CN113655151B

CN113655151B - Construction of amino acid characteristic spectrum of animal traditional Chinese medicine standard decoction and detection of amino acid content of traditional Chinese medicine standard decoction and traditional Chinese medicine formula granule

Info

Publication number: CN113655151B
Application number: CN202111042755.4A
Authority: CN
Inventors: 童培珍; 何嘉莹; 魏梅; 李国卫; 邱韵静; 曾荟; 杨丽; 程学仁; 陈向东; 孙冬梅
Original assignee: Guangdong Yifang Pharmaceutical Co Ltd
Current assignee: Guangdong Yifang Pharmaceutical Co Ltd
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2023-10-03
Anticipated expiration: 2041-09-07
Also published as: CN113655151A

Abstract

The invention relates to construction of an amino acid characteristic map of an animal traditional Chinese medicine standard decoction, and detection of amino acid content of traditional Chinese medicine standard decoction and traditional Chinese medicine formula particles of animal traditional Chinese medicines. The method for constructing the amino acid characteristic spectrum of the invention selects standard decoction of earthworm or stiff silkworm as a test sample, selects an amino acid reference substance of proper kind, further cooperates with proper chromatographic conditions to construct the amino acid characteristic spectrum, uses the amino acid characteristic spectrum as a reference spectrum, and can distinguish different animal medicine standard decoction or/and formula particles by comparing the amino acid characteristic spectrum with the chromatogram of standard decoction or/and formula particles of earthworm or stiff silkworm, ground beetle, periostracum cicada, chicken's gizzard-skin and processed products thereof, turtle shell and processed products thereof. The method for measuring the amino acid content can be used for simultaneously measuring the amino acid content in earthworm or stiff silkworm, ground beetle, cicada slough, chicken's gizzard-skin and processed products thereof, turtle shell and processed products thereof.

Description

Construction of amino acid characteristic spectrum of animal traditional Chinese medicine standard decoction and detection of amino acid content of traditional Chinese medicine standard decoction and traditional Chinese medicine formula granule

Technical Field

The invention belongs to the field of quality standard research of animal traditional Chinese medicines, and particularly relates to construction of an amino acid characteristic map of an animal traditional Chinese medicine standard decoction, and detection of amino acid content of traditional Chinese medicine standard decoction and traditional Chinese medicine formula particles of animal traditional Chinese medicines.

Background

The animal traditional Chinese medicine has a long application history and has extremely important status in the application history of medicines in China. The existing monograph of animal traditional Chinese medicine (Shennong herbal Jing) contains 67 animal medicines such as musk, antelope horn and the like, accounting for 18.36% of the total medicine; 51 animal medicines are also carried in the 2020 edition of Chinese pharmacopoeia, wherein the prescription preparation containing the animal medicines has 461 medicines, and accounts for 30.9 percent of the total amount of the prescription preparation and the single preparation.

The existing identification and analysis means of animal medicines aim at medicinal materials, which are mainly distinguished by character identification and microscopic identification, and standard decoction and formula particles of the animal medicines are prepared by water extraction and concentration, and compared with medicinal materials and decoction pieces, the animal medicines have lost inherent forms, and lack important indexes on the basis of character and microscopic identification in quality control. Eupolyphaga Seu Steleophaga, periostracum Cicadae, endothelium corneum Gigeriae Galli, carapax Trionycis, lumbricus, and Bombyx Batryticatus are common clinical animal drugs, and at present, no effective method is established to distinguish standard decoction or/and formula granule of Eupolyphaga Seu Steleophaga, periostracum Cicadae, endothelium corneum Gigeriae Galli and its processed product, carapax Trionycis and its processed product, lumbricus, and Bombyx Batryticatus.

Disclosure of Invention

Based on the background technology, the main purpose of the invention is to provide a construction method of an amino acid characteristic spectrum of an animal traditional Chinese medicine standard decoction, wherein the amino acid characteristic spectrum constructed by adopting the construction method is used as a reference spectrum, and can distinguish one of earthworm and stiff silkworm from ground beetle, cicada slough, chicken's gizzard-skin and processed products thereof, turtle shell and standard decoction or/and formula particles of the processed products thereof.

The aim of the invention can be achieved by the following technical scheme:

a construction method of an amino acid characteristic map of an animal traditional Chinese medicine standard decoction comprises the following steps:

providing a reference substance solution containing amino acid reference substance, and preparing a test substance solution from standard earthworm decoction or standard stiff silkworm decoction; derivatizing the reference substance solution and the sample solution; detecting the reference substance solution and the sample solution subjected to derivatization by adopting a high performance liquid chromatography method, and introducing a reference substance chromatogram obtained by detection and the sample chromatogram into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain an amino acid characteristic spectrum;

the amino acid reference substance comprises at least one of tyrosine reference substance, methionine reference substance, lysine reference substance and arginine reference substance.

In one embodiment, the amino acid reference further comprises at least one of serine reference, glycine reference, threonine reference, alanine reference, proline reference, valine reference, isoleucine reference, leucine reference, and phenylalanine reference.

In one embodiment, the conditions of the high performance liquid chromatography include: the stationary phase is a C18 chromatographic column;

the mobile phase A is a mixed solution of acetonitrile and sodium acetate solution with the volume ratio of (6.5-7.5) (92.5-93.5), the concentration of sodium acetate contained in the sodium acetate solution is 0.08-0.12 mol/L, and the mobile phase B is a mixed solution of acetonitrile and water with the volume ratio of (3.5-4.5): 1, and gradient elution is adopted.

In one embodiment, the gradient elution procedure comprises: 0-6 min, wherein the volume percentage of the mobile phase A is reduced from 100% to 97%; 6-9 min, wherein the volume percentage of the mobile phase A is 97%; 9-11 min, wherein the volume percentage of the mobile phase A is reduced from 97% to 88%; 11-13 min, wherein the volume percentage of the mobile phase A is 88%; 13-18 min, wherein the volume percentage of the mobile phase A is reduced from 88% to 80%; 18-29 min, wherein the volume percentage of the mobile phase A is reduced from 80% to 72%; 29-33 min, wherein the volume percentage of the mobile phase A is reduced from 72% to 66%; 33-36 min, wherein the volume percentage of the mobile phase A is reduced from 66% to 0%; 36-39 min, wherein the volume percentage of the mobile phase A is 0%.

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the flow rate is 0.8 mL/min-1.2 mL/min.

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the column temperature is 38-42 ℃.

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the detection wavelength is 252 nm-256 nm.

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the sample injection amount is 4-6 mu L.

In one embodiment, the step of preparing the test solution comprises: carrying out protein hydrolysis on the earthworm standard decoction or the stiff silkworm standard decoction, collecting hydrolysate, removing solvent in the hydrolysate, and dissolving the obtained residue with solvent.

