Rana spinosa polypeptide inhibiting angiotensin converting enzyme activity and preparation method and application thereof

Abstract

The present invention discloses a polypeptide of Rana spinosa for inhibiting the activity of angiotensin converting enzyme, a preparation method and an application thereof, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. The polypeptide has the advantages of strong angiotensin converting enzyme inhibition and safety without toxic side effects, and can be used as a drug for lowering blood pressure, thus broadening the added value of frog skin.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a polypeptide of Rana spinosa for inhibiting the activity of angiotensin converting enzyme, and a preparation method and application thereof. Background Art

[0002] The spiny-chested frog is also known as the "living ginseng in mountain springs" because of its dual value of being both edible and medicinal. At present, the main marketing methods for spiny-chested frogs are catering sales and live retail. Most of the frog meat sold on the market is peeled fresh frog meat, and processed products are relatively rare. The industrial chain is simple and the processed products are in a single form. The processing of spiny-chested frogs will produce many by-products such as frog skin, bones, and internal organs. The rational use of by-product resources such as frog skin has a positive impact on the diversification and high-value development of spiny-chested frogs.

[0003] Hypertension, as a "silent killer" of human beings, often leads to cardiovascular and brain diseases such as myocardial infarction and cerebral hemorrhage that seriously endanger human life. Studies have shown that human blood pressure is regulated by many systems, among which the kallikrein-kinin and renin-angiotensin systems play a vital role as two mutually antagonistic systems. The imbalance of the two is one of the main factors causing hypertension. Angiotensin converting enzyme, as a key enzyme in the renin-angiotensin-aldosterone system, plays an important role in regulating blood pressure. At present, the commonly used ACE inhibitors in medicine are amlodipine, rilmenidine, captopril, etc., which can effectively lower blood pressure but also cause side effects such as hypokalemia, cough, edema, headache, etc. As a dietary supplement or adjuvant, food-derived angiotensin converting enzyme inhibitor peptides are pure natural, safe and have no side effects. They play a potential benefit in treatment, such as high stability and low health care costs, and have become a research hotspot in the food field today. Summary of the invention

[0004] To solve the above problems, the embodiments of the present invention provide, in a first aspect, a R. spinosa polypeptide for inhibiting the activity of angiotensin converting enzyme, the amino acid sequence of which is shown in SEQ ID NO:1.

[0005] According to an embodiment of the present invention, a frog spinosa polypeptide for inhibiting angiotensin converting enzyme activity has a molecular weight of 687.3016Da. The polypeptide has the advantages of strong angiotensin converting enzyme inhibition and safety without toxic side effects, and can be used as a blood pressure lowering drug to broaden the added value of frog skin.

[0006] In a second aspect, an embodiment of the present invention provides a method for preparing the above-mentioned Rana spinosa polypeptide that inhibits angiotensin converting enzyme activity, characterized in that it comprises the following steps:

[0007] (1) The frog skin of Rana spinosa was homogenized with distilled water according to the material-liquid ratio of 1 g:15-25 mL, the pH was adjusted to 6.5-7.5, 4%-6% papain was added according to the weight of the frog skin, the enzymatic hydrolysis temperature was adjusted to 40-50°C, the enzymatic hydrolysis was carried out for 4-6 hours, and the enzyme was inactivated with boiling water for 10 minutes;

[0008] (2) adjusting the pH of the enzymatic solution after the enzyme inactivation in step (1) to 1.0-3.0, adding 4%-6% of acidic protease according to the weight of frog skin, adjusting the enzymatic hydrolysis temperature to 45-55° C., and performing enzymatic hydrolysis for 4-6 hours and then inactivating the enzyme in boiling water for 10 minutes;

[0009] (3) Centrifuging the enzymatic hydrolyzate after the enzyme inactivation in step (2), taking the supernatant, and subjecting it to ultrafiltration and chromatography to obtain a Rana spinosa polypeptide that inhibits xanthine oxidase activity.

[0010] According to an embodiment of the present invention, the method performs step-by-step enzymatic hydrolysis on frog skin, and then performs ultrafiltration and chromatography to obtain a Rana spinosa polypeptide capable of inhibiting the activity of angiotensin converting enzyme.

[0011] Optionally, in step (1), the enzymatic activity of papain is ≥200 U / mg.

[0012] Optionally, in step (2), the enzyme activity of the acidic protease is ≥200 U / mg.

[0013] Optionally, in step (3), ultrafiltration is performed using an ultrafiltration tube with a molecular weight cutoff of 3 kD to collect enzymatic hydrolysis products with a molecular weight less than 3 kD.

