Schaffer syngnathus protein-derived oligopeptide as well as screening and application thereof

Abstract

The invention belongs to the technical fields of marine product function application, functional food and biology, and particularly discloses a Schaffer syngnathus protein-derived oligopeptide and application of the Schaffer syngnathus protein-derived oligopeptide. Two preferable peptide sequences are obtained through bioinformatics virtual screening, and the amino acid sequences of the peptide sequences are FVDW and FIFE respectively. The preparation method of the Schaffer syngnathus protein-derived oligopeptide comprises the steps of sequence identification, bioinformatics virtual screening, chemical synthesis, in-vitro RWPE-1 cell viability verification and the like. The bioactive peptide derived from the syngnathus fragile protein can effectively inhibit benign prostatic cell proliferation, and the effect is equivalent to that of a positive drug. The method can be widely applied to research and development in the fields of food, functional nutrient products, health food, biological pharmacy and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of functional application of seafood, functional food and biotechnology, and more specifically, relates to a short peptide derived from the protein of Pieris schrenckii, screening and application thereof. Background Art

[0002] Benign prostatic hyperplasia (BPH) refers to a disease involving benign adenomatous hyperplasia of the prostate around the urethra. This disease is one of the common diseases in middle-aged and elderly men. With the aging of the global population, the incidence of BPH is increasing, and the incidence rate of BPH also increases with age. Studies have shown that the prevalence increases by 10% every decade. The incidence rate in men over 50 years old is as high as 50%, and the incidence rate in men over 80 years old is as high as 80%. Therefore, this disease has seriously affected the quality of life of middle-aged and elderly men and has become a common health problem that urgently needs attention.

[0003] The mechanisms of BPH are not fully understood. Androgens such as testosterone (TP) and dihydrotestosterone (DHT) are believed to be important factors. Therefore, the goals of BPH treatment include normalizing benign prostatic trophism, restoring steroid hormone levels, inhibiting excessive DHT production, reducing inflammatory processes, and limiting the initiators of proliferative processes. Current pharmacological treatments for BPH include α-receptor blockers, PEEIs, and 5α-reductase inhibitors. α-receptor antagonists relax the muscles surrounding the bladder and prostate, making urination easier. However, they have side effects such as headaches, drowsiness, and dizziness, and have no effect on reducing prostate size. Meanwhile, 5α-reductase inhibitors work by inhibiting the activity of 5α-reductase, suppressing DHT production and thus inhibiting prostate overgrowth. However, 5α-reductase inhibitors have also been linked to sexual dysfunction, including erectile dysfunction and decreased or premature ejaculation. Studies have found that the normal prostate is a healthy immune organ composed of a limited number of inflammatory cells. Chronic prostate inflammation may be associated with hormonal changes, infection, dietary or environmental factors, autoimmune reactions, urinary reflux within the prostatic collecting duct, and systemic inflammation associated with metabolic syndrome. Several endogenous peptides, such as vasoactive intestinal peptide (VIP), α-melanocyte-stimulating hormone (aMSH), and adrenomedullin, have emerged as anti-inflammatory agents and are potential new treatments for inflammation. Synthetic and natural peptides, such as sturgeon and oyster hydrolyzed peptides, have been shown to prevent excessive inflammatory responses by inhibiting the production or expression of inflammatory cytokines and signal transduction pathways. Therefore, identifying candidate compounds for ameliorating BPH from active peptides is a viable approach. Against this backdrop, the search for more effective prevention and treatment of BPH, identifying new targets, improving the quality of life of middle-aged and elderly men, and identifying candidate compounds that alleviate BPH symptoms while reducing drug toxicity and side effects has become an urgent market need.

