Polypeptide molecule with ACE (Angiotensin Converting Enzyme) inhibition activity and tumor resistance and preparation method thereof

A technology of inhibiting activity and anti-tumor, which is applied to the preparation method of peptides, anti-tumor drugs, chemical instruments and methods, etc. It can solve the problems of taste disorder and achieve the effect of small molecular weight, novel structure and high blood pressure-lowering activity

Active Publication Date: 2018-06-01
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current antihypertensive drugs that inhibit ACE activity on the market are mainly chemically synthesized drugs such as captopril and enalapril, which have certain side effects on the human body, such as cough, skin itching, taste disturbance, etc.

Method used

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  • Polypeptide molecule with ACE (Angiotensin Converting Enzyme) inhibition activity and tumor resistance and preparation method thereof
  • Polypeptide molecule with ACE (Angiotensin Converting Enzyme) inhibition activity and tumor resistance and preparation method thereof
  • Polypeptide molecule with ACE (Angiotensin Converting Enzyme) inhibition activity and tumor resistance and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]The Monascus mycelium ACE inhibitory peptide dry powder obtained by vacuum freeze-drying was reconstituted in PBS buffer with a concentration of 10mmol / L and a pH of 7.3-7.5; centrifuge the reconstituted ACE inhibitory peptide solution at 3000r / min for 5min , collect the supernatant, and filter it with a 0.22-micron water-based microporous membrane. The obtained filtrate is separated by superdex 30pg Sephadex column chromatography of GE Company, the injection volume is 10mL, the column temperature is 25°C, and the mobile phase is superdex Pure water, flow rate 1mL / min, protein detection wavelength 280nm; collect Monascus mycelia ACE inhibitory peptide active component peaks, numbered as components A-E. Component E with the highest ACE inhibitory activity was centrifuged at 3000r / min for 5min, and the supernatant was collected and filtered through a 0.22 micron water-based microporous membrane. The resulting filtrate was further separated by RP-HPLC. The separation conditi...

Embodiment 2

[0035] The preparation method is the same as in Example 1.

[0036] Dissolve the sample in ultrapure water, centrifuge at 7000 rpm for 15 min, take 10 μL of supernatant and 40 μL of 0.78 mmol / L substrate (hippuryl-L-His-L-Leu dissolved in 0.05 mol / L boric acid buffer at pH 8.3 solution containing 0.3mol / L NaCl) at 37°C for 6min, add 20μL ACE (0.1U dissolved in 1.0mL of the same buffer solution), react at 37°C for 30min, add 80μL 1mol / L HCl solution to terminate the reaction , to room temperature, take 10 μL of the reaction product for detection. Its ACE inhibitory activity was measured.

[0037] The results confirmed that the peptide was a heptapeptide product, the amino acid composition was Asn-Thr-Met-Met-Thr-His-Pro, the molecular weight was 814.493 Da, and the half inhibitory concentration was 0.18 mg / mL.

Embodiment 3

[0039] Using 2-Chlorotrityl Chloride Resin as the starting material, follow the method of solid-phase synthesis to connect sequentially with fluorenylmethoxycarbonyl (Fmoc-) as the protecting group Fmoc-L-Asn(trt)-OH, Fmoc-L-Met- OH, Fmoc-L-Thr(Tbu)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Pro-OH, the reaction was carried out at room temperature to obtain a heptapeptide resin, during which DIEA and HBTU were used in turn as Condensing agent, 20% piperidine DMF as deprotection agent for peptide grafting reaction, single amino acid peptide grafting reaction time 2h, deprotection time 20min, to obtain heptapeptide compound crude product, and then through reverse phase C18 column purification peptide, freeze-dried, obtained The ACE-inhibiting heptapeptide product. The obtained product will be collected and sampled for identification of purity and MS.

[0040] Dissolve the sample in ultrapure water, centrifuge at 7000 rpm for 15 min, take 10 μL of supernatant and 40 μL of 6.5 mmol / L substra...

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Abstract

The invention discloses a polypeptide molecule with ACE (Angiotensin Converting Enzyme) inhibition activity and tumor resistance and a preparation method thereof and belongs to the field of biomedicines and health-care foods. The polypeptide molecule disclosed by the invention is asparagine-threonine-methionine-methionine-threonine-histidine-proline. Two preparation methods of polypeptide comprise(1) a monascus hypha source enzymolysis extraction method: re-dissolving monascus hypha ACE inhibition peptide dry powder with a PBS (Phosphate Buffer Solution); carrying out glucan gel column chromatography; collecting active component peaks of monascus hypha ACE inhibition peptide and determining the ACE inhibition activity; further separating the component with the highest inhibition activityby adopting a RP-HPLC (Reverse Phase-High-performance Liquid Chromatography); collecting the component with the highest ACE inhibition activity from the collected components to obtain the polypeptide;(2) an artificial solid-phase polypeptide synthesis method: taking 2-chlorotrityl chloride2-chlorotriphenylmethyl resin as a carrier, fluorenylmethylcarbonyl (Fmoc-) as an amino protection group, andHBTU and DIEA as a condensing agent and carrying out peptide grafting reaction, so as to obtain inhibition heptapeptide through solid-phase synthesis. The polypeptide molecule and the preparation method thereof have the advantages that the operation is simple and the obtained product has small molecular weight, a novel structure and blood pressure lowering activity and anti-tumor activity.

Description

technical field [0001] The invention relates to the fields of biomedicine and health food, in particular to a polypeptide molecule with ACE inhibitory activity and antitumor activity and a preparation method thereof. Background technique [0002] According to reports, malignant tumors and cerebrovascular diseases have respectively occupied the top two causes of death among Chinese residents, and are major public health problems that threaten human life and health. [0003] As the first killer of human life and health, malignant tumor is due to the malignant proliferation of some cells that are completely unregulated by the body. Therefore, inhibiting the biological activity of tumor cells is the main direction of current anticancer drug research. In recent years, natural and synthetic peptides with anti-tumor activity have attracted much attention. Anti-tumor peptides can destroy the membrane structure of tumor cells, inhibit the proliferation and migration of cancer cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/16C07K1/06C07K1/04C07K1/34C12P21/02A61K38/08A61P35/00A61P9/12C12R1/66
CPCA61K38/00C07K7/06C12P21/02Y02P20/55
Inventor 王伟平何雨峰岳硕豪张华山张军林邓颖
Owner HUBEI UNIV OF TECH
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