In one embodiment, the proteolytic hydrolysis is acid hydrolysis.

In one embodiment, the solvent is hydrochloric acid solution with the concentration of hydrochloric acid of 0.08mol/L to 0.12 mol/L.

In one embodiment, the solvent of the control solution comprises a hydrochloric acid solution having a hydrochloric acid concentration of 0.08mol/L to 0.12 mol/L.

In one embodiment, the derivatizing reagent employed in the derivatization process is phenyl isothiocyanate.

In one embodiment, the profile contains chromatographic peaks for tyrosine, methionine, lysine and arginine.

The invention provides application of the amino acid characteristic spectrum of the animal traditional Chinese medicine standard decoction obtained by the construction in the identification of animal traditional Chinese medicines and traditional Chinese medicine preparations thereof.

In one embodiment, the animal traditional Chinese medicine comprises stiff silkworm, ground beetle, chicken's gizzard-skin and its processed products, turtle shell and its processed products and cicada slough, or the animal traditional Chinese medicine comprises earthworm, ground beetle, chicken's gizzard-skin and its processed products, turtle shell and its processed products and cicada slough.

In one embodiment, the Chinese medicinal preparation comprises a standard Chinese medicinal decoction and Chinese medicinal formula particles.

A method for detecting amino acid content in traditional Chinese medicine standard decoction and traditional Chinese medicine formula particles of animal traditional Chinese medicine comprises the following steps:

providing a reference substance solution containing an amino acid reference substance, and taking a sample to be tested to prepare a sample solution; derivatizing the reference substance solution and the sample solution; detecting the control solution and the sample solution subjected to derivatization by adopting a high performance liquid chromatography method, and determining the content of amino acid in a sample to be detected according to the peak areas of chromatographic peaks in a control chromatogram and a sample chromatogram obtained by detection;

The animal traditional Chinese medicine is Eupolyphaga Seu Steleophaga, periostracum Cicadae, endothelium corneum Gigeriae Galli and its processed product, carapax Trionycis and its processed product, bombyx Batryticatus or Lumbricus.

In one embodiment, the amino acid reference comprises at least one of a tyrosine reference, a methionine reference, a lysine reference, an arginine reference, a serine reference, a glycine reference, a threonine reference, an alanine reference, a proline reference, a valine reference, an isoleucine reference, a leucine reference, and a phenylalanine reference.

In one embodiment, the conditions of the high performance liquid chromatography include: the stationary phase is a C18 chromatographic column; the mobile phase A is a mixed solution of acetonitrile and sodium acetate solution with the volume ratio of (6.5-7.5) (92.5-93.5), the concentration of sodium acetate contained in the sodium acetate solution is 0.08-0.12 mol/L, and the mobile phase B is a mixed solution of acetonitrile and water with the volume ratio of (3.5-4.5): 1, and gradient elution is adopted.

In one embodiment, the step of preparing a test solution comprises: and carrying out proteolysis on the sample to be tested, collecting hydrolysate, removing solvent in the hydrolysate, and dissolving the obtained residue with the solvent.

In one embodiment, the proteolytic hydrolysis is acid hydrolysis.

Compared with the prior art, the invention has the following beneficial effects:

The invention selects standard decoction of earthworm or stiff silkworm as a test sample, selects an amino acid reference substance of proper kind, further matches with proper chromatographic conditions to construct an amino acid characteristic map, uses the amino acid characteristic map as a reference map, and can distinguish different animal medicine standard decoction or/and formula particles by comparing the amino acid characteristic map with the chromatogram of standard decoction or/and formula particles of earthworm or stiff silkworm, ground beetle, cicada slough, chicken's gizzard-skin and processed products thereof, turtle shells and processed products thereof.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram of the specificity of a ground beetle standard decoction;

FIG. 2 is a diagram of the specification of a standard decoction of periostracum Cicadae;

FIG. 3 is a diagram of standard decoction of endothelium corneum Gigeriae Galli;

FIG. 4 is a diagram of standard decoction of fried chicken's gizzard-membrane;

FIG. 5 is a diagram of standard decoction of Lumbricus;

FIG. 6 is a diagram of the specific properties of the turtle shell standard decoction;

FIG. 7 is a diagram of the specificity of the vinegar-turtle shell standard decoction;

FIG. 8 shows the amino acid profile common peaks of 18 batches of standard decoction of endothelium corneum Gigeriae Galli;

FIG. 9 shows the amino acid characteristic spectrum sharing peaks of 17 batches of periostracum Cicadae standard decoction;

FIG. 10 shows the amino acid profile of 18 batches of Lumbricus (limnodrilus) standard decoction with common peaks;

FIG. 11 shows the amino acid profile common peaks of the 15 batches of turtle shell standard decoction;

FIG. 12 shows the amino acid profile common peaks of 21 batches of ground beetle (Eupolyphaga Seu Steleophaga) standard decoction;

FIG. 13 shows amino acid characteristic patterns of 3 batches of stiff silkworm standard decoction with common peaks;

FIG. 14 is a graph showing the assignment of the peak control shared by the 6 animal drug standard decoctions;

a: a reference; b: endothelium corneum Gigeriae Galli; c: cicada slough; d: earthworm (limnodrilus); e: turtle shell; f: ground beetle (ground beetle); h: stiff silkworm; peak 1: serine; peak 2: glycine; peak 3: threonine; peak 4: alanine; peak 5 (S): proline; peak 6: tyrosine; peak 7: valine; peak 8: methionine; peak 9: isoleucine; peak 10: leucine; peak 11: phenylalanine; peak 12: lysine; peak 13: arginine;

Fig. 15 is a hierarchical clustering analysis chart for measuring the content of the standard decoction of 6 animal drugs.

Detailed Description

The present invention will be described in more detail below in order to facilitate understanding of the present invention. It should be understood, however, that the invention may be embodied in many different forms and is not limited to the implementations or embodiments described herein. Rather, these embodiments or examples are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments or examples only and is not intended to be limiting of the invention. As used herein, the optional scope of the term "and/or" includes any one of the two or more related listed items, as well as any and all combinations of related listed items, including any two or more of the related listed items, or all combinations of related listed items.

In the present invention, "first aspect", "second aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of the indicated technical features.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, the numerical range is referred to, and both ends of the numerical range are included unless otherwise specified.

The percentage content referred to in the present invention refers to mass percentage for both solid-liquid mixing and solid-solid mixing and volume percentage for liquid-liquid mixing unless otherwise specified.

The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

In a first aspect, the invention provides a construction method of an amino acid characteristic map of a standard decoction of animal traditional Chinese medicines, which comprises the following steps:

In the invention, the amino acid reference substances comprise one, two, three and four of tyrosine reference substances, methionine reference substances, lysine reference substances and arginine reference substances.

