Anti-epileptic stiff silkworm polypeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and provides an anti-epileptic stiff silkworm polypeptide as well as a preparation method and application thereof. The bombyx batryticatus polypeptide is a peptide fragment with an amino acid sequence as shown in SEQ ID No.1, and is RFAFPAWI (PP6). The method comprises the following steps: preparing a peptide extract in stiff silkworm by simulating gastrointestinal digestion, identifying peptide in stiff silkworm polypeptide by using Nano LC-MS / MS, and carrying out difference analysis; differential peptides with potential anti-epileptic activity are obtained through virtual screening, and the anti-epileptic stiff silkworm polypeptide PP6 with potential anti-epileptic activity is synthesized. PP6 has a neuroprotection effect on PC12 cell neurotoxicity damaged by glutamic acid (Glu) by adjusting a GABA signal channel, and has an anti-epileptic effect on an epileptic mouse induced by pentaerythrityl tetrazole (PTZ). Therefore, PP6 may be a new leading peptide compound for developing epileptic drugs, and the anti-epileptic polypeptide can be widely applied to the fields of medicines, foods and the like to achieve the anti-epileptic target.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an anti-epilepsy polypeptide of cannysia and a preparation method and application thereof. BACKGROUND

[0002] Epilepsy is considered to be one of the most common and most serious chronic brain nervous system diseases, which is caused by various factors. Its characteristic is the imbalance of excitation and inhibition process, leading to unpredictable, unprovoked and recurrent seizures [1]. It is reported that epilepsy affects 5000 to 7000 million people worldwide. Seizure is the onset of uncontrolled spontaneous abnormal electrical activity in the human brain, which can cause changes in consciousness, behavior, memory or sensory perception [2].

[0003] The onset of epilepsy is mainly due to the imbalance of excitation and inhibition of the central nervous system, and the causes of such excitation and inhibition disorders are closely related to neurotransmitters, ion channels, synaptic connections, inflammatory factors, glial cells and glucocorticoids [3]. Glutamate (Glu) is an excitatory neurotransmitter, which enhances the excitability of Glu receptors in neurons. Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter. Glu / GABA metabolic balance affects and maintains the normal physiological activity of the brain [4]. The neurotransmitter metabolism of patients with epilepsy often appears unbalanced, which is characterized by elevated Glu levels and reduced GABA concentration [5]. The GABA signaling pathway mainly includes GABA receptors, glutamate decarboxylase (GADs) and GABA transporters (GATs). GABA receptors can be divided into GABAA, GABAB and GABAC, among which GABAA is most closely related to epilepsy [6]. GADs are key enzymes involved in GABA synthesis, including GAD1 (GAD65) and GAD2 (GAD67). The decrease of GADs level will lead to the weakening of inhibitory neurotransmission, and even may cause neuron death in the epileptic focus [6]. GATs include GAT1, GAT2, GAT3 and GAT4, among which GAT1 and GAT3 are closely related to epilepsy [7, 8]. Therefore, the GABA signaling pathway is usually selected to explore the mechanism of anti-epilepsy effect.

[0004] Cannysia Bombyx batryticatus (BB) is the silkworm moth family insect Bombyx mori Bombyx mori Linnaeus 4-5 instar larvae infected (or artificially inoculated) with Beauveria bassiana Beauveria bassiana(Bals.) Vuillant. BB is a famous traditional Chinese medicine with the function of "restraining convulsion caused by wind", and is often used in the treatment of epilepsy in clinic. Due to its reliable therapeutic effect, it has been used in China for thousands of years. As a widely used animal-derived traditional Chinese medicine, BB has various pharmacological effects, including anticonvulsant, antiepileptic, neurotrophic, antitumor, anticoagulant, antioxidant, antibacterial, antifungal and hypoglycemic effects[9]. According to historical documents, BB was mainly used in the form of powder to treat diseases in ancient times[9]. However, in contemporary clinical practice of traditional Chinese medicine, it is mainly used in the form of decoction to treat diseases. It is worth noting that previous studies have shown that BB powder has a significantly stronger anticonvulsant and antiepileptic effect on mice than BB water decoction[10, 11]. Therefore, the classical use form of BB powder as a medicine to treat diseases has certain scientific rationality

[11] . However, the anti-epilepsy material basis of using BB powder as a medicine is still unclear. As an animal-derived traditional Chinese medicine, the main chemical components of BB are proteins and peptides. Previous studies have shown that BB proteins have significant antiepileptic, anti-apoptotic and neuroprotective effects[12, 13]. Peptides are important components of the therapeutic material basis of animal medicines, which are the intermediate products of protein hydrolysis

[14] . After oral administration of BB powder, the protein components may be hydrolyzed to produce peptide substances under the action of pH and enzymes in the gastrointestinal environment and play an antiepileptic role. However, further research is needed on the mechanism of protein conversion and the active peptide components produced. SUMMARY

[0005] The present application provides an antiepileptic moth cocoon peptide, a preparation method and application thereof, which identifies and virtually screens peptides with potential antiepileptic effects in BB powder, verifies the antiepileptic effect and studies the mechanism of action, provides a basis for elucidating the material basis of BB as a powder medicine for treating epilepsy.

[0006] The present application is implemented by the following technical solutions: an antiepileptic moth cocoon peptide, wherein the moth cocoon peptide is a peptide segment with an amino acid sequence as shown in SEQ ID No. 1, and is RFAFPAWI.

[0007] The present application also provides a method for preparing the antiepileptic moth cocoon peptide, which comprises the following steps: preparing a peptide extract in a moth cocoon by simulating gastrointestinal digestion, identifying peptides in the moth cocoon peptide extract by using Nano LC-MS / MS, and performing differential analysis; obtaining differential peptides with potential antiepileptic activity by virtual screening, and synthesizing the moth cocoon peptide with potential antiepileptic activity.