[0004] According to numerous research reports, sea dragon is a traditional Chinese medicine for tonic, with the effects of warming the kidneys and strengthening yang, dispersing nodules and reducing swelling. The total amount of protein in sea dragon is as high as 50%, making it a high-quality source of protein or peptide active substances. Among them, Xue's sea dragon is a dominant species of Syngnathidae fish distributed in China's coastal waters, with abundant resources. Traditional methods for discovering bioactive peptides include extensive activity evaluation-guided fractionation and purification processes, which are cumbersome, time-consuming, and not easy to succeed. Bioinformatics not only helps predict the formation of bioactive peptides from any known protein source, but also predicts the targets and activities of active peptides, enabling high-throughput and precise screening. Therefore, traditional methods combined with bioinformatics can effectively and quickly screen bioactive peptides. Summary of the Invention

[0005] Based on previous research, the present invention obtains the active peptide components of Xue's sea dragon, constructs a target set and interaction network for Xue's sea dragon peptide to inhibit benign prostatic hyperplasia based on bioinformatics methods, predicts the active peptide sequence, clarifies the core biological process and target, establishes the corresponding RWPE-1 cell model, verifies the active peptide sequence of Xue's sea dragon, and uses multi-omics joint analysis combined with modern pharmacological evaluation methods to clarify the efficacy mechanism of Xue's sea dragon active peptide in inhibiting benign prostatic hyperplasia, which also provides a theoretical basis for the high-value utilization of sea dragon.

[0006] The primary purpose of the present invention is to overcome the above-mentioned problems in the prior art, and firstly provide a short peptide derived from the protein of Heterosaur xuei.

[0007] The second object of the present invention is to provide a short peptide derived from the protein of the dwarf sea dragon for use in preparing a functional product for alleviating benign prostatic hyperplasia, tonifying the kidney and strengthening yang, and overcoming impotence and oligospermia.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A short peptide derived from a schizont protein, wherein the amino acid sequences of the short peptides are as follows:

[0010] i) FVDW (Phe-Val-Asp-Trp);

[0011] ii) FIFE(Phe-Ile-Phe-Glu).

[0012] The present invention also provides a method for screening short peptides derived from the above-mentioned Hesperus schrenkiana protein, comprising the following steps:

[0013] (1) Peptide preparation and sequence identification: The hydrolyzed Hylocereus schrenckii powder was delipidated using neutral protease to prepare Hylocereus schrenckii peptides. The purified peptides were analyzed by liquid chromatography-mass spectrometry and proteomics to identify the amino acid sequence of the small molecular peptides in the fourth (SN4) fraction of the hydrolyzed Hylocereus schrenckii powder using gel separation and proteomics methods.

[0014] (2) Screening of active peptide sequences: All peptide sequences in the fourth (SN4) component were virtually screened using network pharmacology and molecular docking technology, and then the proliferation of RWPE-1 cells was detected by CCK8 assay. The amino acid sequences of active short peptides that inhibit benign prostatic hyperplasia were FVDW and FIFE.

[0015] (3) Cell verification: The above short peptide sequence was synthesized using solid-phase chemical synthesis, and its cell proliferation inhibitory activity was verified using human prostate epithelial cells RWPE-1 in vitro model.

[0016] Preferably, the operation of step (1) is as follows:

[0017] ① Enzymatic hydrolysis:

[0018] Take defatted Sea Dragon powder, add distilled water at a material-liquid ratio of 1:10, adjust the pH to 6.5, preheat at the hydrolysis temperature, add 3000U / g neutral protease, and hydrolyze at 50℃ for 5 hours. After hydrolysis, take out and inactivate the enzyme in a 100℃ boiling water bath for 10 minutes, centrifuge, and centrifuge at 8000r / min for 15 minutes to take the supernatant and collect the supernatant;

[0019] ②Separation

[0020] The collected supernatant was ultrafiltered using 0.2 μm and 5 kDa ultrafiltration membranes in sequence, and the filtrate <5 kDa was concentrated and freeze-dried to obtain the neutral protease-hydrolyzed Hendra schweitzeri polypeptide freeze-dried powder, which was labeled SN.

[0021] ③Purification

[0022] The lyophilized powder of the H. xue'sii polypeptide was hydrolyzed with neutral protease and dissolved, and purified and eluted by Sephadex G-15 column chromatography to obtain 6 component samples (SN1-6). After collection, the samples were concentrated, freeze-dried and stored.

[0023] ④SN1-6 activity verification and flow cytometry analysis

[0024] The CCK8 assay was used to detect the survival rates of RWPE-1 cells treated with SN1-6, and the cell cycle and apoptosis were analyzed by flow cytometry. The fourth component (SN4) with the best inhibitory activity on cell proliferation was saved and screened.

