WO2023082512A1 - Stiff silkworm marker polypeptide and method for identifying stiff silkworms, stiff silkworm aqueous extract products and other stiff silkworm products - Google Patents

Abstract

A stiff silkworm marker polypeptide and a method for identifying stiff silkworms, stiff silkworm aqueous extract products and other stiff silkworm products. The stiff silkworm marker polypeptide comprises a peptide fragment having an amino acid sequence as shown in SEQ ID NO. 1 or/and a peptide fragment having an amino acid sequence as shown in SEQ ID NO. 2. The method for identifying stiff silkworms, stiff silkworm aqueous extract products and other stiff silkworm products comprises a step of using an ultra-high performance liquid chromatography-mass spectrometry method to detect stiff silkworm marker components in a sample to be identified. According to the method, the stiff silkworm marker polypeptide in the sample is detected to identify stiff silkworms and products thereof, thereby solving the problem of difficulty in identifying the traditional Chinese medicine standard decoction, traditional Chinese medicine formula granules, and formulation and preparation from stiff silkworms due to missing appearance characters, and providing a basis for quality control and evaluation of stiff silkworms and products thereof, especially stiff silkworm aqueous extract products.

Description

Bombyx mori characteristic polypeptide and identification method of Bombyx mori, water extract products of Bombyx mori and other Bombyx mori products technical field

The invention belongs to the field of biological technology, and in particular relates to a silkworm characteristic polypeptide and a method for identifying silkworms, silkworm water extract products and other silkworm products.

Background technique

Bombyx mori is the dead body of Bombyx mori Linnaeus 4th to 5th instar larvae infected (or artificially inoculated) with Beauveria bassiana (Bals.) Vuillant. middle grade. Tao Hongjingyun: "When people raise silkworms, there are those who are stiff in the box, even if they are irritable, they are not bad. Now they are small white, which seems to have salinity.""ChuanPingze" is found in all silkworm breeding places today. It is better to use dead white and straight. "Compendium of Materia Medica" contains: "Silkworm disease wind dies, and its color is white, so it is called white stiff silkworm." It is mainly produced in Zhejiang, Jiangsu, Sichuan, Guangdong, Shaanxi and other places. It is more produced in spring and autumn, and the silkworms infected with Beauveria bassiana are dried. The silkworms with thick strips, hard texture, white color and bright cross section are the best. Silkworm has the effects of relieving wind and spasm, dispelling wind and relieving pain, resolving phlegm and resolving stagnation, and is clinically used for liver wind and phlegm, convulsions, convulsions in children, tetanus, and stroke

Wind-heat headache, conjunctival congestion, sore throat, rubella itching, and mumps.

As a commonly used traditional Chinese medicine, silkworm is widely used clinically. There are 175 prescriptions of Chinese patent medicines containing silkworm in "Chinese Pharmacopoeia", "National Standards of Chinese Patent Medicine", "Prescriptions of Traditional Chinese Medicine" and other classics. However, the Research on quality standards is not perfect. The traditional qualitative detection method of silkworm is mainly based on the properties of medicinal materials, infrared spectrum, etc. For example, the quality control of silkworm in the 2020 edition of "Chinese Pharmacopoeia" is limited to traits, microscopic identification, detection items and extracts, and there is no specific regulation for the time being. A qualitative detection method with strong characteristics; Zhao Jianguo established an infrared spectrum identification method of traditional Chinese medicine Bombyx mori in "Fourier Transform Infrared Spectroscopy Identification of Bombyx mori, providing reference for crude drug identification and quality evaluation of Bombyx mori.

At present, the specific qualitative method of Bombyx mori mainly adopts DNA barcoding technology, for example, Jia Jing, Shi Chunlin et al. realized the detection of Bombyx mori based on PCR in "Research on DNA Barcode Identification of Commercially Available Animal Medicinal Materials Bombyx mori". Although PCR technology has strong specificity and high sensitivity, because PCR technology uses universal primers, after the amplification is completed, it is necessary to sequence the amplified product and compare the sequencing result with the sequence in the gene database to determine the species , there are many steps, it takes a long time, and special equipment is needed, so it is difficult to be used in the production of silkworms. In particular, there are limitations in the scope of application of the traditional specific qualitative methods exemplified above, which are mainly applicable to silkworm medicinal materials, but not suitable for specific qualitative identification of silkworm products (including silkworm water extract products), such as standard The preparation process of decoctions and traditional Chinese medicine formula granules is subjected to high temperature, during which specific DNA fragments are damaged, and the complex matrix makes DNA extraction more difficult. Therefore, how to specifically identify the silkworm and its products so as to meet the needs of quality control is an urgent technical problem to be solved, and it is also a technical problem that urgently needs to be overcome in the field of quality control of silkworm water extract products.

Contents of the invention

Based on the above background technology, the main purpose of the present invention is to provide a characteristic polypeptide of Bombyx mori, which can be used for specific identification of Bombyx mori and its products, especially water extract products of Bombyx mori. The identification of the Bombyx mori and its products can be realized by determining whether the characteristic polypeptide of Bombyx mori exists in the silkworm.

The purpose of the present invention can be achieved through the following technical solutions:

A characteristic polypeptide of Bombyx mori, the characteristic polypeptide of Bombyx mori comprises a peptide segment whose amino acid sequence is shown in SEQ ID No.1 or/and a peptide segment whose amino acid sequence is shown in SEQ ID No.2.

In one embodiment, the amino acid sequence is the peptide segment shown in SEQ ID No.1, the m/z of the parent ion is 823.36, and the m/z of the product ions are 1070.47 and 1345.57.

In one embodiment, the amino acid sequence is the peptide segment shown in SEQ ID No.2, the m/z of the parent ion is 637.29, and the m/z of the product ions are 825.36 and 926.40.

A method for identifying the silkworm, silkworm water extract products and other silkworm products, the identification method includes the step of detecting the characteristic components of the silkworm in the sample to be identified, and the characteristic components of the silkworm are the described Bombyx mori characteristic polypeptide.

