Tetrapeptide for inhibiting dipeptidyl peptidase IV and application thereof
阅读说明：本技术 一种抑制二肽基肽酶iv的四肽及其应用 (Tetrapeptide for inhibiting dipeptidyl peptidase IV and application thereof ) 是由 胡松青 叶伟 侯轶 于 2021-08-31 设计创作，主要内容包括：本发明公开了一种抑制二肽基肽酶Ⅳ的四肽及其应用。本发明的多肽包含4个氨基酸残基,分子量为511.27,理论等电点为3.70,其氨基酸序列为：色氨酸-脯氨酸-亮氨酸-脯氨酸(Trp-Pro-Leu-Pro)。本发明的多肽来源于食用酵母酶解产物,可通过固相合成制备。本发明的多肽具有良好的二肽基肽酶Ⅳ抑制活性,其IC-(50)值为178.90μmol/L,且与低剂量浓度西格列汀(30％抑制率)具有协同抑制作用,可用于降血糖功能药品的开发与制备,具有良好的应用前景。(The invention discloses a tetrapeptide for inhibiting dipeptidyl peptidase IV and application thereof. The polypeptide comprises 4 amino acid residues, has the molecular weight of 511.27, the theoretical isoelectric point of 3.70 and the amino acid sequence of: tryptophan-proline-leucine-proline (Trp-Pro-Leu-Pro). The polypeptide of the invention is derived from an edible yeast enzymolysis product and can be prepared by solid phase synthesis. The polypeptide of the present invention has good dipeptidyl peptidase IV inhibiting activity, IC thereof 50 Has a value of 178.90 μmol-L and has a synergistic inhibition effect with low-dose sitagliptin (30% inhibition rate), can be used for development and preparation of medicines with the function of reducing blood sugar, and has a good application prospect.)
1. A tetrapeptide having DPP-IV inhibitory activity, wherein: the amino acid sequence is tryptophan-proline-leucine-proline (Trp-Pro-Leu-Pro).
2. Use of the tetrapeptide having DPP-IV inhibitory activity according to claim 1 for the preparation of DPP-IV inhibitors or hypoglycemic agents.
3. Use of the tetrapeptide having DPP-IV inhibitory activity according to claim 1 in combination with sitagliptin for the preparation of DPP-IV inhibitors or hypoglycemic agents.
4. Use according to claim 3, characterized in that: the sitagliptin exists in a dosage of 75-85 mu mol/L.
5. A DPP-IV inhibitor characterized by: comprising the tetrapeptide having DPP-IV inhibitory activity according to claim 1.
6. The DPP-IV inhibitor according to claim 5, characterized in that: the DPP-IV inhibitor also contains sitagliptin.
7. The DPP-IV inhibitor according to claim 6, characterized in that: the sitagliptin exists in a dosage of 75-85 mu mol/L.
8. A hypoglycemic agent, which is characterized in that: comprising the tetrapeptide having DPP-IV inhibitory activity according to claim 1.
9. The hypoglycemic agent according to claim 8, wherein: the hypoglycemic drug also contains sitagliptin.
10. The hypoglycemic agent according to claim 9, wherein: the sitagliptin exists in a dosage of 75-85 mu mol/L.
Technical Field
The invention relates to the field of functional foods, in particular to a tetrapeptide for inhibiting dipeptidyl peptidase IV and application thereof.
Background
Type ii diabetes is a metabolic disease characterized by hyperglycemia, can cause severe complications such as microvascular and macrovascular, and seriously harms the physical and mental health of patients. Dipeptidyl peptidase IV (DPP-IV) is becoming an increasingly major target for the treatment of diabetes due to its intestinal insulinotropic effect. The half-life of incretin is improved by inhibiting DPP-IV activity, and the purpose of effectively controlling blood sugar can be achieved.
The artificially synthesized hypoglycemic drug has good hypoglycemic effect, but the side effect is not ignored. Such as hypoglycemia, weight gain, intestinal flatulence and diarrhea. Therefore, research and development of DPP-IV inhibitors with good blood sugar reducing effect and lower toxic and side effects have urgent need for prevention and treatment of type II diabetes.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a tetrapeptide with DPP-IV inhibitory activity. According to the invention, the edible yeast is hydrolyzed by utilizing a bacillus subtilis fermentation enzyme preparation, DPP-IV inhibitory activity is taken as an index, high inhibitory activity components are enriched step by using methods of ethanol aqueous solution extraction and gel filtration chromatography, and the enriched components are identified by LC-MS/MS and screened to obtain tetrapeptide containing tryptophan-proline-leucine-proline (Trp-Pro-Leu-Pro). The tetrapeptide is synthesized by a solid phase, and researches show that the tetrapeptide has good inhibitory activity on DPP-IV.