[0025] RWPE-1 cells were collected at 1×10 5Cells were seeded at a density of 10 cells / mL and allowed to adhere for 24 hours. Cells were treated with SN in medium containing TP and incubated for 24 hours. The cells in each well were harvested and centrifuged at 200 × g for 5 minutes. The cells were washed with PBS and centrifuged again at 200 × g for 5 minutes. Next, the cells were resuspended in 100 μL of binding buffer and stained with 5 μL of Annexin V-FITC and 5 μL of PI. After adding each staining reagent, the samples were incubated on ice in the dark for 15 minutes. Subsequently, 300 μL of binding buffer was added, and apoptotic cells were detected by flow cytometry.

[0026] The cells were initially grown at 1 × 10 5 The cells were seeded into 12-well plates at a density of 10 cells / mL and adhered for 24 hours. Thereafter, the cells were further incubated in fresh TP medium containing PE or FI and incubated for another 24 hours. Subsequently, the cells were harvested, washed and fixed with 70% ethanol in PBS at -20°C. After overnight incubation, the fixed cells were precipitated and stained with propidium iodide (25 μg / mL) at 37°C in the presence of RNase A (40 μg / mL) and 0.1% Triton X-100 under dark conditions. Finally, the cell cycle was analyzed by flow cytometry after a 30-minute incubation period.

[0027] ⑤LC-MS / MS identification of peptide amino acid sequence

[0028] The prepared SN4 sea dragon peptide was added to the lysis buffer (8M urea, 1x Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland), ground with shaking, and lysed on ice for 30 minutes. The supernatant was collected by high-speed centrifugation for 15 minutes (4°C, 15,000 rpm), concentrated to dryness using a centrifugal concentrator, and reconstituted with 100 μl of 100 mM TEAB. Each sample was digested with 5 μl of trypsin (Promega, Madison, WI) at 37°C overnight. The sample was desalted using a C18 desalting column (MonoSpin C18, GL). The eluted peptides were dried using a vacuum concentrator and stored at -80°C until mass spectrometry analysis.

[0029] The desalted, lyophilized peptides were reconstituted in solvent A (0.1% formic acid in water) and analyzed by LC-MS / MS equipped with an online nanospray ionization source. The system consisted of an EASY-nanoLC 1200 coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA). A total of 3 μL of sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm × 25 cm. The sample was separated using a 130-min gradient with a flow rate of 250 nL / min and a column temperature of 55°C. The gradient started with 4% phase B, equilibrated for 5 minutes, and then increased to 50% phase B over 120 minutes using a nonlinear gradient, then to 95% phase B over 1 minute, where it was maintained for 9 minutes. The mass spectrometer operated in data-dependent acquisition (DDA) mode, automatically switching between MS and MS / MS acquisition. (Mass spectrometry parameters were set as follows: ①MS: scan range (m / z): 350-1500; resolution: 120000; AGC target: 8×10 5 ; Maximum injection time: 50 ms. ②HCD-MS / MS: Resolution: 30000; AGC target: 1×10 5 The raw data were analyzed using Spectronaut X for peptide identification and relative quantification, yielding 3471 short peptide amino acid sequences.

[0030] Preferably, the operation of step (2) is as follows:

[0031] ①Build a potential target library for benign prostatic hyperplasia

[0032] GeneCards, OMIM, DisGeNET, and TTD (Therapeutic Target Database) databases were used to search for relevant targets using "benign prostatic hyperplasia" as a keyword. The databases were merged, duplicate targets were removed, and targets with correlation scores higher than the median were selected from GeneCards as potential targets of the benign prostatic hyperplasia database. Then, differentially expressed genes were analyzed online using GEO2R on the GSE132714 dataset in NCBI to obtain the up-regulated and down-regulated genes in the dataset. The potential targets of the benign prostatic hyperplasia database and the up-regulated and down-regulated genes in the GSE132714 dataset were merged to form the benign prostatic hyperplasia target library S1.

[0033] ②Constructing a functional target library of Xue's sea dragon peptide

[0034] The peptide sequences of the identified SN2, SN3, and SN4 components were used for target prediction using SEA (http: / / sea.bkslab.org), and after screening out targets with similarity coefficients lower than 0.5, they were used as the functional target set S2 of the Xue's sea dragon peptide.

