A characteristic peptide of cordyceps and application thereof

Abstract

The application discloses a characteristic peptide of Pheretima guangxiensis and application thereof, and belongs to the technical field of biological medicines. The characteristic peptide is LLNYVDYK, EFTEEQLR, LQAELAR, ALETQVK, FVVLK, ARDDPHCCSAQDR or GILAADESTATIGK. The characteristic peptide is highly specific, and a detection method thereof is simple in operation and high in sensitivity, so that whether the Pheretima guangxiensis component is contained in the animal medicine can be accurately identified, and a scientific method is provided for guaranteeing the quality of the Pheretima guangxiensis and products thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a characteristic peptide of *Pheretima aspergillum* and its applications. Background Technology

[0002] Guangdilong is included in the 2020 edition of the Chinese Pharmacopoeia. It is a single species, namely the earthworm *Pheretima asiatica*, belonging to the family Lepididae. Pheretima aspergillum (E. perrier); Guangdilong (Earthworm) mainly originates from Guangdong, Guangxi, and Fujian provinces. Guangdilong is salty and cold in nature, and enters the liver, spleen, and bladder meridians. It has the effects of clearing heat and calming convulsions, unblocking meridians, relieving asthma, and promoting diuresis. Modern pharmacological studies have shown that earthworm has antitussive and antiasthmatic effects, fibrinolytic and anticoagulant effects, anti-liver fibrosis effects, antihypertensive and lipid-lowering effects, anti-inflammatory and analgesic effects, anti-tumor effects, and sedative and anticonvulsant effects. Research on the material basis of Guangdilong shows that proteins, peptides, amino acids, nucleosides, fatty acids, and inorganic elements are its main components. The quality standards for Guangdilong are not perfect. Guangdilong medicinal materials, compound Guangdilong granules, and prepared medicines containing Guangdilong lack specific identification and content determination methods. There is a phenomenon in the medicinal material market of mixing other types of earthworm.

[0003] Studies have shown that DNA barcoding technology based on COI sequences can be used to identify adulterants of *Pheretima aspergillum* (Guangdi Necrophora). However, the high-temperature extraction process of traditional Chinese medicine can affect the integrity of DNA, thus impacting the accuracy of DNA barcoding identification. Compared to DNA molecules, the primary sequence information of proteins and peptides is better preserved under high-temperature extraction conditions. This invention detects *Pheretima aspergillum* components by detecting characteristic peptides. This method provides a reference and basis for *Pheretima aspergillum* quality research and is conducive to further improving the quality standards of *Pheretima aspergillum* and its products. Summary of the Invention

[0004] Technical problem solved: To address the above-mentioned technical problems, this invention provides a characteristic peptide of *Pheretima aspergillum* and its application. This characteristic peptide has strong specificity, and the detection method is simple to operate and highly sensitive. It can accurately identify whether *Pheretima aspergillum* is present in earthworm animal medicines, providing a scientific method to ensure the quality of *Pheretima aspergillum* and its products.

[0005] Technical solution: A characteristic peptide of *Earthworm*, wherein the characteristic peptide is: Leu-Leu-Asn-Tyr-Val-Asp-Tyr-Lys, Glu-Phe-Thr-Glu-Glu-Gln-Leu-Arg, Leu-Gln-Ala-Glu-Leu-Ala-Arg, Ala-Leu-Glu-Thr-Gln-Val-Lys, Phe-Val-Val-Leu-Lys, Ala-Arg-Asp-Asp-Pro-His-Cys-Cys-Ser-Ala-Gln-Asp-Arg or Gly-Ile-Leu-Ala-Ala-Asp-Glu-Ser-Thr-Ala-Thr-Ile-Gly-Lys.

[0006] Application of a characteristic peptide of *Pheretima aspergillum* in the detection of *Pheretima aspergillum*.

[0007] Preferably, the application includes the following steps: (1) Prepare the characteristic peptide of Guangdilong as a solution as a reference; (2) The earthworm sample to be tested is digested with trypsin to obtain an enzymatic hydrolysate. The enzymatic hydrolysate and the reference solution in step (1) are injected into a liquid chromatography-mass spectrometry instrument and detected using multiple reaction monitoring mode. If the same ion pair as the reference is detected in the enzymatic hydrolysate, and the retention time of the ion is consistent with that of the reference, and its daughter ion is consistent with that of the reference, then the earthworm sample to be tested contains earthworm component.

