Active peptide for inhibiting AMPA receptor and preparation method and use thereof

• US 10,800,810 B2
• Filed: 12/06/2017
• Issued: 10/13/2020
• Est. Priority Date: 07/08/2015
• Status: Active Grant

• Current Assignee: China National Research Institute of Food and Fermentation Industries
• Original Assignee: China National Research Institute of Food and Fermentation Industries
• Inventors: Cai, Muyi, Gu, Huaiyu, Lu, Jun, Pan, Xingchang, Ma, Yong, Chen, Liang
• Primary Examiner(s): Orwig, Kevin S
• Application Number:
  US15/834,026
• Publication Number:
  US 20180094028A1
• Time in Patent Office: 1,042 Days
• Field of Search: None
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: C07K 1/12 by hydrolysis , i.e. solvolysis in general
• CPC Class Codes: C07K 1/14 Extraction; Separation; Purification
• CPC Class Codes: C07K 1/34 by filtration, ultrafiltration or reverse osmosis
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C07K 5/1021 with the first amino acid being acidic
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12Y 304/22002 Papain (3.4.22.2)

Abstract

Provided is an active peptide for inhibiting an AMPA receptor and a preparation method and use thereof. The method for preparing the active peptide comprises the following steps: 1) soaking a salmon skin and crushing, adding water and beating, and then adjusting pH to 6.5-7.5; 2) subjecting to a first enzymolysis using a neutral protease; 3) subjecting to a second enzymolysis using papain enzyme and then inactivating enzyme; and 4) centrifuging the enzymatic hydrolysate, and then subjecting the centrifuged supernatant to membrane filtration, concentration and decoloration, to prepare the active peptide. The active peptide contains a tetrapeptide with an amino-acid sequence of Glu-Gly-Ala-Arg. The tetrapeptide has good solubility, can selectively inhibit neuronal synaptic transmission caused by an AMPA receptor, and has a significant antiepileptic effect.

Claims

1. A method for preparing an active peptide, comprising the following steps:

1) soaking a salmon skin in an alkaline solution for 5-20 h and crushing, adding water and beating, and then adjusting the pH of the solution to 6.5-7.5, to obtain a slurry;

2) subjecting the slurry to a first enzymolysis by using a neutral protease, to obtain a first enzymatic hydrolysate;

3) subjecting the first enzymatic hydrolysate to a second enzymolysis by using papain enzyme and then inactivating the enzymes from the enzymolysis steps, to obtain a second enzymatic hydrolysate; and

4) centrifuging the second enzymatic hydrolysate to obtain a centrifuged supernatant, and then subjecting the centrifuged supernatant to membrane filtration, concentration and decoloration, to prepare the active peptide;

wherein the active peptide comprises a tetrapeptide with the amino-acid sequence of SEQ ID NO;

17.

2. The method according to claim 1, wherein the alkaline solution is a 0.1-0.5% NaOH solution, and the mass/volume ratio of the salmon skin to the alkaline solution is from 1:

(2-4).

3. The method according to claim 1, wherein the concentration of the neutral protease is 50-500 U/g, the concentration of the papain is 100-1000 U/g, and an amount ratio of the neutral protease to the papain is 1:

(1-3).

4. The method according to claim 1, wherein the temperature of the first enzymolysis is from 30-60°

C., the time of the first enzymolysis is 4-6 h, the temperature of the second enzymolysis is from 30-60°

C., and the time of the second enzymolysis is 1-3 h.

5. The method according to claim 3, wherein the temperature of the first enzymolysis is from 30-60°

C., the time of the first enzymolysis is 4-6 h, the temperature of the second enzymolysis is from 30-60°

C., and the time of the second enzymolysis is 1-3 h.

6. The method according to claim 1, wherein the membrane filtration is with a ceramic membrane with a pore diameter of 50-1000 nm.

7. The method according to claim 1, further comprising:

separating and purifying the active peptide by using reversed phase high performance liquid chromatography, to obtain the tetrapeptide with the amino acid sequence of SEQ ID NO;

17.