In one of the embodiments, the step of detecting the characteristic components of the silkworm in the sample to be identified comprises:

Take the sample to be identified and the characteristic components of the silkworm respectively to prepare a test solution and a reference solution; use ultra-high performance liquid chromatography mass spectrometry to detect the test solution and the reference solution .

In one of the embodiments, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of the ultra-high performance liquid chromatography include:

Stationary phase: C18 chromatographic column;

Mobile phase: mobile phase A is acetonitrile, mobile phase B is an aqueous solution of formic acid containing 0.08% to 0.12% by volume of formic acid, and the elution method adopts gradient elution.

In one embodiment, the gradient elution conditions include: 0min-3min, the volume percentage of the mobile phase A is 3%; 3min-8min, the volume percentage of the mobile phase A increases from 3% to 5%; 8min～10min, the volume percentage of described mobile phase A is 5%; 10min～18min, the volume percentage of described mobile phase A rises to 7% by 5%; 18min～19min, the volume percentage of described mobile phase A is by 7% % rises to 90%; 19min～21min, the volume percentage of the mobile phase A is 90%.

In one of the embodiments, in the ultra-high performance liquid chromatography-mass spectrometry method, the ultra-high performance liquid chromatography conditions further include: the column temperature is 28°C to 32°C.

In one embodiment, in the ultra-high performance liquid chromatography-mass spectrometry method, the ultra-high performance liquid chromatography conditions further include: a flow rate of 0.25 mL/min-0.35 mL/min.

In one of the embodiments, in the ultra-high performance liquid chromatography-mass spectrometry method, the ultra-high performance liquid chromatography conditions further include: the injection volume is 4.5 μL-5.5 μL.

In one of the embodiments, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of mass spectrometry include: electrospray positive ion mode; multiple reaction monitoring scan; capillary voltage: 0.45kV～0.55kV; ion source temperature: 480°C ~520°C; Solvent gas flow rate: 850L/hr~950L/hr.

In one of the embodiments, the preparation of the reference substance solution includes the following steps: taking the characteristic components of the silkworm and dissolving them with ammonium bicarbonate solution.

In one of the embodiments, the ammonium bicarbonate solution contains 0.8%-1.2% by mass of ammonium bicarbonate.

In one of the embodiments, the preparation of the test solution includes the following steps: extracting the sample to be identified in ammonium bicarbonate solution, collecting the extract, and adding trypsin for enzymatic hydrolysis.

In one of the embodiments, the ammonium bicarbonate solution contains 0.8%-1.2% by mass of ammonium bicarbonate.

In one of the embodiments, the silkworm water extract product comprises standard decoction of silkworm silkworm, standard decoction of silkworm fried silkworm, formula granules of traditional Chinese medicine of silkworm silkworm, formula granules of Chinese medicine fried silkworm silkworm and epilepsy flat tablets.

In one embodiment, the other silkworm products include Zhongfeng Huichun Pills, Rupi Sanjie Capsules, Yixian Pills and Xiaoer Qizhen Pills.

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

The present invention provides a silkworm characteristic polypeptide, which can be used for the specific identification of silkworm and its products, especially water extract products of silkworm, by detecting whether the characteristic polypeptide of silkworm exists in the sample to be identified, that is, The identification of the silkworm and its products can be realized. The present invention solves the problem of difficult identification due to lack of appearance properties of water extract products such as standard Chinese medicine decoction of silkworm, traditional Chinese medicine formula granules and prescription preparations, and is simple to operate. It provides a basis for quality control and evaluation of extract products.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

Fig. 1 is the chromatogram, primary mass spectrogram and secondary mass spectrogram of peptide A and artificially synthesized polypeptide;

Figure 2 is the chromatogram, primary mass spectrogram and secondary mass spectrogram of peptide B and artificially synthesized polypeptide;

Figure 3 is the exclusive mass spectrum of fried silkworm traditional Chinese medicine formula granules (m/z 823);

Figure 4 is the exclusive mass spectrogram (m/z 637) of fried silkworm traditional Chinese medicine formula granules;

Figure 5 is the characteristic verification MRM mass spectrum (m/z 823);

Figure 6 is the characteristic verification MRM mass spectrum (m/z 637);

Figure 7 is the reference substance, silkworm standard decoction, scarab worm standard decoction, bread worm standard decoction, tortoise shell standard decoction, wood beetle standard decoction, earthworm standard decoction, cicada slough standard decoction, chicken gold standard Characteristic verification MRM mass spectrogram (m/z 823) of decoction and leech standard decoction;

Figure 8 is the reference substance, silkworm standard decoction, scarab worm standard decoction, bread worm standard decoction, tortoise shell standard decoction, wood beetle standard decoction, earthworm standard decoction, cicada slough standard decoction, chicken gold standard Characteristic verification MRM mass spectrogram (m/z 637) of decoction and leech standard decoction;

Fig. 9 is the MRM mass spectrogram (m/ z 823);

Figure 10 is the MRM mass spectrogram (m/ z 637);

Fig. 11 is the MRM mass spectrogram (m/z 823) of the prescription preparation containing silkworm;

Fig. 12 is the MRM mass spectrogram (m/z 637) of the prescription preparation containing silkworm.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in more detail below. It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are only for the purpose of describing specific embodiments or examples, and are not intended to limit the present invention. The optional range of the term "and/or" used herein includes any one of two or more of the relevant listed items, and also includes any and all combinations of the relevant listed items, and any and all of the Combinations include combinations of any two of the related listed items, any more of the related listed items, or all of the related listed items.

In the present invention, "the first aspect", "the second aspect" and so on are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or quantity, nor as implying the importance or importance of the indicated technical features. quantity.

In the present invention, the technical features described in open form include closed technical solutions consisting of the enumerated features, as well as open technical solutions including the enumerated features.

In the present invention, when referring to a numerical interval, unless otherwise specified, both endpoints of the numerical interval are included.

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

The percentage concentration involved in the present invention refers to the final concentration unless otherwise specified. The final concentration refers to the proportion of the added component in the system after the component is added.

