A characteristic polypeptide, reagent kit, and application for species identification of leeches and Hirudo nipponia.

Abstract

This invention belongs to the field of biological detection, specifically relating to a characteristic polypeptide, a reagent kit, and their applications for species identification of leeches and *Hirudo medicinalis*. The sequences of the characteristic polypeptides are: Pep1: ISSIKNLFESDDDDYFFENK; Pep2: QTDLDQVKR. This invention screened two characteristic polypeptides that can be used to identify *Hirudo medicinalis* and *Hirudo medicinalis*, exhibiting strong specificity and robustness; they can stably detect target ion pairs. This invention establishes a technical system for species identification and adulteration / weight gain detection of leech medicinal materials. The method is highly specific and sensitive, not only solving the problem of species confusion under current pharmacopoeia standards but also providing a new technical means for the quality supervision of leech medicinal materials, and providing strong technical support for improving the quality control level of leech medicinal materials.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a characteristic polypeptide, a reagent kit, and the application of leeches for species identification between leeches and pheasants. Background Technology

[0002] Leeches, also known as Japanese medicinal leeches ( Hirudo nipponia Whitman (HN) is an important animal-derived traditional Chinese medicine with a long history of medicinal use, first recorded in the "Shennong's Classic of Materia Medica". The Chinese Pharmacopoeia (2020 edition) stipulates that leech is the dried whole body of leeches, water leeches or willow leaf leeches, which are generally collected in summer and autumn. After being killed by boiling water, they are dried in the sun or at low temperature.

[0003] leech( Hirudo nipponia Whitman (Japanese medicinal leech) is a blood-sucking leech belonging to the genus Hirudo in the family Hirudinaceae. It is a legally recognized species recorded in traditional Chinese medicine literature and included in the current Chinese Pharmacopoeia (2020 edition). Japanese medicinal leeches are rich in various functional proteins with anticoagulant effects, such as hirudin, NLP-1, antistasin, saratin, and destabilase. They possess pharmacological activities including antitumor, antithrombotic, antiatherosclerotic, and lipid-regulating effects, and have therefore attracted considerable attention.

[0004] In comparison, Hirudo medicinalis (Hirudo medicinalis) Poecilobdella manillensis The *Hirudo medicinalis* (also known as the "golden-edged leech") is a blood-sucking leech belonging to the genus *Hirudo* in the family Hirudinaceae. Pharmacological studies have shown that the main bioactive components of *Hirudo medicinalis* are natural hirudin and hirudin, along with amino acids, fatty acids, and various trace elements. Natural hirudin is its signature active ingredient. However, compared to *Hirudo medicinalis* from Japan, there are significant differences in their efficacy and market value.

[0005] Identification of the origin of traditional Chinese medicinal materials has always been a challenging issue in traditional medicinal material research. While various identification methods (such as morphological observation, microscopic features, thin-layer chromatography, and content determination) have been applied to relevant standards in the Chinese Pharmacopoeia (2020 edition) for leeches, these methods still have significant shortcomings in distinguishing different leech species, especially in identifying genuine and counterfeit products. Currently, the main problems in leech identification are as follows: ① Although there are many leech species, their genetic material and related proteins are highly similar, lacking sufficiently obvious specific indicators, making it difficult to capture subtle differences between species; ② During protein extraction from leech samples, the thick connective tissue and high solution viscosity often lead to low extraction efficiency, thus affecting the acquisition of high-resolution mass spectrometry data and subsequent screening of differentially expressed peptides.

[0006] Because Japanese medicinal leeches and pheasant leeches are extremely similar in appearance and physicochemical properties, existing pharmacopoeia testing methods are unable to accurately distinguish their origins, posing a challenge to the identification of genuine and counterfeit medicinal materials. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention provides a leech ( Hirudo nipponia Whitman (HN) is a characteristic polypeptide used to distinguish it from other species of Hirudo medicinalis.

[0008] The present invention also provides a kit containing the above-described characteristic polypeptides.

