WO2017004811A1 - 一种抑制ampa受体的活性肽及其制备方法和应用 - Google Patents

一种抑制ampa受体的活性肽及其制备方法和应用 Download PDF

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WO2017004811A1
WO2017004811A1 PCT/CN2015/083571 CN2015083571W WO2017004811A1 WO 2017004811 A1 WO2017004811 A1 WO 2017004811A1 CN 2015083571 W CN2015083571 W CN 2015083571W WO 2017004811 A1 WO2017004811 A1 WO 2017004811A1
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active peptide
preparation
egar
tetrapeptide
enzymatic hydrolysis
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PCT/CN2015/083571
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English (en)
French (fr)
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蔡木易
顾怀宇
鲁军
潘兴昌
蔡松
马勇
段松伟
陈亮
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中国食品发酵工业研究院
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Priority to CN201580077695.6A priority Critical patent/CN107428800B/zh
Priority to PCT/CN2015/083571 priority patent/WO2017004811A1/zh
Priority to JP2018500399A priority patent/JP6670922B2/ja
Publication of WO2017004811A1 publication Critical patent/WO2017004811A1/zh
Priority to US15/834,026 priority patent/US10800810B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22002—Papain (3.4.22.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an active peptide, in particular to an active peptide which inhibits AMPA receptor, and a preparation method and application thereof.
  • Epilepsy is a chronic disease in which transient abnormal discharge of brain neurons leads to transient brain dysfunction. The main symptom is repeated, unstoppable sputum, which has become the second most common disease in neurology after headache in China. About 1-2% of the world's people suffer. Although more than 15 antiepileptic drugs are available in most countries, 20-30% of patients with epilepsy cannot effectively control sputum at the time of onset.
  • AMPA receptor inhibitors may have the effect of preventing or treating epilepsy, and drugs that inhibit or attenuate AMPA receptors may inhibit neuronal over-excitation, thereby protecting neurons and inhibiting sputum.
  • the typical AMPA receptor competitive inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX) can function in the pentylenetetrazol (PTZ) model.
  • PTZ pentylenetetrazol
  • the invention provides an active peptide for inhibiting AMPA receptor and a preparation method and application thereof, which have good solubility and safety, and can selectively inhibit synaptic transmission of neurons caused by AMPA receptor, and have significant anti-epileptic effect. .
  • a first aspect of the invention provides a method for preparing an active peptide, comprising the steps of:
  • the active peptide contains a tetrapeptide (abbreviated as EGAR) having an amino acid sequence of Glu-Gly-Ala-Arg.
  • EGAR tetrapeptide
  • the squid skin may be soaked with an alkaline solution having a mass content of 0.1-0.5%, and the immersion is mainly used for removing the astringency.
  • the mass/volume ratio of the squid skin to the alkaline solution can be controlled to be 1: (2-4), that is, 1 kg of squid skin is soaked with 2-4 L of an alkaline solution, and the slurry concentration is too high (quality)
  • the volume ratio is >1:2)
  • the fluidity is poor, the enzymatic hydrolysis efficiency is lowered, and when the concentration is too low (mass volume ratio ⁇ 1:4), the subsequent treatment volume is large, and the cost is correspondingly increased; in addition, the soaking time can be 5-20 hours.
  • the present invention does not strictly limit the alkaline solution used for immersion.
  • the squid skin can be immersed in a NaOH solution having a mass content of 0.2%, and the mass/volume ratio of the squid skin to the NaOH solution can be 1: 3, the soaking time can be 12h.
  • the amount of the neutral protease may be controlled to be 50-500 U/g, and the amount of the papain may be 100-1000 U/g.
  • the amount of the enzyme is based on the weight of the squid skin, and the ratio of the neutral protease to the papain may be 1: (1-3).
  • the enzymatic hydrolysis of the slurry made from the squid skin by neutral protease and papain in turn facilitates the degradation of macromolecular proteins into small molecular peptides, especially dipeptides to hexapeptides, which is beneficial to improve the solubility and absorption of the active peptides. Sex.
  • the temperature of the first enzymatic hydrolysis can be controlled to be 30 to 60 ° C, the time of the first enzymatic hydrolysis is 4 to 6 h, the temperature of the second enzymatic hydrolysis is 30 to 60 ° C, and the time of the second enzymatic hydrolysis is 1 ⁇ 3h; the short time of the first enzymatic hydrolysis and the second enzymatic hydrolysis is not conducive to the sufficient degradation of the protein, and the excessive time may lead to the production of unfavorable substances (such as bitter and astringent substances), so the above enzymatic hydrolysis time is preferred.
  • the enzyme can be inactivated by a conventional method in the art, for example, at 100 to 120 ° C for 10 to 20 minutes.
  • the rotation speed of the centrifugation in the step 4) can be controlled to be 2000 to 6000 r/min, and the centrifugation can be performed by using conventional equipment such as a decanter centrifuge, a tube centrifuge, or the like.
  • the membrane filtration can be carried out using a ceramic membrane having a pore diameter of 50 to 1000 nm; when membrane filtration, the absolute pressure of membrane filtration can be controlled to be 0.2 to 0.4 MPa, and the temperature is 30 to 80 °C.
  • Membrane filtration can further retain the larger molecular weight components of the enzymatically produced product, thereby ensuring the solubility and absorption of the active peptide.
  • the concentration can be carried out by a conventional method, for example, a double effect falling film evaporator can be used for evaporation concentration, and the vapor pressure at the time of evaporation concentration can be controlled to be 0.1 ⁇ 0.02 MPa, and the evaporation temperature is 40 to 80 ° C.
  • the concentration of the concentrated liquid is 25-30 Baume; in addition, it can be decolorized by a conventional decolorizing agent, and the decolorizing agent can be, for example, activated carbon powder, and the mass ratio of the decolorizing agent to the filtrate can be (10-30): 100, decolorizing
  • the temperature can be controlled at 40 to 70 ° C, for example At 55 ° C, the decolorization time can be 30 to 90 min, and the decolorization can be carried out under stirring.
  • the decolorizing agent can be removed by conventional means such as filtration, such as plate and frame filtration. Further, after decolorization, sterilization and drying may be carried out to prepare an active peptide, and drying may be, for example, spray drying.