[0035] ③Constructing a target set of Xue's Hailong peptide to inhibit benign prostatic hyperplasia

[0036] The functional target library of the hailong peptide in S1 and S2 was intersected with the target library of benign prostatic hyperplasia, and the obtained intersection database was used as the target dataset of Xue's hailong peptide inhibiting prostatic hyperplasia.

[0037] ④ Screening of core gene targets of Xue's Hailong peptide for inhibiting prostatic hyperplasia

[0038] A protein interaction network was constructed on STRING (https: / / cn.string-db.org), and the core modules and top 50 key genes in the protein interaction network were obtained using the CytoHubba plug-in of Cytoscape software. The corresponding gene targets were used as the core gene targets of Xue's Hailong peptide, an active peptide for inhibiting benign prostatic hyperplasia.

[0039] ⑤Molecular docking

[0040] Potential peptides were screened using computer simulations, and the affinity between the peptides identified through virtual screening and potential target proteins was evaluated through molecular docking. Peptides identified through bioinformatics were docked with the target proteins using Autodock vina (v1.2.x) to screen potential peptides. Human target proteins SRC (PDB ID: 3D7T), AKT1 (PDB ID: 4EJN), ITGA4 (PDB ID: 3V4V), and ITGB3 (PDB ID: 3FCS) were downloaded from the PDB protein database. Peptide ligands were constructed using Kingdraw v 3.0. Prior to docking, water molecules were removed from the conformation using AutoDock Tools-1.5.7, and the protein was hydrogenated to further optimize the 3D structure. The 3D conformation of the target compound at minimum energy was calculated, and the interaction between the ligand and the docked receptor was analyzed. Potential active compounds were evaluated based on binding energy scores.

[0041] Preferably, the operation of step (3) is as follows:

[0042] ① Cell culture:

[0043] RWPE-1 (ATCC) cells were cultured in RPMI medium supplemented with 100 mg / mL penicillin / streptomycin and 10% FBS at 37°C with 5% CO2 for 24 h. The culture medium was then replaced with fresh medium containing 5 μM testosterone propionate (TP) to induce cell proliferation. Samples (positive control finasteride, 17 potentially active peptides) were added to the culture medium simultaneously with TP.

[0044] ②Cell viability detection:

[0045] RWPE-1 cells (density 2-3×10 4 The cells were seeded in 96-well plates (100 μg / well) and incubated in RPMI plus 10% FBS for 24 h. The cells were then incubated in fresh TP-medium containing samples at various concentrations for another 24 / 48 h. The cell viability was determined using a CCK8 assay.

[0046] The present invention further provides the use of the short peptide in the preparation of functional foods, health products and medicines, wherein the function is to inhibit benign prostatic hyperplasia.

[0047] Preferably, the concentration of the short peptide in the functional product is 1 to 100 μg / mL.

[0048] The present invention also protects a biological product comprising an active ingredient and other excipients, wherein the active ingredient is either or both of the short peptides shown in SEQ ID NO. 1 or SEQ ID NO. 2. Furthermore, the active ingredient comprises an amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 with one or more amino acids substituted, deleted, or added, and having the same function.

[0049] Preferably, the biological product is used in the preparation of a product for preventing and treating benign prostatic hyperplasia, tonifying the kidneys and strengthening yang, or overcoming impotence and oligospermia. More preferably, the product is a health product, a nutritional product, a special medical food, or a medicine.

[0050] The product may also include other additives or excipients that do not affect the activity of the short peptide; the additives may be pigments such as tartrazine and sunset yellow, or food additives such as acesulfame potassium and sucralose as sweeteners; the excipients may be starch, powdered sugar, dextrin, or mannitol. The dosage form of the product is selected from any one of capsules, tablets, granules, powders, and enteric-coated preparations.