[0014] Optionally, in step (3), the chromatography is to use Sephadex G-15 gel chromatography column to perform column chromatography on the ultrafiltered enzymatic hydrolysate: the loading concentration is 20-40 mg / mL, the loading volume is 2-4 mL, the flow rate is 980 μL / min, one tube is collected every 10 minutes, and the absorbance curve is detected at 220 nm and drawn.

[0015] Optionally, the components after chromatography are purified by reverse-phase high-performance liquid chromatography, and the reverse-phase high-performance liquid chromatography conditions are: injection volume 10-20 μL; using a C18 reverse chromatographic column, the column temperature is 25-30°C, the mobile phase: A is water containing 0.1% trifluoroacetic acid, B is acetonitrile, gradient elution: 0-40min, acetonitrile concentration from 0 to 50%, elution rate 1.0mL / min; ultraviolet detection wavelength 280nm.

[0016] According to an embodiment of the present invention, the present invention further proposes the use of the above-mentioned Rana spinosa polypeptide in the preparation of antihypertensive drugs, so as to achieve high value-added comprehensive utilization of Rana spinosa.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Column chromatography elution diagram of the enzymatic ultrafiltration product according to an embodiment of the present invention;

[0019] Figure 2 is a reverse phase high performance liquid chromatogram according to an embodiment of the present invention;

[0020] Figure 3 is a mass spectrum of a Rana spinosa polypeptide according to an embodiment of the present invention;

[0021] Figure 4 The conformation of the Rana spinosa polypeptide in the ACE pocket, the binding mode in the ACE active site, and the interaction plane diagram according to the embodiments of the present invention;

[0022] Figure 5 is the angiotensin converting enzyme inhibitory activity of the ultrafiltration fraction according to the example of the present invention, and ad indicates that the difference between the groups is extremely significant (P<0.05);

[0023] Figure 6 The cytotoxicity of the Rana spinosa polypeptide according to the examples of the present invention to LO2 cells. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0025] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0026] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0027] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0028] Example 1

[0029] (1) The skin of Rana spinosa was homogenized with distilled water at a solid-liquid ratio of 1 g:15 mL, and the pH was adjusted to 7.0. 4% papain (enzyme activity ≥ 200 U / mg) was added based on the weight of the skin. The enzymatic hydrolysis temperature was adjusted to 50°C. After enzymatic hydrolysis for 4 h, the enzyme was inactivated with boiling water for 10 min.

[0030] (2) The pH of the enzymatic solution after the enzyme inactivation in step (1) was adjusted to 2.0, 6% acid protease (enzyme activity ≥ 200 U / mg) was added based on the weight of frog skin, and the enzymatic hydrolysis temperature was adjusted to 45° C. After the enzymatic hydrolysis treatment for 4 h, the enzyme was inactivated in boiling water for 10 min.

[0031] (3) After the enzyme is inactivated in step (2), the enzymatic hydrolyzate is cooled to room temperature, centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant is collected after centrifugation.

[0032] (4) The supernatant of step (3) was separated and graded using ultrafiltration tubes with a molecular weight cutoff of 3 kD and 10 kD to obtain three components: QSPH-I (<3 kD), QSPH-II (3-10 kD), and QSPH-III (>10 kD).

[0033] (5) The QSPH-Ⅰ enzymatic hydrolysate after ultrafiltration in step (4) was purified by column chromatography using a Sephadex G-15 gel chromatography column (specification: Φ1.6×60 cm). The loading concentration was 40 mg / mL, the loading volume was 4 mL, the flow rate was 980 μL / min, one tube was collected every 10 min, and the absorbance curve was detected at 220 nm. The results are shown in the figure. Figure 1 As shown, fractions 1 to 4 were collected, freeze-dried, and then tested for their angiotensin converting enzyme inhibitory activity. Among them, tubes 11-13 were fractions with the highest angiotensin converting enzyme inhibitory activity, which were gel chromatography hydrolysates.

[0034] (6) The gel chromatography hydrolysate was prepared into a 50 μg / mL solution with double distilled water, and purified by reverse phase high performance liquid chromatography. Based on the angiotensin converting enzyme inhibitory activity, a polypeptide Gly-Phe-Gly-Phe-Gly-Gly-Phe with angiotensin converting enzyme inhibitory activity was obtained; the reverse phase high performance liquid chromatography conditions were: injection volume 10 μL; a C18 reverse phase chromatography column (Acclaim PepMap RSLC, 75 μm×25 cm C18-2 μm ); column temperature was 30°C; mobile phase: A was water containing 0.1% trifluoroacetic acid, B was acetonitrile; gradient elution: acetonitrile concentration was from 0 to 50% in 0-40min; elution rate was 1.0mL / min, and UV detection wavelength was 280nm. The reverse HPLC results are shown in the figure Figure 2 As shown, the single peak with a retention time of 20.812 min is the most active peptide.