[0009] This invention also provides the application of the above-mentioned characteristic polypeptides in the identification of leeches and pheasant leeches.

[0010] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a characteristic polypeptide for species identification of leeches and horn beetles, the sequence of which is: Pep1: ISSIKNLFESDDDDYFFENK; Pep2: QTDLDQVKR.

[0011] The present invention also provides a kit containing the above-described characteristic polypeptides.

[0012] Another object of the present invention is to provide the application of the above-mentioned characteristic polypeptide in the species identification of leeches and pheasant leeches, including the following steps: (1) Sample preparation: Crush the sample to be tested, weigh the sample powder, add denaturing buffer and DTT solution, shake well, oscillate, take out and cool to room temperature, centrifuge, measure the supernatant, desalt and enzymatically digest, terminate the enzymatic digestion reaction, centrifuge, take the supernatant, and obtain the test sample. (2) The test samples were detected and identified by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry.

[0013] Preferably, in step (1), the specific process is as follows: crush the sample to be tested, weigh 50 mg of the sample powder to be tested, add 5 mL of denaturing buffer and 1 mL of DTT solution, shake well, place at 90℃ and shake for 4 hours, take out and cool to room temperature, centrifuge, measure the supernatant, desalt and enzymatically digest the sample, take out and cool to room temperature, terminate the enzymatic digestion reaction, centrifuge, take the supernatant, and obtain the sample.

[0014] Preferably, the denaturing buffer consists of: 6 M guanidine hydrochloride, 1 M Tris, 2.5 mM ethylenediaminetetraacetic acid, with the pH adjusted to 8.0 by adding concentrated hydrochloric acid; the concentration of the DTT solution is 0.5 M; the desalting and enzymatic hydrolysis are performed as follows: 500 μL of water is added to 500 μL of supernatant, which is then added to a 3 kDa ultrafiltration centrifuge tube, centrifuged at 12000 rpm for 10 minutes, the lower layer is discarded, and 500 μL of 1% NH4HCO3 solution and 10 μL of 10 mg / mL bovine trypsin solution are added, followed by enzymatic hydrolysis at 37°C for 12 hours.

[0015] Preferably, in step (2), the conditions for ultra-high performance liquid chromatography-triple quadrupole mass spectrometry are: The chromatographic column was an Agilent SB C. 18 RRHD (2.1 × 100 mm, 1.8 μm), column temperature 43℃, flow rate 0.3 mL / min, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile solution, gradient elution is performed.

[0016] Mass spectrometry detector, electrospray ionization (ESI) source, positive and negative ion modes, multiple reaction monitoring (MRM) was performed; sheath gas flow rate 46 L / hr; auxiliary gas flow rate 850 L / hr; spray voltage 3.5 kV; ion source temperature 550℃; auxiliary gas temperature 400℃; cone voltage 30 V; collision voltage 35 V; solvent delay 0–3 min and 14–20 min.

[0017] Preferably, the gradient elution program is as follows: 0-9 min, 3%B → 7.5%B; 9-13 min, 7.5%B → 25%B; 13-14 min, 25%B → 90%B; 14-17 min, 90%B; 17-17.5 min, 10%B → 97%B; 17.5-21 min, 97%B; the injection volume is 5 μL. Preferably, the characteristic ion pairs of the characteristic polypeptide are: Pep1: 618.53→147.08, 270.14; Pep2: 551.79→175.13, 286.18.

[0018] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry was used for detection and identification. The specific judgment principle during the process is as follows: if the characteristic ion peak of Pep 1 can be detected in the sample, the sample is considered to be Hirudo nipponiae; if the characteristic ion peak of Pep 2 can be detected in the sample but there is no characteristic ion peak of Pep 1, the sample is considered to be Hirudo nipponiae.