In one embodiment, the amino acid reference further comprises at least one of serine reference, glycine reference, threonine reference, alanine reference, proline reference, valine reference, isoleucine reference, leucine reference, and phenylalanine reference (including one, two, three, four, five, six, seven, eight, and nine).

In one embodiment, the conditions of the high performance liquid chromatography include:

the stationary phase is a C18 chromatographic column;

the mobile phase A is a mixed solution of acetonitrile and sodium acetate solution with the volume ratio of (6.5-7.5) (92.5-93.5), the concentration of sodium acetate contained in the sodium acetate solution is 0.08-0.12 mol/L, and the mobile phase B is a mixed solution of acetonitrile and water with the volume ratio of (3.5-4.5): 1, and gradient elution is adopted. The construction method comprises the following steps: c18 columns include, but are not limited to, thermo Acclaim C18 columns (4.6 mm. Times.250 mm,5 μm), phenomenex Luna columns (4.6 mm. Times.250 mm,5 μm), kromasil 100-5C18 columns (4.6 mm. Times.250 mm,5 μm), and the like; mobile phase A can be acetonitrile-0.08 mol/L sodium acetate solution (6.5:93.5), acetonitrile-0.1 mol/L sodium acetate solution (7:93), acetonitrile-0.12 mol/L sodium acetate solution (7.5:92.5); mobile phase B was acetonitrile-water (3.5:1), acetonitrile-water (4:1), acetonitrile-water (4.5:1).

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the flow rate is 0.8mL/min to 1.2mL/min (for example, 0.8mL/min, 1mL/min, 1.2mL/min may be used).

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the column temperature is 38℃to 42℃and may be, for example, 38℃to 40℃to 42 ℃.

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the detection wavelength is 252nm to 256nm (for example, 250nm, 254nm, 256nm may be used).

In one embodiment, the conditions of the high performance liquid chromatography further comprise: the sample amount is 4. Mu.L to 6. Mu.L (for example, 4. Mu.L, 5. Mu.L, 6. Mu.L may be used).

In one embodiment, the proteolytic hydrolysis is acid hydrolysis.

In one embodiment, the solvent is hydrochloric acid solution with the concentration of hydrochloric acid of 0.08mol/L to 0.12mol/L (for example, 0.08mol/L, 0.1mol/L and 0.12 mol/L).

In one embodiment, the solvent of the control solution comprises a hydrochloric acid solution (e.g., 0.08mol/L, 0.1mol/L, 0.12 mol/L) having a hydrochloric acid concentration of 0.08mol/L to 0.12 mol/L.

In a second aspect, the invention provides application of the animal traditional Chinese medicine standard decoction amino acid characteristic spectrum constructed as above in identification of animal traditional Chinese medicines and traditional Chinese medicine preparations thereof.

In one embodiment, the animal traditional Chinese medicine comprises one of stiff silkworm and earthworm, and at least one of ground beetle, chicken's gizzard-skin and its processed products, turtle shell and its processed products, and cicada slough. For example, can be used for distinguishing stiff silkworm, ground beetle, chicken's gizzard-skin and its processed products, carapax Trionycis and its processed products, and standard decoction of periostracum Cicadae; can also be used for distinguishing standard decoction of Lumbricus, eupolyphaga Seu Steleophaga, endothelium corneum Gigeriae Galli and its processed product, carapax Trionycis and its processed product, and periostracum Cicadae; can also be used for the identification between the formulated granules of the animal medicines.

The main components of the animal traditional Chinese medicine are macromolecular components such as protein, amino acid, nucleotide, grease and the like, and the animal traditional Chinese medicine lacks common secondary metabolism micromolecular components in the plant medicine, so that certain difficulty is brought to the quantitative index selection characteristic of the animal traditional Chinese medicine.

Ground beetle, cicada slough, chicken's gizzard-membrane and its processed products (fried chicken's gizzard-membrane), earthworm, turtle shell and its processed products (vinegar turtle shell), stiff silkworm are animal traditional Chinese medicines commonly used in modern Chinese medicine clinic, however, in the current edition of Chinese pharmacopoeia, no content measurement item exists in the quality standards of the animal traditional Chinese medicines. The quantification of animal drugs by modern scholars has mostly focused on the determination of hydrolyzed amino acids, traditional assays such as: liang the content of 16 amino acids in endothelium corneum Gigeriae Galli is determined by pre-column derivatization RP-HPLC; he Xinrong and the like adopt OPA as a derivative reagent, and simultaneously determine the content of 17 amino acids in female and male ground beetles; liao Pengying and the like adopt phthalic aldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) as pre-column derivatization reagents, and simultaneously determine the content of 18 amino acids in carapax Trionycis products and products; four amino acids of L-hydroxyproline, glycine, alanine and L-proline are determined under the content determination item of tortoise shell glue in the edition 2020 of Chinese pharmacopoeia.

In the case of the above-mentioned animal traditional Chinese medicines such as ground beetles, cicada sloughs and the like, although the content measurement technology of amino acids is mature, the content measurement technology is mainly concentrated on medicinal materials, the application of the content measurement technology in standard decoction or formula particles of the medicinal materials is blank, and the quality control and quality assurance of the standard decoction and formula particles lack effective means.

Based on the above, as a third aspect of the present invention, the present invention provides a method for detecting amino acid content in a traditional Chinese medicine standard decoction and a traditional Chinese medicine formula granule of an animal traditional Chinese medicine, the method comprising the following steps:

In one embodiment, the amino acid reference comprises at least one of tyrosine reference, methionine reference, lysine reference, arginine reference, serine reference, glycine reference, threonine reference, alanine reference, proline reference, valine reference, isoleucine reference, leucine reference and phenylalanine reference (including one, two, three, … … thirteen cases).

In one embodiment, the conditions of the high performance liquid chromatography include: the stationary phase is a C18 chromatographic column; the mobile phase A is a mixed solution of acetonitrile and sodium acetate solution with the volume ratio of (6.5-7.5) (92.5-93.5), the concentration of sodium acetate contained in the sodium acetate solution is 0.08-0.12 mol/L, and the mobile phase B is a mixed solution of acetonitrile and water with the volume ratio of (3.5-4.5): 1, and gradient elution is adopted. The detection method comprises the following steps: c18 columns include, but are not limited to, thermo Acclaim C18 columns (4.6 mm. Times.250 mm,5 μm), phenomenex Luna columns (4.6 mm. Times.250 mm,5 μm), kromasil 100-5C18 columns (4.6 mm. Times.250 mm,5 μm), and the like; mobile phase A can be acetonitrile-0.08 mol/L sodium acetate solution (6.5:93.5), acetonitrile-0.1 mol/L sodium acetate solution (7:93), acetonitrile-0.12 mol/L sodium acetate solution (7.5:92.5); mobile phase B was acetonitrile-water (3.5:1), acetonitrile-water (4:1), acetonitrile-water (4.5:1).