[0051] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0052] The present invention discloses the screening of short peptides derived from the protein of Scheherazade and its application. The peptides derived from the protein of Scheherazade for inhibiting benign prostatic hyperplasia are obtained by sequence identification, bioinformatics virtual screening and peptide synthesis, and are combined with human prostate epithelial cells RWPE-1 to conduct in vitro model verification of their activity. Cell experiments have shown that FVDW (Phe-Val-Asp-Trp) and FIFE (Phe-Ile-Phe-Glu) have significant effects on inhibiting the proliferation of human prostate epithelial cells RWPE-1 at low concentrations, and the effect is better than finasteride at low concentrations. Therefore, the two preferred peptide sequences derived from the protein of Scheherazade of the present invention can be applied to the research and development in the fields of food, functional nutritional products and health food. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 (A) is the six fractions (SN1-6) obtained by purification and elution of SN component Xue's Hailong peptide by Sephadex G-15 column chromatography, (B) is the effect of SN1-6 on RWPE-1 cell viability, (C) (1-8) is the anti-proliferative activity and cell apoptosis of SN on TP-induced human prostate cells, C(9) is the proportion of cells in G1, S and G2 phases after SN1-6 intervened in RWPE-1 cell viability, D(1-8) and D(9) are the anti-proliferative activity and cell cycle of SN on TP-induced human prostate cells and the apoptosis index after SN1-6 intervened in RWPE-1 cell viability, respectively;

[0054] Figure 2 (A), (B), and (C) are the molecular weight distribution, peptide length distribution, and quantitative proportion of each amino acid in the Hailong active peptide library, respectively;

[0055] Figure 3 (A) is the difference volcano plot analysis of the GSE132714 dataset, (B) is the intersection of the Hailong peptide target and the benign prostatic hyperplasia disease target, (C) Cytoscape 3.8.2 constructs the PPI map, and (D) CytoHubba plug-in identifies the TOP50 key gene targets;

[0056] Figure 4 The effect of peptide intervention on RWPE-1 cell viability. DETAILED DESCRIPTION

[0057] The following further describes specific embodiments of the present invention. It should be noted that the descriptions of these embodiments are intended to aid understanding of the present invention and do not constitute limitations of the present invention. Furthermore, the technical features involved in the various embodiments of the present invention described below may be combined with one another as long as they do not conflict with one another.

[0058] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are all conventional methods; the materials and reagents used are commercially available unless otherwise specified; the equipment used is all conventional experimental equipment unless otherwise specified.

[0059] Example 1 Preparation, separation and purification of the Xue's stilbone peptide SN for inhibiting benign prostatic hyperplasia according to the present invention

[0060] Take Xue's sea dragon powder (300g) and place it in a beaker. Add petroleum ether (30-60) at a material-liquid ratio of 1:5 for degreasing. Stir for 30s, let it stand for 2h (stir once every half an hour during this period), and then filter. Use a rotary evaporator to recover the solvent from the petroleum ether part, and collect the defatted sea dragon powder.

[0061] Take the defatted sea dragon powder, add distilled water at a material-liquid ratio of 1:10, adjust the pH to 8.0 with 1M NaOH solution, add neutral protease (3000U / g) for enzymatic hydrolysis, and hydrolyze at 55°C for 5 hours. Remove and inactivate the enzyme in a 100°C boiling water bath for 10 minutes. Centrifuge at 8000r / min for 15 minutes, and collect the supernatant. Ultrafiltration is performed using 0.2μm and 5kDa ultrafiltration membranes in sequence. The filtrate <5kDa is collected, concentrated, freeze-dried, and stored at -20°C. The defatted sea dragon powder is enzymatically hydrolyzed with neutral protease, and the resulting hydrolysates are labeled SN, which are then collected and freeze-dried for storage.

[0062] The neutral protease hydrolyzed lyophilized powder of the Heterocarpus schrenkiana polypeptide was dissolved and purified by Sephadex G-15 column chromatography to obtain component samples. After collection, the components were concentrated and freeze-dried to screen the benign prostate proliferation inhibitory activity of each component. The SN enzymatic hydrolyzate was separated by Sephadex-G15 gel to obtain six components: SN1, SN2, SN3, SN4, SN5, and SN6 (such as Figure 1 (as shown in A).