[0019] This invention uses trypsin to enzymatically digest samples, acquires peptide information using Nano LC-Orbitrap MS high-resolution mass spectrometry, analyzes the mass spectrometry data using PEAKS Studio 11 and statistical methods, screens for species-specific peptides, and verifies their specificity using multiple reaction monitoring (MRM) mode of liquid chromatography-triple quadrupole mass spectrometry. A quantitative detection method based on the characteristic peptide peak area ratio is also established to determine whether Japanese medicinal leeches are adulterated with Hirudo medicinalis.

[0020] The beneficial effects of this invention are as follows: (1) The present invention screened two characteristic polypeptides that can be used to distinguish between Japanese medicinal leech and pheasant leech. They have strong specificity and good durability and can stably detect the target ion pair. (2) This invention establishes a technical system for species identification and adulteration weight gain identification of leech medicinal materials. The method is highly specific and sensitive. It not only solves the problem of species confusion under the current pharmacopoeia standards, but also provides a brand-new technical means for the quality supervision of leech medicinal materials, and provides strong technical support for improving the quality control level of leech medicinal materials. Attached Figure Description

[0021] Figure 1 High-resolution mass spectra of proteins from Japanese medicinal herbs; Figure 2 High-resolution mass spectrum of proteins from the order Hirudinales; Figure 3 High-resolution mass spectrum of proteins from the order Hirudo species; Figure 4 High-resolution mass spectrum of proteins from the order Gynostemma pentaphyllum; Figure 5 Venn diagram showing peptide overlap in leech samples; numbers in the diagram represent the number of peptides identified; HN—Japanese medicinal leech; PM—Hymenopterus spp.; WP—Hymenopterus spp.; WA—Hymenopterus spp. Figure 6 mass-to-charge ratio ( m / z The second-order mass spectrum of the two charges (618.53) and the assignment of b and y ions; Figure 7 Secondary mass spectrum of mass-to-charge ratio (m / z, two charges) 551.79 and assignment of b and y ions; Figure 8 Pep 1 (618.53→147.08, 270.14) specificity experiment, A is H2O, B is matrix effect, C is Japanese medicinal leech, D is pheasant leech, E is broad-bodied golden thread leech, F is willow leaf leech; Figure 9Pep 2 (551.79→175.13, 286.18) specificity experiment, A is H2O, B is matrix effect, C is Japanese medicinal leech, D is pheasant leech, E is broad-bodied golden thread leech, F is willow leaf leech. Detailed Implementation

[0022] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0023] Example 1 1. Instrument, Reagent and Sample Information 1.1 Instruments Thermo EASY-nLC 1000 nanoliter liquid chromatograph (Thermo Scientific, San Jose, CA, USA), Thermo Scientific Orbitrap-Fusion high-resolution mass spectrometer (Thermo Scientific, San Jose, CA, USA), AB Triple Quad 6500 + High performance liquid chromatography-mass spectrometry system (AB SCIEX), XSE205DU 0.001 g electronic balance (Mettler Toledo).

[0024] 1.2 Reagents Trypsin (Sigma-Aldrich, batch number: SLBG6452V), guanidine hydrochloride, tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT, analytical grade, Aladdin Reagent (Shanghai) Co., Ltd.), iodoacetamide (IAA, analytical grade, Aladdin Reagent (Shanghai) Co., Ltd.), ammonium bicarbonate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), and acetic acid were all analytical grade. Formic acid (chromatographic grade, batch number E2128234) was a product of Shanghai Aladdin Biochemical Technology Co., Ltd.; acetonitrile (chromatographic grade, batch number 222721) was a product of Thermo Fisher Scientific (China) Co., Ltd.; distilled water was a product of Guangzhou Watsons Food & Beverage Co., Ltd.

[0025] 1.3 Sample Information One hundred batches of leeches were collected, including 23 batches of Japanese medicinal leeches, 10 batches of pheasant leeches, 62 batches of broad-bodied golden thread leeches, and 5 batches of willow leaf leeches. Specific sample information is shown in Table 1.