The amino acid types and contents of different animal traditional Chinese medicines have certain difference, and the influence of other components of the animal traditional Chinese medicines is added, so that the method for measuring the amino acid content of one animal traditional Chinese medicine can not necessarily meet the requirement of the other animal traditional Chinese medicine, therefore, no report that one amino acid detection method can be simultaneously applied to the measurement of the amino acid of a plurality of animal traditional Chinese medicines is provided, and no report that the method is simultaneously applied to the measurement of the amino acid in a plurality of animal traditional Chinese medicine standard decoction/formula particles is provided. According to the detection method provided by the invention, through optimization of chromatographic conditions, the method can be simultaneously applied to measurement of the content of different types of amino acids in standard decoction and formula particles of ground beetles, periostracum cicadae, endothelium corneum gigeriae galli, fried endothelium corneum gigeriae galli, earthworms, turtle shells, vinegar turtle shells and stiff silkworms, so that a content measurement control means is provided for quality control of the standard decoction and formula particles of the 7 animal traditional Chinese medicines, the cost of quality standard research process and later sample inspection is greatly saved, and a reference is provided for the content measurement method of the amino acids in the standard decoction/formula particles of other animal traditional Chinese medicines.

In one embodiment, the proteolytic hydrolysis is acid hydrolysis. The acid type and the acidolysis conditions used in the acid hydrolysis are not particularly limited in the present invention, and for example, a 6mol/L hydrochloric acid solution may be used for hydrolysis at 150℃for 3 hours. Preferably, the acid hydrolysis is performed in a hydrolysis tube, and compared with the traditional hydrolysis performed in an ampoule bottle (for example, the measurement of tortoise-shell glue, donkey-hide gelatin and deer-horn glue amino acid carried by one part of Chinese pharmacopoeia of 2020 edition is performed at 150 ℃ by using the ampoule bottle, the ampoule bottle is easy to burst, and huge experimental safety accidents exist), the acid hydrolysis method is safer.

The steps involved in the identification method of the present invention are not limited in order.

In the present invention, the modes of obtaining the standard decoction of ground beetle, cicada slough, chicken's gizzard-skin and its processed products, earthworm, turtle shell and its processed products, stiff silkworm or earthworm include but are not limited to the following exemplary modes:

taking 100g of ground beetle/chicken's gizzard-membrane/earthworm decoction pieces, placing the decoction pieces into an electric ceramic pot, adding water for decoction twice, adding 8 times of water for the first time, soaking for 30 minutes, boiling with strong fire (power 500W), keeping micro-boiling with slow fire (power 200W) for 30 minutes, filtering the decoction with a 350-mesh screen while the decoction is hot, and rapidly cooling the filtrate with cold water. Adding 6 times of water into the second decoction, boiling with strong fire (power 500W), keeping micro-boiling with slow fire (power 200W) for 25 min, filtering the decoction with 350 mesh sieve, rapidly cooling the filtrate with cold water, and mixing the two decoctions. Transferring the decoction into a round bottom flask, concentrating at low temperature under reduced pressure by adopting a rotary evaporator (temperature: 65 ℃ C.; vacuum degree: minus 0.08MPa to minus 0.1 MPa), rotating at 60 revolutions per minute, and concentrating to a volume of about 100mL; precisely sucking 2mL of fluid extract under magnetic stirring, packaging in 10mL penicillin bottle, transferring to vacuum freeze dryer, lyophilizing, taking out, and rolling aluminum cap.

Taking 100g of cicada slough decoction pieces, placing into an electric ceramic pot, adding water for decoction twice, adding 14 times of water for the first time, soaking for 30 minutes, boiling with strong fire (power 500W), keeping micro-boiling with slow fire (power 200W) for 30 minutes, filtering the decoction with a 350-mesh screen, and rapidly cooling the filtrate with cold water. Adding 12 times of water into the second decoction, boiling with strong fire (power 500W), keeping micro-boiling with slow fire (power 200W) for 25 min, filtering the decoction with 350 mesh sieve, rapidly cooling the filtrate with cold water, and mixing the two decoctions. Transferring the decoction into a round bottom flask, concentrating at low temperature under reduced pressure by adopting a rotary evaporator (temperature: 65 ℃ C.; vacuum degree: minus 0.08MPa to minus 0.1 MPa), rotating at 60 revolutions per minute, and concentrating to a volume of about 100mL; precisely sucking 2mL of fluid extract under magnetic stirring, packaging in 10mL penicillin bottle, transferring to vacuum freeze dryer, lyophilizing, taking out, and rolling aluminum cap.

Taking 100g of turtle shell decoction pieces, placing into an electric ceramic pot, adding water for decoction twice, adding 8 times of water for the first time, soaking for 30 minutes, boiling with strong fire (500W), keeping micro-boiling with slow fire (200W) for 60 minutes, filtering the decoction with a 350-mesh screen, and rapidly cooling the filtrate with cold water. Adding 6 times of water for the second time, boiling with strong fire (500W), keeping micro-boiling with slow fire (200W) for 40 min, filtering the decoction with 350 mesh sieve, cooling the filtrate with cold water, and mixing the decoctions. Transferring the decoction into a 5000mL round bottom flask, and concentrating under reduced pressure and low temperature (temperature: 65 ℃ C.; vacuum degree: 0.10 MPa) to 100mL of fluid extract by adopting a rotary evaporator; under the magnetic stirring, subpackaging into 10mL brown penicillin bottles, wherein the subpackaging volume of each bottle is 2mL, half-plugging, transferring into a vacuum freeze dryer for freeze drying after subpackaging, taking out, and rolling an aluminum cover.

In the invention, the prescription granule of ground beetle, cicada slough, chicken's gizzard-skin and its processed products, earthworm, turtle shell and its processed products, stiff silkworm or earthworm can be obtained by the conventional method.

The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials are commercially available unless otherwise indicated.

Example 1: methodological verification

1. Instrument, reagent and reagent

1.1 instrument: thermo high performance liquid chromatography (U3000, siemens technologies Co., ltd.), thermo Acclaim C18 column (4.6 mm. Times.250 mm,5 μm), phenomenex Luna column (4.6 mm. Times.250 mm,5 μm), kromasil 100-5C18 column (4.6 mm. Times.250 mm,5 μm), waters high performance liquid chromatography (Waters ARC, waters Co., ltd.), one-ten-thousandth balance (ME 204E, metrer-Toli Co., ltd.), one-per-million flat (XP 26, metrer-Toli Co., ltd.), ultra pure water system (Milli-QDirect, merck Co., ltd.).