  • the method for preparing an active peptide provided by the present invention may further comprise: separating and purifying the active peptide by reverse-phase high performance liquid chromatography to obtain a tetrapeptide having the amino acid sequence of Glu-Gly-Ala-Arg.
  • a second aspect of the invention provides an active peptide which is produced according to the preparation method described in any of the above.
  • the active peptide having a molecular weight of less than 1000 Da has a mass content of >80%.
  • the active peptide further includes one or more of the following polypeptides: WYN, NTTM, NGGGGS, PALH, AGGP, QK, MADT, NK, NPR, TQ, RGF, NAGK, SR, QGAK, YSAP, DAGK, GR, SSP, KR, AK, GGH, DSGDG, AGPS, GAAGR, AP, VDGK, RER, PQ, GPR, GPQG, TGVE, ARGGK, VR, LN, VTGK, GHAGE, VGGK, GHGR, SPGAG, FTE, AGGPLG, TGGPK, GAGGMT, AAGPGL, VEKEKH, TGPK, LQ, SGGE, NVG, GPAG, PNH, PH, VL, LIE and TPT
  • EGAR in the active peptide exhibits significant affinity with the AMPR receptor, which is capable of selectively inhibiting AMPA receptor-mediated synaptic transmission in mouse hippocampal neurons. And reduce the post-synaptic currents of hippocampal neurons, but does not affect the electrophysiological function of NMDA receptors in hippocampal neurons; at the same time, it can inhibit the excessive excitability of overexcited neurons.
  • PTZ-stimulated epilepsy mice were relieved of epileptic symptoms after administration of EGAR, thereby exhibiting significant antiepileptic effects.
  • the third aspect of the present invention provides a tetrapeptide which inhibits an AMPA receptor, and the amino acid sequence is Glu-Gly-Ala-Arg.
  • a fourth aspect of the invention provides the use of the above active peptide for the preparation of an anti-epileptic food or medicament.
  • a fifth aspect of the present invention provides the use of the above-described AMPA-inhibiting tetrapeptide in the preparation of an anti-epileptic food or medicament.
  • a sixth aspect of the invention provides a method of treating epilepsy comprising administering to a patient having epilepsy a medicament comprising a therapeutically effective amount of a tetrapeptide having the amino acid sequence Glu-Gly-Ala-Arg.
  • Figure 1 is a RP-HPLC chromatogram of the active peptide prepared in Example 1.
  • 2A to 2D are computer simulations of the binding of EGAR to AMPA receptors.
  • 3A to 3G are electrophysiological maps of EPSC and mEPSC of mouse hippocampal slices.
  • 4A-4D are electrophysiological maps of AMPAR mediated EPSC of mouse hippocampal slices.
  • 5A-5H are electrophysiological maps of MGAR selectively inhibiting mPARC mediated by AMPAR.
  • 6A to 6D are electrophysiological maps of EGAR selectively inhibiting current induced by AMPA.
  • 7A and 7B are electrophysiological maps of EGAR inhibiting epileptic discharge of hippocampal neurons induced by hypomagnesium.
  • FIGS 8A and 8B show the effect of EGAR on PTZ-induced mouse epilepsy.
  • TTX, PTX, CNQX, APV, NMDA, AMPA from TOCRIS Biological Sciences
  • 5 kg of squid skin was made up to 15 L with a 0.2% NaOH solution, soaked at room temperature for 12 h, and drained.
  • the fish skin was minced, beaten with 5 times by weight of deionized water, and adjusted to a pH of 7.0 to prepare a slurry.
  • Neutral protease ie 400 U/g salmon skin
  • papain was added to the hydrolyzate according to the amount of 0.2% (ie 400 U / g squid skin), continued to hydrolyze at 60 ° C for 2 h, after the end of enzymatic hydrolysis at 100 ° C conditions for 10 minutes.
  • the enzymatic hydrolysate after centrifugation was centrifuged, and the centrifuged supernatant was subjected to membrane filtration using a ceramic membrane having a pore size of 200 nm (from Xiamen Star Film Co., Ltd.), followed by an R-151 concentrator (from BUCHI, Switzerland).
  • the membrane-filtered supernatant is concentrated to a concentration of 30 Baume, and then activated carbon is added to the concentrate according to the amount of 20%, and the mixture is decolorized at 55 ° C for 1 hour. After removing the activated carbon by filtration, the supernatant is spray-dried to obtain 620 g of active peptide.
  • the active peptide prepared above was diluted to a concentration of 2 mg/mL with deionized water using RP-HPLC (XBridge) BEH130, 4.6*250nm, Waters, USA) Gradient elution, the elution conditions are as follows:
  • Injection volume 50 ⁇ L
  • the AMBER11 simulation kit is used for molecular dynamics simulation and data analysis.
  • the all-atom charge field (AMBERFF03) which shows a good balance between the spiral and the slice results, is used to delineate the peptide.
  • the water solvent is clearly indicated by the TIP3P model.
  • the parameters generated by EGAR are as follows: After geometric optimization, the electrostatic potential of EGAR is obtained at the HF/6-31G** level. Part of the charge comes from the use of a restricted electrostatic potential (RESP) method to match the gas phase electrostatic potential, EGAR Other mechanical parameters of the molecule are taken from the AMBER GAFF parameter set.
  • the AMBER anterior chamber tool was used to generate the interaction of ligand deletions.
  • the system was first minimized using the steepest descent algorithm in 2000 steps, and then the system was simulated with a 5 nanosecond MD using the NPT.
  • the pressure was coupled to 1 bar with an anisotropic connection time of 1.0 ps and the temperature during the simulation was maintained at 300 K with a coupling time of 0.1 PS.
  • Long-range static electricity is calculated by the method of particle network Ewald (PEM).
  • SHAKE is used to limit the bonds connecting hydrogen atoms so that the time step in the simulation is 2.0fs.
  • Two fixed points of 0.8 and 1.2 nm were used for the evaluation of non-bond interactions, respectively.
  • MM-GBSA was used to estimate the binding energy at 300K.
  • the CPU (2.0 GHz) of the 192 AMD Opteron (tm) processor was used for the above calculation.
  • FIGS. 2A to 2D are computer simulations of the binding of EGAR to AMPA receptors.
