CN103755781B - There is hypotensive and the bifunctional dipeptides GD of reducing blood-fat and uses thereof - Google Patents

There is hypotensive and the bifunctional dipeptides GD of reducing blood-fat and uses thereof Download PDF

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CN103755781B
CN103755781B CN201310744081.1A CN201310744081A CN103755781B CN 103755781 B CN103755781 B CN 103755781B CN 201310744081 A CN201310744081 A CN 201310744081A CN 103755781 B CN103755781 B CN 103755781B
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dipeptides
hmg
fat
ace
hypotensive
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CN103755781A (en
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王伟
王楠
张玉
杨玉英
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Zhejiang Academy of Agricultural Sciences
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Zhejiang Academy of Agricultural Sciences
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Abstract

The invention belongs to biological technical field, particularly an energy is combined with Zinc metallopeptidase Zace1, suppresses its dipeptides that is active, that also can suppress the activity of 3-hydroxy-3-methylglutaric acid list acyl coenzyme A (HMG-CoA) reductase enzyme.Specifically, the invention discloses one and have the hypotensive and bifunctional dipeptides GD of reducing blood-fat, the aminoacid sequence of this dipeptides GD is: Gly-Asp.The present invention also also discloses above-mentioned dipeptides GD and is preparing the application in ace inhibitory peptide and/or HMG-CoA reductase inhibiting peptide.

