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CN-122011104-A - Characteristic peptide segment for identifying male and female ground beetles, and detection method and application thereof

CN122011104ACN 122011104 ACN122011104 ACN 122011104ACN-122011104-A

Abstract

The invention discloses a characteristic peptide segment for identifying male and female ground beetles and a detection method thereof, and the characteristic peptide segment for identifying male and female ground beetles, which has a sequence of Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys、Ile-Val-Pro-Ile-Val-Glu-Pro-Glu-Val-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Arg、Thr-Gly-Ala-Ile-Val-Asp-Val-Pro-Val-Gly-Asp-Glu-Leu-Leu-Gly-Arg, is screened out through a large number of experiments, has high specificity and can be used for identifying medicines containing medicinal materials of male ground beetles. The detection method of the male ground beetle characteristic peptide fragment provided by the invention screens out the optimal enzymolysis condition, the chromatographic condition and the mass spectrum condition through a large number of experiments, and the whole method is simple to operate, accurate to judge, favorable for quality control of ground beetle medicines and has important significance for quality control of the medicines containing ground beetles.

Inventors

  • LIU RUI
  • GE HUI
  • LIU PING
  • GUO SHENG
  • DUAN JINAO

Assignees

  • 南京中医药大学

Dates

Publication Date
20260512
Application Date
20260227

Claims (9)

  1. 1. The characteristic peptide segment for identifying male and female ground beetles is characterized by comprising any one or a combination of the following P1-P3; P1: Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys P2: Ile-Val-Pro-Ile-Val-Glu-Pro-Glu-Val-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Arg P3: Thr-Gly-Ala-Ile-Val-Asp-Val-Pro-Val-Gly-Asp-Glu-Leu-Leu-Gly-Arg The reference peptide PR Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asp-Gly-Thr-Ile-Thr-Thr-Lys.
  2. 2. A detection method for identifying characteristic peptide fragments of male and female ground beetles, which is characterized by comprising the following steps: (1) Preparing the characteristic peptide fragments P1-P3 and the reference peptide PR as reference substance solutions; (2) And (3) after carrying out enzyme digestion on male and female ground beetle samples to be detected by trypsin, respectively injecting enzymolysis liquid and the characteristic peptide fragment reference substance solution in the step (1) into a liquid chromatograph-mass spectrometer, taking characteristic peptide fragments P1-P3 and reference peptide PR as references, adopting a multi-reaction monitoring mode, and selecting the ion pairs as follows: P1:657.9→781.4、657.9→668.2; P2:710.4→639.5、710.4→739.6; P3:806.1→955.2、806.1→343.1; PR:615.6→649.5、615.6→823.1; if the ion pair is detected, the retention time of the ions is consistent with that of the characteristic peptide fragment reference substance, the sub-ions are consistent with that of the reference substance, and the relative content of the ion pair of the characteristic peptide fragments P1-P3 of the male and the reference peptide PR ion pair is detected to be higher than that of the female.
  3. 3. The method for detecting the characteristic peptide fragments for identifying the male and female ground beetles according to claim 2, wherein the enzyme digestion method is characterized in that a sample of the ground beetles to be detected is taken, tris-HCl buffer solution is added for re-dissolution, ultrasound is carried out until the sample is completely dissolved, 1600 mug of protein is taken, tris-HCl buffer solution is added for dilution, trypsin is added, and incubation is carried out in a constant-temperature water bath.
  4. 4. The method for detecting the characteristic peptide fragments for identifying the ground beetles according to claim 3, wherein the enzyme digestion method comprises the steps of taking 30 mg of each sample of the ground beetles to be detected, adding 1mL of precooled 4% SDS solution, grinding, homogenizing, centrifuging to obtain a supernatant, BCA measuring the protein content, taking 1600 mug of protein, adding Tris-HCl buffer with the pH of 8.8 and the concentration of 50mM to dilute to 200 mul, incubating with DTT and IAA, precipitating with 80% acetone at 20 ℃ to 4h, centrifuging to discard the supernatant, volatilizing the acetone, re-dissolving with 8M urea, diluting with Tris-HCl buffer with the pH of 8.8 and the concentration of 50mM to 1M, Adding 0.1 μg/μl trypsin 40 μl, incubating in 37 deg.C constant-temperature water bath for 12 hr, desalting with Seppak C 18 desalting column, volatilizing, and redissolving with 10% acetonitrile.
  5. 5. The detection method for identifying characteristic peptide fragments of male ground beetles according to claim 2, wherein the liquid phase condition of detection by a liquid chromatograph is that a chromatographic column is 1.7 mu m acquisition UPLC 18 column, the specification is 2.1 mu m multiplied by 100 mm, the loading amount is 2 mu l, the flow rate is 0.3 ml/min, a mobile phase A is acetonitrile containing 0.1% formic acid, a mobile phase B is water containing 0.1% formic acid, the elution mode is 0-2 min, 10-30% A linear gradient elution, 2-6 min, 30-50% A linear gradient elution, 6-7 min,50% A isocratic elution, triple quadrupole mass spectrum is adopted, the mass spectrum parameters are that the ion source temperature is 500 ℃, the ionization voltage is 5500V, the desolvation temperature is 500 ℃, the ion source gas is 1,60 psi, and the ion source gas is 2,60 psi; the ion pairs for MRM mode are: 。
  6. 6. The use of the characterizing peptide fragment of claim 1 for identifying male and female ground beetles.
  7. 7. Use of the characterizing peptide fragment of claim 1 for identifying a marker of male ground beetle.
  8. 8. The use according to claim 6 or 7, wherein the relative content of characteristic peptide fragment P1 ion pair 657.9 > 781.4, P2 ion pair 710.4 > 639.5, P3 ion pair 806.1 > 955.2 and reference peptide ion pair PR 615.6 > 823.1 in the male Eupolyphaga are above 10.0, 537.3, 126.2 respectively.
  9. 9. The use according to claim 6 or 7, wherein the relative content of characteristic peptide fragment P1 ion pair 657.9 > 781.4, P2 ion pair 710.4 > 639.5, P3 ion pair 806.1 > 955.2 and reference peptide ion pair PR 615.6 > 823.1 in female Eupolyphaga are below 1.7, 294.2, 50.3 respectively.