  • the morphology of the EGAR-AMPAR complex in Figure 2A indicates that EGAR binds to the S1S2 domain of AMPAR;
  • Figure 2B shows that the Arg219 and Gly73 residues of AMPAR form hydrogen bonds with EGAR;
  • Figure 2C shows the EGAR and AMPAR in the 5000ps simulation.
  • the animal testing protocol was approved by the Animal Care and Use Committee of Sun Yat-sen University and complies with the guidelines for the use of laboratory animals by national health agencies.
  • ACSF contains 124 mM sodium chloride, 2.5 mM potassium chloride, 2 mM calcium chloride, 2 mM magnesium chloride, 1.25 mM sodium dihydrogen phosphate, 26 mM sodium bicarbonate and 10 mM glucose, with 95% O 2 / 5% CO 2 is saturated to a pH of 7.2-7.4.
  • mice 2-3 weeks old, weighing 15-20 grams, each housed in a cage at 20-22 ° C, free access to food and water, and placed in a 12-hour light/dark cycle .
  • EGAR solution The artificially synthesized EGAR lyophilized powder was dissolved in 115 ⁇ L of distilled water as a stock solution. The test solution was diluted with the above ACSF to the final concentration of 100 ⁇ M, 50 ⁇ M, 20 ⁇ M, 1 ⁇ M EGAR solution for use.
  • Internal solution contains 140 mM potassium gluconate, 5 mM sodium chloride, 1 mM calcium chloride, 2 mM MgATP, 10 mM EGTA and 10 mM HEPES, pH 7.2-7.4, were sterile filtered prior to recording.
  • External solution containing 140 mM sodium chloride, 5 mM potassium chloride, 1.5 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES and 10 mM glucose, pH 7.2-7.4, sterile filtration prior to recording.
  • Hippocampus was taken from C57BL/6 mice for whole-cell recording. Mice were deeply anesthetized with 20% urethane before decapitation, then their brains were quickly removed, immersed in ice-cold ACSF, then the brain was trimmed and mounted on a vibratory slicer (Leica VT1000A, Germany). A 350 micron coronal section. The sections were incubated with ACSF for at least 1 hour at room temperature prior to recording.
  • the hippocampal slices were transferred to an underwater recording chamber (Warner instrument). In the recording chamber, sections were continuously perfused with ACSF at a rate of 3 ml/min, and subjects who could not distinguish between the control group and the treatment group were subjected to whole cell recording of neurons in the hippocampal CA1 region. Neurons were identified by morphology using an infrared differential interference contrast microscope (BX51W, Olympus, Japan). A 1.5 mm (outer diameter) ⁇ 0.86 mm (inside diameter) borosilicate microtube was drawn from a flame Brownian electrode drawing apparatus (P-97, Sartre instrument) using a 4-stage drawing protocol to create a resistance of 6- 9 megohm recording electrode. The electrode for recording was filled with the above internal liquid, and whole cell recording was performed, and the above external liquid was also used.
  • the voltage clamp recording of excitatory postsynaptic currents was performed using a bipolar PTFE insulated crucible, and a platinum microelectrode (AM system) was induced at a frequency of 0.0167 Hz. Recordings were taken with a Multiclamp 700B amplifier, filtered at 10 kHz, sampled at 200 microseconds, recorded on a personal computer using pClamp 10.2 software, and analyzed using Axon instruments Digidata 1440A and pClamp 10.2 (Molecular Devices).
  • FIGS. 3A to 3G are electrophysiological maps of EPSC and mEPSC of mouse hippocampal slices.
  • Figure 3A shows sample traces of EPSCs before and after EGAR administration from pyramidal cells in the CA1 region; bar graph of EPSC amplitude in Figure 3B shows control vs.
  • FIG. 3G is a record of pyramidal cells mEPSC in CA1; the histogram in Figure 3D shows that treated with EGAR compared to controls There was no change in the mEPSC frequency; the cumulative probability of the mEPSC event interval in Figure 3E showed no difference between the control group and the EGAR treatment group at each concentration; the bar graph of Figure 3F The amplitudes of mEPSCs in the EGAR treatment group shown at 20 ⁇ M (**P ⁇ 0.03), 50 ⁇ M (**P ⁇ 0.03), and 100 ⁇ M (**P ⁇ 0.03) were all decreased; the cumulative probability of the mEPSC amplitude of FIG. 3G showed the concentration of each concentration. The amplitude of the EGAR treatment group was lower than that of the control group (KS test, p ⁇ 0.05).
  • EGAR excitatory postsynaptic currents
  • mEPSC microscopic excitatory postsynaptic currents
  • FIGS. 4A-4D are electrophysiological maps of AMPAR mediated EPSC of mouse hippocampal slices.
  • Figure 4A shows the representative trajectory before and after the addition of 20 ⁇ M EGAR in the hippocampal CA1 region treated with 50 ⁇ M D-APV (antagonist of NMDA receptor);
  • Figure 4B shows treatment with 10 ⁇ M CNQX (competitive antagonist of non-NMDA receptor)
  • the CA1 region of the hippocampal slices was further added to the representative trajectory before and after 20 ⁇ M EGAR; the bar graph of Figure 4C showed that the EPSCs were significantly more significant than the AMPAR-mediated EPSC amplitude of the treatment group with 50 ⁇ M D-APV and 20 ⁇ M EGAR simultaneously.
  • EGAR is capable of selectively inhibiting excitatory postsynaptic current (EPSC) mediated by AMPAR.
  • FIG. 5A-5H are electrophysiological maps of MGAR selectively inhibiting mPARC mediated by AMPAR.
  • Figure 5A shows sample traces of AMPAR-mediated mEPSCs in the control and EGAR-treated groups
  • the cumulative probability of mEPSCs amplitude in Figure 5E Compared with the control group, the amplitude of the EGAR treatment group was reduced (KS test, p ⁇ 0.05
  • EGAR selectively inhibits microminiature excitatory postsynaptic currents (mEPSC) mediated by AMPA.
  • NMDA-induced current recording 100 ⁇ M of NMDA was added to the above external solution; and for AMPA-induced current recording, 20 ⁇ M of AMPA was added to the above external solution.
  • FIG. 6A to 6D are electrophysiological maps of EGAR selectively inhibiting current induced by AMPA.