1.2 reagent: hydrochloric acid (Guangzhou chemical reagent factory), anhydrous sodium acetate (Shanglong science Co., ltd.), phenyl isothiocyanate (Michael reagent) are all analytically pure; acetic acid (Tianjin, mimo chemical reagent Co., ltd.), acetonitrile (Merck, inc.), triethylamine (Tianjin, mimo chemical reagent Co., ltd.) were all HPLC chromatographic pure, and water was ultrapure water (laboratory self-made).

1.3 reagents: glycine (China food and drug inspection institute, lot number: 140689-201605, content: 100%); alanine (China food and drug inspection institute, lot number: 140680-201604, content: 100%); proline (China food and drug inspection institute, lot number: 140677-201808, content: 99.9%); valine ((China food and drug inspection institute, lot number: 140681-201603, content: 99.5%); phenylalanine (China food and drug inspection institute, lot number: 140676-201706, content: 100%).

2. Detection method

2.1 chromatographic conditions

A Thermo Acclaim C18 (4.6mm.times.250 mm,5 μm) column was selected;

acetonitrile-0.1 mol/L sodium acetate solution (pH value is adjusted to 6.5 by acetic acid) (7:93) is taken as a mobile phase A, acetonitrile-water (4:1) is taken as a mobile phase B, and gradient elution is carried out according to the stipulations in the following table; the flow rate is 1.0mL per minute; column temperature is 40 ℃; the detection wavelength is 254nm; the sample loading was 5. Mu.L.

TABLE 1 gradient elution Table

2.2 preparation of control solution

Precisely weighing appropriate amounts of glycine reference substance, alanine reference substance, proline reference substance, phenylalanine reference substance and valine reference substance, and respectively adding 0.1mol/L hydrochloric acid solution to obtain reference substance solutions with appropriate concentration.

2.3 preparation of sample solutions

Taking a proper amount of standard decoction, grinding, taking a proper amount of standard decoction, precisely weighing, placing the standard decoction into an amino acid hydrolysis tube, precisely adding 10mL of 6mol/L hydrochloric acid solution, respectively placing the amino acid hydrolysis tube and the amino acid hydrolysis tube for hydrolysis for 3 hours at 150 ℃, taking out, cooling, filtering, transferring filtrate into an evaporation dish, washing the hydrolysis tube and filter residues with 10mL of water for several times, filtering, merging the filtrate into the evaporation dish, evaporating to dryness, dissolving the residue with 0.1mol/L hydrochloric acid solution, transferring the residue into a 25mL measuring flask, fixing the volume to a scale, and shaking uniformly to obtain the product.

Precisely measuring 5mL of each of the sample solution and the reference solution, respectively placing in 25mL measuring flask, adding 2.5mL of acetonitrile solution of 0.1mol/L Phenyl Isothiocyanate (PITC) and 2.5mL of acetonitrile solution of 1mol/L triethylamine, shaking, standing at room temperature for 1 hour, adding 50% acetonitrile to scale, and shaking. Taking 10mL, adding 10mL of n-hexane, shaking, standing for 10 minutes, taking the lower layer solution, filtering, and taking the subsequent filtrate to obtain the product.

3. Investigation of specificity

Respectively taking standard decoction and blank solvent (namely 0.1mol/L hydrochloric acid solution) of ground beetle, periostracum Cicadae, endothelium corneum Gigeriae Galli, parched endothelium corneum Gigeriae Galli, lumbricus, carapax Trionycis, and vinegar carapax Trionycis, and preparing into sample solution and blank solvent solution according to the sample solution preparation method under item "2.3" of this example; preparing a reference solution according to the reference solution preparation method under item 2.2 of the present example; 5 mu L of each of the sample solution, the control solution and the blank solvent solution was precisely sucked and injected into a liquid chromatograph, and the chromatographic conditions were developed and analyzed in accordance with the "2.1" item of this example.

The results show that the standard decoction sample chromatograms of ground beetle, cicada slough, chicken's gizzard-membrane, fried chicken's gizzard-membrane, earthworm, turtle shell and vinegar turtle shell have the same chromatographic peak at the retention time corresponding to the corresponding reference sample chromatograms, and the blank solvent has no interference, which indicates that the method has good specificity (see figures 1-7).

4. Linear investigation

Precisely weighing appropriate amounts of glycine reference substance, alanine reference substance, proline reference substance, phenylalanine reference substance and valine reference substance, and preparing into linear reference substance stock solution with appropriate concentration with 0.1mol/L hydrochloric acid solution respectively.

Then precisely measuring 5mL of each reference stock solution, respectively placing the reference stock solutions into 25mL measuring flasks, respectively adding 2.5mL of acetonitrile solution of 0.1mol/L Phenyl Isothiocyanate (PITC) and 2.5mL of acetonitrile solution of 1mol/L triethylamine, shaking uniformly, standing at room temperature for 1 hour, adding 50% acetonitrile to the scale, and shaking uniformly. Transferring to a separating funnel, adding 25mL of n-hexane, shaking, standing for 10 min, and taking the lower layer solution to obtain the derived reference stock solution.

Precisely measuring a proper amount of the derived reference substance stock solution, and preparing reference substance linear solutions with different concentrations by using a 50% acetonitrile solution.

The linear solutions of the above control were precisely aspirated, 5. Mu.L of the sample was sequentially introduced according to the chromatographic conditions set under item "2.1" of this example, and the chromatographic peak areas were recorded. The peak area is taken as an ordinate (y), the concentration of the reference substance is taken as an abscissa (x), and a standard curve is drawn, and the result is as follows:

TABLE 2 Linear investigation of Glycine results

TABLE 3 Ala Linear investigation results

TABLE 4 proline linearity investigation results

TABLE 5 phenylalanine linearity test results

TABLE 6 results of linear investigation of valine

The results showed that the linear regression equation for glycine was: y= 0.8827x-0.2427, correlation coefficient r=1.0000, indicates that the linear relationship between glycine concentration and peak area is good in the range of concentration from 3.135 μg/mL to 313.50 μg/mL; the linear regression equation for alanine is: y= 0.6830x-0.1979, correlation coefficient r=1.0000, indicating that the linear relation between alanine concentration and peak area is good in the range of concentration of 2.099 μg/mL to 209.94 μg/mL; the linear regression equation for proline is: y=0.6181x+0.2566, and a correlation coefficient r=1.0000, which shows that the linear relation between the proline concentration and the peak area is good in the range of the concentration of 2.758 mu g/mL-275.80 mu g/mL; the linear regression equation for phenylalanine is: y= 0.4512x-0.0794, correlation coefficient r=1.0000, indicates that phenylalanine concentration has good linear relation with peak area in the range of concentration of 2.786 μg/mL to 278.64 μg/mL; the linear regression equation for valine is: y= 6957.2x-35380 and correlation coefficient r=0.9994, which shows that the valine concentration and peak area have good linear relationship in the range of 3.692 μg/mL to 738.409 μg/mL.

