Thrombolytic activity marker peptide of earthworm and its application in the determination of thrombolytic activity of earthworm

Abstract

The present invention discloses a marker peptide for thrombolytic activity of earthworms and its application in determining the thrombolytic activity of earthworms. The marker peptides for thrombolytic activity of earthworms include: marker peptides 1 to 11 for thrombolytic activity of Earthworms, whose amino acid sequences are shown in SEQ ID NOs: 1 to 11; and marker peptides 3 and 11 for thrombolytic activity of Earthworms, whose amino acid sequences are shown in SEQ ID NOs: 3 and 11. The present invention screened out 11 marker peptides for thrombolytic activity from Earthworms, the contents of which showed a strong positive correlation with the thrombolytic activity of earthworms, and thus could be used for determining the thrombolytic activity of Earthworms; two of the 11 marker peptides showed a strong correlation with the thrombolytic activity of Earthworms, and thus could be used for determining the thrombolytic activity of Earthworms.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis and detection, in particular to a marker peptide of earthworm thrombolytic activity and an application thereof in determining the earthworm thrombolytic activity. Background Art

[0002] Earthworms are a traditional Chinese medicine first recorded in the Shennong Bencao Jing (Shennong Bencao Jing) and commonly used to treat cardiovascular and cerebrovascular diseases such as stroke. They are known for their heat-clearing and tranquilizing properties, promoting blood circulation and unblocking meridians, and relieving asthma and promoting urination. The 2020 edition of the Chinese Pharmacopoeia lists earthworms as derived from the dried bodies of Pheretima aspergillum (E. Perrier), Pheretima vulgaris Chen, Pheretima guillelmi (Michaelsen), or Pheretima guillelmi (Michaelsen), all of the family Pheretimaceae. The former is commonly known as "Guangdilong," while the latter three are commonly known as "Hudilong." The 2020 edition of the Chinese Pharmacopoeia, Part I, includes 41 formulated earthworm-containing formulas. Among them, 3 have the effect of clearing away heat and calming the nerves, accounting for 7.32% of the total preparations; 3 have the effect of relieving asthma and promoting diuresis (7.32%); 3 have the effect of calming the liver and suppressing yang and reducing hypertension (7.32%); the remaining 32 mainly use the effect of earthworms to promote blood circulation and dredge the meridians, accounting for 78.04% of the total preparations. They are mainly used for patients with clinical symptoms such as difficulty in flexing and extending the legs and feet, cramps in the hands and feet, or soreness of the waist and knees, dark lips, dark red tongue or ecchymosis, etc. with blood stasis symptoms. Severe cases may experience hemiplegia, numbness on one side of the body, crooked mouth and tongue, slurred speech and other stroke symptoms.

[0003] Earthworms are rich in proteins, peptides, amino acids, and nucleosides. Lumbrokinase, a fibrinolytic kinase extracted from earthworms, is the primary thrombolytic agent. It is also known as earthworm fibrinolytic enzyme (EFE). Lumbrokinase standards are a group of enzymes isolated from the earthworm Eisenia foetida. Currently, thrombolytic activity in earthworms is typically measured using the agarose fibrinolytic plate assay, using lumbrokinase standards as a control. The fibrinolytic activity is determined by measuring the diameter of the fibrinolytic zone. This method, which measures the overall activity of a class of lumbrokinases, suffers from poor specificity, linearity, and stability.

[0004] In recent years, based on proteomics research, the establishment of quality control methods using characteristic peptides as indicators has opened up new prospects for improving the quality control standards of animal-based traditional Chinese medicines. Patent CN111303263A reported that 5 characteristic peptides were used to identify the species of common earthworm species, including Pheretima ginseng, Pheretima tongguan, and Pheretima baoningensis. The applicant's research group previously studied the proteomic differences between earthworm species and screened out 6 ion pairs for identifying the authenticity of medicinal earthworms. They can accurately distinguish between Pheretima ginseng, Pheretima tongguan, and Pheretima ctenophora, and can also identify common counterfeit earthworms such as Pheretima baoningensis and Pheretima zhiliensis. (Gu, Y., Zhang, J., Sun, J., Yu, H., Feng, R., Mao,

[0005] However, current methods for identifying characteristic peptides can only be used to identify earthworm species and cannot detect the activity of the herb. The thrombolytic activity of earthworms on the market varies widely, and some even show no activity. Therefore, identifying a set of marker peptides that accurately reflect the thrombolytic activity of earthworms and establishing an activity assay are crucial for quality control of earthworms, regulating the earthworm market, and ensuring clinical drug safety. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide a marker peptide for the thrombolytic activity of earthworms and its application in determining the thrombolytic activity of earthworms.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a group of marker peptides of thrombolytic activity of earthworms, including

[0009] Pheretima thrombolytic activity marker peptides 1 to 11, whose amino acid sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 11;

[0010] The amino acid sequences of the thrombolytic activity marker peptides 3 and 11 of Hudilong are shown in SEQ ID NO: 3 and SEQ ID NO: 11.

[0011] Furthermore, the earthworms are common earthworms and William earthworms.