Another object of the present invention is to provide the use of the above tetrapeptides having DPP-IV inhibitory activity.
Still another object of the present invention is to provide a DPP-IV inhibitor or a hypoglycemic agent.
The purpose of the invention is realized by the following technical scheme:
a tetrapeptide with DPP-IV inhibitory activity has an amino acid sequence of Trp-Pro-Leu-Pro.
The tetrapeptide having DPP-IV inhibitory activity can be prepared by means of conventional techniques in the art, for example, by solid phase synthesis.
The tetrapeptide with DPP-IV inhibitory activity is applied to the preparation of DPP-IV inhibitors or hypoglycemic drugs.
The tetrapeptide with DPP-IV inhibitory activity and sitagliptin are applied to preparation of DPP-IV inhibitors or hypoglycemic drugs.
In the application, sitagliptin exists in a low dosage, specifically 75-85 mu mol/L, more preferably 75-81 mu mol/L, and even more preferably 81 mu mol/L.
The dosage of the tetrapeptide with DPP-IV inhibitory activity is 25-300 mu mol/L.
A DPP-IV inhibitor containing the tetrapeptide having DPP-IV inhibitory activity.
The DPP-IV inhibitor also contains sitagliptin, wherein the sitagliptin exists in a low dose, specifically 75-85 mu mol/L, and more preferably 81 mu mol/L.
A hypoglycemic agent comprising the tetrapeptide having DPP-IV inhibitory activity.
The hypoglycemic drug also contains sitagliptin, wherein the sitagliptin exists in low dose, specifically 75-85 mu mol/L, and more preferably 81 mu mol/L.
Compared with the prior art, the invention has the following advantages and effects:
1. the tetrapeptide of the invention has good DPP-IV inhibitory activity and half inhibition rate IC of DPP-IV50It was 178.90. mu. mol/L.
2. The tetrapeptide and sitagliptin with low dose concentration have synergistic inhibition effect on DPP-IV activity.
3. The method is a micromolecular polypeptide, the structure is easy to regulate and control, and the micromolecular polypeptide is easy to synthesize and modify so as to obtain better activity and has obvious application potential.
4. The tetrapeptide is derived from enzymolysis products of edible yeast, and the edible yeast is listed as 'generally recognized as safe' (GRAS) food ingredients released by FDA, so that the safety is high.
Drawings
FIG. 1 is a gel filtration chromatographic separation pattern.
FIG. 2 is a solid phase synthesis of a tetrapeptide WPLP chromatogram.
FIG. 3 is a solid phase synthesis of a tetrapeptide WPLP mass spectrum.
FIG. 4 is a graph showing the result of analysis of the inhibition rate of DPP-IV by WPLP at different concentrations.
FIG. 5 is a Lineweaver-Burk double reciprocal plot of polypeptide WPLP against DPP-IV.
FIG. 6 is a graph showing the combined inhibitory effect of the polypeptide WPLP and sitagliptin, a commonly used hypoglycemic agent.
FIG. 7 is a graph showing the DPP-IV inhibitory activity of a synthetic polypeptide derived from yeast zymolyte.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The bacillus subtilis HU528 in this embodiment is stored in the Guangdong province microbial strain storage center of the Guangzhou microbial research institute of No. 59 building and No. 5 building of the Middleya Zhonglu 100, Guangzhou city, with the storage number being GDMCC NO: 60364, which is disclosed in chinese patent application CN 201810668986.8.
1. Preparation of enzyme preparations
Inoculating HU528 strain on solid culture medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) by plate-streaking method, culturing at 37 deg.C in an inverted incubator for 6 hr, selecting single colony, and transferring to seed culture medium (1% tryptone, 0.5% yeast extract, 1% sucrose, 0.0001% MgSO 5)4，0.0001％CuSO4pH 7.0) and then cultured at 37 ℃ for 6h (OD: 200 r/min)600About 1.5). The activated bacterial solution was inoculated into a 5L fermentor (0.75% glucose, 1.5% soybean meal, 0.075% CaCl) at 1.0% (v/v)20.045% NaCl), fermenting and culturing for 54h under the conditions that the liquid loading is 70%, the fermentation temperature is 37 ℃ and the ventilation volume is 4L/min, centrifuging the obtained fermentation liquid for 10min at 4 ℃ and 8000 Xg, and removing thalli, wherein the fermentation supernatant is the enzyme preparation.