[0063] Example 2 In vitro cell validation, flow cytometric analysis of cell apoptosis and cell cycle and sequence identification

[0064] The effects of the six components of SN on the viability of RWPE-1 cells were analyzed by CCK8 assay. Figure 1 (B) It can be seen that among the six SN components, SN4 has a more obvious inhibitory effect on cell proliferation activity than other SN components, and this component has a good inhibitory activity on abnormal proliferation of RWPE-1 cells in the low concentration range of 50-100 μg / mL.

[0065] In order to clarify whether SN components inhibit cell proliferation by affecting cell apoptosis or cell cycle, flow cytometry was used to analyze cell apoptosis and cell cycle.

[0066] The RWPE-1 cells induced by TP were treated with the same concentrations of SN1-6 components, and the apoptosis results were shown in Figure 2. Figure 1 As shown in C(1-9), compared with the finasteride group, the apoptosis rate of cells treated with the SN component was significantly decreased, indicating that the SN group had no significant effect on the cell apoptosis process and had low toxicity. Figure 1 D(1-8) shows representative cell cycle diagrams of cells treated with SN1-6. Figure 1 D(9) The results showed that the S phase and G2 phase ratios of the SN4 group were significantly different from those of the finasteride group and other SN groups. The S phase ratio was significantly increased, while the G2 phase ratio was significantly decreased, indicating that TP-induced RWPE-1 cells treated with SN4 were arrested in the S / G2 phase.

[0067] The best-performing SN4 group was analyzed by nano-HPLC-QTOF-MS / MS. The raw data were analyzed by PeaksStudio for peptide identification and relative quantitative analysis to obtain short peptide sequences. From this, a library of Hailong active peptides for inhibiting benign prostatic hyperplasia was constructed. 3471 peptides were identified, and the relative content of peptides <1kDa was greater than 89% ( Figure 2 A), with tetrapeptides being the most abundant ( Figure 2 B), the relative content of hydrophobic amino acids phenylalanine and tryptophan is the highest, followed by branched-chain amino acids leucine and isoleucine, the relative content of which is greater than 10% ( Figure 2 C).

[0068] Example 3 Network pharmacology combined with molecular docking virtual screening of active peptides that inhibit benign prostatic hyperplasia

[0069] (1) Constructing a dataset of targets for Hailong peptide to inhibit benign prostatic hyperplasia

[0070] GeneCards, OMIM, DisGeNET, and TTD databases were used to search for relevant targets using the keywords benign prostatic hyperplasia and BPH. The databases were merged, duplicate targets were removed, and targets with correlation scores higher than the median were selected from GeneCards. At the same time, data mining was performed on the GEO database. The differentially expressed genes were analyzed using GEO2R online analysis for the dataset that met the requirements of this study (GSE132714) (see Figure 3 A) The up-regulated and down-regulated genes were merged with the targets screened from the previous disease database, and after removing duplicate targets, a library of 5696 potential targets for benign prostatic hyperplasia was obtained.

[0071] The 3471 identified Hailong peptide sequences were imported into the SEA (https: / / sea.bkslab.org) online website in Smile format for target prediction. After filtering out targets with similarity coefficients "<0.5" and Target IDs containing "HUMAN", 261 Hailong peptide predicted targets were obtained and used as the Hailong peptide functional target set. The intersection of the above potential BPH disease targets and potential Hailong peptide-inhibited BPH gene targets was obtained as follows: Figure 3 B shows 118 potential sea dragon active peptides inhibiting BPH gene targets.

[0072] (2) PPI construction and key gene identification and peptide sequence

[0073] The 118 potential BPH gene targets inhibited by the sea dragon active peptides obtained by the intersection were input into the protein network interaction network constructed in string. The filtering conditions were set to "confidence score (confidence) 0.9 and no textmining", and a PPI network containing 118 nodes and 76 edges was obtained (PPI enrichment p-value <1.0×10 -16 ).

[0074] Export the short tabular text from the string and perform visualization analysis in Cytoscape. According to the PPI network obtained by the above steps ( Figure 3 C), using the CytoHubba plug-in in Cytoscape to obtain the top 50 key gene targets ( Figure 3 D), virtual screening obtained the target sites and active peptide sequences of Hailong that inhibit BPH (as shown in Table 1).