[0026] Table 1. Information on leech medicinal samples 2 Experimental Methods 2.1 Sample Preparation One hundred batches of leech samples were pulverized, and 50 mg of the powder was weighed out. 5 mL of denaturing buffer (6 M guanidine hydrochloride, 1 M Tris, 2.5 mM ethylenediaminetetraacetic acid, pH adjusted to 8.0 with concentrated hydrochloric acid) and 1 mL of DTT (0.5 M) solution were added. The mixture was shaken well and incubated at 90°C for 4 h. After cooling to room temperature, the mixture was centrifuged (12000 rpm, 10 min). 500 μL of the supernatant was collected and desalted and enzymatically digested using a 3 kDa ultrafiltration centrifuge tube (500 μL of water was added, centrifuged at 12000 rpm for 10 min, the lower layer was discarded, 500 μL of 1% NH4HCO3 solution and 100 μL of bovine trypsin solution (10 mg / mL) were added, and the mixture was incubated at 37°C for 12 h). After cooling to room temperature, the enzymatic digestion was terminated at 100°C, centrifuged, and the supernatant was collected.

[0027] 2.2 Nanoliter Liquid Chromatography-High Resolution Mass Spectrometry (NLC-HDMS) Determination Conditions 2.2.1 EASY-nLC 1000 chromatographic conditions Using Thermo Acclaim PepMap C 18 The chromatographic column (75 μm × 15 cm, 3 μm, Thermo Scientific, San Jose, CA, USA) was used at a flow rate of 300 nL / min. Mobile phase A was a 0.1% formic acid aqueous solution containing 2% acetonitrile, and mobile phase B was a 0.1% formic acid aqueous solution containing 98% acetonitrile. Gradient elution was used (0–1 min, 1%B→6%B; 1–96 min, 6%B→22%B; 96–113 min, 22%B→30%B; 113–117 min, 30%B→95%B; 117–120 min, 95%B). The injection volume was 1 μL.

[0028] 2.2.2 Orbitrap-Fusion High-Resolution Mass Spectrometer Conditions The conditions for high-resolution mass spectrometry analysis were as follows: the ion source was a Nanospray Flex; the analysis was performed in positive ion mode with a spray voltage of 2300 V; the ion transport capillary temperature was 275 °C; and the S-Lens transmission efficiency was set to 60%. Primary mass spectrometry was performed on an Orbitrap mass analyzer with a resolution of 60,000 m / z and an acquisition range of 350–1550 m / z. Secondary mass spectrometry was performed on an Orbitrap mass analyzer using fast scan mode. The top parameters were: velocity data-dependent mode for precursor ion selection, HCD mode for fragmentation, and fragment energy (NCE) set to 40%.

[0029] 2.3 Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry determination conditions 2.3.1 Ultra-high performance liquid chromatography conditions The chromatographic column was an Agilent SBC. 18 RRHD (2.1 × 100 mm, 1.8 μm), column temperature 43℃, flow rate 0.3 mL / min, mobile phase A was 0.1% formic acid solution, mobile phase B was acetonitrile solution, gradient elution was performed (0~9 min, 3%B→7.5%B; 9~13 min, 7.5%B→25%B; 13~14 min, 25%B→90%B; 14~17 min, 90%B; 17~17.5 min, 10%B→97%B, 17.5~21 min, 97%B); injection volume was 5 μL.

[0030] 2.3.2 Triple Quadrupole Mass Spectrometry Conditions Mass spectrometry was used with an electrospray ionization (ESI) source in both positive and negative ion modes, and multiple reaction monitoring (MRM) was performed. The sheath gas flow rate was 46 L / hr; the auxiliary gas flow rate was 850 L / hr; the spray voltage was 3.5 kV; the ion source temperature was 550 °C; and the auxiliary gas temperature was 400 °C. The cone voltage was 30 V, and the collision voltage was 35 V. The solvent delay was 0–3 min and 14–20 min. The characteristic peptide ion information used in this experiment is shown in Table 2.

[0031] Table 2 Ion pair information of characteristic polypeptides 3 Experimental Results 3.1 Selection of characteristic ion pairs for leech samples High-resolution mass spectrometry (HMS) was used to acquire data from samples of *Hirudo medicinalis*, *Hirudo nipponia*, *Hirudo scabra*, and *Hirudo salina*. The HMS acquisition results are shown below. Figure 1 — Figure 4 As shown.