2. Enzyme activity assay
The protease activity is determined by adopting a Folin phenol method according to the national standard GB/T23527-2009. Tyrosine standard curves were first plotted against different concentrations of tyrosine (y ═ 0.0077x, R2＝0.999) The specific method for measuring the protease activity of the sample is as follows. The fermentation broth was centrifuged at room temperature (8000 Xg, 5min) and the resulting supernatant was the crude enzyme. Taking 1mL of crude enzyme solution diluted by a certain time into a sample tube and a blank tube, adding 1mL of casein solution (2%, w/v) into the sample tube, reacting for 10min at 55 ℃, adding 2mL of 0.4M trichloroacetic acid to stop the reaction, and carrying out water bath at 55 ℃ for 20 min; the blank tube was first treated with 2mL of 0.4M trichloroacetic acid in a water bath at 55 ℃ for 10min to inactivate the protease, and then with 1mL of casein solution (2%, w/v) in a water bath at 55 ℃ for 20 min. The blank tube and the laboratory tube were centrifuged at room temperature (8000 Xg, 5min), 1mL of the supernatant was aspirated, and 5mL of 0.4M Na was added2CO3And 1mL of a Folin phenol reagent, developing the mixture for 20min at 55 ℃, and adjusting the mixture to zero by using a blank tube to measure the absorbance at 680 nm. The measured OD was passed through a standard curve to obtain the corresponding tyrosine concentration, and the enzyme activity was calculated. Definition of enzyme activity unit: under the conditions of 55 ℃ and pH 7.0, the enzyme amount required by hydrolyzing casein to generate 1 mu g of tyrosine in 1min is 1 enzyme activity unit and is expressed by U/mL. The protease activity in the enzyme preparation is measured to be 4200U/mL.
3. Preparation, separation and identification of yeast-derived DPP-IV inhibitory peptide
Adding the enzyme preparation into edible yeast powder (from Wuzhou pharmaceutical industry Co., Ltd., Guangdong, dried yeast, 2017, 6 months) according to the enzyme adding amount of 8000U/g yeast powder, and supplementing sterile water until the volume ratio (material-liquid ratio, w/v) of the edible yeast powder to the mixed liquid is 1: 10. The pH of the mixture was adjusted to 8.0. Performing enzymolysis reaction at 55 deg.C for 4.5h, inactivating enzyme at 100 deg.C for 10min, centrifuging to obtain supernatant, and freeze drying the supernatant to obtain yeast zymolyte lyophilized powder.
Adding 60% (v/v) ethanol water solution into yeast zymolyte lyophilized powder, stirring, standing at 4 deg.C for 12 hr, centrifuging at room temperature (2500 × g, 20min), concentrating supernatant at 55 deg.C under reduced pressure to remove ethanol, and freeze drying. Then, the freeze-dried powder obtained by extracting the ethanol water solution is separated by a Sephadex Peptide 10/300GL chromatographic column, ultrapure water is taken as eluent, the elution flow rate is 0.5mL/min, and the 2 nd component F is detected and collected under the detection wavelength of 214nm2(FIG. 1).
Identification of the above F by LC-MS/MS2Component (b) one sequence was found to be tryptophan-proline-leucine-proline (Trp-Pro-Leu-Pro) tetrapeptide derived from Saccharomyces cerevisiae (strain ATCC 204508/S288c) alcohol dehydrogenase II, protein accession No.: p00331. The theoretical isoelectric point of the small molecule tetrapeptide is 3.70. The molecular weight of the small molecular polypeptide is 511.27 g/mol.
Example 2
The tetrapeptide is prepared by artificial synthesis by adopting Fmoc solid phase synthesis method. According to the amino acid residue composition of the tetrapeptide, various amino acids (Fmoc-Trp, Fmoc-Pro and Fmoc-Leu) with Fmoc-protecting groups at amino terminals are used as raw materials, and carboxyl of the Fmoc-Pro is connected with high molecular resin (Wang resin) through covalent bonds; adding 20% (v/v) piperidine Dimethylformamide (DMF), reacting for 0.5h, and removing the amino protecting group Fmoc-; adding excessive Fmoc-Leu, taking Hydroxybenzotriazole (HOBT) as a condensing agent, and reacting for 2h at 30 ℃ to condense the carboxyl of the Fmoc-Leu with the active amino of Pro on the resin; and (3) repeating the deprotection and condensation reaction, sequentially connecting the rest other amino acids, cracking the tetrapeptide from the resin, separating and purifying by using a C18 column, and freeze-drying to obtain the DPP-IV inhibitory tetrapeptide. The liquid chromatogram (fig. 2) analysis shows that the purity of the small molecule polypeptide synthesized by the method is 99.95%. The liquid chromatography-mass spectrometry (FIG. 3) shows that the sequence of the synthesized polypeptide is tryptophan-proline-leucine-proline (Trp-Pro-Leu-Pro).