[0012] Furthermore, the mass spectrometry conditions for the thrombolytic activity marker peptides 1 to 11 are:

[0013]

[0014]

[0015] The second aspect of the present invention is to provide the use of the above earthworm thrombolytic activity marker peptide in the determination of earthworm thrombolytic activity.

[0016] The third aspect of the present invention is to provide a method for determining the thrombolytic activity of earthworms based on the above-mentioned earthworm thrombolytic activity marker peptide, characterized in that it comprises the following steps:

[0017] Step 1: Process the sample to be tested to obtain a peptide mixture to be tested;

[0018] Step 2: Using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to detect the polypeptide mixture to be tested, and obtaining a mass spectrum of the thrombolytic activity marker peptide of the test sample.

[0019] Furthermore, the chromatographic conditions for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer detection are:

[0020] Analytical column: CORTECS C18 column, 2.7 μm, 2.1 × 50 mm;

[0021] Injection volume: 1 μl;

[0022] Flow rate: 0.2 ml / min;

[0023] Mobile phase: Mobile phase A is 0.1% formic acid-water solution, mobile phase B is acetonitrile,

[0024] Gradient elution: 0-10 min, 2-35% B; 30-32 min, 35-90% B.

[0025] Furthermore, the mass spectrometry conditions for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer detection are: spray voltage 4000 V, gas temperature 270° C., flow rate 16 L / min, Nebulizer 45 psi, and MRM mode.

[0026] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0027] The present invention uses fresh earthworms as the research object, conducts label-free proteomics research on earthworm tissues and body cavity fluids, and TMT-labeled proteomics research on earthworm drying temperature. By systematically comparing the changes in up-regulated and down-regulated related proteins and peptides, 11 marker peptides related to thrombolytic activity were screened out, and their contents have a strong positive correlation with the activity measurement results.

[0028] The present invention screened out 11 thrombolytic activity marker peptides from Pheretima ginseng, the contents of which showed a strong positive correlation with the thrombolytic activity of earthworms, and could be used for the determination of the thrombolytic activity of Pheretima ginseng; among them, two had a strong correlation with the thrombolytic activity of earthworms, and could be used for the determination of the thrombolytic activity of earthworms. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The fibrinolytic enzyme spectrum characterizes the fibrinolytic activity of earthworm tissue and body cavity fluid;

[0030] Figure 2 The effect of different drying temperatures on the fibrinolytic activity protein of earthworms;

[0031] Figure 3 It is the Heatmap analysis for identifying non-redundant peptides in samples with different drying temperatures;

[0032] Figure 4 This is the DMRM chromatogram of the marker peptide of thrombolytic activity of Pheretima quinata;

[0033] Figure 5 It is the DMRM chromatogram of the marker peptide of thrombolytic activity of Pheretima serrata - continued;

[0034] Figure 6 This is the DMRM chromatogram of the marker peptide of thrombolytic activity of Pheretima ctenoides;

[0035] Figure 7 This is the DMRM chromatogram of the marker peptide of thrombolytic activity of Pheretima cyclophila;

[0036] Figure 8 It is the specificity verification of the thrombolytic activity marker peptide;

[0037] Figure 9 It is the content of marker peptides and fibrinolytic activity of Guangdilong medicinal material;

[0038] Figure 10 is the correlation coefficient r value of the Pearson correlation analysis between the content of marker peptides in Hudilong medicinal materials and fibrinolytic activity;

[0039] Figure 11 It is the thrombolytic activity and marker peptide content of Hudilong medicinal materials;

[0040] Figure 12 is the correlation coefficient r value of the Pearson correlation analysis between the content of marker peptides in Hudilong medicinal materials and fibrinolytic activity;

[0041] Figure 13 This is the EIC graph of the Dilong medicinal material sample in Example 1 of the present invention;

[0042] Figure 14 This is the EIC graph of the Dilong medicinal material sample in Example 2 of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0044] The present invention screened out 11 thrombolytic activity marker peptides from Pheretima serrata. Their contents showed a strong positive correlation with the thrombolytic activity of earthworms and can be used to determine the thrombolytic activity of Pheretima serrata. Among them, two showed a strong correlation with the thrombolytic activity of Pheretima serrata and can be used to determine the thrombolytic activity of Pheretima serrata. The specific content is as follows:

[0045] 1. Screening of characteristic peptides

[0046] Shotgun proteomics and TMT labeling proteomics strategies were used to enzymatically hydrolyze the extracted earthworm protein to obtain a peptide mixture. After chromatographic column separation, the mixture was analyzed by tandem mass spectrometry. Protein identification was performed by searching the database to determine protein information, peptide sequence, source protein, and other information. Statistical methods were used to screen for thrombolytic activity marker peptides.

[0047] 1.1 Extraction and determination of protein from earthworm tissue and coelomic fluid

[0048] 1) Extraction of earthworm coelomic fluid and tissue protein: 20 mg of earthworm coelomic fluid and tissue powder were added to 400 μl of 50 mM NH4HCO3 extraction solution, ultrasonically extracted at 37°C for 40 min, centrifuged, and the solid was added to 200 μl of 50 mM NH4HCO3 extraction solution and ultrasonically extracted at 37°C for 30 min. Centrifuged, and the supernatants were combined to obtain earthworm protein.