The temperature parameters in the present invention, unless otherwise specifically limited, allow either constant temperature treatment or treatment within a certain temperature range. The isothermal treatment allows the temperature to fluctuate within the precision of the instrument control.

In recent years, with the development of proteomics, the 2020 edition of "Chinese Pharmacopoeia" has applied the characteristic ions and characteristic peptides of donkey-hide gelatin to the identification and content determination of donkey-hide gelatin. This method is faster, more accurate and more specific . However, the study of characteristic polypeptides in Bombyx mori and its products is still blank. For this reason, the present invention utilizes LC-MS technology, combined with proteomics, to screen out two sections of characteristic polypeptides of Bombyx mori, which can be applied to the specificity of Bombyx mori (including Bombyx mori medicinal materials, Bombyx mori decoction pieces, and fried Bombyx silkworm pieces). It can also be used for identification of silkworm products, including water extract products of silkworm silkworm and specific identification of silkworm components in other silkworm products. It provides a basis for the quality control and evaluation of Bombyx mori and its products, especially Bombyx mori water extract products.

The silkworm water extract products described in the present invention are mainly used for traditional Chinese medicine products prepared from silkworm water extracts, including but not limited to: standard decoction of silkworm silkworm, standard decoction of silkworm fried silkworm, formula granules of traditional Chinese medicine of silkworm silkworm, fried silkworm silkworm Silkworm traditional Chinese medicine formula granules and prescription preparations (such as Epilepsy Tablets).

The other silkworm products described in the present invention are mainly used for traditional Chinese medicine products prepared in other pharmaceutical forms (such as silkworm crushed products) other than silkworm water extracts, including but not limited to Zhongfeng Huichun Pills, Rupi Sanjie Capsules, Medical Epilepsy Pills and Xiaoer Qizhen Pills.

Technical scheme of the present invention comprises:

In a first aspect, the present invention provides a characteristic polypeptide of Bombyx mori, which comprises a peptide segment (denoted as "peptide A") with an amino acid sequence as shown in SEQ ID No. 1 or/and an amino acid sequence such as SEQ ID No. 1 The peptide indicated by ID No.2 (referred to as "peptide B").

SEQ ID No.1: GGSVSSTGSSSNTDSSTK;

SEQ ID No. 2: GHLGTVSSTGSTSNTDSSSK.

In one example, the amino acid sequence is the peptide shown in SEQ ID No.1, the m/z of the parent ion is 823.36, and the m/z of the product ions are 1070.47 and 1345.57.

In one example, the amino acid sequence is the peptide segment shown in SEQ ID No.2, the m/z of the parent ion is 637.29, and the m/z of the product ions are 825.36 and 926.40.

In the second aspect, the present invention provides a method for identifying the silkworm, the water extract product of the silkworm and other silkworm products, the identification method includes the step of detecting the characteristic components of the silkworm in the sample to be identified, and the silkworm The characteristic component is the above-mentioned characteristic polypeptide of Bombyx mori.

The characteristic components of the silkworm described in the present invention refer to the unique components of the silkworm compared with other traditional Chinese medicinal materials.

In one example, the step of detecting components characteristic of the silkworm in the sample to be identified comprises:

Take the sample to be identified and the characteristic components of the silkworm respectively to prepare a test solution and a reference solution; use ultra-high performance liquid chromatography mass spectrometry to detect the test solution and the reference solution .

In one of the examples, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of the ultra-high performance liquid chromatography include:

Stationary phase: C18 chromatographic column;

Mobile phase: mobile phase A is acetonitrile, mobile phase B is an aqueous solution of formic acid containing 0.08% to 0.12% by volume of formic acid, and the elution method adopts gradient elution.

The C18 chromatographic column described in the present invention includes but is not limited to Phenomenes Titank C18 chromatographic column (1.7 μm, 2.1 mm×150 mm).

In one example, the gradient elution conditions include: 0min-3min, the volume percentage of the mobile phase A is 3%; 3min-8min, the volume percentage of the mobile phase A increases from 3% to 5%; 8min ~10min, the volume percentage of the mobile phase A is 5%; 10min～18min, the volume percentage of the mobile phase A rises from 5% to 7%; 18min～19min, the volume percentage of the mobile phase A increases from 7% Increase to 90%; 19min-21min, the volume percentage of the mobile phase A is 90%.

In one example, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of the ultra-high performance liquid chromatography also include: the column temperature is 28°C to 32°C, such as 28°C, 29°C, 30°C, 31°C, 32°C.

In one example, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of the ultra-high performance liquid chromatography further include: the flow rate is 0.25mL/min～0.35mL/min, such as 0.25mL/min, 0.30mL/min min, 0.35mL/min.

In one example, in the ultra-high performance liquid chromatography-mass spectrometry method, the ultra-high performance liquid chromatography conditions further include: the injection volume is 4.5 μL-5.5 μL, for example, 4.5 μL, 5 μL, 5.5 μL.

In one example, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of mass spectrometry include: electrospray positive ion mode; multiple reaction monitoring scan; capillary voltage: 0.45kV～0.55kV (such as 0.45kV, 0.5kV, 0.55kV); ion source temperature: 480℃～520℃ (such as 480℃, 490℃, 500℃, 510℃, 520℃); solvent gas flow rate: 850L/hr～950L/hr (such as 850L/hr, 900L/hr hr, 950L/hr).

Adopting the above-mentioned detection conditions has the following advantages: shortening the detection time and saving the detection cost; optimization of the mass spectrometry conditions increases its response, etc.

In one example, the preparation of the reference substance solution includes the following steps: taking the characteristic components of the silkworm and dissolving them with ammonium bicarbonate solution.

In one example, the ammonium bicarbonate solution contains ammonium bicarbonate in a mass percentage of 0.8% to 1.2%, such as 0.8%, 0.9%, 1.0%, 1.1%, and 1.2%.

In one example, the preparation of the test solution includes the following steps: placing the sample to be identified in an ammonium bicarbonate solution for extraction, collecting the extract, and adding trypsin for enzymatic hydrolysis.