  • Figure 6A is a representative trace of the patch clamp experiment, which shows that the inward current induced by 20 ⁇ M AMPA is reduced in the presence of EGAR
  • the horizontal bar indicates the application time of the drug
  • the upper and middle traces refer to AMPA treatment and AMPA and 20 ⁇ M EGAR, respectively.
  • Co-processing the traces of the lower layer show that after washing EGAR, the AMPA reaction part is restored to the control level, and the chamber voltage is clamped at -70 mV; the bar graph of Fig.
  • Figure 6C is the effect of 20 ⁇ M EGAR on NMDA-evoked current;
  • magnesium ions were replaced by a sodium ion of equal osmotic concentration.
  • FIG. 7A and 7B are electrophysiological maps of EGAR inhibiting epileptic discharges of hippocampal neurons induced by hypomagnesium.
  • Membrane capacitance pF 20.64 ⁇ 0.79 19.07 ⁇ 0.67 Amplitude, mV 89.43 ⁇ 0.2 87.61 ⁇ 1.18 Action potential half width, ms 0.95 ⁇ 0.01 0.99 ⁇ 0.01 Frequency, Hz 14.11 ⁇ 3.84 13.53 ⁇ 2.42 Threshold potential, mV -46.26 ⁇ 0.5 -45.61 ⁇ 0.2
  • EGAR EGAR anticonvulsant effect
  • mice The behavior of the mice was observed immediately after injection of PTZ for 30 minutes. Two well-trained observers who had previously unclear results analyzed the behavior of the mice. The mice were placed in clear plastic cages and observed for 30 minutes. The cage was thoroughly washed with a wet/dry cloth and 70% ethanol to remove any olfactory cues. Animals that exhibit violent jumps and forelimb clonic or more serious behaviors (violent, clonic, and fallen) are considered to have epilepsy at the edge of the mouse brain. Seizures were scored according to the following criteria: 0, no response; 1, no movement; 2, gustatory movement and grasping; 3, tremor; 4, head shaking; 5, violent jumping and forelimb clonic; 6, violent jumping, clonic and falling Next; 7, death.
  • Figures 8A and 8B show the effect of EGAR on PTZ-induced mouse epilepsy.
  • Figure 8A shows the behavioral scores of 5 groups of mice treated with PBS, 1 mg/kg, 10 mg/kg, and 100 mg/kg EGAR, respectively;
  • EGAR can alleviate PTZ-induced seizure symptoms and prolong the onset of seizures in mice.

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Abstract

本申请提供了一种抑制AMPA受体的活性肽及其制备方法和应用。所述活性肽的制备方法包括如下步骤：1）将鲑鱼皮浸泡后破碎，加水打浆，调pH值至6.5-7.5；2）采用中性蛋白酶进行第一酶解；3）采用木瓜蛋白酶进行第二酶解后灭酶；4）将酶解液离心后，对上清液进行膜过滤、浓缩、脱色，制得活性肽。所述活性肽中含有氨基酸序列为Glu-Gly-Ala-Arg的四肽。该四肽的溶解性好，可选择性地抑制神经元由AMPA受体引起的突触传递，具有抗癫痫的作用。

Description

一种抑制AMPA受体的活性肽及其制备方法和应用 技术领域
本发明涉及一种活性肽，特别是涉及一种抑制AMPA受体的活性肽及其制备方法和应用。
背景技术
癫痫是一种大脑神经元突发性异常放电导致短暂的大脑功能障碍的慢性疾病，主要症状为反复的、无法抑制的痉挛，其在中国已经成为神经科仅次于头痛的第二大常见病，全世界大约有1-2％的人深受其害。尽管大部分国家已经可以提供15种以上的抗癫痫药物，然而仍有20-30％的癫痫症病人在发作时无法有效地控制痉挛。