[0049] 2) Extraction of earthworm protein at different drying temperatures: 40 mg of earthworm coelomic fluid and tissue powder were added to 800 μl of 50 mM NH4HCO3 extract, extracted by ultrasonication at 37°C for 40 min, centrifuged, and the solid was added to 400 μl of 50 mM NH4HCO3 extract and extracted by ultrasonication at 37°C for 30 min. Centrifuged, the supernatants were combined, 400 μl of the solution was added to methanol-chloroform (1:1, v:v) solution, vortexed, and placed at 4°C overnight (12 hours). Centrifuged, and the upper layer was taken to obtain the freeze-dried earthworm thrombolytic protein.

[0050] 3) Reductive Alkylation: Take 100-200 μg of protein and add 200 μl of cold tris-phenol to each sample. Vortex at room temperature for 20 minutes. Precipitate overnight with five times the volume of 100 mM ammonium acetate in methanol prepared at -20°C. Centrifuge and remove the supernatant. Dissolve the solid in urea (8 M, 1% SDS) and remove the urea by ultrafiltration. Add 50 mM DTT (dissolved in 50 mM ammonium bicarbonate, prepared immediately before use, to a final DTT concentration of 25 mM) and incubate at 95°C for 30 minutes. Add 50 mM IAA (dissolved in 50 mM ammonium bicarbonate, prepared immediately before use, to a final IAA concentration of 50 mM) and incubate in the dark for 40 minutes.

[0051] 4) Trypsin digestion, desalting, and enrichment: Dissolve trypsin in 1 ml of acetic acid to a concentration of 100 μg / ml. Each sample was digested with trypsin (approximately 1:50, w / w) at 37°C for 18 hours. The reaction was terminated by acidification with 10% TFA to pH 2-3. The sample was lyophilized, phosphate buffer (pH 7.8) was added (200 μl), and digested with trypsin (approximately 1:50, w / w) at 45°C for 4 hours. C18 desalting: Buffer A: 38.4 ml H2O, 1.6 ml 5% TFA; Buffer B: 6.4 ml H2O, 32 ml ACN, 1.6 ml 10% TFA. Activate a C18 column with 50% ACN, pipette buffer A through the column, pipette the sample onto the column, desalt with buffer A, and extract peptides by pipetting 200 μl of buffer B. Repeat this process once. The product was spin-dried at room temperature in a vacuum centrifugal concentrator, 0.1% FA 100 μl was added for re-dissolution, and the supernatant was removed by centrifugation and placed in a plastic liquid phase vial.

[0052] 1.2 Determination of thrombolytic activity of earthworms

[0053] 1) Characterization of Lumina Protein Activity by Fibrinolytic Enzymography: Fibrinolytic zymography is a polyacrylamide gel-based assay for measuring fibrinolytic activity. Fibrinolytic proteins are separated according to their molecular weight. During incubation, the fibrin in the gel is enzymatically degraded. Fibrinolytic activity is characterized by measuring the specific proteolytic activity of the sample. Proteins with fibrinolytic activity appear as clear, transparent bands on the zymogram. Fibrinogen (final concentration 0.5 mg / mL) and thrombin (0.012 BP / mL) were added to a homemade 12% separating gel. After electrophoresis, the gel was destained by soaking in 2.5% Triton X-100 solution for 30 minutes. The gel was then incubated in PBS buffer (pH 7.4) at 37°C for 30 minutes, stained with Coomassie Brilliant Blue R250 overnight, and eluted with methanol-acetic acid-water (4:1:5, v:v:v) until a clear, transparent band was apparent. Images were taken using a Bio-Rad XR+.

[0054] The protein plasmin spectrum of the tissues and body fluids of Pheretima spp., Pheretima vulgaris and Pheretima ctenophora was shown in Figure 1The coelomic fluid showed fibrinolytic activity, with distinct bands that differed from those of lumbrokinase. The distribution of fibrinolytic proteins in the coelomic fluid varied significantly between species, with the majority being between 15kDa and 55kDa. The fibrinolytic protein bands in P. truncatum and P. vulgaris were more abundant, ranging from 15kDa to 35kDa. Tissues, however, showed no fibrinolytic activity. Label-free proteomics was performed using P. truncatum tissues and coelomic fluid to screen for marker peptides of thrombolytic activity in earthworms.

[0055] 2) Characterization of earthworm thrombolytic activity using agarose-fibrin plate: 250 mg of agarose gel was placed in a clean small beaker, and 30 ml of PBS buffer (pH 7.4) was added. Microwave-heated for 1 minute until completely dissolved to obtain an agarose solution. This solution was then kept warm in a 55°C waterbath and set aside. 50 mg of fibrinogen was dissolved in 30 ml of PBS buffer to obtain a fibrinogen solution. This solution was then kept warm in a 55°C waterbath and set aside. 1 ml of 4 BP / ml thrombin solution was added to the agarose solution and mixed thoroughly. The preheated fibrinogen solution was then slowly added, mixed thoroughly, and poured into a 15 cm diameter Petri dish. The solution was allowed to solidify for 1 hour before use. 5 μl of earthworm protein solution (concentration greater than 1 mg / ml) was loaded onto the plate. A 48,000 U / ml stock solution of lumbrokinase control was prepared by adding 0.5 ml of water. A series of dilutions were prepared, and 5 μl of the solution was loaded onto the plate. A standard curve was constructed with Log U as the ordinate and the area of ​​the lysis zone as the abscissa.