In one example, the ammonium bicarbonate solution contains ammonium bicarbonate in a mass percentage of 0.8% to 1.2%, such as 0.8%, 0.9%, 1.0%, 1.1%, and 1.2%.

It can be understood that, in the process of preparing the test solution, suitable preparation conditions can be selected according to the type of the sample to be identified, such as a suitable extraction method (for example, the silkworm medicinal material and decoction pieces can adopt the method of reflux extraction, The standard decoction of silkworm and the standard decoction of fried silkworm can adopt the mode of ultrasonic extraction, etc.), an appropriate amount of trypsin.

It can be understood that the silkworm aqueous extract products include, but are not limited to: standard decoction of silkworm, standard decoction of fried silkworm, Chinese medicine formula granules of silkworm, fried silkworm Chinese medicine formula granules and epilepsy tablets.

It can be understood that the other silkworm products include, but are not limited to: Zhongfeng Huichun Pills, Rupi Sanjie Capsules, Yixian Pills and Xiaoer Qizhen Pills. It should be noted that there is no sequence limitation for the steps involved in the identification method of the present invention.

In the present invention, standard decoction of silkworm, standard decoction of fried silkworm, standard decoction of scarab worm, standard decoction of bread worm, standard decoction of tortoise shell, standard decoction of chicken gold, standard decoction of leech, standard decoction of cicada slough , Dilong standard decoction, ground beetle standard decoction, the preparation process includes but not limited to the following exemplified processes:

(1) Standard decoction of silkworm, standard decoction of fried silkworm, standard decoction of scarab worm, standard decoction of bread worm, standard decoction of leech, standard decoction of earthworm, standard decoction of ground beetle: take 100g of decoction pieces, put In an electric ceramic pot, add water to decoct twice, add 8 times the amount of water to the first decoction, soak for 60 minutes, boil with strong fire (500W) and keep it at a low fire (200W) for 30 minutes, and the decoction passes through a 350-mesh sieve Filter while it is hot, and the filtrate is quickly cooled with cold water. Add 6 times the amount of water for the second time, heat it to a boil with high fire and keep it at a low heat for 25 minutes. Filter the decoction while it is hot with a 350-mesh sieve, cool the filtrate quickly with cold water, and combine the two decoctions. Transfer the decoction to a 2000ml round bottom flask, use a rotary evaporator to concentrate under reduced pressure and low temperature (temperature: 65°C; vacuum degree: -0.10MPa) to 150ml of extract; under magnetic stirring, divide into 10ml brown vials , the volume of each bottle is 2ml, half-stoppered, transferred to a vacuum freeze dryer to freeze-dry, taken out, rolled on an aluminum cap, and obtained.

(2) Guijia standard decoction: take 100g of decoction pieces, add water to decoct twice, add 8 times the amount of water to the first decoction, soak for 30 minutes, boil with strong fire (power 500W) and then change to simmer fire (power 200W) and decoct for 60 minutes Minutes, use a 200-mesh sieve to filter while it is hot, and the filtrate is quickly cooled with cold water; add 6 times the amount of water to the second decoction, boil with a strong fire (power 500W), then change to a simmer (power 200W) and decoct for 40 minutes, and use a 200-mesh sieve. Filtrate hot, and cool the filtrate quickly with cold water; combine the two filtrates, concentrate under reduced pressure to a volume of about 100ml, dispense into 10ml vials, and dispense 2ml into each vial, vacuum freeze-dry, take out, and roll an aluminum cap to obtain the product.

(3) Chicken Gold Standard Decoction: Take 100g of decoction pieces, put them in an electric ceramic pot, add water to decoct twice, add 9 times the amount of water for the first decoction, soak for 30 minutes, boil with a strong fire (power 500W) and then simmer (Power 200W) Keep boiling slightly for 30 minutes, filter the decoction while it is hot through a 350-mesh sieve, and quickly cool the filtrate with cold water. Add 7 times the amount of water for the second time, boil with high fire (power 500W) and keep boiling for 25 minutes with slow fire (power 200W), filter the decoction while it is hot with a 350-mesh sieve, cool the filtrate quickly with cold water, and combine the two decoctions . Transfer the decoction to a round-bottomed flask, use a rotary evaporator to concentrate under reduced pressure and low temperature (temperature: 65°C; vacuum degree: -0.08MPa～-0.1MPa), rotate at a speed of 50～90 rpm, and concentrate to a volume of about 100ml ; Under magnetic stirring, precisely absorb 2ml of the decoction and evenly distribute it in 10ml vials, transfer to a vacuum freeze dryer to freeze-dry, take it out, and roll the aluminum cap to get it.

(4) Cicada standard decoction: take 100g of cicada decoction pieces, put them in an electric ceramic pot, add water to decoct twice, add 14 times the amount of water for the first decoction, soak for 30 minutes, boil with a strong fire (power 500W) and then simmer ( power 200W) keep boiling slightly for 30 minutes, the decoction is filtered through a 350-mesh sieve while it is still hot, and the filtrate is quickly cooled with cold water. Add 12 times the amount of water for the second time, boil with high fire (power 500W) and keep boiling for 25 minutes with slow fire (power 200W), filter the decoction while it is hot with a 350-mesh sieve, cool the filtrate quickly with cold water, and combine the two decoctions . Transfer the decoction to a round-bottomed flask, use a rotary evaporator to concentrate under reduced pressure and low temperature (temperature: 65°C; vacuum degree: -0.08MPa～-0.1MPa), rotate at a speed of 50～90 rpm, and concentrate to a volume of about 100ml ; Under magnetic stirring, precisely absorb 2ml of the liquid extract and evenly distribute it in 10ml vials, transfer to a vacuum freeze dryer to freeze-dry, take it out, and roll an aluminum cap to get it.

The test methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

Example 1. Screening and verification of characteristic polypeptides

1 Preparation of the test solution

(1) Refer to the following methods to prepare the test solution of silkworm medicinal material, silkworm decoction pieces, fried silkworm decoction pieces, scarab worm fresh products and bread worm fresh products respectively:

Take about 1g of the powder of this product, weigh it accurately, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, reflux for 10min, take it out, let it cool, and use 1% ammonium bicarbonate solution to make up for the lost weight. Shake well.