[0075] Network pharmacology prediction can obtain potential biological targets with inhibitory effects on BPH, and the potential core pharmacological targets are identified as CASP3, SRC, CASP7, CASP8, BRCA1, XIAP, CASP9, PLG, BIRC2, CASP10, CDK4, BIRC3, AKT1, STAT3, CCND1, CCNA2, CDK2, MMP9, MMP2, ITGB3, ITGB1, ITGAV, ITGA4, PRSS1, FOS, GZMB, RAD51, MMP13, MMP1, MDM2, F2, MME, XPNPEP2, HDAC1, DPP4, ACE2, F12, TNFRSF10A, PLAU, PLAT, PLK1, CBX4, MMP14, MMP3, CTSL, CTSB, CTRB1, CASP1, and PPARG.

[0076] Table 1 Top 50 BPH inhibitory targets and active peptide sequences based on network pharmacology

[0077]

[0078]

[0079] (3) Molecular docking

[0080] The peptide compounds obtained by virtual screening were molecularly docked with the target protein receptors ITGB3 (PDB: 3FCS), ITGA4 (PDB: 3V4V), SRC (PDB: 3D7T), and AKT1 (PDB: 4EJN). The results are shown in Table 2. WFDK, FYEGY, FVDW, FLQG, QIEW, GYLF, VGPVGPVG, DFY, FAVY, FDW, FIFE, RIF, FALF, FSR, FLSYPH, FGREDRF, RGDYSDYQ, and VIHDFPQHY have good affinity with the receptor proteins.

[0081] Table 2 Virtual screening of peptides and docking with receptor protein molecules

[0082]

[0083]

[0084] Example 4: RWPE-1 Cell Verification of Virtual Screening of Peptides for Inhibiting Benign Prostatic Hyperplasia Activity

[0085] The effects of 17 peptides screened virtually on RWPE-1 cell viability were analyzed again by CCK8. Figure 4 It can be seen that in the experiment of 17 peptides intervening in RWPE-1 cell viability, FVDW (Phe-Val-Asp-Trp) and FIFE (Phe-Ile-Phe-Glu) have good inhibitory activity on abnormal proliferation of RWPE-1 cells at low concentrations of 6.25μg / mL and 12.5μg / mL, respectively. Compared with other peptides, the intervention of FVDW and FIFE in RWPE-1 cell viability is only 12.77±1.29% and 15.01±0.70%, respectively, and the inhibitory effect on benign prostate proliferation activity is the most significant. Compared with the cell viability of RWPE-1 cells treated with 100μg / mL finasteride, which is 9.86±4.19%, the effect is comparable.

[0086] Obviously, the specific implementation scheme described above is only a further detailed description of the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific example of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A short peptide derived from a protein of Heterosaurus xuei, characterized in that: The amino acid sequence of the short peptide is any one or a combination of the following sequences: i) FVDW, the sequence of which is shown in SEQ ID NO. 1; ii) FIFE, the sequence is shown in SEQ ID NO.

2.

2. The short peptide according to claim 1, characterized in that The short peptide also contains an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, wherein one or more amino acids are replaced, deleted or added, and the sequence has the same function.

3. Use of the short peptide derived from the Hesperus schrenckii protein according to claim 1 in the preparation of a functional product for preventing and treating benign prostatic hyperplasia.

4. The use according to claim 3, characterized in that The concentration of the short peptide in the functional product is 1 to 100 μg / mL.

5. A biological product, characterized in that The biological product is: i) active ingredient; the active ingredient is: any one or two of the short peptides shown in SEQ ID NO.1 or SEQ ID NO.2; ii) Additives or excipients that do not affect the function of the active ingredient.

6. The biological product according to claim 5, characterized in that The active ingredient contains an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, with one or more amino acids substituted, deleted or added, and has the same function.

7. Use of the biological product according to claim 5 in the preparation of a product for preventing, improving or treating benign prostatic hyperplasia, tonifying the kidney and strengthening yang, or overcoming impotence and oligospermia.

8. The use according to claim 7, characterized in that The product is a health product, nutritional product, special medical food or medicine.

9. The use according to claim 8, characterized in that The dosage form of the product is selected from any one of capsules, tablets, granules, powders and enteric-coated preparations.

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