robustness investigation

[0153] Investigation of chromatographic column model: The Amaranthus retroflexus L. seeds solution prepared in Example 2 was detected by HPLC according to the method of Example 1. The difference from Example 1 was only that three types of chromatographic columns shown in Table 9 were used. The results are shown in Figure 28 (Agilent Eclipse Plus C18 chromatographic column), Figure 29 (Phenomenex luna C18 chromatographic column) and Figure 30 (Lichrospher C18 chromatographic column). The results show that the method has good robustness to the chromatographic column.

[0154] Investigation of high performance liquid chromatograph model: The Amaranthus retroflexus L. seeds solution prepared in Example 2 was detected by HPLC according to the method of Example 1. The difference from Example 1 was only that different detection instruments shown in Table 10 were used. The results are shown in Figure 31 (Waters high performance liquid chromatograph), Figure 32 (Agilent high performance liquid chromatograph) and Figure 33(Shimadzu LC high performance liquid chromatograph), verified by ultraviolet spectrum, can achieve good separation, and the spectral characteristics of the sample chromatographic peaks are basically the same as those of the reference substance, indicating that the method provided by the present invention has good durability.

[0155] Table 9 Investigation on the durability of different types of chromatographic columns

[0156]

[0157] Table 10 Investigation on the durability of high performance liquid chromatographs of different brands

[0158]

[0159] In summary, to effectively solve the problem of adulteration of Celosiae Semen, based on the systematic analysis of the chemical components of Celosiae Semen and Amaranthi Retroflexi Semen, the present invention established the chromatographic fingerprint of Celosiae Semen and its adulterant Amaranthi Retroflexi Semen by high performance liquid chromatography, searched for and found the characteristic components in Amaranthi Retroflexi Semen that are different from Celosiae Semen, namely the differential components, and established a method for constructing the characteristic chromatogram of Amaranthi Retroflexi Semen and a method for identifying Amaranthi Retroflexi Semen adulterated in Celosiae Semen.

[0160] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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. Application of the characteristic spectrum of Amaranthus retroflexus obtained by the construction method according to any one of claims 1 to 4 in the identification of Amaranthus retroflexus seeds.

6. A method for identifying adulteration of Amaranthus retroflexus seeds in Amaranthus chinensis seeds, comprising the following steps: The Cercidiphyllum fruit to be tested is 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 high performance liquid chromatography (HPLC) detection include: the chromatographic column is a C18 chromatographic column; the mobile phase system includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution, with the volume fraction of formic acid in the aqueous formic acid solution being 0.09 - 0.11%; the detection wavelength is 275 nm; the elution method is gradient elution, and the gradient elution program includes: from 0 to 15 min, the volume fraction of mobile phase A increases from 9.5 - 10.5% to 19.5 - 20.5%; from 15 to 33 min, the volume fraction of mobile phase A increases from 19.5 - 20.5% to 27.5 - 28.5%; from 33 to 38 min, the volume fraction of mobile phase A increases from 27.5 - 28.5% to 89.5 - 90.5%; from 38 to 44 min, the volume fraction of mobile phase A is 89.5 - 90.5%. Compare the spectrum of the sample to be tested with the characteristic spectrum of Amaranthus retroflexus seeds obtained by the construction method described in any one of claims 1 - 4. If there are no chromatographic peaks with the same retention time in the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus seeds, then the Celosia argentea L. seeds to be tested do not contain Amaranthus retroflexus seeds; if there are chromatographic peaks with the same retention time in the spectrum of the sample to be tested and the characteristic spectrum of Amaranthus retroflexus seeds, and the ultraviolet spectra are consistent, then the Celosia argentea L. seeds to be tested contain Amaranthus retroflexus seeds.

7. The authentication method according to claim 6, wherein The volume fraction of methanol in the methanol aqueous solution is 75 - 85%. The ratio of the mass of the Celosia argentea L. seeds to be tested to the volume of the methanol aqueous solution is 1 g : 20 - 30 mL.

8. The identification method according to claim 6 or 7, characterized in that, The extraction includes heating under reflux extraction, and the extraction time is 55 - 65 min.

9. The identification method according to claim 6, wherein The column temperature for the high performance liquid chromatography detection is 20 - 30 °C, the detector is a diode array detector, the flow rate of the mobile phase is 0.9 - 1.1 mL / min, and the injection volume is 5 - 15 μL.

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