[0032] High-resolution mass spectrometry data were imported into PEAKS Studio 11 software. The "Hirudo" database (downloaded from uniport) was selected for data alignment analysis. The analysis parameters were set as follows: query type was homology matching, variable modifications included: Carbami-domethylation (57.02), oxidation (M) (15.99), acetylation (K) (42.01), precursor ion mass error tolerance was 15.0 ppm, fragment mass error tolerance was 0.02 Da, maximum number of missed cuts was 3, maximum number of variable post-translational modifications (PTM) per peptide was 6, de novo sequencing score (%) threshold was 15, and charge filtering range was 2-8.

[0033] A total of 679 peptides matching the database were identified in samples from *Hirudo medicinalis*, *Hirudo phalloides*, *Hirudo scabra*, and *Salix viminalis*. Specifically, 278 peptides were identified from *Hirudo medicinalis*, 241 from *Hirudo phalloides*, 265 from *Hirudo scabra*, and 265 from *Salix viminalis*. Venn diagram analysis was performed on the identified peptides, and the results are shown below. Figure 5 Characteristic peptides with specific properties were selected and further validated using liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS). The final characteristic peptides and peptide secondary mass spectra, along with the assignments of b and y ions, are shown in [reference needed]. Figure 6 and Figure 7 .

[0034] 3.2 Method Establishment Using SCIEX AB Triple Quad 6500 + The specificity of the characteristic peptides was verified using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), and an MRM method was established. The product ion with the highest response intensity was analyzed and selected as the daughter ion. The specificity and robustness of the method were then investigated. The characteristic ion pairs for leech samples were obtained: Pep 1: 618.53→147.08, 270.14; Pep 2: 551.79→175.13, 286.18. The specificity was: Pep 1 was *Hirudo medicinalis*, and Pep 2 was *Hirudo medicinalis* and *Hirudo nipponia*. Therefore, the following judgment criteria were established: if the characteristic ion peak of Pep 1 was detected in the sample, the sample was considered to be *Hirudo medicinalis*; if the characteristic ion peak of Pep 2 was detected in the sample but the characteristic ion peak of Pep 1 was not detected, the sample was considered to be *Hirudo nipponia*.

[0035] 3.3 Sample Test Results This method was applied to 100 batches of collected leech samples. After processing according to method 2.1, the samples were subjected to liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) to extract the ion chromatograms of each characteristic polypeptide. In all batches, Pep 2 characteristic ion peaks were detected in 23 batches of *Hirudo medicinalis* but not in 10 batches of *Hirudo nipponia*. Pep 1 characteristic ion peaks were detected in all 10 batches of *Hirudo pungens*. No corresponding characteristic ion peaks were detected in 62 batches of *Hirudo floribunda* and 5 batches of *Hirudo willowleaf*.

[0036] 3.4 Methodological Examination 3.4.1 Specificity Pep 1 was detected in *Hirudo nipponia* samples but not in other leech samples. (See [link to relevant documentation]). Figure 8 Pep 2 was detected in samples of Japanese medicinal leeches and Hirudo medicinalis, but not in other samples. (See [link to relevant documentation]). Figure 9 The results show that the method has good specificity.

[0037] 3.4.2 Durability The robustness test results are shown in Table 3. All three chromatographic columns showed significant separation differences, indicating that the method has good robustness.

[0038] Table 3. Results of Methodological Durability Tests This invention screens for species-specific characteristic peptides as chemical markers, enabling accurate and efficient differentiation between *Hirudo nipponia* and *Hirudo apiacea* samples. Compared to traditional identification methods such as morphological identification and thin-layer chromatography, this method can accurately distinguish samples at the protein level, and is simpler to operate and more sensitive.

