CN104651437A

CN104651437A - Blood fat reducing active peptide separated from codium fragile and application thereof

Info

Publication number: CN104651437A
Application number: CN201510099688.8A
Authority: CN
Inventors: 刘红卫
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-03-07
Filing date: 2015-03-07
Publication date: 2015-05-27

Abstract

The invention provides a polypeptide separated from codium fragile, a preparation method of the polypeptide and a method for treating hyperlipidemia by using the polypeptide. The peptide provided by the invention has the function of reducing blood fat. In addition, the invention further provides a method for treating hyperlipidemia by using active medicines.

Description

Hypolipidemic activity peptide be separated in thorn Korean pine and uses thereof

Technical field

What the present invention relates to is biological technical field, is specifically related to the bioactive peptide being separated the treatment hyperlipidemia obtained from thorn Korean pine.

Background technology

Cardiovascular disorder occupy first or second of the cause of death in China, and wherein the morbidity of coronary heart disease and mortality ratio rise in certain areas.Existing evidence illustrates in the morbidity of coronary heart disease has some Hazard Factor to play an important role, and comprises hypercholesterolemia, hypertension, smoking and diabetes.Hyperlipemia (dyslipdemia) generally refer to total cholesterol in blood (TC), LDL-C (LDL-C), triglyceride level (TG) overrun and (or) highdensity lipoprotein-cholesterol (HDL-C) is low.Low hdl mass formed by blood stasis is also important Hazard Factor.In above each factor, hypercholesterolemia is taken seriously most, and its clinical meaning confirms repeatedly, increasing for a long time with blood cholesterol, and the incidence of coronary event increases; Long-term control blood cholesterol in suitable level, can prevention of arterial atherosis, and reduce blood cholesterol and can alleviate atherosclerotic plaque, reduce coronary event.In recent years, the understanding of the meaning of hypertriglyceridemia in atherosclerosis is deepened.Therefore, the control carrying out hyperlipidaemia in many people becomes the important step of atherosclerosis control.Rational diet regulates preventing and treating hyperlipidaemia very important with life, and most people can reduce blood fat through this method.The lipid-regulation medicine of recent new development partly can have been kept on a diet and treated out of contior hyperlipidaemia.

Applicant is found by research, and thorn is tested by mouse feeding by Korean pine, and this algae has the effect reducing blood fat preferably.Be the effect what material plays lipopenicillinase to further study, by extracting experiment, applicant has found that a series of little peptide has the effect of good reducing blood-fat actually.

Summary of the invention

The present invention relates to a class reducing blood-fat polypeptide on the one hand.Its structure has following form:

CHS-03：PSDESPRLVPFAKGSGGRVL 20

CHS-07：CRPGSRILDCQQIVPPA 17

CHS-10：IIHIMMEDGSSGSSIVIVIV 20

CHS-12：QQMFSVRGGSCCDMNNRS 18

CHS-15：MMGSFVIIPPGSSRVMCT 18

CHS-17：GSMRIVVGSMNNSSRSVSPH 20

CHS-20：HAMRIVVMMPHIIISSSRVFS 21

CHS-21：CCACPHMGSRPPHIHHQQCC 20

CHS-23：VVMMHIGSRPVNVPPIIPVAG 21

CHS-24：GGSFVMMTHPACPGSVGSSR 20

CHS-27：INNGSPHIPPGSRVCPCCAAQP 22

On the other hand, the invention provides a kind of method being separated described polypeptide, thorn Korean pine is first used PBS buffer solution for cleaning by (1), then under specific 32 degrees celsius, is prepared into rough liquid with the homogenate of 13mmol/L HCl solution; (2) by centrifugal 20 minutes with 7000 revs/min for the rough liquid of step (1) gained, collect supernatant liquor, by supernatant liquor three layers of filtered through gauze, then add papoid (enzyme activity is 800AU/g) 1kg, continue insulation and constantly stir to carry out enzyme digestion reaction 3h, obtain enzymolysis solution.Be separated with dextran G-50 (Sephadex G-50) (1.5cm × 90cm), 18mmol/L HCl eluant solution, flow velocity 1.3mL/ minute, collect eluted product, regulator solution is to pH6.8, and 10000 revs/min centrifugal 15 minutes, and supernatant liquor lyophilize is for subsequent use; Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reclaim the band of small-molecular-weight, through checking, obtain 35 little polypeptide, confirm wherein have 11 to have good biologic activity through overtesting.

On the other hand, the invention provides a kind of pharmaceutical composition, described composition contains body polypeptide as above.

Pharmaceutical composition as above, it is also containing also comprising one or more pharmaceutically acceptable carriers; Preferably, described pharmaceutically acceptable carrier is selected from: water-soluble filler, pH adjusting agent, stablizer, water for injection and osmotic pressure regulator;

More preferably, described water-soluble filler is selected from following one or more: N.F,USP MANNITOL, low molecular dextran, sorbyl alcohol, polyoxyethylene glycol, glucose, lactose and semi-lactosi;

More preferably, described pH adjusting agent is the acceptable acid of physiology, alkali and/or salt, is preferably selected from following one or more:

Non-volatile acid as Citric Acid, phosphoric acid, lactic acid, tartrate or hydrochloric acid, alkali as potassium hydroxide, sodium hydroxide or potassium hydroxide or ammonium hydroxide, salt as sodium carbonate or salt of wormwood or ammonium carbonate salts, sodium bicarbonate or saleratus or hydrogen-carbonate ammonium salt;

More preferably, described stablizer is be selected from following one or more: EDTA-2Na, Sulfothiorine, Sodium Pyrosulfite, S-WAT, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, L-glutamic acid, polyethylene glycol 6000, Macrogol 4000, sodium lauryl sulphate or Tutofusin tris etc., further preferably from one or more in Sodium Pyrosulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, Tutofusin tris;

More preferably, described osmotic pressure regulator is selected from sodium-chlor and/or Repone K.