Filter through a 0.22 μm microporous membrane, take 1 mL of the subsequent filtrate, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

(2) prepare respectively the test solution of standard decoction of silkworm and fried standard decoction of silkworm with reference to the following methods:

Take about 0.1g of the powder of this product, weigh it accurately, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30min, take it out, let it cool, and use 1% Ammonium bicarbonate solution to make up the weight, shake well.

Filter through a 0.22 μm microporous membrane, take 1 mL of the filtrate, add 50 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

(3) Prepare the test solution of the silkworm traditional Chinese medicine formula granules with reference to the following method:

Take an appropriate amount of this product, grind it finely, take about 0.5g, accurately weigh it, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 60min, take it out, put it Cold, make up the weight with 1% ammonium bicarbonate solution, shake well.

Filter through a 0.22 μm microporous membrane, take 1 mL of the subsequent filtrate, add 300 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

(4) Prepare the test solution of fried silkworm traditional Chinese medicine formula granules with reference to the following method:

Take an appropriate amount of this product, grind it finely, take about 0.5g, accurately weigh it, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, and ultrasonically treat it (power 250W, frequency 40kHz) for 30min, take it out, put it Cold, make up the weight with 1% ammonium bicarbonate solution, shake well.

Filter through a 0.22 μm microporous membrane, take 500 μL of the subsequent filtrate, add 300 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare it before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

2 data collection

The samples were collected by Vanquish ultra-high performance liquid chromatography and Q-Exactive ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap combined high-resolution mass spectrometer (ThermoFisher, USA).

Chromatographic conditions and mass spectrometry conditions are as follows:

Chromatographic conditions: chromatographic column: Waters BEH C18 chromatographic column (1.7μm, 2.1mm×150mm); mobile phase: acetonitrile-0.1% formic acid aqueous solution, gradient elution according to Table 1; column temperature: 30°C; flow rate: 0.30mL/ min; injection volume: 5 μL.

Table 1. Gradient elution table

Mass spectrometry conditions: electrospray positive ion mode (ESI ⁺ ); sheath gas flow: 35Arb; auxiliary gas flow: 10Arb; spray voltage: 3.80kV; capillary temperature: 350°C; detector temperature: 350°C; collision energy: 30%; FullMS+ddms2 mode for full scan, PRM mode for confirmation.

3 Peptide Screening

Import the collected mass spectrometry data into Proteome Discoverer 2.4 analysis software, use the Uniprot_bombyx database to search and match, and screen to obtain the specific polypeptide sequence of Bombyx mori. The polypeptide sequence is handed over to the biological company for synthesis. Peptide specific information is as follows:

Table 2. Sequences and monitored transitions of the two polypeptides

Table 3. The ion matching information table of the polypeptide GGSVSSTGSSSNTDSSTK sequence

#1	b +	b 2+	Seq.	y +	y 2+	#2
1	58.02874	29.51801	G	the	the	18
2	115.05020	58.02874	G	1588.69328	794.85028	17
3	202.08223	101.54475	S	1531.67181	766.33955	16
4	301.15065	151.07896	V	1444.63979	722.82353	15
5	388.18267	194.59498	S	1345.57137	673.28932	14
6	475.21470	238.11099	S	1258.53934	629.77331	13
7	576.26238	288.63483	T	1171.50732	586.25730	12
8	633.28385	317.14556	G	1070.45964	535.73346	11
9	720.31587	360.66158	S	1013.43817	507.22272	10
10	807.34790	404.17759	S	926.40614	463.70671	9

11	894.37993	447.69360	S	839.37412	420.19070	8
12	1008.42286	504.71507	N	752.34209	376.67468	7
13	1109.47054	555.23891	T	638.29916	319.65322	6
14	1224.49748	612.75238	D.	537.25148	269.12938	5
15	1311.52951	656.26839	S	422.22454	211.61591	4
16	1398.56154	699.78441	S	335.19251	168.09989	3
17	1499.60921	750.30825	T	248.16048	124.58388	2
18	the	the	K	147.11280	74.06004	1

Table 4. Ion matching information table of polypeptide GHLGTVSSTGSTSNTDSSSK sequence

#1	b +	b 2+	b 3+	Seq.	y +	y 2+	y 3+	#2
1	58.02874	29.51801	20.01443	G	/	/	/	20
2	195.08765	98.04746	65.70074	h	1852.85190	926.92959	618.28882	19
3	308.17172	154.58950	103.39542	L	1715.79299	858.40013	572.60252	18
4	365.19318	183.10023	122.40258	G	1602.70893	801.85810	534.90783	17
5	466.24086	233.62407	156.08514	T	1545.68746	773.34737	515.90067	16
6	565.30927	283.15827	189.10794	V	1444.63979	722.82353	482.21811	15
7	652.34130	326.67429	218.11862	S	1345.57137	673.28932	449.19531	14
8	739.37333	370.19030	247.12929	S	1258.53934	629.77331	420.18463	13
9	840.42101	420.71414	280.81185	T	1171.50732	586.25730	391.17396	12
10	897.44247	449.22487	299.81901	G	1070.45964	535.73346	357.49140	11
11	984.47450	492.74089	328.82968	S	1013.43817	507.22272	338.48424	10
12	1085.52218	543.26473	362.51224	T	926.40614	463.70671	309.47357	9
13	1172.55421	586.78074	391.52292	S	825.35847	413.18287	275.79101	8
14	1286.59713	643.80220	429.53723	N	738.32644	369.66686	246.78033	7
15	1387.64481	694.32604	463.21979	T	624.28351	312.64539	208.76602	6
16	1502.67175	751.83952	501.56210	D.	523.23583	262.12155	175.08346	5
17	1589.70378	795.35553	530.57278	S	408.20889	204.60808	136.74115	4
18	1676.73581	838.87154	559.58345	S	321.17686	161.09207	107.73047	3
19	1763.76784	882.38756	588.59413	S	234.14483	117.57605	78.71980	2
20	/	/	/	K	147.11280	74.06004	49.70912	1

4 verification

According to the information in the above table, the above-mentioned peptides and artificially synthesized peptides screened were confirmed by PRM mode, and the secondary mass spectrum was obtained.