[0008] Further, the method comprises the following steps: (1) Degreasing of silkworm: The silkworm is crushed and passed through a No. 4 sieve, then 10 times the amount of petroleum ether is added to the dry powder, and the silkworm is degreased by reflux at 60-90℃ twice, each time for 1 hour. Then, it is centrifuged at 5000g for 10 minutes and the residue is dried at 60℃ to obtain the degreased silkworm. (2) Preparation of peptide extract: The defatted silkworm sample was digested in vitro to prepare peptide extract. The pH value of the defatted silkworm sample was adjusted to 3.0 with 1M HCl, and pepsin was added at 37℃ for 2h digestion. Then, 1M NaHCO3 was added to adjust the pH value to 7.5, and trypsin was added at 37℃ for 2h digestion. After digestion, the sample was placed in a boiling water bath for 5min to inactivate it. The sample was centrifuged at 5000g at 4℃ for 10min. The precipitate obtained was the peptide extract BPP. (3) Nano-liquid chromatography-tandem mass spectrometry (Nano LC-MS / MS) analysis: BPP was placed in 0.1% formic acid solution, washed with the tip of a C-18 rotating column, and analyzed by Nano LC MS / MS on a Q-Exactive HF mass spectrometer; the original mass spectrometry file was analyzed and searched by Proteome Discoverer 2.5 with the following specified parameter settings: no enzyme; variable modification including oxidation M; peptide mass tolerance set to 10 ppm, fragment mass tolerance set to 0.02 Da; matching analysis was performed with the silkworm, mulberry and Beauveria bassiana protein databases NCBInr and UniProt by the search software; the same number of bait sequences were also searched for to estimate the false discovery rate; the false discovery rate score obtained by the search was ≤0.05, which was considered a valid identification result; (4) Predicting peptide activity: The online tool PeptideRanker was used to predict the bioactivity of the identified peptides. Peptides with a bioactivity score ≥0.8 were selected as potential bioactive peptides. Peptides with a sequence length <20 were selected for toxicity prediction using ToxinPred. (5) Molecular docking model prediction and screening of anti-epileptic peptides: GABA was downloaded from the RCSB protein database. A Crystal structures of R (PDB ID: 4COF) and NMDAR (PDB ID: 1Y20) were obtained; amino acids were linked into peptide sequences using ChemDraw 20.0 software, and 3D structure files of the peptides were generated using Chem3D 20.0 software; MOL2 format was converted to PDBQT format using OpenBabel 3.1.1 for future integration; the obtained PDBQT protein receptor files were purified using AutoDock Vina to remove all water molecules and unnecessary substructures, and hydrogen atoms were added; the peptides were also processed in the same way, and docking simulations were performed using the protein as the receptor and the peptide as the ligand to obtain the appropriate minimum binding energy. Molecular visualization and analysis were performed using PyMOL software. (6) Synthesis of peptides: Predicted peptides were synthesized according to the docking results, and the purity of the synthesized peptides was identified by HPLC to be >95%.

[0009] The application also provides use of the anti-epilepsy polypeptide of Cantharis in the preparation of a drug for protecting PC12 cells damaged by Glu. The anti-epilepsy polypeptide of Cantharis significantly increases the content of GABA in PC12 cells damaged by Glu and reduces the levels of IL-1β, IL-4, GRα and TNF-α. The concentration of the anti-epilepsy polypeptide of Cantharis is 40-160 µM.

[0010] The application also provides use of the anti-epilepsy polypeptide of Cantharis in the preparation of a drug for improving the epileptic behavior of a PTZ-induced epileptic mouse.

[0011] The anti-epilepsy polypeptide of Cantharis significantly improves the behavior of a PTZ-induced epileptic mouse by regulating the GABA signaling pathway.

[0012] Further, the anti-epilepsy polypeptide of Cantharis significantly increases the content of GABA in the brain tissue of a PTZ-induced mouse and reduces the levels of Glu and TNF-α. The dose of the anti-epilepsy polypeptide of Cantharis is 40-80 mg / kg, and the drug is administered for 15-20 days.