[0008] Preferably, the liquid chromatography conditions used in the liquid chromatography-mass spectrometry (LC-MS) instrument are as follows: mobile phase A is formic acid, mobile phase B is formic acid acetonitrile; elution with a linear gradient of 5%B to 25%B for 0-15 min, linear gradient elution with a linear gradient of 25%B to 50%B for 15-20 min, and linear gradient elution with a linear gradient of 50%B to 5%B for 20-21 min.

[0009] Preferably, the liquid chromatography-mass spectrometry (LC-MS) instrument uses triple quadrupole mass spectrometry for detection.

[0010] Preferably, the precursor ions used in the liquid chromatography-mass spectrometry (LC-MS) instrument are 514.27, 526.25, 400.73, 394.72, 303.20, 529.88, or 673.86.

[0011] Preferably, the earthworm sample to be tested is an earthworm animal medicine, which includes *Pheretima asiatica*, *Pheretima spp.*, *Pheretima spp.*, *Pheretima hainanensis*, and *Pheretima hainanensis*.

[0012] Beneficial effects: The seven characteristic peptides of *Pheretima aspergillum* screened by this invention have high specificity and can be used to identify medicines containing *Pheretima aspergillum*.

[0013] The method for detecting characteristic peptides of *Pheretima aspergillum* provided by this invention is simple to operate, accurate in judgment, and can accurately distinguish characteristic peptides of *Pheretima aspergillum*, which is beneficial to the quality control of *Pheretima aspergillum* drugs and is of great significance for ensuring the quality of drugs containing *Pheretima aspergillum*. Attached Figure Description

[0014] Figures 1-7 The XIC diagrams show the characteristic peptides of different earthworms. Among them, (A) is Guangdilong; (B) is Hudilong (Pheretima spp.); (C) is Hudilong (Pheretima spp.); (D) is Hudilong (Pheretima spp.); and (E) is Hainan giant earthworm. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0016] Example 1 This invention screened a characteristic peptide from *Pheretima aspergillum*, the characteristic peptide being: Leu-Leu-Asn-Tyr-Val-Asp-Tyr-Lys, Glu-Phe-Thr-Glu-Glu-Gln-Leu-Arg, Leu-Gln-Ala-Glu-Leu-Ala-Arg, Ala-Leu-Glu-Thr-Gln-Val-Lys, Phe-Val-Val-Leu-Lys、 Ala-Arg-Asp-Asp-Pro-His-Cys-Cys-Ser-Ala-Gln-Asp-Arg or Gly-Ile-Leu-Ala-Ala-Asp-Glu-Ser-Thr-Ala-Thr-Ile-Gly-Lys.

[0017] See the sequence list: Example 2 The following steps were taken to detect characteristic polypeptides in various earthworm-derived medicinal products: (1) Take 10 mg each of *Pheretima hainanensis*, *Pheretima wieldiensis*, *Pheretima hainanensis*, and *Earthworm hainanensis*, and add 1 ml of 50 mM 4 wt.% SDS-Tris-HCl buffer (pH 8.8) to each sample. Incubate overnight at 65°C. For BCA protein content determination, take 200 μg of protein, add an appropriate amount of DTT at a volume ratio (protein:DTT / 10:1), incubate at 37°C for 0.5 h, then add IAA to 20 mmol / L, alkylate in the dark for 0.5 h, and finally add 5.5 times the total volume of 80 vt.% acetone (ice acetone), and precipitate overnight (-20°C). Use 80 The precipitate was washed with 1% acetone, and then 200µl of urea (8mol / L) was added to dissolve the precipitate. The urea concentration was diluted to below 1mol / L with Tris-HCl buffer solution. Finally, 2µg of 1% trypsin was added by mass percentage and the mixture was enzymatically hydrolyzed for 12 hours.

[0018] (2) Prepare 1 ml of a characteristic peptide reference solution with a concentration of 200 ng / ml by dissolving the pure characteristic peptide described in Example 1.

[0019] (3) The enzymatic hydrolysate from each dragon in step (1) and the characteristic peptide reference solution in step (2) were analyzed by liquid chromatography-mass spectrometry (LC-MS). The LC conditions were as follows: the column was a 3.5µm Select HSS T3 column with a specification of 4.6×150mm; the sample loading volume was 2µl; the flow rate was 0.4ml / min; the mobile phase A was 0.1 vt.% formic acid; the mobile phase B was 0.1 vt.% formic acid acetonitrile; the elution was carried out with a linear gradient of 5%B to 25%B for 0~15min, 25% to 50%B for 15~20min, and 50% to 5%B for 20~21min; the column temperature was 30℃.