目前，很多关于癫痫的研究旨在寻找更加安全有效的可抑制神经元兴奋的药物，其在调节哺乳动物中枢神经系统生理和病理状态下的神经元活动中起到关键作用，尤其是在α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体(AMPA受体，AMPAR)参与的兴奋传递活动中。已经证实，AMPA受体的过度兴奋是细胞内钙离子浓度过高的主要原因，腹腔注射和脑室注射AMPA均可引起动物模型痉挛，这表明了AMPA在痉挛产生中的作用，因此AMPA受体是一个理想的药物靶点。
现有资料表明，AMPA受体抑制剂可能具有预防或治疗癫痫的作用，抑制或减弱AMPA受体的药物可抑制神经元过度兴奋，从而保护神经元和抑制痉挛。典型的AMPA受体竞争型抑制剂2,3-二羟基-6-硝基-7-氨磺酰基苯并[f]喹喔啉(NBQX)可在戊四氮(PTZ)模型中发挥作用，但其溶解度低，可能会在肾中沉淀，尽管加入极性成分的NBQX派生物溶解度有所提高，然而其透过血脑屏障的能力有所降低。因此，开发一种溶解性好、安全、有效的AMPA受体抑制剂用作抗癫痫药物成为亟待解决的问题。
发明内容
本发明提供一种抑制AMPA受体的活性肽及其制备方法和应用，其溶解性好、安全，并且可选择性地抑制神经元由AMPA受体引起的突触传递，具有显著的抗癫痫作用。
本发明的第一方面提供一种活性肽的制备方法，包括如下步骤：
1)将鲑鱼皮浸泡后，破碎，加水打浆，调pH值至6.5-7.5后，制得浆料；
2)采用中性蛋白酶对所述浆料进行第一酶解，制得第一酶解液；
3)采用木瓜蛋白酶对所述第一酶解液进行第二酶解，灭酶后，制得第二酶解液；
4)将所述第二酶解液离心后，对离心上清液进行膜过滤、浓缩、脱色，制得活性肽；
所述活性肽中含有氨基酸序列为Glu-Gly-Ala-Arg的四肽(简称EGAR)。
在本发明中，可以采用质量含量为0.1-0.5％的碱性溶液对所述鲑鱼皮进行浸泡，浸泡主要用于去除腥味。在浸泡时，可以控制所述鲑鱼皮与碱性溶液的质量/体积比为1：(2-4)，即1kg鲑鱼皮用2-4L的碱性溶液进行浸泡，浆料浓度过高(质量/体积比＞1：2)时流动性差，酶解效率降低，而浓度过低(质量体积比＜1：4)时后续处理体积大，成本相应增加；此外，浸泡时间可以为5-20h。本发明对浸泡所采用的碱性溶液不作严格限制，在一实施方式中，可以采用质量含量为0.2％的NaOH溶液对鲑鱼皮进行浸泡，鲑鱼皮与NaOH溶液的质量/体积比可以为1：3，浸泡时间可以为12h。
进一步地，在进行所述第一酶解和第二酶解时，可以控制所述中性蛋白酶的用量为50～500U/g，所述木瓜蛋白酶的用量为100～1000U/g，上述两种酶的用量均是基于鲑鱼皮的重量，并且所述中性蛋白酶与木瓜蛋白酶的用量比可以为1：(1-3)。依次采用中性蛋白酶和木瓜蛋白酶对鲑鱼皮制成的浆料进行酶解有利于使大分子蛋白质降解为小分子多肽，特别是二肽至六肽，从而有利于提高活性肽的溶解性和吸收性。
此外，可以控制所述第一酶解的温度为30～60℃，第一酶解的时间为4～6h，所述第二酶解的温度为30～60℃，第二酶解的时间为1～3h；第一酶解和第二酶解的时间过短不利于蛋白的充分降解，而时间过长可能导致不利物质(例如苦味涩味物质)产生，因此以上述酶解时间为宜。并且，可以采用本领域常规方法进行所述灭酶，例如在100～120℃的温度下灭酶10～20分钟。
进一步地，可以控制步骤4)中所述离心的转速为2000～6000 r/min，离心可采用常规设备进行，例如卧螺离心机、管式离心机等。并且，可以采用孔径为50～1000nm的陶瓷膜进行所述膜过滤；膜过滤时，可控制膜过滤的绝对压力为0.2～0.4MPa，温度为30～80℃。膜过滤可进一步地截留酶解产物中分子量较大的成分，从而保证活性肽的溶解性和吸收性。
在本发明中，可以采用常规方法进行所述浓缩，例如可采用双效降膜蒸发器进行蒸发浓缩，并且可控制蒸发浓缩时的蒸汽压为0.1±0.02MPa，蒸发温度为40～80℃，浓缩液的浓度为25-30波美；此外，可以采用常规脱色剂进行脱色，脱色剂例如可以为活性碳粉，脱色剂与滤液的质量配比可以为(10～30)：100，脱色的温度可控制在40～70℃，例如 55℃，脱色时间可以为30～90min，脱色可在搅拌下进行。在脱色后，可通过过滤等常规方式去除脱色剂，例如板框过滤。进一步地，在脱色后可进行灭菌和干燥，从而制得活性肽，干燥例如可以为喷雾干燥。
进一步地，本发明提供的活性肽的制备方法还可以包括：采用反相高效液相色谱对所述活性肽进行分离纯化，制得所述氨基酸序列为Glu-Gly-Ala-Arg的四肽。
本发明的第二方面提供一种活性肽，按照上述任一所述的制备方法制得。
进一步地，本发明的活性肽中，分子量小于1000Da的肽的质量含量＞80％。此外，该活性肽中还包括如下多肽中的一种或多种：WYN、NTTM、NGGGGS、PALH、AGGP、QK、MADT、NK、NPR、TQ、RGF、NAGK、SR、QGAK、YSAP、DAGK、GR、SSP、KR、AK、GGH、DSGDG、AGPS、GAAGR、AP、VDGK、RER、PQ、GPR、GPQG、TGVE、ARGGK、VR、LN、VTGK、GHAGE、VGGK、GHGR、SPGAG、FTE、AGGPLG、TGGPK、GAGGMT、AAGPGL、VEKEKH、TGPK、LQ、SGGE、NVG、GPAG、PNH、PH、VL、LIE和TPT。
在对该活性肽进行功能学研究时发现，该活性肽中的EGAR表现出与AMPR受体的显著配对性，其能够选择性地抑制小鼠海马神经元由AMPA受体介导的突触传递，并且降低海马神经元突触后电流，但不影响海马神经元NMDA受体的电生理功能；同时，其能够抑制过度兴奋的神经元的过度兴奋性。此外，在小鼠癫痫模型中，PTZ激发的癫痫小鼠在给予EGAR治疗后，癫痫症状得到缓解，从而表现出显著的抗癫痫作用。
鉴于此，本发明的第三方面提供一种抑制AMPA受体的四肽，氨基酸序列为Glu-Gly-Ala-Arg。
本发明的第四方面提供一种上述活性肽在制备抗癫痫食品或药物中的应用。
本发明的第五方面提供一种上述抑制AMPA受体的四肽在制备抗癫痫食品或药物中的应用。
本发明的第六方面提供一种治疗癫痫的方法，包括向癫痫患者施用含有治疗有效量的氨基酸序列为Glu-Gly-Ala-Arg的四肽的药物。