[0013] The application aims to identify, synthesize, virtually screen and verify the anti-epilepsy peptides in BB powder (BPP) and BB water decoction (BDP) peptide extracts. Peptides are identified using Nano LC-MS / MS, and BPP and BDP are subjected to differential analysis. Subsequently, differential peptides with potential anti-epilepsy activity between BPP and BDP are obtained through virtual screening, including unique peptides in BPP and up-regulated peptides. Peptides with potential anti-epilepsy activity are synthesized. Then, the neuroprotective effect of the synthesized peptides on the neurotoxicity of Glu-damaged PC12 cells is explored, and the potential mechanism is studied. Finally, the anti-epilepsy effect of the active peptides is verified using a PTZ-induced epileptic mouse model. The application is conducive to further elucidating the material basis of BB for treating epilepsy and providing theoretical support and research ideas for the development of new anti-epilepsy drugs based on traditional Chinese medicine. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Timeline for mouse experiments; Figure 2 Amino acid frequency based on peptide percentage; in the figure: (A) molecular weight distribution of BPP, (B) molecular weight distribution of BDP, (C) peptide length of BPP, (D) peptide length of BDP, (E) distribution of terminal amino acids of BPP and BDP, (F) statistical results of hydrophilic / hydrophobic amino acids in BPP and BDP; Figure 3Figure 6. The distribution of differential peptides in BPP and BDP, (A) the distribution of differential peptides in BPP and BDP, (B) the volcano plot of differential peptides in BPP and BDP (Note: the left green circle represents down-regulation, and the right red circle represents up-regulation), (C) the PCA plot of differential peptides in BPP and BDP (Note: each point in the figure represents a sample, and the powder is red and the decoction is green), (D) the heat map of different peptides in BPP and BDP (Note: each column represents a sample, and each row corresponds to a peptide segment. The color indicates that the peptides with similar patterns of quantitative data are adjacent rows), (E) the OPLS-DA plot of differential peptides in BPP and BDP (Note: each point in the figure represents a sample, and the red color represents the powder and the green color represents the decoction), (F) the OPLS-DA verification plot of differential peptides in BPP and BDP (Orange represents R2Y random grouping model, purple represents Q2, and black arrow represents R2X, R2Y and Q2 values of the original model); Figure 4 Molecular docking of GABA / NDA receptors and BBPs; Figure: (A) PFAGLF (PP1) - GABA, (B) PFGAIF (PP4) - GABA, (C) RFAFPAWI (PP6) - GABA, (D) WFAGFS (PP7) - GABA, (E) FGDPFKGYK (PP8) - GABA, (F) SDAASSEDGFWWW (PP10) - GABA, (G) SWFVTPF (PP2) - NMDA, (H) WGAFSF (PP3) - NMDA, (I) PFGAIF (PP4) - NMDA, (J) AWAAGFGR (PP5) - NMDA, (K) RFAFPAWI (PP6) - NMDA, (L) WFAGFS (PP7) - NMDA, (M) AGAGFGPG (PP9) - NMDA; Figure 5 Protective effect of BBPs on survival rate of PC12 cells damaged by Glu; Figure: (A) Effect of BBPs on survival rate of PC12 cells damaged by Glu, (B) Effect of BBPs on survival ability of control PC12 cells, (C) Effect of 24-hour pretreatment of BBPs on survival rate of PC12 cells damaged by Glu. These values are expressed as mean ± standard deviation (n = 6). Compared with the control group, #P < 0.05 and ##P < 0.01; compared with the model group, P < 0.05 and P < 0.01; Figure 6Mean ± SD, n = 6. #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group. P < 0.05 and P < 0.01; (B) is the effect of PP6 on the expression of GABAA-Ral, GAD65, GAD67, GATl and GAT3 mRNA in Glu-injured PC12 cells (mean ± SD, n = 6). #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group. P < 0.05 and P < 0.01; (C) is the effect of BBPs on the expression of GABAA-Ral, GAD65, GAD67, GATl and GAT3 protein in Glu-injured PC12 cells (mean ± SD, n = 6). #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group. P < 0.05 and P < 0.01; Figure 7 Histological analysis (100x and 400x); Figure: (A) H&E staining, (B) Nissl staining, (C) the average optical density value of CA1 and CA3 positive cells. #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group. P < 0.05 and P < 0.01; Figure 8 Mean ± SD, n = 6. #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group. P < 0.05 and P < 0.01; (B) is the effect of PP6 on the expression of GABAA-Ral, GAD65, GAD67, GATl and GAT3 mRNA in PTZ-induced mouse brain tissues (mean ± SD, n = 6). #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group. P < 0.05 and P<0.01; (C) shows the effect of BBPs on the expression of GABAA-Rα1, GAD65, GAD67, GAT1, and GAT3 proteins in PTZ-induced mouse brain tissue (mean ± standard deviation, n=6). Compared with the control group, #P<0.05 and ##P<0.01; compared with the model group, P<0.05 and P<0.01. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited by them are incorporated herein by reference. Equivalent techniques of the specific embodiments described herein, which can be understood by those skilled in the art through routine experiments, are included in this application. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the instruments and equipment used in the following embodiments are standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0017] I. Reagents and Antibodies: Fried silkworm pupae (batch numbers D2206113, D2208029, D2207093) were purchased from Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd.; artificial gastric fluid (batch number 0020A17) and artificial intestinal fluid (batch number 1122A23) were purchased from Beijing Regen Biotechnology Co., Ltd.; formic acid was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and acetonitrile was purchased from Thermo Fisher Scientific, Inc. DYY-6C electrophoresis apparatus power supply (Beijing Liuyi Instrument Factory); mini-P4 vertical electrophoresis tank (Jinan Junyi Biotechnology Co., Ltd.); QuantStudio TM3. Real-time quantitative PCR system (Thermo Fisher Scientific, USA). Pentyltetrazolate (batch number: P103065, Shanghai Aladdin Biochemical Technology Co., Ltd.); Sodium valproate (batch number: C15971233, Shanghai Maclean Biochemical Technology Co., Ltd.); GABA (batch number: L231217465), 5-hydroxytryptamine (5-HT, batch number: L231217474), interleukin-6 (IL-6, batch number: L240822147), tumor necrosis factor-α (TNF-α, batch number: L240822182), glucocorticoid receptor α (GRα) ELISA kit (batch number: L240822195, Wuhan Yunclone Technology Co., Ltd.); glutamate (Glu) kit (batch number: 20231216, Nanjing Jiancheng Bioengineering Institute). Glutamate dehydroxylase 65 (GAD 65, batch number: 00063948, Wuhan Sanying Biotechnology Co., Ltd.); β-actin and HRP-labeled secondary antibodies (batch numbers: AC230702004, AC2408111994, Wuhan Saiwei Biotechnology Co., Ltd.); GAT 3 (batch number: PS08046S, Abimate Pharmaceutical Technology Co., Ltd.); Gamma-aminobutyric acid receptor α1 (GABAA-Rα1, batch number: BB04145144, Beijing Bio-Sens Biotechnology Co., Ltd.); Glutamate decarboxylase 67 (GAD 67) and Gamma-aminobutyric acid carrier protein 1 (GAT 1) antibodies (batch numbers: 4000001879, 1600290101, Aibote Biotechnology Co., Ltd.).

[0018] II. Experimental Methods: 1. Preparation of BB peptide: BB was crushed and ground and passed through a No. 4 sieve. BB dry powder was defatted by adding 10 times petroleum ether (60-90℃) and refluxed twice for 1h each time. Then it was centrifuged at 5000g for 10 minutes and the residue was dried at 60℃ to obtain defatted BB powder called BP. Then the defatted BB powder was extracted with 30 times the volume of distilled water by reflux for 2 hours and centrifuged at 10000rpm for 15 minutes. Then the supernatant was concentrated and freeze-dried to obtain BB decoction powder BD. In vitro gastrointestinal digestion was simulated for BP and BD samples to prepare peptide extracts. The peptide extraction was optimized based on the previously reported method

[16] . The pH of the mixture of BP samples was adjusted to 3.0 with 1M HCl, pepsin was added (37℃, 2h), the pH was adjusted to 7.5 by adding 1M NaHCO3, and then trypsin (37℃, 2h) was added to promote trypsin digestion. After digestion, it was taken out and placed in a boiling water bath for 5min for inactivation. Subsequently, the sample was centrifuged at 5000g for 10 min at 4°C to obtain the peptide extract of BP (BPP). The peptide extract of BD (BDP) was obtained using the same method as described above.

[0019] 2. Nano-Liquid Chromatography-Tandem Mass Spectrometry (Nano LC-MS / MS) Analysis: BPP and BDP were placed in 0.1% formic acid solution, washed with the tip of a C-18 rotating column, and then analyzed separately by Nano LC MS / MS on a Q-Exactive HF mass spectrometer. The raw mass spectrometry files were analyzed and searched using Proteome Discoverer 2.5 with the following parameter settings: enzyme-free; variable modifications included oxidation (M). Peptide mass tolerance was set to 10 ppm, and fragment mass tolerance was set to 0.02 Daltons. Matching analysis was performed using the search software against silkworm, mulberry, and Beauveria bassiana protein databases (NCBInr, UniProt, etc.). The spectra were also searched for the same number of bait sequences to estimate the false discovery rate. A false discovery rate score ≤0.05 obtained through the search was considered a valid identification result.

[0020] 3. Peptide Activity Prediction: The online tool PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the bioactivity of the identified peptides. A higher score indicates a greater likelihood of them being bioactive peptides. Peptides with a bioactivity score ≥0.8 were selected as potential bioactive peptides. The bioactivity of a peptide is closely related to its molecular weight (MW). Peptides with lower molecular weights are more likely to exhibit bioactivity when crossing the intestinal barrier. Bioactive peptides typically consist of no more than 20 amino acid residues; therefore, peptides with a sequence length <20 were selected for further study. ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) was used for toxicity prediction.