[0056] The samples of earthworms were freeze-dried (FD) and dried at 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 12 hours. The thrombolytic activity was characterized by plasminogram and plasminogen plate assay. As the drying temperature increased, a decrease in thrombolysis was observed in both assays. The plasminogram results showed that the fibrinolytic bands became lighter when the temperature was above 70°C, and the thrombolytic activity decreased significantly; however, the distribution of plasminogen bands was consistent in samples dried at different temperatures ( Figure 2 ).

[0057] Fibrinolytic activity can be quantitatively measured using a plasminogen plate. In this study, lumbrokinase control samples were used as a control. After drying at 80°C for 12 hours, fibrinolytic activity decreased by approximately 50%, and by 80% at 100°C. High-temperature drying significantly affects the thrombolytic activity of earthworms. Labeled proteomics studies were conducted using earthworm samples that had been freeze-dried and dried at 40°C, 60°C, 80°C, and 100°C to screen for marker peptides for thrombolytic activity.

[0058] 1.3 Chromatographic and mass spectrometry conditions

[0059] An UltiMate 3000 Nano RSLC-Orbitrap Fusion Lumos high-resolution mass spectrometer (Thermo Fisher, USA) was used with a Thermo Acclaim PepMap RSLC C18 2 μm analytical column (50 μm × 15 cm) and a Thermo Acclaim PepMap C18 3 μm pre-column (75 μm × 2 cm). The injection volume was 1 μl, the flow rate was 300 nl / min, the mobile phase A was (0.1% formic acid-water solution), and the mobile phase B was (acetonitrile / 0.1% formic acid-water solution = 80 / 20). The gradient elution was as follows: 0-5 min, 5% B; 5-90 min, 5-80% B; 91-100 min, 80% B; 100-105 min, 80%-5% B; 105-120 min, 5% B. Mass spectrometry conditions were: spray voltage 2.0 kV, ion transfer capillary temperature 320°C, high-energy induced dissociation (HCD) fragmentation mode, normalized collision energy 30%, resolution settings: primary 120 K, secondary 30 K, precursor ion scan range (m / z): 350–1500, dynamic data-dependent acquisition (DDA) was used for secondary mass spectrometry, and the "Top Speed" algorithm was used to select primary ions for HCD fragmentation scanning. Data acquisition and processing were performed using Xcalibur 4.0 software.

[0060] 1.4 Protein identification and preliminary screening of characteristic peptides

[0061] 1) Proteomic results identification

[0062] Proteome Discoverer 2.2 software analysis parameters: Nano LC-Orbirap Fusion Lumos mass spectrometry data were imported into PD2.2 software, and the Seuqest HT algorithm was used to analyze the MS 2Mass spectra were processed using a theoretical protein database derived from the earthworm transcriptome as the search library. Seuqest HT parameters were set as follows: trypsin enzyme, two missed cleavages allowed, minimum peptide length of 6, precursor ion mass tolerance of 5 ppm, and fragment ion mass tolerance of 0.02 Daltons. Fixed modifications included carbamidomethyl, 57.02 Da (C); variable modifications (peptide termini): pro-hydroxypro, 15.99 Da (P); oxidation, 15.99 Da (M); and lyshydroxy-lys, 15.99 Da (K). Variable modifications (protein termini): acetyl, 42.01 Da (N-terminus). Peptide match error rates were determined using a target-decoy strategy combined with Percolator modeling of correct and incorrect matches. A q-value chi-square of 0.01, determined by Percolator, was used to filter data at the peptide match level to control for false discoveries.

[0063] 2) Screening of marker peptides for thrombolytic activity

[0064] The selected thrombolytic activity marker peptides should have the following characteristics: (1) the number of amino acids is between 6 and 25; (2) the peptide segment is unmodified or has a fixed modification; (3) it can be effectively ionized, and the signal is stable and has a certain intensity.

[0065] 3) Screening of thrombolytic activity marker peptides based on label-free proteomics studies of earthworm tissue and coelomic fluid

[0066] Based on the purpose of discovering fibrinolytic activity markers, the present invention screened candidate targets from the following two aspects: one is to screen from non-redundant peptides downregulated in tissues, and the screening conditions are non-redundant peptides with abundance >E+8, detected in three biological replicates, and ratio variability (%) >100; the other is to screen from downregulated lumbrokinase.

[0067] 4) Screening of thrombolytic activity marker peptides based on TMT-labeled proteomics studies

[0068] Proteome Discoverer 2.4 software was used to analyze the heatmap of the identified non-redundant peptides. The clustering results showed that freeze-dried, 40℃ and 60℃ dried were clustered into one group, and 80℃ and 100℃ dried were clustered into one group ( Figure 3The Heatmap had two significantly downregulated regions, containing 132 non-redundant peptides. Marker peptides associated with thrombolytic activity were screened according to the following method: peptides with a drying ratio between 40°C and 60°C between 0.67 and 1.2, and a drying ratio between 80°C and 100°C less than 0.67.