附图说明
图1为实施例1制备的活性肽的RP-HPLC色谱图。
图2A至图2D为EGAR与AMPA受体结合的计算机模拟图。
图3A至图3G为小鼠海马切片的EPSC和mEPSC的电生理图。
图4A至图4D为小鼠海马切片的由AMPAR介导的EPSC的电生理图。
图5A至图5H为EGAR选择性抑制由AMPAR介导的mEPSC的电生理图。
图6A至图6D为EGAR选择性抑制由AMPA诱发的电流的电生理图。
图7A和图7B为EGAR抑制低镁外液诱导的海马神经元的癫痫样放电的电生理图。
图8A和图8B显示EGAR对PTZ诱导的小鼠癫痫的影响。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明的附图和实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明各实施例所采用的原料及其来源：
中性蛋白酶：来自诺维信生物技术有限公司；
TTX、PTX、CNQX、APV、NMDA、AMPA：来自TOCRIS生物科学；
木瓜蛋白酶、PTZ：来自Sigma Aldrich公司；
人工合成的EGAR：来自上海强耀有限公司。
实施例1制备活性肽
1、制备活性肽
将5kg鲑鱼皮用质量含量为0.2％的NaOH溶液定容至15L，常温下浸泡12h，沥干。将鱼皮绞碎，用5倍重量的去离子水打浆，调pH值至7.0后，制得浆料。
按照0.1％的用量向上述浆料中加入中性蛋白酶(即400U/g鲑鱼皮)，在50℃条件下酶解5个小时，然后按照0.2％的用量向酶解液中加入木瓜蛋白酶(即400U/g鲑鱼皮)，在60℃条件下继续酶解2h，酶解结束后在100℃条件下灭酶10分钟。
对灭酶后的酶解液进行离心，并采用孔径为200nm的陶瓷膜(来自厦门世达膜公司)对离心上清液进行膜过滤，随后采用R-151浓缩仪(来自瑞士BUCHI公司)将膜过滤后的清液浓缩至浓度为30波美，再按照20％的用量向浓缩液中添加活性炭，在55℃条件下脱色1小时，过滤去除活性碳后，对清液进行喷雾干燥，得到620g活性肽。
2、活性肽的结构鉴定
用去离子水将上述制备的活性肽稀释至浓度为2mg/mL，利用RP-HPLC(XBridge BEH130，4.6*250nm，美国Waters公司)进行梯度洗脱，洗脱条件如下：
流动相A：V(水):V(三氟乙酸)＝100:0.1；
流动相B：V(乙腈):V(水):V(三氟乙酸)＝80:20:0.1；
检测波长：UV220nm；
流速：0.6mL/min；
柱温：32℃；
进样体积：50μL；
梯度程序：0-10min，流动相B：0％-5％；10-20min，流动相B：5％-5％；20-35min，流动相B：5％-9％；35-45min，流动相B：9％-13％；45-60min，流动相B：13％-13％；60-70min，流动相B：13％-70％；70-90min，流动相B：70％-70％。
如图1所示，对上述制备的活性肽梯度洗脱的过程中出现11个主要吸收峰，收集该11个主要吸收峰对应的洗脱液，并利用Q-TOF2质谱仪(英国Micromass公司)对各组分中的多肽进行序列分析，结果见表1。
表1各组分中的多肽序列
组分编号 多肽序列
1 WYN、NTTM、NGGGGS、PALH
2 AGGP、QK、MADT、NK、NPR
3 TQ、RGF、NAGK、SR、QGAK
4 YSAP、DAGK、GR、SSP、KR、AK、GGH、DSGDG
5 AGPS、GAAGR、AP、VDGK、RER
6 PQ、GPR、GPQG、TGVE、ARGGK、EGAR
7 VR、LN、VTGK、GHAGE、VGGK、GHGR
8 SPGAG、FTE、AGGPLG、TGGPK、GAGGMT
9 AAGPGL、VEKEKH、TGPK、LQ
10 SGGE、NVG、GPAG、PNH、PH
11 VL、LIE、TPT
实施例2分子动力学模拟试验
AMBER11模拟套件用于分子动力学模拟和数据分析。全原子点电荷力场(AMBERFF03)，显示着螺旋和片层结果之间的良好平衡，被用来描绘肽。水溶剂明确由TIP3P模型表示。EGAR生成的参数如下：几何优化后，EGAR的静电势在HF/6-31G**级别处获得。部分电荷来源于使用受限制的静电电势(RESP)方法来匹配气相静电势，EGAR 分子的其它力学参数取自AMBER GAFF参数集。使用AMBER前室工具生成配体缺失的相互作数。在2000个步骤中采用最速下降算法首先最小化该系统，然后用NPT全体进行5纳秒的MD模拟该系统。用1.0ps的各向异性连接时间将压力联接至1巴，并用0.1PS的偶联时间使模拟期间的温度保持在300K。长程静电用粒子网状埃瓦尔德(PEM)的方法进行计算。SHAKE被用来限制连接氢原子的键，从而使在模拟中时间步长为2.0fs。0.8和1.2纳米的两个定点，分别用于非键相互作用的评价。然后，采用MM-GBSA估计300K时的结合能。192AMD Opteron(tm)处理器的CPU(2.0GHz)被用于上述计算。
图2A至图2D为EGAR与AMPA受体结合的计算机模拟图。其中，图2A中EGAR-AMPAR复合体的形态表明EGAR结合到AMPAR的S1S2结构域；图2B显示，AMPAR的Arg219和Gly73残基与EGAR形成氢键；图2C为在5000ps模拟中EGAR和AMPAR之间的距离，三个重复模拟中，该AMPAR的RMSD保持稳定在大约1.75埃，表明该复合物是相对稳定的；图2D为EGAR和AMPAR((Glu-OE1)-219Arg，(Glu-OE2)-219Arg，Arg-73Gly)之间的三个强氢键的距离的时间依赖性，氢供体和受体之间的距离小于3.5埃。
上述结果表明：EGAR能够稳定地与AMPA受体结合。
实施例3动物试验
该动物试验方案被中山大学的动物护理和使用委员会批准，并符合国家卫生机构的实验动物使用准则。
1、试剂、动物及材料
ACSF：含有124mM的氯化钠，2.5mM的氯化钾，2mM的氯化钙，2mM的氯化镁，1.25mM的磷酸二氢钠，26mM的碳酸氢钠和10mM的葡萄糖，用95％O2/5％CO2饱和至pH值7.2-7.4。
C57BL/6小鼠：2-3周龄，重15-20克，各自圈养在20-22℃的笼内，自由获取食物和水，并使其处于在12小时的明/暗循环的环境内。