Description

Characteristic peptide segment for identifying male and female ground beetles, and detection method and application thereof Technical Field The invention relates to a detection and identification method of animal drugs, in particular to a characteristic peptide segment for identifying male and female ground beetles, a detection method and application thereof. Background Eupolyphaga Seu Steleophaga, is a dried body of female Eupolyphaga sinensis Walker Eupolyphaga SINENSIS WALKER or Eupolyphaga Seu Steleophaga Steleophaga plancyi (Boleny) belonging to family Eupolyphaga Seu Steleophaga, which is originally found in Shennong's herbal channels, is listed as a medicinal material with moderate medicinal value, and has been conventionally promoted for its effects of promoting blood circulation, removing blood stasis, removing thrombus, and promoting tendon and bone healing. Eupolyphaga Seu Steleophaga is recorded in 2025 edition of Chinese pharmacopoeia, and has effects in removing blood stasis, promoting reunion of fractured bones, and treating traumatic injury, fracture due to injury, amenorrhea due to blood stasis, puerperal abdominal pain due to obstruction, and lump. Modern pharmacological research shows that ground beetle has wide pharmacological effects in resisting tumor, easing pain, resisting blood coagulation, promoting fracture healing and the like. The ground beetle substance basic research shows that the ground beetle main chemical components are protein, amino acid, polysaccharide, volatile oil and the like. According to the regulations of Chinese pharmacopoeia, only female woodlouse is approved for medicinal use, and the confusion of female woodlouse exists in the market, so that the male and female individuals are difficult to distinguish by the traditional method. The invention screens out three characteristic peptide markers of male ground beetles through experiments, can be used for detecting whether male worms are contained in female worms, and can also detect the adulteration proportion of male worms in different ground beetle samples. The research result provides a practical molecular tool for quality control and standardization of ground beetle medicinal materials. Disclosure of Invention The invention mainly aims to provide a characteristic peptide segment for identifying male ground beetles and a detection method thereof aiming at the problems and the defects. The ground beetle characteristic peptide fragment provided by the invention has strong specificity. The provided method is simple to operate and high in sensitivity, can accurately identify whether female ground beetle medicinal materials contain male ground beetle components, and provides a scientific method for guaranteeing the quality of ground beetles and products thereof. The technical scheme adopted by the invention is as follows: The characteristic peptide segment is used for identifying male and female ground beetles and comprises any one or combination of the following P1-P3; P1: Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys P2: Ile-Val-Pro-Ile-Val-Glu-Pro-Glu-Val-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Arg P3: Thr-Gly-Ala-Ile-Val-Asp-Val-Pro-Val-Gly-Asp-Glu-Leu-Leu-Gly-Arg。 The reference peptide PR Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asp-Gly-Thr-Ile-Thr-Thr-Lys. The P1-P3 and the reference peptide PR are replaced by amino acid abbreviations with the following formats: P1: EMSWIADTYAK P2: IVPIVEPEVLPDGDHDLDR P3: TGAIVDVPVGDELLGR PR: EAFSLFDKDGDGTITTK The reference peptide PR adopted by the invention is contained in both male and female ground beetles and has equivalent content, and the reference peptide is adopted by the invention for homogenizing the relative content of male and female characteristic peptides, so that the content of the characteristic peptide segments P1-P3 in the male and female ground beetles can be detected more accurately. A detection method for identifying characteristic peptide fragments of ground beetles, comprising the steps of: (1) Preparing a reference substance solution from characteristic peptide segments P1-P3 for identifying ground beetles and a reference peptide PR; (2) After enzyme digestion is carried out on an animal drug sample of ground beetles to be detected by trypsin, the enzymolysis liquid and the characteristic peptide fragment mixed reference substance solution for identifying the ground beetles in the step (1) are injected into a liquid-mass spectrometer, characteristic peptide fragments P1-P3 and reference peptide PR are used as references, a multi-reaction monitoring mode is adopted, and ion pairs are selected as follows: P1:657.9→ 781.4 (for relative content detection), 657.9→ 668.2; p2:710.4 → 639.5 (for relative content detection), 710.4 → 739.6; P3:806.1→ 955.2 (for relative content detection), 806.1→ 343.1; PR 615.6-649.5, 615.6-823.1 (for relative content detection); Detecting, if the ion pair is detected, and the retention time of the ions is consistent with that of a cha