[0020] Triple quadrupole mass spectrometry was used, with the following parameters: ion source temperature 500℃, ionization voltage 5500V, and desolventization temperature 500℃. Ion source gas 1, 60 psi; ion source gas 2, 60 psi; ion pairs in MRM mode: m / z 514.27 double charge → 801.38, DP = 80, CE = 10; m / z double charge 526.25 → 545.30, DP = 60, CE = 20; m / z 400.73 double charge → 559.32, DP = 50, CE = 15; m / z 394.72 double charge → 604.33, DP = 80, CE = 10; m / z 303.20 double charge → 359.26, DP = 60, CE = 10; m / z 529.88 double charge → 458.20, DP = 100, CE = 10; m / z 673.86 double charge → 418.27, DP = 100, CE = 40.

[0021] See results Figures 1-7 The same ion peaks as the characteristic peptide reference solution could be detected in the Guangdilong sample, while none were detected in other samples. That is, the same parent ion pairs and daughter ions as those in the reference solution were detected in the enzymatic hydrolysate, and the retention time of the ions was consistent with that of the reference solution. This indicates that the present invention can specifically detect the characteristic peptide components in Guangdilong, thereby distinguishing it from other earthworm animal drugs.

Claims

1. A characteristic peptide of *Pheretima aspergillum*, characterized in that, The characteristic peptides are: Leu-Leu-Asn-Tyr-Val-Asp-Tyr-Lys, Glu-Phe-Thr-Glu-Glu-Gln-Leu-Arg, Leu-Gln-Ala-Glu-Leu-Ala-Arg, Ala-Leu-Glu-Thr-Gln-Val-Lys, Phe-Val-Val-Leu-Lys, Ala-Arg-Asp-Asp-Pro-His-Cys-Cys-Ser-Ala-Gln-Asp-Arg or Gly-Ile-Leu-Ala-Ala-Asp-Glu-Ser-Thr-Ala-Thr-Ile-Gly-Lys.

2. A method for detecting *Pheretima aspergillum*, characterized in that, The method includes the following steps: (1) The characteristic peptide of *Pheretima aspergillum* as described in claim 1 is prepared into a solution as a reference standard; (2) The earthworm sample to be tested is digested with trypsin to obtain an enzymatic hydrolysate. The enzymatic hydrolysate and the reference solution in step (1) are injected into a liquid chromatography-mass spectrometry instrument and detected using multiple reaction monitoring mode. If the same ion pair as the reference is detected in the enzymatic hydrolysate, and the retention time of the ion is consistent with that of the reference, and its daughter ion is consistent with that of the reference, then the earthworm sample to be tested contains earthworm component.

3. The method for detecting *Pheretima aspergillum* according to claim 2, characterized in that, The liquid chromatography conditions used in the liquid chromatography-mass spectrometry (LC-MS) instrument are as follows: mobile phase A is formic acid, mobile phase B is formic acid acetonitrile; elution is performed with a linear gradient of 5%B to 25%B for 0-15 min, elution with a linear gradient of 25%B to 50%B for 15-20 min, and elution with a linear gradient of 50%B to 5%B for 20-21 min.

4. The method for detecting *Pheretima aspergillum* according to claim 2, characterized in that, The liquid chromatography-mass spectrometry (LC-MS) instrument uses triple quadrupole mass spectrometry for detection.

5. The method for detecting *Pheretima aspergillum* according to claim 2, characterized in that, The precursor ion used for the LC-MS / MS detection of Leu-Leu-Asn-Tyr-Val-Asp-Tyr-Lys was 514.27, for Glu-Phe-Thr-Glu-Glu-Gln-Leu-Arg it was 526.25, for Leu-Gln-Ala-Glu-Leu-Ala-Arg it was 400.73, for Ala-Leu-Glu-Thr-Gln-Val-Lys it was 394.72, and for Phe-Val-Val-Leu-Lys it was... The precursor ion used was 303.

20. The precursor ion used for detecting Ala-Arg-Asp-Asp-Pro-His-Cys-Cys-Ser-Ala-Gln-Asp-Arg was 529.

88. The precursor ion used for detecting Gly-Ile-Leu-Ala-Ala-Asp-Glu-Ser-Thr-Ala-Thr-Ile-Gly-Lys was 673.

86.

6. The method for detecting *Pheretima aspergillum* according to claim 2, characterized in that, The earthworm samples to be tested are earthworm animal medicines, including *Pheretima asiatica*, *Pheretima spp.*, *Pheretima spp.*, *Pheretima hainanensis*, and *Pheretima hainanensis*.

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