Example 3
Adding 25 μ L sample and 50 μ L DPP-IV enzyme solution (purchased from Sigma, cat # D3446) with concentration of 200ng/mL into 96-well enzyme label plate, mixing, incubating at 37 deg.C for 10min, adding 25 μ L substrate Gly-Pro-AMC (final concentration of 0.5mmol/L), collecting fluorescence readings by enzyme label every minute under kinetic mode, and measuring for 15-30min (37 deg.C, λ)ex＝360/λem460 nm). 100mmol/L Tris-HCl buffer (pH 8.0) was used as a blank in place of DPP-IV enzyme solution, and the putative DPP-IV inhibitory tripeptide Diprotin A (IPI) was used as a positive control, with 3 replicates for each set of experiments.
Data from each well was plotted as "fluorescence value versus time", and two time points were selected within the linear range of the plot(T1And T2) And its corresponding fluorescence value (FLU)1And an FLU2) Thereby, the slope of the image is obtained. The formula for calculating the relative inhibition ratio of DPP-IV is shown below.
In the formula, T1And T2: two time points selected within the linear range of the graph; FLU1And an FLU2: time T1And T2The corresponding fluorescence value; slopeEC: the slope of the control group; slopeSM: slope of experimental group.
semi-Inhibitory Concentration (IC)50) Is the concentration of the sample required when the DPP-IV inhibition reaches 50%. Respectively preparing sample solutions with different concentrations, measuring DPP-IV inhibition rate, fitting by using the sample concentration as an abscissa and the DPP-IV inhibition rate as an ordinate through a nonlinear curve, and calculating IC50The value is obtained.
As can be seen from FIG. 4, the tetrapeptide of the invention has inhibitory effect on DPP-IV under different concentrations, and the IC of the tetrapeptide can be calculated50The value was 178.90. mu. mol/L. The small molecular polypeptide has good DPP-IV inhibitory activity and can be used for developing hypoglycemic drugs.
Example 4
In the DPP-IV inhibitory activity experiment, different concentration gradients of a substrate Gly-Pro-AMC are respectively configured: 0.0625mmol/L, 0.0833mmol/L, 0.125mmol/L, 0.25mmol/L, and 0.5 mmol/L. The inhibition of DPP-IV by tetrapeptide WPLP at 100. mu. mol/L and 200. mu. mol/L at different substrate concentrations was determined. The Lineweaver-Burk double reciprocal number graph (figure 5) is drawn, and the result shows that the inhibition type of the polypeptide WPLP on DPP-IV belongs to anti-competitive inhibition.
Example 5
Concentration-inhibition curves of sitagliptin and WPLP were plotted, and the Equivalent Doses (ED) of sitagliptin at concentrations of 81nmol/L (30% inhibition concentration), 124nmol/L (50% inhibition concentration) and 196nmol/L (70% inhibition concentration) corresponding to WPLP were 91. mu. mol/L, 177. mu. mol/L and 319. mu. mol/L, respectively.
The concentrations of the fixed sitagliptin are 81nmol/L, 124nmol/L and 196nmol/L, and then the fixed sitagliptin is compounded with WPLPs of 25 mu mol/L, 50 mu mol/L, 100 mu mol/L, 150 mu mol/L, 200 mu mol/L, 250 mu mol/L and 300 mu mol/L respectively to determine the DPP-IV inhibition rate of each compounding system. And (3) drawing a combined effect curve of sitagliptin and WPLP by taking the WPLP concentration as an abscissa and the DPP-IV inhibition rate of the compound system as an ordinate. The combined effect curve is compared with the base line (concentration-inhibition curve of the inhibitory polypeptide WPLP), and the combined effect curve is synergistic above the base line, antagonistic below the base line, and additive when the combined effect curve is superposed. As shown in fig. 6, the inhibition effect of sitagliptin at low dose concentration (30% inhibition rate) and WPLP was synergistic, while the combined inhibition effect of sitagliptin at medium and high dose concentration (50% and 70% inhibition rate) and WPLP was shown to be additive.
Example 6
During the present study F from example 1 was obtained by LC-MS/MS2186 polypeptides were identified in the fractions. Several of the polypeptides were synthesized by solid phase synthesis and investigated for their DPP-IV inhibitory activity by the method of example 3, and the DPP-IV inhibitory activity at a concentration of 0.40mmol/L is shown in FIG. 7. The tetrapeptide WPLP can be found to have the best inhibitory activity on DPP-IV, is remarkably superior to other polypeptides, and has outstanding effects, so that the polypeptide DPP-IV of the invention is unpredictable.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.