Further aspect of the present invention also provides the application of aforementioned polypeptides in the medicine of preparation treatment hyperlipidemia.

Further, described pharmaceutical composition is preferably injection, is more preferably freeze-dried powder or injection of solution agent.

Another aspect of the present invention also provides a kind of pharmaceutical composition, comprises mutant polypeptide described above.

Preferably, acceptable adjunct ingredient is also comprised.

The advantage of invention is:

Provide a peptide species and can improve hypolipidemic activity, there is good stability simultaneously.

Embodiment

The separation of embodiment 1 polypeptide

(1) thorn Korean pine is first used PBS buffer solution for cleaning, then under specific 32 degrees celsius, be prepared into rough liquid with the homogenate of 13mmol/L HCl solution; (2) by centrifugal 20 minutes with 7000 revs/min for the rough liquid of step (1) gained, collect supernatant liquor, by supernatant liquor three layers of filtered through gauze, then add papoid (enzyme activity is 800AU/g) 1kg, continue insulation and constantly stir to carry out enzyme digestion reaction 3h, obtain enzymolysis solution.Be separated with dextran G-50 (Sephadex G-50) (1.5cm × 90cm), 18mmol/L HCl eluant solution, flow velocity 1.3mL/ minute, collect eluted product, regulator solution is to pH6.8, and 10000 revs/min centrifugal 15 minutes, and supernatant liquor lyophilize is for subsequent use; Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reclaim the band of small-molecular-weight, obtain the little corresponding amino acid whose sequence of polypeptide of wherein 35 by order-checking.

Embodiment 2 polypeptide is on the impact of lipid of mice

By described aminoacid sequence by synthetic, then carry out mouse experiment by the conventional mouse model for hypolipidemic medicine screening, confirm wherein have 11 to have the active reducing blood fat preferably through overtesting.

In addition, be " a kind of improvement spectrophotometry detecting HMG-CoA reductase activity " of 201110073398.8 according to the patent No. published, described 35 little peptides reduced accordingly the detection of blood fat effect, record result as follows:

As can be seen from the result of experiment, described polypeptide all has the effect reducing blood fat preferably.

The stable experiment of embodiment 3 polypeptide in Mice Body

By described polypeptide, be expelled in Mice Body with the dosage of 0.5mg/ml/kg, through the internal metabolism of a week, get mouse tail blood, detected by SDS-PAGE, analysis obtains, peptide concentration remaining in blood still on average can reach 0.1mg/ml/kg, this absolutely proves, polypeptide all has good body internal stability, may be used for follow-up pharmaceutical use.

Above result clearly states, and the invention provides one and can be used for treating hyperlipidaemic conditions, the especially new peptide medicine of hyperlipidemia.There is the prospect for the treatment of blood-lipoids disease preferably.

Sequence table

<110> Liu Hong defends

<120> stings hypolipidemic activity peptide be separated in Korean pine and uses thereof

<160> 12

<210> 1

<211> 38

<212> PRT

<213> artificial sequence

HSQGTFTSDYSKYLDEQAAKEFIAFFLMNTKRNRNNIA

<210> 2

<211> 20

<212> PRT

<213> artificial sequence

<400> CHS-03

PSDESPRLVPFAKGSGGRVL

<210> 3

<211> 17

<212> PRT

<213> artificial sequence

<400> CHS-07

CRPGSRILDCQQIVPPA

<210> 4

<211> 20

<212> PRT

<213> artificial sequence

<400> CHS-10

IIHIMMEDGSSGSSIVIVIV

<210> 5

<211> 18

<212> PRT

<213> artificial sequence

<400> CHS-12

QQMFSVRGGSCCDMNNRS

<210> 6

<211> 18

<212> PRT

<213> artificial sequence

<400> CHS-15

MMGSFVIIPPGSSRVMCT

<210> 7

<211> 20

<212> PRT

<213> artificial sequence

<400> CHS-17

GSMRIVVGSMNNSSRSVSPH

<210> 8

<211> 21

<212> PRT

<213> artificial sequence

<400> CHS-20

HAMRIVVMMPHIIISSSRVFS

<210> 9

<211> 20

<212> PRT

<213> artificial sequence

<400> CHS-21

CCACPHMGSRPPHIHHQQCC

<210> 10

<211> 21

<212> PRT

<213> artificial sequence

<400> CHS-23

VVMMHIGSRPVNVPPIIPVAG

<210> 11

<211> 20

<212> PRT

<213> artificial sequence

<400> CHS-24

GGSFVMMTHPACPGSVGSSR

<210> 12

<211> 22

<212> PRT

<213> artificial sequence

<400> CHS-27

INNGSPHIPPGSRVCPCCAAQP

Claims

1. a reducing blood-fat polypeptide, is characterized in that: from thorn Korean pine.

2. reducing blood-fat polypeptide as claimed in claim 1, its sequence had is as shown in any one of SEQ ID NO:1-12.

3. reduce the method for blood lipid level in the mammalian hosts suffering from hyperlipidaemic conditions, described method comprises to the oral pharmaceutical composition containing the polypeptide shown in claim 1-2 of this host.

4. contain the peptide described in claim 1-2, their analogue or the pharmaceutical composition of stand-in or its mixture that can accept carrier on physiology.

5. the purposes of the polypeptide described in claim 1-2 in the pharmaceutical composition for the preparation of reduction blood fat.

6., according to the purposes of claim 5, using wherein is oral or intravenous injection.

7., according to the purposes described in any one of claim 5-6, wherein said host suffers from hyperlipidaemic conditions.