EGAR溶液：将人工合成的EGAR冻干粉溶解于115μL蒸馏水中作为储备溶液，试验时将储备溶液用上述ACSF稀释至最终浓度分别为100μM、50μM、20μM、1μM的EGAR溶液备用。
内液：含有140mM的葡糖酸钾，5mM的氯化钠，1mM的氯化钙，2mM的MgATP， 10mM的EGTA和10mM的HEPES，pH7.2-7.4，在记录前使用无菌过滤。
外液：含有140mM的氯化钠，5mM的氯化钾，1.5mM的氯化钙，1mM的氯化镁，10mM的HEPES和10mM的葡萄糖，pH7.2-7.4，在记录前使用无菌过滤。
2、脑组织准备
从C57BL/6小鼠中取海马做全细胞记录。断头之前用20％的乌拉坦对小鼠进行深度麻醉，接着迅速除去其大脑，浸入冰冷的ACSF，随后对大脑进行修整，并将其安装在震动切片机(Leica VT1000A，德国)上切成350微米的冠状切片。记录前将切片在室温用ACSF孵育至少1小时。
3、电生理实验
将海马切片转移至水下记录室(Warner仪器)。在记录室，以3ml/分钟的速度用ACSF连续灌注切片，并使不能区分对照组和处理组的实验者进行海马CA1区神经元全细胞记录。用红外线微分干涉相衬显微镜(BX51W，Olympus，日本)通过形态学识别神经元。使用4阶段拉制方案由火焰布朗电极拉制仪(P-97，萨特仪器)将1.5毫米(外径)×0.86毫米(内径)的硼硅酸盐微管拉制成电阻介于6-9兆欧的记录电极。用于记录的电极填充上述内液，进行全细胞记录还使用上述外液。
兴奋性突触后电流(EPSCs)的电压钳记录采用双极聚四氟乙烯绝缘铱，铂微电极(AM系统)在0.0167赫兹的频率诱发。记录用Multiclamp 700B放大器获取，在10kHz过滤，在200微秒采样，使用pClamp10.2软件记录到一台个人计算机上，并使用Axon仪器Digidata 1440A和pClamp10.2(Molecular Devices)进行分析。
分别进行下述试验，结果值用平均值±S.E.M来表示。除累积概率的数据通过K-S检验的手段进行了分析以外，生物数据的统计分析都使用T检验。使用SPSS 13.0软件进行所有统计分析。
(1)记录经EGAR处理的切片的EPSCs，同时以不经EGAR处理的切片的EPSCs作为对照，将10μM的PTX加到外液中分离出mEPSCs。
图3A至图3G为小鼠海马切片的EPSC和mEPSC的电生理图。其中：图3A显示从CA1区锥体细胞中记录的给予EGAR前后EPSCs的样本痕迹；图3B中EPSC幅度的条形图显示对照组与在20μM(**P<0.03)、50μM(**P<0.03)和100μM(**P<0.03)EGAR处理组之间的显著差异；图3C为CA1区锥体细胞mEPSC的记录；图3D的直方图显示，与对照相比，用EGAR处理的切片在mEPSC频率没有变化；图3E的mEPSC事件间期的累积概率显示对照组和各浓度的EGAR处理组没有区别；图3F的条形图显 示在20μM(**P<0.03)、50μM(**P<0.03)和100μM(**P<0.03)EGAR处理组的mEPSC的幅度均降低；图3G的mEPSC幅度的累积概率显示各浓度的EGAR处理组的幅度与对照组相比有所降低(K-S检验，p<0.05)。
上述结果表明：在离体海马脑片实验中，一定浓度的EGAR能够抑制兴奋性突触后电流(EPSC)和微小型兴奋性突触后电流(mEPSC)。
(2)在加入50μM的D-APV到ACSF后阻断NMDA受体介导的EPSCs的成分和将10μM的CNQX加入到ACSF后阻断AMPAR介导的EPSCs后，两者剩下的EPSCs也被记录。将10μM的PTX加到外液中分离出mEPSCs。10μM的CNQX以及20μMEGAR和50μMD-APV(NMDA受体的拮抗剂)以及20μMEGAR加入到ACSF后，也记录了mEPSCs。通过加入10μM的CNQX或50μM的D-APV到ACSF，NMDAR介导的或AMPAR介导的mEPSCs被分离出。
图4A至图4D为小鼠海马切片的由AMPAR介导的EPSC的电生理图。其中：图4A显示经50μM D-APV(NMDA受体的拮抗剂)处理的海马CA1区再加入20μM EGAR前后的代表轨迹；图4B显示经10μM CNQX(非NMDA受体的竞争性拮抗剂)处理的海马切片的CA1区再加入20μM EGAR前后的代表轨迹；图4C的条形图表明同时加入50μM的D-APV和20μM EGAR的处理组与对照组的AMPAR介导的EPSC幅度相比，EPSCs显著降低(***P<0.01，t检验中，n＝6)；图4D的条形图显示当给予神经元10μM CNQX处理后再加入20UM EGAR的处理组与对照组相比，NMADR介导的EPSC幅度相比，EPSCs幅度没有改变(P>0.05，t检验，n＝6)。
上述结果表明：EGAR能够选择性抑制由AMPAR介导的兴奋性突触后电流(EPSC)。
(3)对于NMDAR介导的电流记录，向上述外液中添加100μM的NMDA；对于AMPAR介导的电流记录，向上述外液中添加20μM的AMPA。
图5A至图5H为EGAR选择性抑制由AMPAR介导的mEPSC的电生理图。其中：图5A显示对照组和EGAR处理组的AMPAR介导的mEPSCs的样本痕迹；图5B的条形图显示，与对照组相比，经EGAR处理神经元AMPAR介导的mEPSCs频率没有变化(P>0.05，t检验，N＝6)；图5C中mEPSC事件间期的累计概率表示在对照组和EGAR处理组之间的频率没有差异(K-S检验，p>0.05，n＝6)；图5D的条形图表示从EGAR处理过的神经元记录AMPAR介导的mEPSCs的幅度与对照组相比有下降(*P<0.05，t检验，n＝6)；图5E的mEPSCs幅度的累积概率表示与对照组相比，EGAR处理组的幅度降低(K-S检验，p<0.05，n＝6)；图5F显示经NMDA(100μM)处理 后的CA1神经元的NMDA受体介导的mEPSCs再加入EGAR(20μM)前后的代表痕迹；图5G的条形图显示100μM NMDA再加20μM EGAR对NMDAR介导的mEPSCs幅度影响的量化效应，组别之间无差异显著(P>0.05，t检验，n＝6)；图5H的条形图显示100μM NMDA再加20μM EGAR对NMDAR介导的mEPSCs频率影响的量化效应，组别之间无差异显著(P>0.05，t检验，N＝6)。