[0021] 4. Molecular docking model for predicting antiepileptic peptides: Molecular docking is used to assess the interaction between peptides and GABA. AInteraction between R and NMDAR receptors. Crystal structures of GABAAR (PDB ID: 4COF) and NMDA (PDB ID: 1Y20) were downloaded from the RCSB Protein Database (http: / / www.rcsb.org / ). Amino acids were linked into peptide sequences using ChemDraw 20.0 software, while Chem3D 20.0 software was used to generate 3D structural files for the peptides. This process included selecting the lowest energy conformation and saving in MOL format. The MOL2 format was converted to PDBQT format using OpenBabel 3.1.1 for future integration. The obtained PDBQT protein receptor files were purified using AutoDock Vina (http: / / autodock.scripps.edu / ), removing all water molecules and unnecessary substructures, and adding hydrogen atoms. The peptides underwent the same treatment, and docking simulations were performed using the protein as the receptor and the peptide as the ligand to obtain the appropriate minimum binding energy, which was visualized. The binding energy (kcal / mol) value represents the binding strength between the receptor and the peptide; the lower the binding energy, the more stable the interaction between the ligand and the receptor. Finally, PyMOL software was used for molecular visualization and analysis.

[0022] 5. Peptide Synthesis: Based on the docking results, 10 predicted peptides were chemically synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. (Shanghai, China). HPLC analysis confirmed that the purity of the synthesized peptides was >95%.

[0023] 6. Cell Culture: PC12 cells were cultured at 37°C in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin in an incubator containing 5% CO2. Cells were passaged every 2 to 3 days, and experiments were conducted using 3 to 5 passages of cells.

[0024] 7. Cell viability assay: Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. PC12 cells (5 × 10⁻⁶) were... 4 PC12 cells were seeded at 5 × 10⁶ cells / ml in 96-well plates and grown for 24 hours. To assess the effect of Glu and BBPs on cell viability, PC12 cells were seeded at 5 × 10⁶ cells / ml in 96-well plates and grown for 24 hours. 4Cells were seeded at a density of 1 / ml in 96-well plates with different concentrations of Glu (14, 16, 18, 20, and 22 mM) and BBP (20, 40, 80, and 160 µM) for 24 hours. Cell viability was then determined using the MTT assay as described previously. The culture medium was removed, MTT solution was added, and the plates were incubated at 37°C for 4 hours. Optical density (OD) values ​​were then measured at 570 nm using a Gen 5 multimicroplate reader (Biotek, Winooski, VT, USA). Each experiment was repeated six times. Cell viability is expressed as a percentage of the OD values ​​of normal (untreated) cells.

[0025] 8. Animal grouping and treatment: SPF KM mice (20±2 g) were purchased from Spiford (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019–0010, and housed in a controlled environment (temperature 21±2℃, humidity 55±10%) with alternating 12-hour light / dark cycles. The mice had free access to food and water.

[0026] According to the literature report

[15] , a chronic PTZ-ignited model was established in mice to evaluate the therapeutic effect of PP6 on epilepsy. Except for the control group, all other mice were intraperitoneally injected with PTZ (35 mg / kg) every 48 hours and their behavioral changes were observed for 30 min. The control group was intraperitoneally injected with an equal volume of physiological saline. According to the modified Racine grading system, if a grade 4 or higher seizure occurred for 3 consecutive days, it was considered a successful ignition. The modeling period was 21 days, and mice that were not ignited after 21 days were discarded. The mice that were successfully modeled were divided into: model group (PTZ), sodium valproate group (VPA, 250 mg / kg, ip), low-dose PP6 group (PP6-L, 20 mg / kg, ip), medium-dose PP6-M group (40 mg / kg, ip) and high-dose PP6 group (PP6-H, 80 mg / kg, ip). During the administration period, the mice that were successfully modeled were intraperitoneally injected with PTZ 35 mg / kg on days 5, 10, 15 and 20 to maintain the ignition state, and the administration period was 20 days. During the observation period on days 5, 10, 15, and 20 after administration, the latency of the first myoclonic or generalized tonic-clonic seizure was recorded, as well as the seizure stage in mice.

[0027] Grade 0: No seizures at all; Grade 1: Facial twitching (including whisker twitching, blinking, rhythmic chewing, wet dog-like shaking, etc.); Grade 2: Facial twitching, neck muscle spasms (rhythmic head nodding and tail wagging); Grade 3: Facial twitching, neck muscle spasms, forelimb clonus (frequent ear-scratching and cheek-scratching); Grade 4: Facial twitching, neck muscle spasms, forelimb clonus, hindlimb standing; Grade 5: Facial twitching, neck muscle spasms, limb twitching, body dorsiflexion, rigidity, and falling.

[0028] 9. Histopathological evaluation: Fresh brain tissue was collected and fixed with 4% paraformaldehyde. The brain tissue was then dehydrated and embedded in paraffin. Sections (4 μm) were prepared using a cryostat and then stained with hematoxylin and eosin (H&E) and Nissl stain (toluidine blue) according to standard procedures. The staining results of the hippocampus in each group of mice were observed under a microscope.

[0029] 10. Enzyme-Linked Immunosorbent Assay (ELISA): Determine the levels of GABA, IL-1β, IL-4, Glu, GRα, and TNFα in PC12 cells or brain tissue using an ELISA kit, following the manufacturer's instructions. Measure absorbance using a Gen 5 multi-plate reader (Biotek, Winooski, VT, USA).

[0030] 11. Real-time quantitative polymerase chain reaction (RT-PCR) detection: Total RNA was extracted from PC12 cells or brain tissue using Trizol reagent according to the manufacturer's instructions, and its purity and concentration were determined by absorbance at 260 and 280 nm. Subsequently, 2 μg of RNA was reverse transcribed into cDNA using the RevertAid first-strand cDNA synthesis kit. The relative abundance of genes of interest was determined using the 2-ΔCt method on a QuantStudio3 PCR instrument (Applied Biosystems, Shanghai, China), with GAPDH or β-actin used as internal controls. Primer sequences are shown in Table 1.

[0031] Table 1: RT-qPCR primer sequences 12. Western Blot Detection: Total protein was extracted from PC12 cells or brain tissue, and protein concentration was determined using the BCA protein assay reagent. Subsequently, 30 μg of protein was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h at ambient temperature. After blocking, the sample was incubated overnight at 4°C with antibodies GAPDH or β-actin, GABAA-Rα1, GAD 65, GAD67, GAT 1, and GAT 3. The membrane was then washed four times with TBST buffer and incubated with secondary antibody for 1 h, followed by four more washes with TBST buffer. Protein expression levels were quantified using an imaging system and ImageJ software.