5. Stability investigation

Taking a proper amount of standard decoction of each animal medicine, preparing a sample solution according to the sample solution preparation method under the item "2.3" of the embodiment, preparing chromatographic conditions under the item "2.1" of the embodiment, injecting samples at different time points within 0 to 24 hours respectively, wherein the injection volume is 5 mu L, recording the peak area of each amino acid, and calculating the RSD value of the peak area, wherein the result is as follows:

TABLE 7 results table of results of determination of Standard decoction content stability of Eupolyphaga Seu Steleophaga

Table 8, stability test results table for content measurement of periostracum Cicadae standard decoction

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Table 9 and table of results of examination of stability of standard decoction of endothelium corneum Gigeriae Galli

Table 10 and table of results of examination of stability of content measurement of standard decoction of fried chicken's gizzard-membrane

Table 11, table of results of examination of stability of standard decoction of Lumbricus

Table 12, results table of stability study of turtle shell standard decoction content measurement

Table 13, table of results of examination of stability of content measurement of standard decoction of vinegar turtle shell

The results showed that the peak area RSD of the amino acids in each test solution was less than 3.0%, indicating good stability of the test solutions over 18 hours.

6. Repeatability investigation

Taking a proper amount of standard decoction of the same batch of animal medicines, precisely weighing, and parallelly weighing 6 parts, and preparing 6 parts of test solution according to the test solution preparation method under item 2.3 of the embodiment. The content of each amino acid and the RSD value were calculated by measuring the chromatographic conditions under the condition of "2.1" in this example, and the results were as follows:

Table 14 table ground beetle (ground beetle) standard decoction content determination repeatability test result table

Table 15, cicada slough standard decoction content determination repeatability investigation result table

Table 16, table of repeated investigation results of chicken's gizzard-membrane standard decoction content measurement

Table 17, repeated investigation result table for content measurement of standard decoction of fried chicken's gizzard-membrane

Table 18 table of the results of repeated investigation of standard decoction content measurement of Lumbricus

TABLE 19 repeated investigation result table for turtle shell standard decoction content measurement

Table 20 table of repeated investigation results of content measurement of standard decoction of vinegar turtle shell

The results show that the same batch of samples are repeatedly measured for 6 times, and the content RSD of each component is less than 3.0%, which shows that the repeatability of the analysis method is good.

7. Intermediate precision investigation

Other analysts operate on different dates and different chromatographs, respectively taking the same batch of ground beetle (ground beetle) standard decoction, cicada slough standard decoction, chicken's gizzard-membrane standard decoction, fried chicken's gizzard-membrane standard decoction, earthworm standard decoction, turtle shell standard decoction and vinegar turtle shell standard decoction, precisely weighing 6 parts in parallel, and preparing 6 parts of test sample solutions according to the test sample solution preparation method under the item '2.3' of the embodiment. The content of each amino acid and the RSD value were calculated by measuring the chromatographic conditions under the condition of "2.1" in this example, and the results were as follows:

Table 21, intermediate precision investigation result table for ground beetle standard decoction content measurement

Table 22, cicada slough standard decoction content measurement intermediate precision investigation result table

Table 23 and table of intermediate precision investigation results of chicken's gizzard-membrane standard decoction content measurement

Table 24 and intermediate precision investigation result table for content measurement of fried chicken's gizzard-membrane standard decoction

Table 25, intermediate precision investigation result table for standard decoction content measurement of earthworm

Table 26, turtle shell standard decoction content measurement intermediate precision investigation result table

Table 27 Table A results table of intermediate precision investigation of content determination of Vinegar turtle Shell Standard decoction

The results show that the same batch of samples was run by different personnel on different instruments at different times and the measurement was repeated 6 times with an intermediate precision RSD limit of <5% for each amino acid content. Thus, different analysts operate on different dates and with different chromatographs, and the method has good intermediate precision.

8. Accuracy investigation

Taking a proper amount of standard decoction of each animal medicine (half of the preparation amount of the sample solution under the item of 2.3 in the embodiment), precisely weighing, placing into an amino acid hydrolysis tube, paralleling 3 groups, respectively adding a proper amount of amino acid reference substances (50%, 100 and 150% of the sample weighing amount) into each group, respectively adding 6mol/L hydrochloric acid solution, respectively placing for hydrolysis for 3 hours at 150 ℃, taking out, cooling, filtering, transferring the filtrate into an evaporation dish, washing the hydrolysis tube and filter residues with 10mL of water for a plurality of times, filtering, merging the filtrate into the evaporation dish, evaporating to dryness, dissolving the residues with 0.1mol/L hydrochloric acid solution, transferring into a 25mL measuring flask, fixing the volume to a scale, and shaking uniformly to obtain the medicine.

Precisely measuring 5mL of the sample solution, placing in a 25mL measuring flask, adding 2.5mL of acetonitrile solution of 0.lmol/L Phenyl Isothiocyanate (PITC) and 2.5mL of acetonitrile solution of lmol/L triethylamine, shaking, standing at room temperature for 1 hour, adding 50% acetonitrile to the scale, and shaking. Taking 10mL, adding 10mL of n-hexane, shaking, standing for 10 minutes, taking the lower layer solution, filtering with a 0.22 mu m filter membrane, and taking the subsequent filtrate to obtain the product. And (3) measuring according to the chromatographic conditions under the item of 2.1, respectively injecting samples, measuring the content of each amino acid in the sample solution, and calculating the sample injection recovery rate.

Table 28 ground beetle (ground beetle) standard decoction alanine sample recovery rate investigation result table (n=9)

Table 29 ground beetle (ground beetle) standard decoction proline sample recovery ratio investigation result table (n=9)

Table 30 table ground beetle (ground beetle) standard decoction phenylalanine sample recovery rate investigation result table (n=9)

Table 31, cicada slough standard decoction glycine sample recovery rate investigation result table (n=9)

Table 32, cicada slough standard decoction alanine sample recovery rate investigation result table (n=9)

Table 33, cicada slough standard decoction proline sample recovery rate investigation result table (n=9)

Table 34, cicada slough standard decoction phenylalanine sample recovery rate investigation result table (n=9)

Table 35, endothelium corneum Gigeriae Galli standard decoction glycine sample recovery rate investigation result table (n=9)

Table 36, chicken's gizzard-membrane standard decoction alanine sample recovery rate investigation result table (n=9)

Table 37, membrane standard decoction proline sample recovery rate investigation result table (n=9)

Table 38, endothelium corneum Gigeriae Galli standard decoction phenylalanine sample recovery rate investigation result table (n=9)