By comparing the retention time and secondary ion fragments of the artificially synthesized peptides and the sample screening peptides at the same mass-to-charge ratio, the results show that the retention time and secondary ion fragments of the above two characteristic peptides can be in one-to-one correspondence with the artificially synthesized peptides. , indicating that the sequences of the screened polypeptides are all correct sequences. See Figures 1 and 2.

Embodiment 2, verification of characteristic polypeptide and sample determination

1 Preparation of the test solution

(1) Refer to the following methods to prepare silkworm medicinal materials, silkworm decoction pieces, fried silkworm decoction pieces, chafer medicinal materials, mealworm medicinal materials, tortoise shell medicinal materials, gallinaceous gold medicinal materials, leech medicinal materials, cicada slough medicinal materials, earthworm medicinal materials, and wood beetle medicinal materials The test solution:

Take about 1g of the powder of this product, weigh it accurately, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, reflux for 10min, take it out, let it cool, and use 1% ammonium bicarbonate solution to make up for the lost weight. Shake well.

Filter through a 0.22 μm microporous membrane, take 1 mL of the subsequent filtrate, add 200 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

(2) Prepare standard decoction of silkworm, standard decoction of fried silkworm, standard decoction of scarab worm, standard decoction of mealworm, standard decoction of tortoise shell, standard decoction of chicken gold, standard decoction of leeches, The test solution of cicada slough standard decoction, earthworm standard decoction, and ground beetle standard decoction:

Take about 0.1g of the powder of this product, weigh it accurately, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30min, take it out, let it cool, and use 1% Ammonium bicarbonate solution to make up the weight, shake well.

Filter through a 0.22 μm microporous membrane, take 1 mL of the filtrate, add 50 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare it before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

(3) Prepare the test solution of the silkworm traditional Chinese medicine formula granules with reference to the following method:

Take an appropriate amount of this product, grind it finely, take about 0.5g, accurately weigh it, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 60min, take it out, put it Cold, make up the weight with 1% ammonium bicarbonate solution, shake well.

Filter through a 0.22 μm microporous membrane, take 1 mL of the subsequent filtrate, add 300 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

(4) Prepare the test solution of fried silkworm traditional Chinese medicine formula granules with reference to the following method:

Take an appropriate amount of this product, grind it finely, take about 0.5g, accurately weigh it, put it in a conical flask, add 50mL of 1% ammonium bicarbonate solution, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30min, take it out, put it Cold, make up the weight with 1% ammonium bicarbonate solution, shake well.

Filter through a 0.22 μm microporous membrane, take 500 μL of the subsequent filtrate, add 300 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare it before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

(5) Prepare the test solution of the prescription preparations (Zhongfeng Huichun Pills, Rupi Sanjie Capsules, Yixian Pills, Epilepsy Tablets, Xiaoer Qizhen Pills) according to the following method:

Take about 5g of the powder of this product, weigh it accurately, put it in a conical flask, add 25mL of 1% ammonium bicarbonate solution, weigh it, reflux for 30min, take it out, let it cool, and use 1% ammonium bicarbonate solution to make up for the lost weight. Shake well.

Filter through a 0.22 μm microporous membrane, take 500 μL of the subsequent filtrate, add 50 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 mL, and prepare before use), Shake well, 37 ℃ constant temperature enzymatic hydrolysis for 12 hours, as the test solution.

2 Preparation of reference substance solution

Take an appropriate amount of peptide A and peptide B reference substances, weigh them accurately, add 1% ammonium bicarbonate solution to prepare a mixed reference solution containing 2 μg per 1 ml.

3 Chromatography and mass spectrometry conditions

The samples were determined by Waters Xevo TQ-S Micro triple quadrupole liquid mass spectrometer (Waters Corporation). Chromatographic conditions and mass spectrometry conditions are as follows:

Chromatographic conditions: Chromatographic column: Phenomenes Titank C18 chromatographic column (1.7μm, 2.1mm×150mm); mobile phase: acetonitrile-0.1% formic acid aqueous solution, gradient elution according to the table below; column temperature: 30°C; flow rate: 0.30 mL/min; injection volume: 5 μL.

Table 5. Gradient elution table

Mass spectrometry conditions: electrospray positive ion mode (ESI+); MRM mode, that is, multi reaction monitoring scanning (multi reaction monitoring); capillary voltage: 0.50kV; ion source temperature: 500°C; solvent flow rate: 900L/hr. Cone voltage and collision energy are shown in the table below.

Table 6. Collision energy and cone voltage of monitored ion pairs

4 methodological verification (verify the identification method of the present invention with fried silkworm traditional Chinese medicine formula granules)

(1) Specificity inspection

Weigh an appropriate amount of fried silkworm Chinese medicine formula granules, and prepare the fried silkworm Chinese medicine formula granules for the test solution according to the method under "1 Preparation of test solution"; The blank solvent solution of the traditional Chinese medicine formula granules; and then prepare the reference substance solution according to the preparation method under "2 Preparation of the reference substance solution".

Precisely draw 5 μl each of the test solution, the reference solution and the blank solvent solution, inject it into the liquid mass spectrometer, and analyze it according to the "3 chromatography and mass spectrometry conditions". The results are shown in Figure 3 and Figure 4.

The results showed that no characteristic ion peaks were detected at the corresponding retention time of the peptide A and peptide B reference substance chromatograms of the blank solvent test solution spectrum of the fried silkworm traditional Chinese medicine formula granules, indicating that the blank solvent paired the characteristic ions in the method. There is no interference in the detection of pairs, and the method is specific.

(2) Inspection of instrument precision

Precisely draw the reference substance solution prepared under "2 Preparation of reference substance solution", repeat the sample injection 6 times according to the chromatographic conditions and mass spectrometry conditions under "3 Chromatography and mass spectrometry conditions", record the peak area, calculate the peak area RSD value, and the result See Table 7.