[0069] 1.5 Characteristic peptide verification

[0070] Thrombolytic activity marker peptides initially screened using Proteome Discoverer software were validated by triple quadrupole mass spectrometry. Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (Agilent 1290 / Agilent 6495) was used on an Agilent CORTECS C18 column (2.7 μm, 2.1 × 50 mm). The injection volume was 1 μl, the flow rate was 0.2 ml / min, and the mobile phases A and B were 0.1% formic acid in water and B, respectively. The gradient elution was: 2-35% B over 0-10 min; 35-90% B over 30-32 min. Mass spectrometry conditions included a spray voltage of 4000 V, a gas temperature of 270°C, a flow rate of 16 L / min, a nebulizer at 45 psi, and multiple reaction monitoring (MRM) mode. Data were acquired and processed using Mass Hunter software. The specificity of the initially screened characteristic peptides was verified, and the results showed that 11 marker peptides could distinguish the activity of Pheretima serrata tissue and body fluid. Further verification in other species showed that 4 marker peptides could distinguish the activity of Pheretima serrata tissue and body fluid, and 5 marker peptides could distinguish the activity of Pheretima serrata tissue and body fluid. Figure 5-7 shown.

[0071] Table 1 Sequences of marker peptides for thrombolytic activity of earthworms and mass spectrometry conditions

[0072]

[0073] 2. Methodological Validation of the Determination Method for the Content of Thrombolytic Activity Marker Peptide

[0074] The present invention used a synthetic marker peptide of dragon thrombolytic activity as a control to investigate the linearity and range, precision, repeatability, stability and accuracy of the method. The results are as follows:

[0075] 2.1 Specificity test

[0076] In order to investigate the specificity of the established method, the specificity was investigated by comparing the retention time of the ion pairs with those of the synthetic marker peptide reference substances. The synthetic marker peptides were identified and the blank solvent had no interference. Figure 8 The results showed that the established method had good specificity.

[0077] 2.2 Standard curve

[0078] Accurately weigh an appropriate amount of synthetic peptide reference substance, and use water to prepare a mixed reference substance solution with a concentration of 5 μg / ml. Accurately measure an appropriate amount of the mixed reference substance solution into a volumetric flask of a certain volume to prepare a series of standard solutions. Pipette 1 μl of each reference substance solution for analysis. Draw a standard curve with the reference substance concentration (μg / ml) as the abscissa X and the peak area as the ordinate Y, and calculate the regression equation (Table 2).

[0079] Table 2 Linear relationship

[0080]

[0081] 2.3 Accuracy

[0082] Nine samples were taken and, before enzymatic hydrolysis, 11 thrombolytic activity marker peptide reference substances were precisely spiked with concentrations equivalent to 50% (low concentration level), 100% (medium concentration level), and 150% (high concentration level) of the original amount. Samples were prepared according to the enzymatic hydrolysis method used in the test sample preparation and used to determine the recovery rates of the spiked samples to investigate the accuracy of the method. The recovery rates of the 11 thrombolytic activity marker peptides are shown in Table 6-6. The ranges for PEP.1, PEP.2, PEP.3, and PEP.7 were 84.3% to 113.1%, 99.6% to 118.9%, 83.9% to 117.5%, and 85.5% to 108.5%, respectively. The RSDs were all less than 11%, indicating that the method had good accuracy. The recoveries of PEP.4, PEP.5, PEP.6, PEP.8, PEP.9, PEP.10, and PEP.11 ranged from 76.4% to 123.9%, 102.2% to 149.4%, 99.7% to 138.8%, 99.7% to 138.8%, 80.3% to 133.0%, 80.3% to 133.0%, 111.6% to 215.3%, 81.2% to 133.4%, and 101.3% to 199.7%, respectively, with relatively high RSDs. The recoveries of PEP.5 and PEP.11 were higher at low concentrations, but better at medium and high concentrations.

[0083] 2.4 Precision

[0084] Precisely measure 1 μl of the mixed reference solution and inject it six times for analysis. Peak areas are recorded. The results are shown in Table 3. Six consecutive analyses revealed an RSD of 11.82 for PEP.3 (GEFPWQLSMTR), demonstrating acceptable repeatability. The RSDs for the areas of the remaining 10 thrombolytic activity marker peptides were all less than 8%, demonstrating good parallelism for the reference solution.

[0085] Table 3 Precision investigation

[0086]

[0087] 2.5 Repeatability

[0088] Six 40mg samples of earthworms were accurately weighed and prepared according to the test solution preparation method described in the main text. The results are shown in Table 4. The relative standard deviation of the marker peptide PEP.3 in the six samples was 10.43%, indicating that the repeatability of the method is generally acceptable. The relative standard deviation of the peak area / sample weight (mg) for the remaining 10 marker peptides was less than 6.0%, indicating good repeatability of the method (Table 4).