上述结果表明：EGAR能够选择性抑制由AMPA介导微小型兴奋性突触后电流(mEPSC)。
(4)对于NMDA诱发的电流记录，向上述外液中添加100μM的NMDA；对于AMPA诱发的的电流记录，向上述外液中添加20μM的AMPA。
图6 A至图6D为EGAR选择性抑制由AMPA诱发的电流的电生理图。其中：图6A为膜片钳实验代表痕迹，其显示由20μM AMPA诱发的内向电流在EGAR存在下会减少，单杠表明药物的应用时间，上层和中层的痕迹分别指AMPA处理以及AMPA与20μM的EGAR共同处理，下层的痕迹显示冲刷EGAR后，AMPA反应部分恢复到对照水平，该小室电压钳位在-70mV；图6B的条形图表示20μM AMPA加20μM EGAR对AMPA峰值电流的量化影响，组别之间的差异是显著的(***P<0.01，t检验中，n＝6)；图6C为20μM EGAR对NMDA诱发电流的影响；图6D的条形图表示100μM NMDA加上20μM EGAR对NMDA峰值电流的量化影响，组别之间无差异显著(P>0.05，t检验，n＝6)。
上述结果表明：EGAR能够选择性抑制由AMPA诱发的电流。
(5)在标称零镁的实验中，镁离子由一种渗透浓度相等的钠离子代替。
图7 A和图7B为EGAR抑制低镁外液诱导的海马神经元的癫痫样放电的电生理图。其中：图7A显示在有、无20μM EGAR的处理的情况下，低镁诱发的峰值电流，以及清洗后的恢复的样本痕迹；图7B的条形图表示20μM EGAR对低镁引起的峰值电流频率的量化影响，组别之间的差异是显著的(*P<0.05，t检验中，n＝6)。
上述结果表明：EGAR能够抑制低镁外液诱导的海马神经元的癫痫样放电。
(6)测试EGAR对神经元的电生理性质的影响，结果见表2。
表2 EGAR对神经元的电生理性质的影响
  对照组 20uM EGAR处理组
静息电位，mV -68.57±2.16 -65.57±1.23
输入电阻，MΩ 163.71±7.29 164.57±9.96
膜电容，pF 20.64±0.79 19.07±0.67
幅度，mV 89.43±0.2 87.61±1.18
动作电位半宽度，ms 0.95±0.01 0.99±0.01
频率,Hz 14.11±3.84 13.53±2.42
阈电位，mV -46.26±0.5 -45.61±0.2
表2结果表明：EGAR对CA1神经元的电生理参数无显著影响。
4、PTZ诱发癫痫发作的模型
为了确定EGAR抗惊厥效果而评估了给予PTZ诱发癫痫发作的模型EGAR治疗后癫痫的程度。EGAR比PTZ早30分钟注射到动物体内。动物被随机分为4组，10个1组。各组给予分别0、1、10和100毫克/千克的EGAR。所有动物腹膜内注射PTZ的剂量为55毫克/千克，这个剂量在全部的对照动物都能产生阵挛性发作。
注射PTZ后立即观察小鼠的行为30分钟。两个训练有素的对之前结果不清楚的观察者分析了小鼠的行为。将小鼠放置于透明塑料笼，并观察30分钟。笼子用湿/干布彻底清洗和70％的乙醇除去任何嗅觉线索。表现出暴跳和前肢阵挛或更严重的行为(暴跳，阵挛和倒下)的动物被认为小鼠大脑边缘癫痫。癫痫发作根据以下标准进行评分：0，无反应；1，不动；2，味觉运动和抓；3，震颤；4，头晃动；5，暴跳和前肢阵挛；6，暴跳，阵挛和倒下；7，死亡。
图8A和图8B显示EGAR对PTZ诱导的小鼠癫痫的影响。其中：图8A分别显示用PBS、1毫克/千克、10毫克/千克和100毫克/千克EGAR处理的5组小鼠的行为评分；图8B的条形图分别显示PBS、1毫克/千克、10毫克/千克和100毫克/千克EGAR在癫痫发作潜伏期的量化影响，PBS处理组与1毫克/千克、10毫克/千克和100毫克/千克EGAR处理组之间的差异是显著的(**P<0.03，***P<0.01，#p<0.05，t检验中，n＝6)。
上述结果表明：在行为学实验中，EGAR能够减轻PTZ诱导的小鼠癫痫的抽搐症状和延长发作的潜伏期。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (11)

  1. 一种活性肽的制备方法，其特征在于，包括如下步骤：
    1)将鲑鱼皮浸泡后，破碎，加水打浆，调pH值至6.5-7.5后，制得浆料；
    2)采用中性蛋白酶对所述浆料进行第一酶解，制得第一酶解液；
    3)采用木瓜蛋白酶对所述第一酶解液进行第二酶解，灭酶后，制得第二酶解液；
    4)将所述第二酶解液离心后，对离心上清液进行膜过滤、浓缩、脱色，制得活性肽；
    所述活性肽中含有氨基酸序列为Glu-Gly-Ala-Arg的四肽。
  2. 根据权利要求1所述的制备方法，其特征在于，采用质量含量为0.1-0.5％的碱性溶液对所述鲑鱼皮进行浸泡，并且控制所述鲑鱼皮与碱性溶液的质量/体积比为1：(2-4)，所述浸泡时间为5-20h。
  3. 根据权利要求1所述的制备方法，其特征在于，所述中性蛋白酶的用量为50～500U/g，所述木瓜蛋白酶的用量为100～1000U/g，并且所述中性蛋白酶与木瓜蛋白酶的用量比为1：(1-3)。
  4. 根据权利要求1或3所述的制备方法，其特征在于，控制所述第一酶解的温度为30～60℃，第一酶解的时间为4～6h；控制所述第二酶解的温度为30～60℃，第二酶解的时间为1～3h。
  5. 根据权利要求1所述的制备方法，其特征在于，采用孔径为50～1000nm的陶瓷膜进行所述膜过滤。
  6. 根据权利要求1所述的制备方法，其特征在于，还包括：采用反相高效液相色谱对所述活性肽进行分离纯化，制得氨基酸序列为Glu-Gly-Ala-Arg的四肽。
  7. 一种活性肽，其特征在于，按照权利要求1至6任一所述的制备方法制得。
  8. 一种抑制AMPA受体的四肽，其特征在于，氨基酸序列为Glu-Gly-Ala-Arg。
  9. 权利要求7所述的活性肽在制备抗癫痫食品或药物中的应用。
  10. 权利要求8所述的抑制AMPA受体的四肽在制备抗癫痫食品或药物中的应用。
  11. 一种治疗癫痫的方法，其特征在于，包括向癫痫患者施用含有治疗有效量的氨基酸序列为Glu-Gly-Ala-Arg的四肽的药物。
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