[0032] 13. Statistical Analysis: All experiments were performed in triplicate. Data are reported as mean ± standard deviation. One-way ANOVA was performed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA, USA) to assess differences between groups. PA value <0.05 is considered statistically significant.

[0033] III. Experimental Results 1. Identification of BB precursor proteins: LC-MS / MS was used to identify peptides in BPP and BDP. The amino acid sequences of the peptides in BPP and BDP were identified as 6972 and 4264, respectively. These peptides were retrieved from 4377 precursor proteins using protein databases of *Bombyx mori* and *Beauveria bassiana*. The main precursor proteins were fibrin heavy chain (P05790), serine 1-like proteins (A0A8R2QX88, A0A8R2QUV5, A0A8R2QUR5, A0A8R2DLE6), and myosin heavy chain proteins (A0A8R2QYX1, A0A8R22). LWE6, A0A8R1LVN9, A0A8R20QX97), serine protease 1 (P07856), aryl protein (Q1HPP4, G9I6Y1), sex-specific storage protein 1 (P09179, P20613), serine 1B (Q17240), fibrin light chain (Q7JYG3, Q9BIF8), silkworm storage protein (H9JHM9), apolipoprotein (A0A8R2AKU9, G1UIS8), actin (A0A8R20M3B1, S5M4E2, J4UN10), heparan sulfate proteoglycan (A0A8R1R6D9, A0A8R2M5B7), low molecular weight 30 kDa lipoprotein 19G1 precursor (C7A8A3) and 30K proteins (E5EVW2, H9B436, H9B440), etc.

[0034] 2. Peptide analysis in BBP and BDP: The molecular weight (MW) distribution and peptide length of the peptides identified by BB were evaluated, and the results are as follows: Figure 2 As shown, the molecular weight distribution of peptides in BPP and BDP is mainly between 500 and 1000, with peptides between 500 and 2000 accounting for 91.02% and 93.99% of the total, respectively. In BPP, most of the identified peptides were 6, 7, and greater than 10 amino acids in length, accounting for 16.31%, 15.3%, and 43.98%, respectively. Similarly, in BDP, peptides with lengths of 6, 7, and greater than 10 amino acids were also the most common, accounting for 20.19%, 17.99%, and 36%, respectively.

[0035] In addition, the C-terminal and N-terminal peptides in BPP and BDP were analyzed. Figure 2 E). The most abundant amino acid residues at the C-terminus of BPP and BDP peptides are Ala, Ile, Val, Gly, and leu. The most abundant amino acids at the N-terminus are Arg, Lys, Leu, and Phe. The frequencies of hydrophilic amino acids are as follows: Figure 2As shown in Figure F, statistical analysis revealed that hydrophilic amino acids appeared 41,103 times in BPP and 24,028 times in BDP, including Arg, Asn, Asp, Gln, Glu, Gly, His, Ser, Thr, and Lys. In contrast, hydrophobic amino acids appeared 36,787 times and 20,324 times, including Ala, Ile, Leu, Met, Phe, Pro, Trp, Tyr, and Val. This indicates that the peptides in BB are more hydrophilic.

[0036] Venn diagrams of peptides identified in BPP and BDP are shown below. Figure 3 As shown in Figure A, the amino acid sequences of the peptides identified in BPP and BDP are 6972 and 4264, respectively. There are 4015 identical peptides in BPP and BDP. BPP contains 2957 unique peptides, and BDP contains 249 unique peptides.

[0037] By analyzing raw LC-MS data, the signal intensity of each peptide can be measured and quantified. After normalization, these peptides can be quantitatively compared across different samples. A total of 10,675 peptides were identified from BPP and BDP, with a fold difference ≥2.0 or <0.05 (P<0.05) as the criterion. Figure 3 As shown in the BF analysis, compared with BDP, the expression levels of 2047 identical peptides in BPP were significantly upregulated, while the expression levels of 426 peptides in BPP were downregulated. Hierarchical cluster analysis was performed on the peptides. Figure 3 (D) The results showed good biological repeatability within the variety, and there were significant differences between the BPP and BDP groups.

[0038] 3. Peptide Grading and Activity Prediction: Previous studies have shown that BB powder has significantly stronger anticonvulsant and antiepileptic effects than BB decoction. Therefore, compared with BDP, the activity of 2957 unique peptides and 2047 upregulated peptides in BPP was screened and predicted. Forty-three peptides with a ranking score ≥0.8, peak area response value >200, and amino acid count ≤20 were selected for further study. Toxicity is an important criterion for assessing peptide medicinal applicability. Toxicity screening was used to identify these 43 potentially bioactive peptides, and all 43 peptides were determined to be non-toxic.

[0039] 4. Molecular docking: Molecular docking is a widely used method for studying interactions between molecules and predicting their most likely binding modes and intermolecular interactions. Further identification of peptides with anti-epileptic activity is possible. Molecular docking simulations are performed to evaluate the interaction of selected peptides with GABA. A The binding affinity of R and NMDAR. Using AutoDock-Vina software, 43 potentially bioactive peptides were bound to GABA. A R and NMDAR molecular docking.

[0040] The molecular docking results are shown in Table 2. A binding energy less than 0 kcal / mol is considered effective docking; the smaller the value, the more stable the interaction between the peptide and the receptor protein. As shown in Table 2, the binding energies of 43 peptides are less than -5, indicating that these 43 peptides can effectively bind to GABA. A R and NMDAR proteins dock. Results showed that the 43 screened peptides were compatible with GABA. A R and NMDAR have good compatibility.

[0041] Table 2: Molecular docking scores of peptides Peptides derived from BPP, namely PFAGLF (PP1), PFGAIF (PP4), RFAFPAWI (PP6), WFAGFS (PP7), FGDPFKGYK (PP8), and SDAASEDGFWW (PP10), were selected because they exhibit strong GABA binding affinity. We also selected SWFVTPF (PP2), WGAFSF (PP3), PFGAIF (PP4), AWAGFGR (PP5), and RFAF PAWI. The PyMol program was used to visualize and analyze their docking conformations, such as... Figure 4 As shown.