Table 39, sample recovery rate of glycine in standard decoction of fried chicken's gizzard membrane survey result table (n=9)

Table 40, sample recovery rate of alanine of standard decoction of fried chicken's gizzard-membrane (n=9)

Table 41, sample recovery rate of standard decoction of fried chicken's gizzard-membrane proline investigation result table (n=9)

Table 43, carapax Trionycis Standard decoction glycine sample recovery ratio investigation result table (n=9)

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Table 44, turtle shell standard decoction proline sample recovery ratio investigation result table (n=9)

Table 45, turtle shell standard decoction valine sample recovery ratio investigation result table (n=9)

Table 46 table of results of investigation of glycine sample recovery rate of vinegar turtle shell standard decoction (n=9)

Table 47 table of results of investigation of proline sample recovery rate of vinegar-turtle shell standard decoction (n=9)

Table 48, sample recovery ratio investigation result table of vinegar-turtle shell standard decoction valine (n=9)

The results show that the recovery rate of all the sample amino acids is in the range of 92% -105%, which indicates that the recovery rate is good.

Example 2: establishment of amino acid characteristic spectrum and common peak attribution

1. Instrument, reagent and reagent

1.3 reagents: glycine (China food and drug inspection institute, lot number: 140689-201605, content: 100%); alanine (China food and drug inspection institute, lot number: 140680-201604, content: 100%); proline (China food and drug inspection institute, lot number: 140677-201808, content: 99.9%); valine ((China food and drug assay institute, batch No. 140681-201703, content: 99.5%), phenylalanine (China food and drug assay institute, batch No. 140676-201706, content: 100%), threonine (China food and drug assay institute, batch No. 140682-201302, content 99.9%), tyrosine (China food and drug assay institute, batch No. 140609-201914, content 99.9%), isoleucine (China food and drug assay institute, batch No. 140683-201302, content 99.9%), phenylalanine (China food and drug assay institute, batch No. 140676-201706, content: 100%), L-lysine (Chem Faces Inc, batch No. CFS201902, content: 98%), methionine (Sichuan Vickers Biotechnology Co., ltd., batch No. wkq20061207, content: 98%)

2. Method for establishing

2.1 chromatographic conditions

A Thermo Acclaim C18 (4.6mm.times.250 mm,5 μm) column was selected; acetonitrile-0.1 mol/L sodium acetate solution (pH value is adjusted to 6.5 by acetic acid) (7:93) is taken as a mobile phase A, acetonitrile-water (4:1) is taken as a mobile phase B, and gradient elution is carried out according to the stipulations in the following table; the flow rate is 1.0mL per minute; column temperature is 40 ℃; the detection wavelength is 254nm; the sample loading was 5. Mu.L.

TABLE 49 gradient elution Meter

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2.2 preparation of control solution

Precisely weighing serine reference, glycine reference, threonine reference, alanine reference, proline reference, tyrosine reference, valine reference, methionine reference, isoleucine reference, leucine reference, phenylalanine reference, lysine reference and arginine reference, and adding 0.1mol/L hydrochloric acid solution to obtain reference solutions with proper concentration.

2.3 preparation of sample solutions

2.4 assignment of reference peaks and consensus peaks

Taking 18 batches of chicken's gizzard-membrane standard decoction, 17 batches of cicada slough standard decoction, 18 batches of earthworm (limnodrilus ginseng) standard decoction, 15 batches of turtle shell standard decoction, 21 batches of ground beetle (ground beetle) standard decoction and 3 batches of stiff silkworm standard decoction samples, preparing a test sample solution under the item "2.3" according to the embodiment, measuring according to the chromatographic condition under the item "2.1", and introducing the obtained chromatographic data into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (national formulary committee, 2012.0 edition) for result analysis: the chicken' S gizzard-membrane determines 12 common peaks, the cicada slough determines 12 common peaks, the earthworm (limnodrilus ginseng) determines 13 common peaks, the turtle shell determines 9 common peaks, the ground beetle (ground beetle) determines 12 common peaks (see fig. 8-12), wherein the peak 5 (proline) has better separation degree and higher response, so the peak 5 (proline) is used as a reference peak (namely S peak); the comparison of the retention time of chromatographic peaks of the control substances and the combination of DAD spectrum analysis show that 13 characteristic peaks are identified, each characteristic peak is identified as a graph (see fig. 13 and 14), and the detailed information of the chromatographic peaks in the standard decoction of 6 animal medicaments is shown in the following table.

Comparison of amino acid chromatogram peak information of standard decoction of 50 and 6 animal drugs

2.5 similarity evaluation

The method comprises the steps of adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.0 version) recommended by the national formulary committee to respectively perform data processing on 89 batches of standard decoction sample amino acid characteristic patterns of chicken's gizzard-skin standard decoction, cicada slough standard decoction, ground beetle (ground beetle) standard decoction, turtle shell standard decoction and earthworm (limnodrilus) standard decoction, adopting an average number and a time window of 0.1, automatically matching, respectively taking the 5 animal medicine standard decoction sharing modes as comparison characteristic patterns, calculating similarity coefficients, wherein the similarity of the animal medicine standard decoction of the same variety is higher than 0.95, and the similarity results show that chemical components among different batches of the animal medicine standard decoction of the same variety have better consistency.

Table 51, similarity evaluation results of 7 animal decoction pieces Standard decoction samples of different varieties

3. Sample amino acid content detection application

Table 52, amino acid content comparison of decoction of various kinds

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Hierarchical clustering analysis: carrying out cluster analysis on the 92 batches of standard decoction samples by using SPSS 20.0 software, adopting average number connection among groups, taking square Euclidean distance as a distance formula of sample similarity, and collecting six types of samples when the Euclidean distance is 4, wherein DL-YC-01-DL-YC-18 are collected as I types, and the standard decoction is earthworm (limnodrilus holmiq.) standard decoction; JNJ-T-01-JNJ-T-18 is classified as class II, and is a standard decoction of chicken's gizzard-membrane; CT-T-01-CT-T-17 are gathered into class III, which is a cicada slough standard decoction; BJ-T-01-BJ-T-15 are collected into IV class, and are turtle shell standard decoction; JC-T-15 to JC-T-17 are concentrated into class V, and are standard decoction of stiff silkworm; TBC-T-01-TBC-T-21 are classified into VI types and are ground beetle standard decoction, as shown in figure 15.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. The construction method of the animal traditional Chinese medicine standard decoction amino acid characteristic map is characterized by comprising the following steps:

providing a reference substance solution containing an amino acid reference substance, and preparing a sample solution by taking a standard decoction; derivatizing the reference substance solution and the sample solution; detecting the reference substance solution and the sample solution subjected to derivatization by adopting a high performance liquid chromatography method, and introducing a reference substance chromatogram obtained by detection and the sample chromatogram into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain an amino acid characteristic spectrum;

the amino acid reference substance comprises at least one of tyrosine reference substance, methionine reference substance, lysine reference substance and arginine reference substance;

the step of preparing the test solution includes: carrying out protein hydrolysis on the standard decoction, collecting hydrolysate, removing solvent in the hydrolysate, and dissolving the obtained residue with solvent;

the derivatization reagent adopted in the derivatization treatment is phenyl isothiocyanate;

the conditions of the high performance liquid chromatography include: the mobile phase A is a mixed solution of acetonitrile and sodium acetate aqueous solution with the volume ratio of (6.5-7.5) (92.5-93.5), the concentration of sodium acetate contained in the sodium acetate aqueous solution is 0.08 mol/L-0.12 mol/L, and the mobile phase B is a mixed solution of acetonitrile and water with the volume ratio of (3.5-4.5): 1, and gradient elution is adopted;

