CN104497105A

CN104497105A - Pentapeptide KLPGF with auxiliary hyperglycemic function

Info

Publication number: CN104497105A
Application number: CN201410693316.3A
Authority: CN
Inventors: 于志鹏; 赵文竹; 励建荣
Original assignee: Bohai University
Current assignee: Bohai University
Priority date: 2014-11-20
Filing date: 2014-11-20
Publication date: 2015-04-08

Abstract

The invention belongs to the biotechnical field, especially relates to a pentapeptide with an auxiliary hyperglycemic function, and concretely relates to a pentapeptide KLPGF capable of inhibiting the activity of alpha-glucosidase and inhibiting the activity of alpha-amylase. The amino acid sequence of the pentapeptide KLPGF is Lys-Leu-Pro-Gly-Phe. The pentapeptide can be used to develop auxiliary hpyerglycemic drugs and functional foods.

Description

There is the pentapeptide KLPGF of auxiliary hyperglycemic function

Technical field

The invention belongs to biological technical field, be specifically related to one and can be combined with alpha-glucosidase and energy Inhibiting α-glucosidase activity, also can be combined the pentapeptide KLPGF with auxiliary hyperglycemic function that also can suppress alpha-amylase activity with α-amylase.

Background technology

Improving constantly and poor eating habits in recent years along with human living standard, cause a lot of hyperglycemic patients, diabetes prevalence sharply rises, and the research such as the pathomechanism of diabetes is extremely urgent, and people are in the urgent need to effectively reducing the activeconstituents of blood sugar for human body.

Glossy ganoderma and Cordyceps sinensis are taken as hypoglycemia healthcare food by people for a long time, but bring heavy economical load to patient and family members due to expensive raw material price.

The active ingredient of current Oral anti-diabetic develops rapidly, as alpha-glucosidase inhibitor and aldose reductase inhibitor etc.Research finds that the many enzymes in body play an important role in adjustment blood sugar process, can be used as antidiabetic action target spot, for the effective constituent of development treatment diabetes provides effective way.

Suppress the activity of α-amylase and alpha-glucosidase to be considered to control an available strategy of diabetes, become new approaches of auxiliary hyperglycemic activeconstituents exploitation.Acarbose is as a kind of new oral antidiabetic drug, and mainly competitive inhibition glycoside lytic enzyme in enteron aisle, acarbose is to the 503nhibiting concentration (IC of alpha-glucosaccharase enzyme inhibition activity ₅₀) be 60.8 μm of ol L ^-1.

From this angle, increasing research all concentrates on finds more effective enzyme inhibitors, and then develops more effective antidiabetic functional component.

Along with deepening continuously of research, research finds that animals and plants polysaccharide has hypoglycemic effect, but rarely has report about the research of biologically active peptides hypoglycemic activity.

The effect important in vital process of bioactive peptide is gradually by people are familiar with, it participates in receptor-mediated signal transduction as neurotransmitter, neuregulins and hormone, affect many important biochemical functions in organism, as blood pressure regulation and blood glucose regulation etc., and bioactive peptide is easily absorbed by the body, have the great interest that the feature such as chemosynthesis and chemically modified causes people simultaneously concurrently, also will promote deepening continuously about the research of bioactive peptide effect of lowering blood sugar.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of pentapeptide KLPGF with auxiliary hyperglycemic function.

Some abbreviations involved in the present invention have following implication:

K：Lys

L：Leu

P：Pro

G：Gly

F：Phe

Lys: Methionin

Leu: leucine

Pro: proline(Pro)

Gly: glycine

Phe: phenylalanine

In order to solve the problems of the technologies described above, the invention provides a kind of pentapeptide KLPGF, its aminoacid sequence is: Lys-Leu-Pro-Gly-Phe.

Pentapeptide KLPGF provided by the invention by suppressing the activity of α-amylase and alpha-glucosidase, and then plays the effect of auxiliary hyperglycemic, and form is white powder.Pentapeptide KLPGF suppresses the 503nhibiting concentration of alpha-amylase activity to be 59.5 ± 5.7 μm of ol L ^-1, the 503nhibiting concentration of Inhibiting α-glucosidase activity is 120.0 ± 4.0 μm of ol L ^-1.

Pentapeptide KLPGF provided by the present invention can entrust Ke Tai bio tech ltd, Shanghai to synthesize and obtain.

Detection method involved in the present invention is as follows:

1. suppress glucosidase activity:

The detection method concrete operation step of Inhibiting α-glucosidase activity is: the alpha-glucosaccharase enzyme solution (10unit/ml getting 5 μ L, be dissolved in the potassium phosphate buffer of 0.1mol/L pH 6.8, pH6.8) 620 μ L 0.1mol/L potassium phosphate buffers (pH value 6.8) are joined successively with the bioactive peptide solution (bioactive peptide is dissolved in 10%DMSO solution) of 10 μ L different concns, at 37.5 DEG C, preheating adds 10 μ L p-nitrophenyl glucoside (pNPG, 10mmol/L) as reaction substrate to start reaction after 20 minutes.

Reaction mixture carries out reaction 30 minutes under being placed in 37.5 DEG C of conditions, finally adds the 1mol/L Na of 650 μ L ₂cO ₃termination reaction, uses UV, visible light sub-ray spectrometer measure at 410nm place and then calculate enzymic activity.