[0035] Example 2

[0036] A C18 reverse phase column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm ), the mobile phase was acetonitrile / water / formic acid = 5 / 95 / 0.1 (v / v), and the mobile phase A was reduced from 95% to 62% within 60 min. The mass spectrometer used the ThermoFisher Q Exactive system (Thermo Fisher, USA) combined with the nanospray Nano Flex ion source, the spray voltage was 1.9 kV, and the ion transfer tube heating temperature was 275 ° C. The mass spectrometer scanning mode was the information-dependent acquisition mode (Data Dependent Analysis, DDA), the primary mass spectrometer scanning range was 100-1500 m / z, the scanning resolution was 70000, and the maximum injection time was 100 ms. In each DDA cycle, up to 20 secondary spectra with charges of 1+ to 3+ were collected, and the maximum injection time of the secondary mass spectrometer ions was 50 ms. The collision cell energy (high energy collision induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 6 seconds.

[0037] Get as Figure 3 In the mass spectrum shown, the ion of the target peptide is selected as the parent ion to collide with the inert gas to break the peptide bond in the peptide chain to form a series of ions. The N-terminal fragment ion series is marked as the b series, and the C-terminal fragment ion series is marked as the y series. A comprehensive analysis of these fragment ion series shows that the amino acid sequence of the peptide is Gly-Phe-Gly-Phe-Gly-Gly-Phe.

[0038] Example 3

[0039] Based on molecular docking, the peptides identified by mass spectrometry were screened for biological activity using the software Autodock (version 4.2). The spatial structure of the peptide was drawn by ChemDraw 11.0 (Cambridge Soft Corporation, Cambridge, Massachusetts, USA). The peptide structure was saved in mol2 format using Open Babel GUI, and the peptide molecular structure was constructed using the Tripos force field energy minimization principle. The protonation state and hydrogen orientation were optimized in Autodock and saved as pdbqt files as ligand files. The Lamarkian genetic algorithm (LGA) was used for docking calculations, and energy range = 4.0, exhaustiveness = 9.0, and num modes = 30.0 were set.

[0040] The 3D structure of angiotensin converting enzyme (ACE) was obtained from the PDB database (PDB ID: 1O86), and the incomplete side chains were replaced using the Dunbrack 2010 rotamer library. The docking coordinates in the ACE structure are x: 40.6559, y: 37.3827, z: 43.3401, and the radius

[0041] The results are as follows Figure 4 As shown, A is the conformation of Gly-Phe-Gly-Phe-Gly-Gly-Phe in the ACE pocket; B. Binding mode in the ACE active site; C. Interaction plane diagram. Studies have reported three pockets that form the active site of angiotensin converting enzyme: S1 (Ala354, Glu384 and Tyr523), S2 (Gln281, His353, Lys511, His 513 and Tyr520) and S1' (Gln162). Ligand molecules that interact with these residues can achieve the effect of reducing enzyme activity through competitive inhibition. Gly-Phe-Gly-Phe-Gly-Gly-Phe forms pi-piT-shaped interactions with the benzene ring of phenylalanine at His383 and Phe512, forms hydrophobic interactions with the benzene ring at Val379 and Val380, and forms hydrogen bonds at Asn277, Gln281, Thr282, His353, Ala356, Glu384, His513, and Tyr523. Therefore, Gly-Phe-Gly-Phe-Gly-Gly-Phe has strong binding ability in both S1 and S2 regions and can competitively inhibit the activity of angiotensin converting enzyme.

[0042] Example 4

[0043] The QSPH-Ⅰ, QSPH-Ⅱ and QSPH-Ⅲ prepared in Example 1 were subjected to an in vitro ACE inhibition experiment: the sample was dissolved in a borate buffer solution (0.1M boric acid mixed with 0.3M sodium chloride, and the pH was adjusted to 8.3 with NaOH). 50μL of the sample was mixed with 100μL 5mMHHL and then bathed in 37℃ water for 10min. Then 50μL 0.1U / mL ACE solution was added and mixed and bathed in 37℃ water for 10min. Finally, 150μL 1M hydrochloric acid was added to terminate the reaction, and 1.5mL ethyl acetate was added and shaken to mix. After centrifugation (4000r / min, 10min), 0.8mL of the ethyl acetate layer solution was taken in a test tube, dried in a 95℃ oven, 4mL of deionized water was added to mix, and the absorbance at 228nm was detected. The control group did not add ACE inhibitors during the reaction but added ACE inhibitors after the reaction. The blank group added 150μL 1M hydrochloric acid before the reaction to terminate the reaction. Captopril was used as the positive control group.