Table 7. Instrument precision result table

The results showed that the same reference solution was injected continuously for 6 times, and the retention time and peak area RSD values of the two pairs of characteristic ion pairs were both less than 3%, indicating that the precision of the instrument was good.

(3) Stability inspection

Take an appropriate amount of fried silkworm Chinese medicine formula granules, and prepare the fried silkworm Chinese medicine formula granules for the test solution according to the method under "Preparation of the test solution", and draw them precisely at 0, 2, 5, 8, 10, and 12 hours respectively. 5 μl was injected into the liquid mass spectrometer, and measured according to the conditions under "3 Chromatography and mass spectrometry conditions", and the stability of the solution was evaluated by the peak area of the ion pair. The measurement results are shown in Table 8.

Table 8. Stability investigation results

The results show that the test solution was injected at 0, 2, 5, 8, 10, and 12 hours respectively, and the RSD values of the two pairs of characteristic ion pair peak areas were in the range of 1.27% to 4.41%, both less than 5%, indicating that the test product The solution has good stability within 12 hours.

(4) Repeated inspection

Take about 0.5 g of the same batch of fried silkworm traditional Chinese medicine formula granules, accurately weigh, weigh 6 parts in parallel, and prepare 6 parts of the test solution according to the method under "1 Preparation of the test solution". According to the conditions under "3 Chromatography and mass spectrometry conditions", the RSD value of the ratio of "peak area/sample weight" was calculated, and the measurement results are shown in Table 9.

Table 9. Repeatability inspection results

The results showed that the same batch of samples was repeatedly measured 6 times, and the RSD values of the ratio of "peak area/sample weight" were all less than 5%, indicating that the method had good repeatability.

(5) Durability inspection

① Investigation of different chromatographic columns

Compared Phenomenon Titank C18 column (2.1mm×150mm, 1.7μm); Waters BEH C18 column (2.1mm×150mm, 1.7μm); Shimadzu Shim-pack C18-AQ column (2.1mm×150mm, 1.9μm) 3 different brands and types of chromatographic columns on the detection of characteristic ion peaks of fried silkworm traditional Chinese medicine formula particles.

Take an appropriate amount of fried silkworm traditional Chinese medicine formula granules, prepare the test solution according to the preparation method of the test solution determined under "2 Preparation of the Reference Substance Solution", accurately draw 5 μl and inject it into the liquid chromatography-mass spectrometer, and press "3 Chromatography and mass spectrometry" The conditions under the item "conditions" were measured, and the experimental results are shown in Table 10.

Table 10. Investigation results of the durability of different chromatographic columns of fried silkworm traditional Chinese medicine formula granules

Note: 1# chromatographic column is Philomens Titank C18 chromatographic column;

2# chromatographic column is Waters BEH C18 chromatographic column;

3# chromatographic column is Shimadzu Shim-pack C18-AQ chromatographic column

The results showed that the three chromatographic columns used could clearly detect the specified two pairs of characteristic ion pairs, indicating that different brands and models of chromatographic columns had no effect on the detection and identification of the characteristic ions of the fried silkworm traditional Chinese medicine formula granules.

② Investigation of different column temperatures

The effects of different column temperatures of 30°C, 28°C, and 32°C on the detection of characteristic ion peaks of the traditional Chinese medicine formula granules of fried silkworm were compared.

Take an appropriate amount of fried silkworm Chinese medicine formula granules, prepare the test solution according to the preparation method of the test solution determined under "1 Preparation of the test solution", accurately draw 5 μl and inject it into the liquid mass spectrometer, press "3 Chromatography and Mass spectrometry conditions" under the conditions for determination, the experimental results are shown in Table 11.

Table 11. The peak area results of the investigation of the durability of fried silkworm traditional Chinese medicine formula granules at different column temperatures

The results showed that the specified two pairs of characteristic ion pairs could be clearly detected using three different column temperature conditions, indicating that a small range of column temperature adjustment had no effect on the detection and identification of the characteristic ions of the fried silkworm traditional Chinese medicine formula granules.

③ Investigation of different flow rates

The effects of different flow rates of 0.30mL/min, 0.27mL/min, and 0.33mL/min on the detection of characteristic ion peaks of the traditional Chinese medicine formula granules of fried silkworm were compared.

Take an appropriate amount of Stir-fried Silkworm Traditional Chinese Medicine Formula Granules (CG2012024), prepare the test solution according to the preparation method of the test solution determined under "1 Preparation of the test solution", and accurately draw 5 μl into the LC-MS instrument, press " 3 Chromatography and mass spectrometry conditions" under the conditions for determination, the experimental results are shown in Table 12.

Table 12. Peak area results of durability investigation of fried silkworm traditional Chinese medicine formula granules at different flow rates

The results showed that the specified two pairs of characteristic ion pairs could be clearly detected using three different flow rate conditions, indicating that the small range adjustment of the flow rate had no effect on the detection and identification of the characteristic ions of the fried silkworm traditional Chinese medicine formula granules.

(6) Summary

To sum up, the specificity of mass spectrometric identification analysis method for fried silkworm traditional Chinese medicine formula granules was investigated, and the characteristic peaks were not interfered by solvent peaks, and the method was exclusive; the injection precision, solution stability and repeatability of the method were verified, and the results All meet the corresponding requirements. And the durability of the method was investigated. It shows that different chromatographic columns, different flow rates and small range of column temperature fluctuations have small fluctuations in the relative retention time of each chromatographic peak, and the method has good durability.

4 Sample Determination - Different Species

From Figure 5 to Figure 7, it can be seen that the silkworm medicinal materials and standard decoction present the same chromatographic peaks as the reference substance, and the medicinal materials and standard soups of mealworm, scarab, tortoise shell, gallinacea, leech, cicada slough, earthworm, and wood beetle There is no chromatographic peak at the corresponding retention time of the drug in the silkworm, indicating that the two peptides found are characteristic peptides of the silkworm, which can be used to distinguish animals with similar shapes from other animal drugs.