[0042] Figure 4 Table 3 shows peptides with potential anti-epileptic activity in relation to GABA. A The number of hydrogen bonds and docking sites of R / NMDAR. (GABA binding) AR, PP1 forms a hydrogen bond with THR-256 at its docking site. Similarly, PP4 also forms a hydrogen bond with THR-256. PP6 forms three hydrogen bonds with both THR-256 and THR-260. PP7 forms two hydrogen bonds with THR-256. PP8 forms three hydrogen bonds with THR-256. Furthermore, PP10 forms five hydrogen bonds with THR-256, THR-263, and ALA-252. PP2 binds to NMDAR and forms six hydrogen bonds with ARG-155, ILE-156, THR-240, THR-241, ALA-232, and GLN-288. Similarly, PP3 also forms six hydrogen bonds with ARG-155, ILE-156, THR-240, THR-241, ALA-232, and GLN-288. PP4 forms two hydrogen bonds with GLU-208 and THR-50. PP5 forms five hydrogen bonds with GLU-31, TYR-64, THR-283, TYR-18, and GLN-288. PP6 forms four hydrogen bonds with GLY-90 and THR-50. PP7 forms five hydrogen bonds with LYS-235, GLU-14, GLU-208, GLU-234, and SER-205. PP9 forms eight hydrogen bonds with THR-50, ASP-49, ASN-48, PRO-47, GLN-61, and LSY-91. The formation of hydrogen bonds with these amino acids significantly stabilizes the enzyme-peptide complex.

[0043] Table 3. Peptides with potential anti-epileptic activity and GABA A Number of hydrogen bonds and docking sites in R / NMDAR 5. Neuroprotective effect of peptides with potential anti-epileptic activity on Glu-damaged PC12 cells: PC12 cells were treated with different concentrations of Glu (14, 16, 18, 20, and 22 mM) and cultured for 24 hours to determine the optimal conditions for Glu induction. Figure 5 As shown in Figure A, Glu treatment significantly reduced cell viability (P<0.01), with viability decreasing to 80.95%, 68.94%, 59.03%, 51.23%, and 37.67%, respectively. This indicates that Glu has a dose-dependent toxic effect on PC12 cells. A Glu concentration of 20 mM was selected as the modeling concentration for this invention.

[0044] Cell viability of peptides with potential anti-epileptic activity against PC12 cells was determined using the MTT assay to assess their cytotoxicity. For example... Figure 5As shown in Figure B, the 10 peptides (10, 20, 40, 80, and 160 μM) showed no cytotoxicity in PC12 cells. Among them, PP3, PP8, and PP9 at concentrations of 20–160 μM improved cell viability within 24 hours. Therefore, peptides at concentrations of 20, 40, 80, and 160 μM were selected for subsequent experiments in this invention.

[0045] In addition, such as Figure 5 As shown in Figure C, PP6 at concentrations ranging from 40 to 160 µM effectively alleviated Glu-induced damage in PC12 cells and exhibited a dose-dependent protective effect (P < 0.05 or P < 0.01). In contrast, other peptides at concentrations ranging from 20 to 160 µM did not show significant protective effects against Glu-injured PC12 cells.

[0046] 6. Effects of PP6 on GABA, IL-1β, IL-4, GRα, Glu, and TNF-α: The effects of PP6 on the levels of GABA, IL-1β, IL-4, GRα, and TNF-α in Glu-damaged PC12 cells are as follows: Figure 6 As shown in Figure A, compared with normal cells, the GABA level in the model cells was significantly decreased (P<0.01), while the levels of IL-1β, IL-4, GRα, and TNF-α were significantly increased (P<0.01). Interestingly, compared with the model cells, PP6 (80 and 160 μM) significantly increased the GABA level (P<0.01) and significantly decreased the levels of IL-1β, IL-4, GRα, and TNF-α (P<0.05 or P<0.01).

[0047] 7. Effects of PP6 on the expression of GABAA-Rα1, GAD 65, GAD 67, GAT 1, and GAT 3 mRNA: To further investigate the mechanism of action of PP6, we measured the expression of related mRNAs in the GABA signaling pathway (GABAA-Rα1, GAD 65, GAD 67, GAT 1, and GAT3). Figure 6 As shown in Figure B, compared with control cells, the mRNA expression of GABAA-Rα1, GAD65, and GAD67 was significantly decreased in Glu-damaged PC12 cells (P<0.01), while the mRNA expression of GAT1 and GAT3 was significantly increased (P<0.01). Treatment with 80 and 160 μM PP6 significantly upregulated the mRNA expression of GABAA-Rα1 and GAD67 (P<0.01), while significantly downregulated the mRNA expression of GAT3 (P<0.01). Furthermore, compared with model cells, 160 μM PP6 significantly enhanced the mRNA expression of GAD65 but decreased the mRNA expression of GAT1 (P<0.05).

[0048] 8. Effects of PP6 on the expression of GABAA-Rα1, GAD65, GAD67, GAT1, and GAT3 proteins: The effects of PP6 on the expression of GABAA-Rα1, GAD65, GAD67, GAT1, and GAT3 proteins in Glu-damaged PC12 cells are as follows: Figure 6 As shown in Figure C. The results indicated that, compared to the normal group, Glu treatment significantly downregulated the protein expression of GABAA-Rα1, GAD65, and GAD67 in PC12 cells, while significantly upregulating GAT1 and GAT3 (P<0.01). Interestingly, PP6 (80 and 160 μM) significantly upregulated the protein expression of GABAA-Rα1, GAD65, and GAD67 (P<0.05), while significantly downregulating GAT3 protein expression. Furthermore, 160 μM PP6 significantly reduced GAT1 protein expression in Glu-damaged PC12 cells (P<0.01).

[0049] 9. Effects of PP6 on PTZ-induced epileptic mice: The effects of PP6 on PTZ-induced epileptic behavior in mice are shown in Tables 4 and 5. Compared with the model group, high-dose (80 mg / kg) PP6 significantly prolonged the latency of PTZ-induced epilepsy in mice 15 days after administration. P The medium dose (40 mg / kg) of PP7 significantly prolonged the latency of epilepsy after 20 days of administration. Furthermore, as shown in Table 5, compared with model mice, the high dose (80 mg / kg) of PP6 significantly reduced the severity of PTZ-induced epilepsy in mice after 15 days of administration (P<0.05). After 20 days of administration, the 40 mg / kg dose of PP6 also significantly reduced the severity of PTZ-induced epilepsy in mice (P<0.01). These results indicate that the antiepileptic effect of PP6 on PTZ-induced epilepsy exhibits time- and dose-dependent characteristics.

[0050] Table 4: Effect of PP6 on the latency of epilepsy in PTZ-induced mice (n=13) Note: # Compared with the control group, P<0.05 and ##P<0.01; compared with the model group, P<0.05 and P<0.01.

[0051] Table 5: Effects of PP6 on the seizure phase in PTZ-induced mice (n=13) Note: # Compared with the control group, P<0.05 and ##P<0.01; compared with the model group, P<0.05 and P<0.01.