[0089] Table 4 Repeatability test

[0090]

[0091]

[0092] 2.6 Stability

[0093] Take the test solution and inject 1 μl of it at 0, 4, 8, 12, 16, and 24 hours for analysis, and record the peak area. The results are shown in Table 5. The relative standard deviation of the sample peak area is less than 6.0%, indicating that the test solution is basically stable within 24 hours.

[0094] Table 5 Stability study of test solution

[0095]

[0096] 3. Correlation analysis between thrombolytic activity marker peptides and fibrinolytic activity

[0097] The content of 11 thrombolytic activity marker peptides was determined in 39 samples using established methods. These included 18 batches of Shanghai earthworms (A1-A16 were common earthworms, of which A1-A4 were home-made, and A17 and A18 were earthworms), 15 batches of Guangdong earthworms (B1-B5 were home-made), and 6 batches of counterfeit earthworms (C1-C6). The thrombolytic activity of the 39 samples was determined using the agarose-fibrin plate method. The correlation between the thrombolytic activity marker peptide content and fibrinolytic activity in the earthworms was determined using the Pearson correlation coefficient method.

[0098] 3.1 Correlation analysis between thrombolytic activity marker peptide and fibrinolytic activity of Dilong

[0099] Analysis of the content of thrombolytic activity marker peptides and fibrinolytic activity in different samples of Pheretima ternatum revealed that ( Figure 9), when the detection levels of PEP.1(TDASNILPNTLQK), PEP.3(GEFPWQLSMTR), PEP.4(VISTDECNR), PEP.5(DSCQGDSGGPLSVK), PEP.7(ATFDATIV), PEP.8(YAINVIGR), PEP.9(WPLDYFIK), PEP.10(VLYPSSGTAQDYSK) and PEP.11(VTMTPAPGLIYR), or PEP.2(TSASNILPNTLQK) and PEP.6(GSVIAGVGSWVVR) were lower than 1.198 and 4.757 μg / g, earthworms had no fibrinolytic activity.

[0100] from Figure 10 As can be seen, the 11 thrombolytic activity marker peptides screened from Pheretima serrata all showed extremely significant positive correlations with the fibrinolytic activity of Pheretima serrata (Pheretima serrata). Pearson correlation coefficients for PEP.3, 7, 8, 9, 10, and 11 were all greater than 0.90 (P < 0.01). This suggests that the thrombolytic activity marker peptides screened from Pheretima serrata are related to their thrombolytic activity. Furthermore, the correlation r values ​​between the 11 screened thrombolytic activity marker peptides ranged from 0.790 to 0.999 (P < 0.01), indicating significant positive correlations. This suggests that the screened thrombolytic activity marker peptides have varying degrees of influence on each other.

[0101] 3.2 Correlation analysis between thrombolytic activity marker peptide and fibrinolytic activity of Hudilong

[0102] Analysis of the thrombolytic activity marker peptide content and fibrinolytic activity in different samples of Pheretima cyclophagnum medicinal materials showed that ( Figure 11 ), all four batches of homemade medicinal materials were active and five marker peptides of thrombolytic activity were detected, and the active medicinal marker peptides PEP.3 (GEFPWQLSMTR) and PEP.11 (VTMTPAPGLIYR) were detected in the Shanghai earthworm medicinal materials (Pheretima common and Pheretima Williamii) with higher fibrinolytic activity; although one batch of counterfeit products had fibrinolytic activity, none of the three batches of counterfeit products had PEP.3 and PEP.11; the other three marker peptides had no obvious pattern in the medicinal materials.

[0103] The Pearson correlation coefficient method was further used to determine the correlation between the content of thrombolytic activity marker peptide and fibrinolytic activity in earthworm medicinal materials. Figure 12It can be seen that the thrombolytic activity marker PEP.3 was significantly correlated with the fibrinolytic activity of common earthworms (r=0.635, P<0.01), and PEP.11 was significantly correlated with the fibrinolytic activity of common earthworms (r=0.878, P<0.01). The thrombolytic activity marker peptides PEP.3 and PEP.11 had different degrees of influence on each other (r=0.677, P<0.01). The thrombolytic activity marker peptides PEP.3 and PEP.11 screened from earthworms can be used to characterize the thrombolytic activity of common earthworms.

[0104] Eleven marker peptides for thrombolytic activity showed strong correlations among each other, indicating that their contents in the medicinal material influenced each other. Therefore, two representative marker peptides were selected for use in medicinal material quality control. PEP.7 exhibited strong correlations with the other marker peptides, with correlation coefficients greater than 0.90 (P < 0.01). Its precision, reproducibility, and accuracy were also good, making it a suitable quality control peptide. Furthermore, PEP.1 showed a strong correlation with P. serrata (r = 0.865, P < 0.01). Its assigned protein is lumbrokinase and fibrinolytic activity protein, but it was not detected in inactive P. serrata. Its precision, reproducibility, and accuracy were also good, making it a suitable quality control peptide for P. serrata. The marker peptides PEP.7 and PEP.1 (associated with fibrinolytic activity protein) were used in quality control of P. serrata to detect the activity of the medicinal material. Both were detected in active P. serrata but not in inactive P. serrata or counterfeit products.