5 Determination of samples - Bombyx mori and its products

From Figure 8 to Figure 10, it can be seen that the silkworm medicinal materials, silkworm decoction pieces, silkworm standard decoction, silkworm traditional Chinese medicine formula granules, fried silkworm decoction pieces, fried silkworm standard decoction, fried silkworm traditional Chinese medicine formula granules and the comparison The consistent chromatographic peaks indicated that the detected two pairs of ion pairs existed in the processed products of Bombyx mori, standard decoction containing Bombyx mori and traditional Chinese medicine formula granules.

Buy Zhongfeng Huichun Pills, Rupi Sanjie Capsules, Yixian Pills, Epilepsy Ping Tablets, and Xiaoer Qizhen Pills from pharmacies, which contain silkworms, and use the original powder as medicine or water extraction process, and use the established silkworm mass spectrometry The identification method was tested, and the results are shown in Figure 11 and Figure 12. The results showed that the components of Bombyx mori can be detected in these five formulations, indicating that the mass spectrometry identification method established can be used for the detection of Bombyx mori components in Bombyx mori and its products.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, which are convenient for a specific and detailed understanding of the technical solution of the present invention, but should not be construed as limiting the protection scope of the invention patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. It should be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent for the present invention shall be determined by the content of the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims (13)

1. A characteristic polypeptide of Bombyx mori, characterized in that the characteristic polypeptide of Bombyx mori comprises a peptide with an amino acid sequence as shown in SEQ ID No.1 or/and a peptide with an amino acid sequence as shown in SEQ ID No.2.
2. The silkworm characteristic polypeptide according to claim 1, wherein the amino acid sequence is a peptide segment as shown in SEQ ID No.1, the m/z of the parent ion is 823.36, and the m/z of the product ion is 1070.47, 1345.57; or/and, the amino acid sequence is the peptide segment shown in SEQ ID No.2, the m/z of the parent ion is 637.29, and the m/z of the product ions are 825.36 and 926.40.
3. A method for identifying silkworms, silkworm water extract products, and other silkworm products, characterized in that the identification method includes the step of detecting characteristic components of silkworms in samples to be identified, and the characteristic components of silkworms are It is the characteristic polypeptide of Bombyx mori according to claim 1 or 2.
4. The identification method of silkworm silkworm, water extract product of silkworm silkworm and other silkworm products according to claim 3, wherein the step of detecting characteristic components of silkworm silkworm in the sample to be identified comprises:

   Take the sample to be identified and the characteristic components of the silkworm respectively to prepare a test solution and a reference solution; use ultra-high performance liquid chromatography mass spectrometry to detect the test solution and the reference solution .
5. The identification method of silkworm silkworm, water extract product of silkworm silkworm and other silkworm products according to claim 4, characterized in that, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of ultra-high performance liquid chromatography include:

   Stationary phase: C18 chromatographic column;

   Mobile phase: mobile phase A is acetonitrile, mobile phase B is formic acid aqueous solution containing 0.08%～0.12% formic acid by volume, and the elution method adopts gradient elution.
6. The identification method of silkworm silkworm, water extract product of silkworm silkworm and other silkworm products according to claim 5, wherein the conditions of gradient elution include:

   0min～3min, the volume percentage of described mobile phase A is 3%;

   3min～8min, the volume percentage of described mobile phase A rises from 3% to 5%;

   8min～10min, the volume percentage of described mobile phase A is 5%;

   10min～18min, the volume percent of described mobile phase A rises from 5% to 7%;

   18min～19min, the volume percent of described mobile phase A rises from 7% to 90%;

   From 19 min to 21 min, the volume percentage of the mobile phase A is 90%.
7. According to the identification method of silkworm silkworm, water extract product of silkworm silkworm and other silkworm products according to any one of claims 4 to 6, it is characterized in that, in the ultra-high performance liquid chromatography-mass spectrometry method, the ultra-high performance liquid phase The chromatographic conditions also include: the column temperature is 28°C-32°C; or/and, the flow rate is 0.25mL/min-0.35mL/min; or/and, the injection volume is 4.5μL-5.5μL.
8. According to the identification method of silkworm silkworm, silkworm water extract product and other silkworm products according to any one of claims 4 to 6, it is characterized in that, in the ultra-high performance liquid chromatography-mass spectrometry method, the conditions of mass spectrometry include : Electrospray positive ion mode; multiple reaction monitoring scan; capillary voltage: 0.45kV～0.55kV; ion source temperature: 480℃～520℃; solvent gas flow rate: 850L/hr～950L/hr.
9. According to the identification method of silkworm silkworm, silkworm water extract product and other silkworm products according to any one of claims 4 to 6, it is characterized in that the preparation of the reference solution comprises the steps of: taking the silkworm silkworm Characteristic ingredients, dissolved in ammonium bicarbonate solution.
10. The method for identifying silkworms, silkworm water extracts and other silkworm products according to claim 9, wherein the ammonium bicarbonate solution contains 0.8% to 1.2% by mass of ammonium bicarbonate.
11. According to the identification method of silkworm silkworm, water extract product of silkworm silkworm and other silkworm products according to any one of claims 4 to 6 and 10, it is characterized in that the preparation of the test solution comprises the following steps: The identified samples were extracted in ammonium bicarbonate solution, and the extract was collected and hydrolyzed with trypsin.
12. The method for identifying silkworms, silkworm water extracts and other silkworm products according to claim 11, characterized in that the ammonium bicarbonate solution contains 0.8%-1.2% by mass of ammonium bicarbonate.
13. The method for identifying silkworms, silkworm water extracts and other silkworm products according to any one of claims 4 to 6, 10 and 12, wherein the silkworm water extracts contain silkworm standard Decoction, fried silkworm standard decoction, silkworm traditional Chinese medicine formula granules, fried silkworm traditional Chinese medicine formula granules and epilepsy tablets; or/and, the other silkworm products include Zhongfenghuichun pills, Rupi Sanjie capsules, medical epilepsy Pills and Xiaoer Qizhen Pills.

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