Description

There is hypotensive and the bifunctional dipeptides GD of reducing blood-fat and uses thereof
Technical field
The invention belongs to biological technical field, particularly an energy is combined with Zinc metallopeptidase Zace1, suppresses it active, also can suppress the dipeptides of the activity of 3-hydroxy-3-methylglutaric acid list acyl coenzyme A (HMG-CoA) reductase enzyme.
Background technology
Zinc metallopeptidase Zace1 (Angiotensin converting enzyme, ACE, EC3.4.15.1, in document, former name has kininaseIl, dipeptidyl carboxypeptidase I etc.) be a kind of two carboxypeptidases, cause a hypertensive key enzyme, it changes into angiotensin II by hydrolytic action hypertensinⅠ, and meanwhile, ACE also can passivation bradykinin, these two kinds effects all can cause vasoconstriction, thus cause hypertension.Therefore ACE is considered to cause a hypertensive important factor.Finding angiotensin converting enzyme inhibitor (ACEI) after deliberation, reaching hypotensive effect by suppressing the activity of ACE.ACE inhibitor is widely used in the diseases such as treatment is cardiovascular, hypertension, heart failure, renal failure.ACE inhibitor finds at first from snake venom, it is found that the ace inhibitory peptide extracted from foodstuff raw material subsequently, as gelatin, casein, fish, Fructus Fici natural gum, α-zein etc. all can be used as the raw material preparing ace inhibitory peptide.
3-hydroxy-3-methylglutaric acid list acyl coenzyme A (HMG-CoA) reductase enzyme is the key enzyme that in body, catalysis 3-hydroxy-3-methylglutaric acid list acyl coenzyme A (HMG-CoA) generates dihydroxymethyl valeric acid (MVA), this step is the rate-limiting step of synthetic cholesterol in body, is also the target spot of current topmost hyperlipidemia clinical medicine.HMG-CoA reductase inhibitor is one of blood fat reducing function composition and drug screening Main Means.
Hypotensive medicine common are:
1., diuretic(s): represent medicine and have that hydrogen chlorine bites throat, phenalgin butterfly suckes, in spiral shell the tenth of the twelve Earthly Branches purport etc.
2., sheet receptor-blocking agent: representing medicine has Pu Cailuoer, metoprolol, atenolol USP 23, nadolol etc.
3., calcium channel blocker: representing medicine has nifedipine, amlodipine, felodipine, nitrendipine, Lacidipine (62 etc.
4., angiotensin converting enzyme inhibitor: representing medicine has captopril, Zinadril Briem, lisinopril, enalapril, Yipingshu etc.
5., a mono-receptor-blocking agent: represent that medicine has croak throat, spy draws mile prolixity etc.
6., hypertensin 11 receptor antagonist: represent medicine and have losartan, pick Sha Tan, irbesartan, Candesartan, Irb, telmisartan etc.
The medicine of reducing blood-fat has:
1, fibrate: this type of medicine has fenofibrate, gemfibrozil, bezafibrate etc.Fibrate drug reducing blood lipid is strong, and rapid-action, the effect of triglyceride reducing is stronger than the effect of decreasing cholesterol.
2, trishydroxymethyl glutaryl-CoA-reductase inhibitors: this type of medicine has lovastatin, simvastatin, general Liprevil etc.This type of medicine is based on decreasing cholesterol, and effect for reducing fat is strong, rapid-action.
3, nicotinic acid class: in this type of medicine, Acipimox is more conventional, the effect reducing serum levels of triglyceride is stronger than reducing cholesterol.
4, polyunsaturated fatty acid class: comprise various plant seed oils.As rubber seed oil, seed of Radix Oenotherae erythrosepalae, the oil of Silymarin seed and the preparation of ocean fish.This kind of medicine has reducing blood-fat and reduces the effect of blood viscosity, but effect is gentleer.
5, Pantethine: be the derivative of coenzyme A, has the effect reducing serum cholesterol, triglyceride and high density lipoprotein increasing-cholesterol.
6, propylene glycol alginate sodium sulfate (PPS): be scoop up with marine alga the heparitin marine drug that thing is raw material.Have and significantly reduce blood viscosity, vasodilation and reduction blood fat, raise the effect of HGD level.Be mainly used in the control of ischemic cardio cerebrovascular diseases.
7, other blood lipid-lowering medicines: test proof as ginkgo class (taponin) and serum levels of triglyceride (TG) can be made significantly to reduce.
Existing medicine report, except Chinese medicine, has hypotensive rarely found with medicine that is reducing blood lipid simultaneously.Because the target that these two kinds of medicines act on is different.
Summary of the invention
The technical problem to be solved in the present invention is to provide one and has hypotensive and the bifunctional dipeptides GD of reducing blood-fat and uses thereof.
In order to solve the problems of the technologies described above, the invention provides a kind of dipeptides GD, the aminoacid sequence of this dipeptides GD is: Gly-Asp.
