CN108059656B

CN108059656B - Small molecule polypeptide and application thereof

Info

Publication number: CN108059656B
Application number: CN201711476938.0A
Authority: CN
Inventors: 李培源; 苏炜; 霍丽妮; 陈睿
Original assignee: Guangxi University of Chinese Medicine
Current assignee: Masala (Shanghai) Biotechnology Co.,Ltd.
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-07-17
Anticipated expiration: 2037-12-29
Also published as: CN108059656A

Abstract

The invention discloses a small molecular polypeptide, the amino acid sequence of which is shown in SEQ ID No.1, and the invention also provides the application of the small molecular polypeptide, the polypeptide of the invention has good α -glucosidase inhibitory activity and antioxidant activity, strong stability, easy structure regulation and control, and obvious application potential.

Description

Small molecule polypeptide and application thereof

Technical Field

The invention relates to a small molecular polypeptide, in particular to a small molecular polypeptide with α -glucosidase inhibitory activity and antioxidant activity.

Background

The present hypoglycemic medicine has the action mechanism of inhibiting α -glucosidase activity capable of catalyzing the last step of starch or cane sugar digestion to produce saccharide matter, and is considered to be an ideal way for controlling type II diabetes mellitus.

Disclosure of Invention

The invention aims to provide a small molecule polypeptide and application thereof, wherein the small molecule polypeptide has strong stability and good α -glucosidase inhibition activity and antioxidant activity.

To achieve these objects and other advantages in accordance with the present invention, a small molecule polypeptide is provided, the amino acid sequence of which is shown in SEQ ID No. 1.

Preferably, the small molecule polypeptide has an isoelectric point of 9.7.

The invention also provides application of the small molecular polypeptide in preparing a medicament for treating diabetes.

The invention also provides an application of the small molecular polypeptide in preparing a medicament for removing free radicals.

The invention at least comprises the following beneficial effects:

1. the polypeptide of the invention has good α -glucosidase inhibitory activity and antioxidant activity.

2. The method is a small molecular polypeptide, has strong stability and an easily regulated structure, and has obvious application potential.

3. Compared with the macromolecular polypeptide, the small molecular polypeptide of the invention has simple and convenient synthesis method and is easy for large-scale production.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a graph of free radical scavenging efficiency as a function of polypeptide concentration for polypeptides of the invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

Example 1

A small molecular polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1;

SEQ ID No.1：ICKKMMKKSTLLQDDIL。

the isoelectric point of the small molecular polypeptide is 9.7.

The molecular weight of the small molecular polypeptide is 2008.54 g/mol.

The preparation method of the small molecule polypeptide comprises the following steps:

selecting amino acid-royal resin as a carrier (resin), fully swelling the resin by using dichloromethane, adding first Fmoc-protected amino acid, washing the resin for several times by using dimethylformamide, removing Fmoc-protected groups by using DB L K with proper concentration, then washing the resin for several times by using dimethylformamide, washing DB L K, weighing a proper condensing agent benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, an activating agent methylmorpholine and a second Fmoc-protected amino acid (Fmoc-Cys-OH) at the C end, carrying out coupling by using an ninhydrin detection method to ensure that the connection is complete, washing the resin for several times by using dimethylformamide, washing residual various residues, the activating agent and the condensing agent, carrying out coupling according to amino acid sequences, removing the last Fmoc-protected groups after all the amino acids are connected, cracking by using a cutting fluid, removing the resin and the amino acid protected groups to obtain a crude product of the micromolecule polypeptide, and sending a mass spectrum to confirm that the molecular weight of the product conforms to a theoretical value of 2008.54 g/mol.

Example 2

And (3) measuring the activity of α -glucosidase in vitro of the small molecule polypeptide.

All tests were carried out using a Microplate reader model E L X808TM Microplate reader (BioTek, USA) at 37 deg.C.

(1) Preparation of an inhibitor stock solution: the inhibitors tested were formulated in 10mM DMSO (DMSO solution of the polypeptide of example 1).

(2) Preparation of enzyme stock solution α -glucosidase was purchased from Sigma, USA, and prepared to 1mg/m L in phosphate buffer pH 6.8.

(3) Preparation of stock solution for substrate p-nitrophenyl glucoside (PNPG) was used as a substrate and purchased from Sigma, and 10mg/m L was prepared using a phosphate buffer solution of pH 6.8.

(4) Preparation of a stop solution: sodium carbonate (Na2CO3) was purchased from shanghai; the solutions were mixed with phosphate buffer (pH 6.8) to give 0.1M Na₂CO₃And (3) solution.

(5) Test volume of each test was 200 μ L pH 6.8 phosphate buffer.

Inhibitor solutions of 10 μ L were added to a 96-well microplate (inhibitor stock solutions were diluted with phosphate buffer solution of pH 6.8), the pH was adjusted to 170 μ L with phosphate buffer solution of pH 6.8, then 10 μ L was added, the microplate was incubated at 37 ℃ for 10min, 20 μ L substrate stock solutions were immediately added, and the change in absorbance at λ 405nm for one minute (slope) was measured immediately after mixing.

(6) And (5) judging a result: the change (slope) in absorbance measured without sample as 100 activity units;

relative enzyme activity (change in absorbance with inhibitor added (slope)/change in absorbance without inhibitor added (slope) × 100,

the concentration of inhibitor when the relative activity of the enzyme is 50 is the IC of the inhibitor₅₀Values, results are shown in table 1:

TABLE 1 IC of polypeptide on α -glucosidase inhibitory activity₅₀Value of

Medicine	IC₅₀(μM)
		Polypeptides	6.59
Acarbose (control)	7.05

From the results of example 2, it can be seen that α -glucosidase inhibitory activity (IC) is exhibited by the polypeptide of the present invention₅₀6.59 μ M) stronger than control acarbose (IC)₅₀7.05 mu M), the compound has strong α -glucosidase inhibition activity, and the invention provides a new idea for researching and developing new antidiabetic drugs.

Example 3

0.1ml of the DMSO solution of the polypeptide of example 1 (0.2, 0.4, 0.6, 0.8. mu.g/ml) was added to 8ml of a 0.004% solution of 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH). Absorbance was measured at the maximum wavelength (517nm) until equilibrium was reached. The clearance calculation formula is as follows:

S％＝(1-A_{sample (I)})/A_{Blank space}× 100% where A is_{Blank space}Absorbance of DPPH solution without addition of a solution of the polypeptide in DMSO, A_{Sample (I)}Absorbance of a DPPH solution to which a DMSO solution of the compound was added. The results are shown in FIG. 1. Therefore, the polypeptide molecule has excellent antioxidant performance, and the DPPH free radical scavenging capacity can reach more than 99.5% at lower concentration.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.

SEQUENCE LISTING

<110> university of traditional Chinese medicine in Guangxi

<120> small molecule polypeptide and use thereof

<130>application

<160>1

<170>PatentIn version 3.5

<210>1

<211>17

<212>PRT

<213> Polypeptides

<400>1

Ile Cys Lys Lys Met Met Lys Lys Ser Thr Leu Leu Gln Asp Asp Ile

1 5 10 15

Leu

Claims

1. A small molecular polypeptide is characterized in that the amino acid sequence of the small molecular polypeptide is shown as SEQ ID No. 1.

2. The small molecule polypeptide of claim 1, wherein said small molecule polypeptide has an isoelectric point of 9.7.

3. Use of the small molecule polypeptide of claim 1 for the preparation of a medicament for the treatment of diabetes.