[0105] Five of the 11 thrombolytic activity marker peptides screened from Pheretima serrata can distinguish between Pheretima vulgaris tissue and body fluid. Further analysis of the five marker peptides in Pheretima serrata revealed a strong correlation between the thrombolytic activity of Pheretima serrata and PEP.11, both of which were not detected in counterfeit products. These peptides are being used for quality control and bioactivity testing of Pheretima serrata.

[0106] Example 1

[0107] A sample of Guangdilong was purchased from the market and identified as Pheretima ginseng according to the DNA barcode molecular identification method of Chinese medicinal materials in Part 9107 of the 2020 edition of the "Chinese Pharmacopoeia".

[0108] 40 mg of Guangdilong medicinal material powder was added to 800 μl of 50 mM NH4HCO3 extraction solution, extracted with ultrasound at 37°C for 40 minutes, and centrifuged. The solid was then added to 400 μl of 50 mM NH4HCO3 extraction solution and extracted with ultrasound at 37°C for 30 minutes. The supernatants were combined to obtain the earthworm protein. Activity was measured on an agarose fibrinogen plate, and the thrombolytic activity was 2395.6 U per mg of the medicinal material.

[0109] Take 100-200 μg of protein and add 200 μl of cold tris-phenol to each volume. Vortex at room temperature for 20 minutes. Precipitate overnight with five times the volume of 100 mM ammonium acetate in methanol prepared at -20°C. Centrifuge, remove the supernatant, dissolve the solid in urea (8 M, 1% SDS) solution, and remove the urea by ultrafiltration. Add 10 μl of 500 mM DTT (dissolved in 50 mM ammonium bicarbonate, prepared immediately before use, to a final DTT concentration of 25 mM) and incubate at 95°C for 30 minutes. Add 20 μl of 500 mM IAA (dissolved in 50 mM ammonium bicarbonate, prepared immediately before use, to a final IAA concentration of 50 mM) and incubate in the dark for 40 minutes.

[0110] Trypsin was dissolved in 1ml of acetic acid to a concentration of 100μg / ml. Each sample was then enzymatically digested with trypsin (approximately 1:50, w / w) at 37°C for 18 hours. The reaction was terminated by acidification with 10% TFA to a pH of 2-3. The sample was lyophilized, 200μl of phosphate buffer (pH 7.8) was added, and Glu-C endonuclease (approximately 1:50, w / w) was added and enzymatically digested at 45°C for 4 hours. C18 desalting was performed using the following methods: Buffer A: 38.4ml H2O, 1.6ml 5% TFA; Buffer B: 6.4ml H2O, 32ml ACN, 1.6ml 10% TFA. A C18 column was activated with 50% ACN, and buffer A was pipetted through the column. The sample was then loaded, desalted with buffer A, and peptides were extracted using 200μl of buffer B. This was repeated once. The product was spin-dried at room temperature in a vacuum centrifugal concentrator, 0.1% FA 100 μl was added for re-dissolution, and the supernatant was removed by centrifugation and placed in a plastic liquid phase vial.

[0111] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (Agilent 1290 / Agilent 6495) was used, using a CORTECS C18 column (2.7 μm, 2.1 × 50 mm) as the analytical column. The injection volume was 1 μl, the flow rate was 0.2 ml / min, and the mobile phases A and B were 0.1% formic acid in water, and B were acetonitrile. The gradient elution was: 2-35% B over 0-10 min; 35-90% B over 30-32 min. Mass spectrometry conditions were: spray voltage 4000 V, gas temperature 270°C, flow rate 16 L / min, nebulizer 45 psi, MRM mode was used, and data acquisition and processing were performed using Mass Hunter software. The resulting MRM chromatogram is shown in Figure 2. Figure 13 As shown, 11 chromatographic peaks of thrombolytic activity marker peptides can be detected simultaneously, and the contents of PEP.1 to PEP.11 are 1.294, 4.948, 1.774, 2.287, 2.306, 12.156, 0.509, 3.333, 1.550, 1.253 and 1.651 μg / g, respectively.

[0112] Example 2

[0113] A sample of Guangdilong was purchased from the market and identified as Pheretima ginseng according to the DNA barcode molecular identification method of Chinese medicinal materials in Part 9107 of the 2020 edition of the "Chinese Pharmacopoeia".

[0114] 40 mg of Guangdilong medicinal material powder was added to 800 μl of 50 mM NH4HCO3 extraction solution, extracted with ultrasound at 37°C for 40 minutes, and centrifuged. The solid was then added to 400 μl of 50 mM NH4HCO3 extraction solution and extracted with ultrasound at 37°C for 30 minutes. The supernatants were combined to obtain the earthworm protein. Activity was measured on an agarose fibrinogen plate, and the thrombolytic activity was 0 U per mg of medicinal material.

[0115] Take 100-200 μg of protein and add 200 μl of cold tris-phenol to each sample. Vortex at room temperature for 20 minutes. Precipitate overnight with five times the volume of 100 mM ammonium acetate in methanol prepared at -20°C. Centrifuge, remove the supernatant, dissolve the solid in urea (8 M, 1% SDS) solution, and remove the urea by ultrafiltration. Add 10 μl of 500 mM DTT (dissolved in 50 mM ammonium bicarbonate, prepared immediately before use, to a final DTT concentration of 25 mM) and incubate at 95°C for 30 minutes. Add 20 μl of 500 mM IAA (dissolved in 50 mM ammonium bicarbonate, prepared immediately before use, to a final IAA concentration of 50 mM) and incubate in the dark for 40 minutes.