The present invention also provides the purposes of above-mentioned dipeptides GD simultaneously: as ace inhibitory peptide and HMG-CoA reductase inhibiting peptide.
Dipeptides GD of the present invention obtains by entrusting the synthesis of gill biochemical (Shanghai) Co., Ltd..
The dipeptides GD that the present invention reports all has inhibit activities for ACE and HMG-CoA reductase two targets, thus shows and have the hypotensive and bifunctional characteristic of reducing blood-fat simultaneously.
Every detection method involved in the present invention is specific as follows:
1, the detection method of ACE inhibitory activity:
ACE is at 37 DEG C, and pH value is that the stand-in Hippuryl-L-Histidyl-L-Leucine (HHL) of catalytic decomposition angiotensin I under the condition of 8.3 produces urobenzoic acid (HA), and this material has charateristic avsorption band at ultraviolet 225nm place; When adding ACE inhibitor, the catalyticing decomposition action of ACE to HHL is suppressed, and the growing amount of urobenzoic acid reduces, by HPLC method, measure to add before and after inhibitor generate the amount of urobenzoic acid change can calculate the size of inhibit activities.
Reaction system is: add the ACE of 20 μ L0.1U/mL, 50 μ L ace inhibitory peptides (i.e. GD dipeptides) successively respectively at 37 DEG C of temperature bath 5min, then the HHL substrate adding 10 μ L5mM starts the catalyzed reaction of ACE, after 37 DEG C of shaking bath 30min, add the HCl termination reaction of 250 μ L1.0moL/L, system solution carries out RP-HPLC and detects the content analyzing urobenzoic acid (HA) after crossing 0.45 μm of filter membrane.Above-mentioned similarity condition, in the borate buffer of 50 μ L0.1moL/L, (NaCl, pH=8.3 containing 0.3moL/L) replaces ACE inhibitor as blank reaction system.
Note: above-mentioned ACE, HHL substrate is all for solvent with the borate buffer of 0.1moL/L (NaCl, pH=8.3 containing 0.3moL/L).
Ace inhibitory peptide (GD dipeptides), is dissolved in the borate buffer of 0.1moL/L with different concns and obtains in (NaCl, pH=8.3 containing 0.3moL/L).
RP-HPLC detects: solvent I is 0.05%(V/V) trifluoroacetic acid (TFA) and triethylamine 0.05%(v/v) (TTA) be dissolved in deionized water, and solvent II is the chromatographically pure second eyeball of 100%.Solvent I is 70%:30%(volume ratio with the ratio of solvent II), flow velocity is 0.5mL/min, and determined wavelength is 225nm, and detecting column temperature is 30 DEG C.
ACE inhibitory activity calculates according to following formula:
I%=（A-B）/A×100%
A: the peak area of urobenzoic acid when not adding small peptide inhibitor;
B: the peak area of urobenzoic acid when adding small peptide inhibitor;
ACE:1U unit definition is, under Standard Test Conditions, at 37 DEG C, catalytic substrate (Hippuryl-L-Histidyl-L-Leucine, HHL) in the 1min time, produce 1 μM of urobenzoic acid consume the amount of ACE.That is, be the activity unit of ACE.
2, the external detection method of reducing blood-fat peptide activity:
Chromatographic condition
c1 8chromatographic column (5 μm, 4.6mm × 250mm).Moving phase is: V (K 2hPO 4-KH 2pO 4): V (methyl alcohol)=85:15, pH7.2, isocratic elution, flow velocity 1mL/min; Determined wavelength 337nm; Sample size 20 μ L; Column temperature 25 DEG C.
Reaction system experimental procedure
In reaction various component add-on and order as table 1, temperature of reaction 37 DEG C of water-baths, add 200 μ L0.5mol/L NaOH solution termination reactions, by the concentration of NADPH in above-mentioned chromatographic condition measure sample after having reacted.Reaction times is determined according to enzyme control group time gradient.
Table 1 reaction system moiety
Remarks illustrate: reaction buffer is the potassium phosphate buffer of 0.1mol/L, and the concentration of HMGR solution is 6.56 μ g/mL, and HMG-CoA strength of solution is 5.23mmol/mL, NADPH strength of solution is 1mg/mL.
The calculating of experimental result
After adding inhibitor, the activity of HMG-CoA reductase is suppressed, and the amount of substrate reactions reduces.Therefore, the change of NADPH amount before and after HPLC assaying reaction can be utilized to evaluate the inhibiting rate of inhibitor to HMG-CoA reductase.Calculation formula is:
R=(S inhibitor-S contrast)/(S blank-S contrast) × 100
In formula, R is inhibiting rate (%); S blank, S contrastand S inhibitorbe respectively blank group, the peak area (mAU.min) of NADPH in enzyme control group and inhibitor group.
Advantage of the present invention and positively effect:
(1) this dipeptides GD can suppress the activity of Zinc metallopeptidase Zace1.
According to aminoacid sequence of the present invention, the synthesis of gill biochemical (Shanghai) Co., Ltd. can be entrusted, thus obtain ace inhibitory peptide of the present invention (or referred to as dipeptides GD).