The gradient elution procedure includes:

0-6 min, wherein the volume percentage of the mobile phase A is reduced from 100% to 97%;

6-9 min, wherein the volume percentage of the mobile phase A is 97%;

9-11 min, wherein the volume percentage of the mobile phase A is reduced from 97% to 88%;

11 min-13 min, wherein the volume percentage of the mobile phase A is 88%;

13 min-18 min, wherein the volume percentage of the mobile phase A is reduced from 88% to 80%;

18 min-29 min, wherein the volume percentage of the mobile phase A is reduced from 80% to 72%;

29 min-33 min, wherein the volume percentage of the mobile phase A is reduced from 72% to 66%;

33 min-36 min, wherein the volume percentage of the mobile phase A is reduced from 66% to 0%;

36 min-39 min, wherein the volume percentage of the mobile phase A is 0%;

the conditions of the high performance liquid chromatography further include: the stationary phase is a C18 chromatographic column;

the animal traditional Chinese medicine comprises carapax Trionycis, periostracum Cicadae and Bombyx Batryticatus.

2. The method for constructing an amino acid profile of an animal traditional Chinese medicine standard decoction according to claim 1, wherein the amino acid reference substance further comprises at least one of serine reference substance, glycine reference substance, threonine reference substance, alanine reference substance, proline reference substance, valine reference substance, isoleucine reference substance, leucine reference substance and phenylalanine reference substance.

3. The method for constructing an amino acid profile of an animal traditional Chinese medicine standard decoction according to claim 1 or 2, wherein the conditions of the high performance liquid chromatography further comprise: the flow rate is 0.8 mL/min-1.2 mL/min; or/and, the column temperature is 38-42 ℃; or/and the detection wavelength is 252 nm-256 nm; or/and the sample injection amount is 4-6 mu L.

4. The method for constructing an amino acid characteristic map of an animal traditional Chinese medicine standard decoction according to claim 1, wherein the proteolysis is carried out by an acid method; or/and the solvent adopts hydrochloric acid solution with the concentration of hydrochloric acid of 0.08mol/L to 0.12 mol/L.

5. The method for constructing amino acid profile of animal traditional Chinese medicine standard decoction according to claim 4, wherein the acid hydrolysis is performed in a hydrolysis tube.

6. The method for constructing amino acid profile of animal traditional Chinese medicine standard decoction according to claim 1, 2 or 4, wherein the solvent of the reference solution comprises hydrochloric acid solution with hydrochloric acid concentration of 0.08 mol/L-0.12 mol/L.

7. The method for constructing amino acid profile of animal traditional Chinese medicine standard decoction according to claim 1, 2 or 4, wherein the profile contains chromatographic peaks of tyrosine, methionine, lysine and arginine.

8. Use of the amino acid profile of the standard decoction of animal traditional Chinese medicine constructed according to any one of claims 1 to 7 in the identification of animal traditional Chinese medicine and traditional Chinese medicine preparation thereof, wherein the traditional Chinese medicine preparation comprises the standard decoction of traditional Chinese medicine and traditional Chinese medicine prescription granule.

9. A method for detecting the amino acid content in a traditional Chinese medicine standard decoction and traditional Chinese medicine formula particles of animal traditional Chinese medicines is characterized by comprising the following steps:

the step of preparing the test solution includes: carrying out proteolysis on the sample to be tested, collecting hydrolysate, removing solvent in the hydrolysate, and dissolving the obtained residue with solvent;

the derivatization reagent adopted in the derivatization treatment is phenyl isothiocyanate; the conditions of the high performance liquid chromatography include:

The mobile phase A is a mixed solution of acetonitrile and sodium acetate solution with the volume ratio of (6.5-7.5) (92.5-93.5), the concentration of sodium acetate contained in the sodium acetate solution is 0.08 mol/L-0.12 mol/L, and the mobile phase B is a mixed solution of acetonitrile and water with the volume ratio of (3.5-4.5): 1, and gradient elution is adopted;

the gradient elution procedure includes:

6-9 min, wherein the volume percentage of the mobile phase A is 97%;

11 min-13 min, wherein the volume percentage of the mobile phase A is 88%;

36 min-39 min, wherein the volume percentage of the mobile phase A is 0%;

the amino acid reference substance comprises glycine reference substance, alanine reference substance, proline reference substance, valine reference substance and phenylalanine reference substance;

10. The method for detecting the amino acid content in the standard decoction and the formula particles of the animal traditional Chinese medicine according to claim 9, wherein the conditions of the high performance liquid chromatography further comprise: the flow rate is 0.8 mL/min-1.2 mL/min; or/and, the column temperature is 38-42 ℃; or/and the detection wavelength is 252 nm-256 nm; or/and the sample injection amount is 4-6 mu L.

11. The method for detecting the amino acid content in the standard decoction and the formula particles of the animal traditional Chinese medicine according to claim 10, wherein the conditions of the high performance liquid chromatography comprise: the flow rate is 1.0mL/min; or/and, the column temperature is 40 ℃; or/and, the detection wavelength is 254nm; or/and the sample injection amount is 5 mu L.

12. The method for detecting the amino acid content in the traditional Chinese medicine standard decoction and the traditional Chinese medicine formula particles of the animal traditional Chinese medicine according to claim 9, wherein the proteolysis adopts an acid method for hydrolysis; or/and the solvent adopts hydrochloric acid solution with the concentration of hydrochloric acid of 0.08mol/L to 0.12 mol/L.

13. The method for detecting the amino acid content in the standard decoction and the traditional Chinese medicine formula particles of the animal traditional Chinese medicine according to claim 9, 11 or 12, wherein the solvent of the reference solution comprises hydrochloric acid solution with hydrochloric acid concentration of 0.08 mol/L-0.12 mol/L.