[0052] Histological analysis results as follows Figure 7 As shown, this includes HE staining ( Figure 7 A) and Nissl staining ( Figure 7 B). Compared with the control group mice, no significant morphological changes were observed in the CA1 region of the hippocampus in the model group mice, while neurons in the CA3 region showed significant morphological abnormalities, including irregular cell morphology, disordered arrangement, nuclear condensation, and a deepened triangular shape, with a significant reduction in the number of neurons. Compared with the model mice, neurons in the CA3 region of the hippocampus in the PP6-M and PP6-H groups were more densely arranged, cell integrity was improved, and the degree of neuronal damage was reduced. In addition, Nissl staining results ( Figure 7 (B and 7C) showed that, in addition to abnormal neuronal cell morphology, the number of Nissl positive cells in the CA3 region of the hippocampus of the model mice was significantly lower than that of the control group, and the average optical density value was significantly reduced (P<0.05). Interestingly, the neurons in the PP6-M and PP6-H groups were relatively orderly arranged, and the number of positive cells in the CA3 region of the hippocampus of the PP6-H group was significantly higher than that of the model group, and the average optical density value of the positive cells was significantly increased (P<0.01).

[0053] 10. Effects of PP6 on the levels of GABA, Glu, IL-1β, GRα, and TNF-α in PTZ-induced mouse brain tissue: Figure 8 As shown in Figure A, compared with normal mice, the GABA level in the model mice was significantly decreased (P<0.05), while the levels of Glu and TNF-α were significantly increased (P<0.01). Compared with the model mice, treatment with PP6 (40 and 80 mg / kg) significantly increased the GABA level in brain tissue (P<0.01), but significantly decreased the levels of Glu and TNF-α (P<0.05 or P<0.01). Furthermore, GABA levels were also significantly increased in mice treated with PP6 at a dose of 20 mg / kg (P<0.05).

[0054] 11. Effects of PP6 on the expression of GABA signaling pathway-related mRNAs in the brain tissue of PTZ-induced epileptic mice. Figure 8As shown in Figure B, compared with the control group, the mRNA expression of GABAA-Rα1, GAD 65, and GAD 67 in the brain tissue of PTZ-induced epileptic mice was significantly downregulated (P<0.05), while the mRNA expression of GAT 1 and GAT 3 was significantly upregulated (P<0.01). Compared with model mice, treatment with PP6 at doses of 40 and 80 mg / kg significantly increased the mRNA expression of GAD 65 and GAD 67 in brain tissue (P<0.05 or P<0.01), while significantly decreasing the mRNA expression of GAT 3 (P<0.01). Furthermore, 80 mg / kg of PP6 significantly enhanced the mRNA expression of GABAA-Rα1 (P<0.05) but decreased the mRNA expression of GAT 1 (P<0.01).

[0055] 12. Effects of PP6 on the expression of GABAA-Rα1, GAD65, GAD67, GAT1, and GAT3 proteins in the brain tissue of PTZ-induced epileptic mice. Figure 8 As shown in Figure C, compared with the control group, the expression of GABAA-Rα1, GAD65, and GAD67 proteins was significantly decreased in the model mice, while the expression of GAT1 and GAT3 was significantly increased (P<0.05). Conversely, after treatment with PP6 at doses of 40 and 80 mg / kg, the protein levels of GABAA-Rα1 and GAD65 were significantly upregulated, while the protein level of GAT3 was significantly downregulated (P<0.05 or P<0.01). In the PP6 80 mg / kg dose group, the protein level of GAD67 was significantly increased (P<0.01), and the protein level of GAT1 was significantly decreased (P<0.05).

[0056] Animal-derived traditional Chinese medicines or foods are rich in proteins or peptides with therapeutic properties, such as antimicrobial peptides[3], antioxidants

[16] , and antihypertensive peptides

[17] . Proteins in BB have shown significant anticonvulsant and antiepileptic properties[12, 13]. However, the complex composition of animal-derived traditional Chinese medicines, coupled with the complex structure of biomolecular proteins, poses a challenge to elucidating the basic components of the pharmacological effects of these animal-derived traditional Chinese medicines. Animal-derived active peptides have become the focus of research and application due to their significant bioactivity, precise targeting specificity, and low incidence of adverse reactions

[18] . Peptidomics is widely used to identify peptides in animal-derived traditional Chinese medicines, such as leeches

[19] and earthworms

[20] . Virtual screening is an efficient and convenient computational simulation technique. The combination of peptidomics and virtual screening methods can be used to identify bioactive peptides, which can reduce workload and labor costs, shorten the development cycle, and improve the success rate of bioactive peptide identification. This invention combines peptidomics and virtual screening to study peptides in BB. This method effectively detects and analyzes the composition of peptides and screens out peptides with potential activity.

[0057] The present invention uses nano-LC-MS / MS technology to detect the peptide composition in BB powder and decoction protein extract. The results showed that there were 6972 peptides in the BPP sample and 4264 peptides in the BDP sample. Among them, 4015 peptides were found to be consistent, accounting for 55.6% of the total peptides. In addition, the BPP sample included 2957 unique peptides, accounting for 41% of the total peptide composition. The results showed that more peptides could be obtained after powdering compared with decoction. Subsequently, virtual screening was performed on the 2957 unique peptides and 2047 upregulated peptides in BPP, and 43 potential active peptides were obtained. Current evidence suggests that among all GABA receptor subtypes, GABAA receptor is most strongly associated with epilepsy. Glu receptor NMDA is also significantly associated with epilepsy

[21] . The interaction between peptides and GABAAR or NMDA receptors was evaluated to further screen peptides with potential anti-epileptic effects

[22] . Ten potential antiepileptic peptides (PP1-PP10) with strong binding affinity to GABAAR or NMDA receptors were selected for further activity validation studies.

[0058] PC12 cells are a widely used model in neurobiology, and research involving PC12 cells covers a range of topics, including neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synapsis

[23] . Glutamate is a major excitatory neurotransmitter in the central nervous system and is essential for excitatory synaptic transmission, neuronal development, and neuroplasticity in adults

[22] . Excess glutamate can have significant effects on neurons by initiating neurotoxic or excitotoxic pathways

[24] . Furthermore, elevated glutamate levels are positively correlated with the severity of epileptic activity

[25] . Glu-damaged PC12 cell injury models are commonly used in in vitro epilepsy studies. Therefore, this invention uses glut-damaged PC12 cells to detect the in vitro anti-epileptic effect of PP1 on PP10. The results showed that PP2, PP3, PP4, PP8, and PP9 promoted PC12 cell proliferation; however, these peptides did not protect against damaged glut in PC12 cells. Interestingly, PP6 was shown to have a neuroprotective effect against the neurotoxicity of glut-damaged PC12 cells. In addition, the PTZ chronic ignition model can effectively simulate the chronic development process of human epilepsy and has become a core tool for epilepsy research

[26] . Therefore, this invention also used a PTZ-induced epilepsy mouse model to verify the anti-epileptic effect of PP6. The results showed that PP6 had a significant anti-epileptic effect on PTZ-induced mice. The precursor protein of PP6 (RFAFPAWI) is a trichosanthin 3-O-acetyltransferase (A0A2N6NVJ1) from Beauveria bassiana.