[0116] Trypsin was dissolved in 1ml of acetic acid to a concentration of 100μg / ml. Each sample was then enzymatically digested with trypsin (approximately 1:50, w / w) at 37°C for 18 hours. The reaction was terminated by acidification with 10% TFA to a pH of 2-3. The sample was lyophilized, 200μl of phosphate buffer (pH 7.8) was added, and Glu-C endonuclease (approximately 1:50, w / w) was added and enzymatically digested at 45°C for 4 hours. C18 desalting was performed using the following methods: Buffer A: 38.4ml H2O, 1.6ml 5% TFA; Buffer B: 6.4ml H2O, 32ml ACN, 1.6ml 10% TFA. A C18 column was activated with 50% ACN, and buffer A was pipetted through the column. The sample was then loaded, desalted with buffer A, and peptides were extracted using 200μl of buffer B. This was repeated once. The product was spin-dried at room temperature in a vacuum centrifugal concentrator, 0.1% FA 100 μl was added for re-dissolution, and the supernatant was removed by centrifugation and placed in a plastic liquid phase vial.

[0117] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (Agilent 1290 / Agilent 6495) was used, using a CORTECS C18 column (2.7 μm, 2.1 × 50 mm) as the analytical column. The injection volume was 1 μl, the flow rate was 0.2 ml / min, and the mobile phases A and B were 0.1% formic acid in water, and B were acetonitrile. The gradient elution was: 2-35% B over 0-10 min; 35-90% B over 30-32 min. Mass spectrometry conditions were: spray voltage 4000 V, gas temperature 270°C, flow rate 16 L / min, nebulizer 45 psi, MRM mode was used, and data acquisition and processing were performed using Mass Hunter software. The resulting MRM chromatogram is shown in Figure 2. Figure 14 As shown, the contents of PEP.1 to PEP.11 are 0, 0, 0, 0, 0, 3.116, 0, 0, 0, 0 and 0 μg / g, respectively.

[0118] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Shanghai Institute for Food and Drug Inspection <120> Thrombolytic activity marker peptide of earthworm and its application in the determination of thrombolytic activity of earthworm <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <400> 1 Thr Asp Ala Ser Asn Ile Leu Pro Asn Thr Leu Gln Lys 1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <400> 2 Thr Ser Ala Ser Asn Ile Leu Pro Asn Thr Leu Gln Lys 1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <400> 3 Gly Glu Phe Pro Trp Gln Leu Ser Met Thr Arg 1 5 10 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 Val Ile Ser Thr Asp Glu Cys Asn Arg 1 5 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <400> 5 Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ser Val Lys 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <400> 6 Gly Ser Val Ile Ala Gly Val Gly Ser Trp Val Val Arg 1 5 10 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Ala Thr Phe Asp Ala Thr Ile Val Arg 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <400> 8 Trp Pro Leu Asp Tyr Phe Ile Lys 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <400> 9 Tyr Ala Ile Asn Val Ile Gly Arg 1 5 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <400> 10 Val Leu Tyr Pro Ser Ser Gly Thr Ala Gln Asp Tyr Ser Lys 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <400> 11 Val Thr Met Thr Pro Ala Pro Gly Leu Ile Tyr Arg 1 5 10

Claims

1. A group of marker peptides of earthworm thrombolytic activity, characterized in that: When the detection object is Pheretima annuli, 11 marker peptides with amino acid sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 11 are used; When the detection object is L. humilis, two marker peptides with amino acid sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 11 are used.

2. The thrombolytic activity marker peptide of earthworm according to claim 1, characterized in that The Shanghai earthworms are common earthworms and William earthworms.

3. The thrombolytic activity marker peptide of earthworm according to claim 1, characterized in that The mass spectrometry conditions for the thrombolytic activity marker peptides 1 to 11 are:

4. A method for determining the thrombolytic activity of earthworms based on the earthworm thrombolytic activity marker peptide according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: Process the sample to be tested to obtain a peptide mixture to be tested; Step 2: Using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to detect the polypeptide mixture to be tested, and obtaining a mass spectrum of the thrombolytic activity marker peptide of the test sample.

5. The method for determining the thrombolytic activity of earthworms according to claim 4, wherein: The chromatographic conditions for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer detection are: Analytical column: CORTECS C18 column, 2.7 μm, 2.1 × 50 mm; Injection volume: 1 μl; Flow rate: 0.2 ml / min; Mobile phase: Mobile phase A is 0.1% formic acid-water solution, mobile phase B is acetonitrile, Gradient elution: 0-10 min, 2-35% B; 30-32 min, 35-90% B.

6. The method for determining the thrombolytic activity of earthworms according to claim 4, wherein: The mass spectrometry conditions for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer detection are as follows: spray voltage 4000 V, gas temperature 270° C., flow rate 16 L / min, nebulizer 45 psi, and MRM mode.

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