[0039] The characteristic peptides of *Hirudo nipponia* provided in this invention originate from a protein family containing ankyrin repeat sequences. These peptides are widely involved in protein-protein interactions in organisms, regulating key physiological processes such as cell signaling and cell adhesion. This invention successfully screened characteristic peptides with high species specificity by optimizing protein extraction and enzymatic digestion conditions, and quantitatively detected them using liquid chromatography-triple quadrupole mass spectrometry (MRM). Experimental results show that detecting the Pep1 ion pair can clearly determine whether a sample is *Hirudo nipponia*, while the combined detection of Pep2 and Pep1 can accurately distinguish *Hirudo jatropha*. The characteristic ion peaks of the target peptides were consistently detected in all batches of leech samples. This method can accurately and efficiently distinguish between *Hirudo jatropha* and *Hirudo nipponia*.

Claims

1. A characteristic polypeptide for species differentiation between Japanese medicinal leech and pheasant leech, characterized in that, The sequences of the characteristic polypeptides are: Pep1: ISSIKNLFESDDDDYFFENK and Pep2: QTDLDQVKR.

2. The application of the characteristic polypeptide as described in claim 1 in the identification of Japanese medicinal leeches and Hirudo medicinalis, characterized in that, Includes the following steps: (1) Sample preparation: Crush the sample to be tested, weigh 50 mg of the sample powder to be tested, add 5 mL of denaturing buffer and 1 mL of DTT solution, shake well, place at 90℃ and shake for 4 hours, take out and cool to room temperature, centrifuge, measure the supernatant, desalt and enzymatically digest the sample, take out and cool to room temperature, terminate the enzymatic digestion reaction, centrifuge, take the supernatant, and obtain the test sample. (2) The test samples were detected and identified using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry; The desalting and enzymatic hydrolysis are as follows: 500 μL of water is added to 500 μL of supernatant, which is then added to a 3 kDa ultrafiltration centrifuge tube. The mixture is centrifuged at 12,000 rpm for 10 minutes, the lower layer is discarded, and 500 μL of 1% NH4HCO3 solution and 10 μL of 10 mg / mL bovine trypsin solution are added. The mixture is then enzymatically hydrolyzed at 37°C for 12 hours. In step (2), the conditions for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry are as follows: The chromatographic column was an Agilent SBC. 18 RRHD, 2.1 × 100 mm, 1.8 μm; column temperature 43℃, flow rate 0.3 mL / min, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile solution, gradient elution is performed; Multiple reaction monitoring was performed using a mass spectrometer detector, an electrospray ionization source, and positive and negative ion modes; the sheath gas flow rate was 46 L / hr; the auxiliary gas flow rate was 850 L / hr; the spray voltage was 3.5 kV; the ion source temperature was 550℃; the auxiliary gas temperature was 400℃; the cone voltage was 30 V; the collision voltage was 35 V; and the solvent delay was 0–3 min and 14–20 min. The gradient elution program was as follows: 0–9 min, 3%B → 7.5%B; 9–13 min, 7.5%B → 25%B; 13–14 min, 25%B → 90%B; 14–17 min, 90%B; 17–17.5 min, 10%B → 97%B; 17.5–21 min, 97%B; the injection volume was 5 μL. During the identification process, the judgment principle is as follows: if the characteristic ion peak of Pep 1 can be detected in the sample, the sample is considered to be Hirudo nipponiae; if the characteristic ion peak of Pep 2 can be detected in the sample but there is no characteristic ion peak of Pep 1, the sample is considered to be Hirudo nipponiae.

3. The application according to claim 2, characterized in that, The denaturing buffer solution consists of 6 M guanidine hydrochloride, 1 M Tris, 2.5 mM ethylenediaminetetraacetic acid, with the pH adjusted to 8.0 by adding concentrated hydrochloric acid; the DTT solution has a concentration of 0.5 M.

4. The application according to claim 2 or 3, characterized in that, The characteristic ion pairs of the characteristic polypeptides are Pep1: 618.53→147.08, 270.14; Pep2: 551.79→175.13, 286.18.

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