[0059] The results of this invention indicate that PP6 can significantly increase GABA levels in Glu-damaged PC12 cells and decrease the levels of IL-1β, IL-4, GRα, and TNF-α. PP6 also significantly increased GABA levels in PTZ-induced mouse brain tissue and decreased Glu and TNF-α levels. Its neuroprotective and antiepileptic effects may be related to the regulation of the GABA signaling pathway. PP6 exerts its antiepileptic effect by balancing GABA and Glu levels and the neuroimmunoendocrine network.

[0060] This invention characterized peptides in BBP and BDP using Nano-LC-MS / MS. Virtual screening was performed to obtain peptides with antiepileptic activity, ultimately selecting peptides PP1 to PP10. Subsequently, the protective effects of these ten peptides on Glu-damaged PC12 cells were investigated. PP2, PP3, PP4, PP8, and PP9 exhibited proliferative effects on PC12 cells. PP6 was shown to have a neuroprotective effect against the neurotoxicity of Glu-damaged PC12 cells. Finally, the antiepileptic effects of the active peptides were verified using PTZ-induced epileptic mice, demonstrating that PP6 significantly improved the behavior of PTZ-induced epileptic mice by regulating the GABA signaling pathway. This invention identifies novel antiepileptic peptides and demonstrates their potential as antiepileptic drugs. Furthermore, it provides substantial evidence for further exploration of powdered administration and antiepileptic activity of BB, and offers new research ideas for the study of other animal herbal peptide components.

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Claims

1. A silkworm polypeptide for anti-epileptic purposes, characterized in that: The silkworm polypeptide is a peptide with the amino acid sequence shown in SEQ ID No. 1, namely: RFAFPAWI, named PP6.

2. A method for preparing the anti-epileptic silkworm polypeptide of claim 1, characterized in that: Peptide extracts from *Bombyx mori* were prepared by simulating gastrointestinal digestion. The peptides in the *Bombyx mori* peptide extracts were identified using Nano LC-MS / MS, and differential analysis was performed. Differential peptides with potential anti-epileptic activity were obtained through virtual screening, and *Bombyx mori* polypeptides with potential anti-epileptic activity were synthesized.

3. The method for preparing anti-epileptic silkworm peptide according to claim 2, characterized in that: Includes the following steps: (1) Degreasing of silkworm: The silkworm is crushed and passed through a No. 4 sieve, then 10 times the amount of petroleum ether is added to the dry powder, and the silkworm is degreased by reflux at 60-90℃ twice, each time for 1 hour. Then, it is centrifuged at 5000g for 10 minutes and the residue is dried at 60℃ to obtain the degreased silkworm. (2) Preparation of peptide extract: The defatted silkworm sample was digested in vitro to prepare peptide extract. The pH value of the defatted silkworm sample was adjusted to 3.0 with 1M HCl, and pepsin was added at 37℃ for 2h digestion. Then, 1M NaHCO3 was added to adjust the pH value to 7.5, and trypsin was added at 37℃ for 2h digestion. After digestion, the sample was placed in a boiling water bath for 5min to inactivate it. The sample was centrifuged at 5000g at 4℃ for 10min. The precipitate obtained was the peptide extract BPP. (3) Nano-liquid chromatography-tandem mass spectrometry (Nano LC-MS / MS) analysis: BPP was placed in 0.1% formic acid solution, washed with the tip of a C-18 rotating column, and analyzed by Nano LC MS / MS on a Q-Exactive HF mass spectrometer; the original mass spectrometry file was analyzed and searched by Proteome Discoverer 2.5, with the following parameter settings: no enzyme; variable modification including oxidation M; peptide mass tolerance set to 10 ppm, fragment mass tolerance set to 0.02 Da; matching analysis was performed with the silkworm, mulberry plant and Beauveria bassiana protein databases NCBInr and UniProt by the search software; the false discovery rate score obtained by the search was ≤0.05, which was considered a valid identification result; (4) Predicting peptide activity: The online tool PeptideRanker was used to predict the bioactivity of the identified peptides. Peptides with a bioactivity score ≥0.8 were selected as potential bioactive peptides. Peptides with a sequence length <20 were selected for toxicity prediction using ToxinPred. (5) Molecular docking model prediction and screening of anti-epileptic peptides: GABA was downloaded from the RCSB protein database. A Crystal structures of R and NMDAR; amino acids were linked into peptide sequences using ChemDraw 20.0 software, and 3D structure files of the peptides were generated using Chem3D 20.0 software; The MOL2 format was converted to PDBQT format using OpenBabel 3.1.1 for integration; the obtained PDBQT protein receptor file was purified using AutoDock Vina, removing all water molecules and unnecessary substructures, and adding hydrogen atoms; the peptide underwent the same treatment, and docking simulations were performed using the protein as the receptor and the peptide as the ligand to obtain the minimum binding energy. Molecular visualization and analysis were performed using PyMOL software; GABA was included. A R's PDB ID: 4COF; NMDAR's PDB ID: 1Y20; (6) Synthetic peptides: Based on the docking results, peptides with potential anti-epileptic activity were synthesized, and the purity of the synthesized peptides was identified by HPLC as >95%.

4. The use of the anti-epileptic silkworm polypeptide of claim 1 in the preparation of a medicament for protecting PC12 cells damaged by Glu.

5. The application according to claim 4, characterized in that: The anti-epileptic silkworm polypeptide significantly increased the GABA content in Glu-damaged PC12 cells and decreased the levels of IL-1β, IL-4, GRα and TNF-α.

6. The application according to claim 4, characterized in that: The concentration of the anti-epileptic silkworm polypeptide is 40-160 µM.

7. The use of the anti-epileptic silkworm polypeptide of claim 1 in the preparation of a drug for improving the epileptic behavior of PTZ-induced epileptic mice.

8. The application according to claim 7, characterized in that: The anti-epileptic silkworm polypeptide significantly improved the behavior of PTZ-induced epileptic mice by regulating the GABA signaling pathway.

9. The application according to claim 7, characterized in that: The anti-epileptic silkworm polypeptide significantly increased GABA content and decreased Glu and TNF-α levels in PTZ-induced mouse brain tissue.

10. The application according to claim 7, characterized in that: The dosage of the anti-epileptic silkworm polypeptide is 40-80 mg / kg, administered for 15-20 days.

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