High-activity blood pressure lowering peptide and preparation method thereof

A blood pressure lowering peptide and high activity technology, applied in the field of high activity blood pressure lowering peptide and its preparation, can solve the problem that natural blood pressure lowering peptide needs to be improved and the like

Active Publication Date: 2012-10-31
BEIJING FORESTRY UNIVERSITY
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AI-Extracted Technical Summary

Problems solved by technology

[0003] However, the current methods for preparing na...
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Abstract

The invention relates to high-activity blood pressure lowering peptide and a preparation method thereof, wherein the high-activity blood pressure lowering peptide has an amino acid sequence shown by SEQ ID NO: 1. The high-activity blood pressure lowering peptide has the advantages of low cost, high angiotensin-converting enzyme inhibition activity and capability of effectively inhibiting the activity of angiotensin-converting enzyme, and can be therefore effectively applied to preparation of medicaments for treating hypertension.

Application Domain

Peptide preparation methodsFermentation +1

Technology Topic

Angiotensin-converting enzymePeptide +8

Image

  • High-activity blood pressure lowering peptide and preparation method thereof
  • High-activity blood pressure lowering peptide and preparation method thereof
  • High-activity blood pressure lowering peptide and preparation method thereof

Examples

  • Experimental program(2)

Example Embodiment

[0050] Example 1:
[0051] First, dissolve defatted walnut powder and water in a ratio of 1:10, hydrolyze for 5 hours at pH 8.0 and hydrolysis temperature of 65 °C, terminate the hydrolysis reaction, and centrifuge the supernatant for acid precipitation at an isoelectric point of 4.5. , the precipitate was collected by centrifugation, washed with water until it became neutral, and then it was quickly stored in vacuum freeze-drying, and its protein content was determined to be 86.32% by Coomassie brilliant blue method.
[0052] Next, utilize protease to carry out hydrolysis respectively, hydrolysis condition:
[0053] 1) Papain: the ratio of material to liquid is 25g/L, the amount of enzyme added is 3% (m/v), the pH is 6, the temperature is 50°C, and the hydrolysis time is 4h.
[0054] 2) Pepsin: the ratio of material to liquid is 25g/L, the amount of enzyme added is 4% (m/v), the pH is 2, the temperature is 55°C, and the hydrolysis time is 4h.
[0055] 3) Alkaline protease: the ratio of material to liquid is 25g/L, the amount of enzyme added is 7% (m/v), the pH is 10, the temperature is 60°C, and the hydrolysis time is 2h.
[0056] Next, the hydrolyzate was subjected to ultrafiltration through ultrafiltration membranes with different molecular weights (MWCO30000, 10000, 5000) at 4 °C, and the obtained filtrate was rapidly vacuum-freeze-dried. Then, the lyophilized powder of each component was prepared into a solution with a concentration of 1 mg/ml to determine the ACE (angiotensin-converting enzyme) inhibitory activity of each component in different molecular weight ranges (the ACE inhibitory activity detection method was as described in the general method). ), and the results are shown in Table 1 below.
[0057] Table 1 ACE inhibitory activity of components in different molecular weight ranges obtained by ultrafiltration
[0058]

Example Embodiment

[0059] Example 2:
[0060] The lyophilized powder of pepsin and alkaline protease with high ACE inhibitory activity and molecular weight less than 5000 obtained in Example 1 was dissolved in 5ml of 10mM Tris-HCl (pH 7.0), and subjected to degassing and membrane treatment , separated and purified by Sephadex G25 Medium column (that is, the first gel chromatography treatment), the elution peak was detected at 280nm and the ACE inhibitory activity of each component was collected and determined (the ACE inhibitory activity detection method was described in the general method), wherein chromatogram see figure 1 , the activity detection results are shown in Table 2 below, and the collected liquid is quickly vacuum freeze-dried.
[0061] Table 2 IC of the ACE inhibitory activity of different components obtained by the first gel chromatography treatment 50
[0062]
[0063] figure 1 Chromatograms of the first gel chromatography treatment during the preparation of highly active blood pressure lowering peptides are shown, where A shows the Sephadex G25 Medium column chromatogram of pepsin hydrolysate, and B shows Sephadex G25 of alkaline protease hydrolyzate Medium column chromatography. like figure 1 As shown in A and B, the abscissa represents the separation time of each component, and the ordinate represents the absorbance value of each component at 280 nm.
[0064] It can be seen from Table 2 that the ACE inhibitory activity of the pepsin hydrolyzate component PP3 is the highest, and the ACE inhibitory activity of the alkaline protease hydrolyzate component PP3 is not limited. Therefore, the pepsin hydrolyzate component PP3 is used for subsequent experiments.
[0065] Next, the highly active pepsin hydrolysate component PP3 is subjected to gel chromatography (ie, the second gel chromatography treatment) for further separation and purification (see the separation results in figure 2 ), and the ACE inhibitory activity of the obtained components was determined (the detection method was as described in the general method), and the results are shown in Table 3 below.
[0066] Table 3 IC of the ACE inhibitory activity of different components obtained by the second gel chromatography treatment 50
[0067] component
[0068] figure 2 The chromatogram of the second gel chromatography treatment during the preparation of the highly active blood pressure lowering peptide is shown. like figure 2 As shown, the abscissa represents the separation time of each component, and the ordinate represents the absorbance value of each component at 280 nm.
[0069] It can be seen from Table 2 that the ACE inhibitory activity of PP3-3 is the highest among the different components obtained by the second gel chromatography. Therefore, the high activity main peak PP3-3 was collected, and the component PP3-3 was used for subsequent experiments.
[0070] Then, the PP3-3 component was dissolved in the mobile phase A, and the PP3-3 was purified by reversed-phase high performance liquid chromatography, so as to obtain a component with higher purity, that is, a highly active blood pressure-lowering peptide, and the obtained high blood pressure lowering peptide was obtained. Active blood pressure lowering peptides are rapidly vacuum freeze-dried. Among them, the results of reversed-phase high performance liquid chromatography treatment are shown in image 3. image 3 Chromatograms of reversed-phase high performance liquid processing during the preparation of highly active blood pressure lowering peptides are shown. like image 3 As shown, the abscissa represents the separation time of each component, and the ordinate represents the absorbance value of each component at 280 nm.
[0071]Then, perform mass spectrometry analysis and amino acid sequencing on the purified hypertensive peptide in sequence, wherein the mass spectrum of the hyperactive hypotensive peptide is as follows Figure 4 As shown in the figure, its molecular weight was determined to be 408.60; the amino acid sequencing showed that the purified highly active blood pressure lowering peptide (ACE inhibitory peptide) had the amino acid sequence shown in SEQ ID NO: 1, namely: tyrosine-glutamic acid- Proline Tyr-Glu-Pro (YEP) has a theoretical molecular weight of 407.43D, and the detected molecular weight is basically consistent with the results of mass spectrometry. In addition, it can be seen from the measurement of ACE inhibitory activity that the ACE inhibition rate of the hyperactive blood pressure lowering peptide obtained in this example is 0.29 μmol/L, indicating that the method for preparing the hyperactive hypotensive peptide of the present invention is effective, and the obtained natural hypotensive The ACE inhibitory activity of the peptide was significant.

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