Measuring principle makes reaction substrate with colourless p-NP-alpha-D-glucose glycosides (pNPG), through alpha-glucosidase hydrolyzing alpha-1, p-NP (pNP) is discharged after 4-glucoside bond, p-NP is yellow in the basic conditions, finally by the criterion of the pNP generation under monitoring 410nm as α-Isosorbide-5-Nitrae-glucosidase activity size.

Bioactive peptide is as follows to alpha-glucosaccharase enzyme inhibition activity calculation formula:

Inhibit activities/%=(ODa-ODb)/ODa × 100

In formula: ODa is the absorbancy measured at 410nm place after having reacted in blank group;

ODb is the absorbancy measured at 410nm place after having reacted in interpolation bioactive peptide group.

2. suppress amylase activity:

Suppression amylase activity measures concrete steps: get the α-amylase aqueous solution (1unit/L) of the 10 μ L concentration bioactive peptide solution different from 10 μ L (bioactive peptide sample 10%DMSO dissolves) and mix preheating 15 minutes, then adds starch solution (being dissolved in the sodium phosphate buffer of 20mol/L pH 6.9) the startup reaction that 500 μ L massfractions are 1%.

Reaction carries out 5 minutes under 37.5 DEG C of conditions, drips 600 μ L DNS reagent (3, the 5-dinitrosalicylic acids containing massfraction 1% and the 0.4mol/L sodium hydroxide solution containing massfraction 12% Seignette salt) termination reaction.

After reaction terminates, reaction mixture is placed in boiling water bath 15 minutes, is then cooled to room temperature at 540nm place UV-2550 spectrophotometric determination.

The measuring principle of amylase inhibiting activity is that to be based upon amylase by Starch Hydrolysis be maltose, and maltose and 3,5-dinitrosalicylic acid generation reduction reaction generate the colour developing group of 3-amido-5-NITROSALICYLIC ACID, and its shade is directly proportional to sugared concentration.

Bioactive peptide calculates by following formula diastatic inhibit activities:

Amylase inhibiting activity/%=(ODa-ODb)/ODa × 100

In formula: ODa is the absorbancy measured at 540nm place after having reacted in blank group;

ODb is the absorbancy measured at 540nm place after having reacted in interpolation bioactive peptide group.

Advantage of the present invention:

1), pentapeptide provided by the invention has the effect suppressing alpha-amylase activity.

2), pentapeptide provided by the invention has the effect of Inhibiting α-glucosidase activity simultaneously.

Embodiment

Below in conjunction with embodiment, the present invention is set forth further, but be not limited thereto.

Embodiment 1, pentapeptide KLPGF are to the inhibit activities of α-amylase:

Determination of activity operation steps: get the α-amylase aqueous solution (1unit/L) of the 10 μ L concentration different from 10 μ L (10 μMs, 20 μMs, 40 μMs, 80 μMs, 160 μMs and 320 μMs) pentapeptide KLPGF solution (dissolving with 10%DMSO) and mix preheating 15 minutes, then adds starch solution (being dissolved in the sodium phosphate buffer of 20mol/L pH 6.9) the startup reaction that 500 μ l massfractions are 1%.Determination of activity reaction conditions: carry out 5 minutes under 37.5 DEG C of conditions, drip 600 μ LDNS reagent (containing 3 of massfraction 1%, the 0.4mol/L sodium hydroxide solution of 5-dinitrosalicylic acid and the Seignette salt containing massfraction 12%) termination reaction, after reaction terminates, reaction mixture is placed in boiling water bath 15 minutes, then be cooled to room temperature to measure at 540nm place UV-2550 spectrophotometer, and then calculate the inhibit activities of bioactive peptide to α-amylase.

Result: pentapeptide KLPGF suppresses the 503nhibiting concentration of alpha-amylase activity to be 59.5 ± 5.7 μm of ol L ^-1.

Embodiment 2, pentapeptide KLPGF are to the inhibit activities of alpha-glucosidase:

Determination of activity operation steps is: the alpha-glucosaccharase enzyme solution (10unit/ml getting 5 μ L, be dissolved in the potassium phosphate buffer of 0.1mol/L pH 6.8, pH6.8) with 10 μ L different concns (10 μMs, 20 μMs, 40 μMs, 80 μMs, 160 μMs and 320 μMs) pentapeptide KLPGF solution (being dissolved in 10%DMSO solution) join 620 μ L0.1mol/L potassium phosphate buffers (pH6.8) successively, at 37.5 DEG C, preheating adds 10 μ L p-nitrophenyl glucoside (pNPG after 20 minutes, 10mmol/L) as reaction substrate to start reaction.Determination of activity reaction conditions: reaction mixture carries out reaction 30 minutes under being placed in 37.5 DEG C of conditions, finally adds the 1mol/LNa of 650 μ L ₂cO ₃termination reaction, uses UV, visible light sub-ray spectrometer measure at 410nm place and then calculate the inhibit activities of bioactive peptide to alpha-glucosidase.

Result: the 503nhibiting concentration of pentapeptide KLPGF Inhibiting α-glucosidase activity is 120.0 ± 4.0 μm of ol L ^-1.

Claims

1. there is the pentapeptide KLPGF of auxiliary hyperglycemic function, it is characterized in that: the aminoacid sequence of described pentapeptide KLPGF is: Lys-Leu-Pro-Gly-Phe.