(2) ace inhibitory peptide of the present invention (or referred to as dipeptides GD) has HMG-CoA reductase inhibit activities simultaneously.
Usage and the consumption of ace inhibitory peptide of the present invention and HMG-CoA reductase inhibiting peptide (or referred to as dipeptides GD) are as follows:
Dipeptides of the present invention is oral type, and consumption is oral, each 1.5g, every day 2 ~ 3 times.
Embodiment
Embodiment 1,
1), the ACE inhibitory activity of dipeptides GD under the concentration of 1.0mg/mL:
Chromatographic condition: solvent I be 0.05% trifluoroacetic acid (TFA) and 0.05% triethylamine (TTA) to be dissolved in deionized water (namely in often liter of solvent I containing the trifluoroacetic acid of 0.5mL and the triethylamine of 0.5mL), solvent II is the chromatographically pure second eyeball of 100%.Solvent I is 70%:30%(volume ratio with the ratio of solvent II), ultimate3000 wears peace liquid chromatograph, and chromatographic column is watersSymmetry C 185 μm of 4.6 × 250mm, flow velocity is 0.5mL/min, sample size 10 μ L, and determined wavelength is 225nm, and detecting column temperature is 30 DEG C.
Detection method: by this dipeptides GD obtained by chemical synthesis, carry out Activity determination (detection method is the same).Now GD concentration is 1.0mg/mL.
Result: the ACE inhibitory activity of dipeptides GD when 1.0mg/mL is 31.48%.
2), the HMG-CoA reductase inhibit activities of dipeptides GD under the concentration of 1.0mg/mL:
Chromatographic condition: c1 8chromatographic column (5 μm, 4.6mm × 250mm).Moving phase is: V (K 2hPO 4-KH 2pO 4): V (methyl alcohol)=85:15, pH7.2, isocratic elution, flow velocity 1mL/min; Determined wavelength 337nm; Sample size 20 μ L; Column temperature 25 DEG C.
Detection method: this dipeptides GD will be obtained by chemical synthesis, carry out Activity determination (detection method is the same).Now GD concentration is 1.0mg/mL.
Result: the HMG-CoA reductase inhibit activities of dipeptides GD when 1.0mg/mL is 44.68%.
Embodiment 2,
1), the ACE inhibitory activity of dipeptides GD under the concentration of 2.0mg/mL:
Chromatographic condition: solvent I be 0.05% trifluoroacetic acid (TFA) and 0.05% triethylamine (TTA) be dissolved in deionized water; Solvent II is the chromatographically pure second eyeball of 100%.Solvent I is that 70%:30%, ultimate3000 wear peace liquid chromatograph with the ratio of solvent II, and chromatographic column is waters Symmetry C 185 μm of 4.6 × 250mm, flow velocity is 0.5mL/min, sample size 10 μ L, and determined wavelength is 225nm, and detecting column temperature is 30 DEG C.
Detection method: by this dipeptides GD obtained by chemical synthesis, carry out Activity determination (detection method is the same).Now GD concentration is 2.0mg/mL.
Result: the ACE inhibitory activity of dipeptides GD when 2.0mg/mL is 55.74%.
2), the HMG-CoA reductase inhibit activities of dipeptides GD under the concentration of 2.0mg/mL:
Chromatographic condition: c1 8chromatographic column (5 μm, 4.6mm × 250mm).Moving phase is: V (K 2hPO 4-KH 2pO 4): V (methyl alcohol)=85:15, pH7.2, isocratic elution, flow velocity 1mL/min; Determined wavelength 337nm; Sample size 20 μ L; Column temperature 25 DEG C.
Detection method: by this dipeptides GD obtained by chemical synthesis, carry out Activity determination (detection method is the same).Now GD concentration is 2.0mg/mL.
Result: the HMG-CoA reductase inhibit activities of dipeptides GD when 2.0mg/mL is 61.20%.
By the inhibition concentration in embodiment 1 and embodiment 2 and activity data, activity and the concentration amount effect relationship of this dipeptides GD are described, this dipeptides GD has ACE inhibitory activity simultaneously and HMG-CoA reductase suppresses to have no report, belongs to the new bifunctional peptide having ACE inhibitory activity and HMG-CoA reductase inhibit activities concurrently.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had, such as the different proteins source separating obtained GD structure of degraded and derivatize structure thereof.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
<110> Zhejiang Academy of Agricultural Science
 
<120> has hypotensive and the bifunctional dipeptides GD of reducing blood-fat and uses thereof
 
<160> 1
 
<210> 1
<211> 2
<212> PRT
<213> artificial sequence
 
<220>
<223> dipeptides GD
 
<400> 1
Gly Asp
 

Claims (1)

1. have the hypotensive and application of the bifunctional dipeptides GD of reducing blood-fat in preparation HMG-CoA reductase inhibiting peptide, the aminoacid sequence of described dipeptides GD is: Gly-Asp.
CN201310744081.1A 2013-12-30 2013-12-30 There is hypotensive and the bifunctional dipeptides GD of reducing blood-fat and uses thereof Expired - Fee Related CN103755781B (en)

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