[0044] ACE inhibition rate (%) = [(A1-A0) / (A1-A2)] × 100%;

[0045] In the formula: A0 is the sample group; A1 is the control group; A2 is the blank group.

[0046] The results are as follows Figure 5 As shown, Gly-Phe-Gly-Phe-Gly-Gly-Phe has a good inhibitory effect on angiotensin converting enzyme.

[0047] The QSPH-Ⅰ prepared in Example 1 was subjected to a cytotoxicity experiment: LO2 cells in the logarithmic growth phase were taken to make a uniform single-cell suspension, counted and diluted to 20,000 / 100 μL with culture medium, and 100 μL was spread on a 96-well plate in each well. After culturing for 12-16 hours, the old culture medium was discarded, and culture medium containing polypeptides at different concentrations (culture medium: polypeptide solution = 99:1) was added to continue culturing for 24 hours. Finally, 100 μL of new culture medium and 15 μL of 0.5 mg / mL thiazolyl blue solution were added to each well under light-proof conditions, incubated in an incubator for 4 hours, and the supernatant was discarded. 150 μL of DMSO was added to each well and shaken in the dark for 10 minutes, and the absorbance was detected at 490 nm. Six parallel wells were set for each group of samples, and a PBS control group was set; the formula for calculating cell survival rate is as follows:

[0048] Cell survival rate (%) = [(A1-A0) / (A2-A0)] × 100%;

[0049] In the formula: A0 is the blank group; A1 is the sample group; A2 is the control group.

[0050] The results are as follows Figure 6As shown, at a concentration of 0.1 to 10 μg / mL, the Gly-Phe-Gly-Phe-Gly-Gly-Phe polypeptide has a growth-promoting effect on LO2 cells, and the cell survival rate is between 98% and 120%.

[0051] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0052] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A polypeptide of Rana spinosa that inhibits the activity of angiotensin converting enzyme, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A method for preparing a polypeptide of Rana spinosa for inhibiting angiotensin converting enzyme activity as claimed in claim 1, characterized in that: The following steps are involved: (1) The frog skin of Rana spinosa was homogenized with distilled water according to the material-liquid ratio of 1 g:15-25 mL, the pH was adjusted to 6.5-7.5, 4%-6% papain was added according to the weight of the frog skin, the enzymatic hydrolysis temperature was adjusted to 40-50°C, the enzymatic hydrolysis was carried out for 4-6 hours, and the enzyme was inactivated with boiling water for 10 minutes; (2) adjusting the pH of the enzymatic solution after the enzyme inactivation in step (1) to 1.0-3.0, adding 4%-6% of acidic protease according to the weight of frog skin, adjusting the enzymatic hydrolysis temperature to 45-55° C., and performing enzymatic hydrolysis for 4-6 hours and then inactivating the enzyme in boiling water for 10 minutes; (3) Centrifuging the enzymatic hydrolysate after the enzyme inactivation in step (2), taking the supernatant, and subjecting the supernatant to ultrafiltration and chromatography to obtain a polypeptide that inhibits the activity of angiotensin converting enzyme; the ultrafiltration is performed by using an ultrafiltration tube with a molecular weight cutoff of 3 kD to collect enzymatic hydrolysate with a molecular weight less than 3 kD.

3. The preparation method according to claim 2, characterized in that: In step (1), the enzymatic activity of papain is ≥200 U / mg.

4. The preparation method according to claim 2, characterized in that: In step (2), the enzyme activity of the acidic protease is ≥ 200 U / mg.

5. The preparation method according to claim 2, characterized in that: In step (3), the chromatography is to use Sephadex G-15 gel chromatography column to perform column chromatography on the ultrafiltered enzymatic hydrolysate: the loading concentration is 20-40 mg / mL, the loading volume is 2-4 mL, the flow rate is 980 μL / min, one tube is collected every 10 minutes, and the absorbance curve is detected at 220 nm and drawn.

6. The preparation method according to claim 5, characterized in that: The components after chromatography were purified by reverse-phase high performance liquid chromatography, and the reverse-phase high performance liquid chromatography conditions were as follows: injection volume 10-20 μL; a C18 reverse chromatographic column was used, the column temperature was 25-30° C., the mobile phases: A was water containing 0.1% trifluoroacetic acid, B was acetonitrile, gradient elution: 0-40 min, acetonitrile concentration from 0 to 50%, elution rate 1.0 mL / min; ultraviolet detection wavelength was 280 nm.

7. Use of the Rana spinosa polypeptide as claimed in claim 1 in the preparation of antihypertensive drugs.

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