CN116199741A - Rana spinosa polypeptide for inhibiting angiotensin converting enzyme activity and preparation method and application thereof - Google Patents

Abstract

The invention discloses a rana spinosa polypeptide for inhibiting the activity of angiotensin converting enzyme, a preparation method and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. The polypeptide has the advantages of strong angiotensin converting enzyme inhibition effect, safety and no toxic or side effect, can be used as a medicine for reducing blood pressure, and widens the added value of frog skin.

Description

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

Technical Field

The invention relates to the technical field of biomedicine, in particular to a rana spinosa polypeptide for inhibiting the activity of angiotensin converting enzyme, a preparation method and application thereof.

Background

The thorn chest frog has dual values of eating and medicinal, and is also called as "live ginseng in mountain spring". At present, the chest frogs are sold in catering and living retail as main marketing modes, most of the chest frogs sold in the market are peeled fresh frog meat, processed products are less common, and the industrial chain is simple and the processed products are single in form. The processing process of the thorn chest frog can generate more byproducts such as frog skin, bones, viscera and the like, and the reasonable utilization of the byproduct resources such as the frog skin and the like has positive influence on diversification and high-value development of the thorn chest frog.

Hypertension is used as a silent killer for human beings, and often causes cardiovascular and brain diseases such as myocardial infarction, cerebral hemorrhage and the like which seriously endanger human lives. Studies have shown that human blood pressure is regulated by a number of systems, of which the kallikrein-kinin and renin-angiotensin systems play a vital role as two systems that antagonize each other, and imbalance between the two is one of the major factors responsible for the onset of hypertension. Angiotensin converting enzyme plays an important role in regulating blood pressure as a key enzyme in the renin-angiotensin-aldosterone system. The ACE inhibitors commonly used in the medicine at present are amlodipine, rimidine, captopril and the like, and the ACE inhibitors can effectively reduce blood pressure and simultaneously produce side effects such as blood potassium reduction, cough, edema, headache and the like. The food source angiotensin converting enzyme inhibitory peptide is used as a dietary supplement or an adjuvant, is purely natural, safe and free of side effects, plays potential benefits of high stability, low medical care cost and the like in treatment, and becomes a research hot spot in the current food field.

Disclosure of Invention

In order to solve the above problems, in a first aspect, the embodiment of the present invention provides a rana spinosa polypeptide inhibiting angiotensin converting enzyme activity, the amino acid sequence of which is as shown in SEQ ID NO: 1.

The molecular weight of the rana spinosa polypeptide for inhibiting the activity of the angiotensin converting enzyme is 687.3016Da, and the polypeptide has the advantages of strong angiotensin converting enzyme inhibition effect, safety and no toxic or side effect, can be used as a medicine for reducing blood pressure, and widens the added value of frog skin.

In a second aspect, the embodiment of the invention provides a method for preparing the rana spinosa polypeptide for inhibiting the activity of angiotensin converting enzyme, which is characterized by comprising the following steps:

(1) Adding distilled water into the Rana spinosa skin according to the feed liquid ratio of 1g to 15-25 mL for homogenization, adjusting the pH value to 6.5-7.5, adding 4-6% papain according to the leather quality of the Rana spinosa, adjusting the enzymolysis temperature to 40-50 ℃, and inactivating enzyme in boiling water for 10min after enzymolysis for 4-6 h;

(2) Adjusting the pH value of the enzyme-deactivated enzymolysis solution in the step (1) to 1.0-3.0, adding 4-6% of acid protease according to the leather quality of the frog, adjusting the enzymolysis temperature to 45-55 ℃, and inactivating the enzyme by boiling water for 10min after the enzymolysis treatment for 4-6 h;

(3) Centrifuging the enzyme-inactivated enzymolysis solution obtained in the step (2), taking supernatant, and performing ultrafiltration and chromatography to obtain the rana spinosa polypeptide inhibiting xanthine oxidase activity.

According to the embodiment of the invention, the frog skin is subjected to stepwise enzymolysis, and then ultrafiltration and chromatography are carried out to obtain the rana spinosa polypeptide capable of inhibiting the activity of angiotensin converting enzyme.

Optionally, in the step (1), the enzyme activity of papain is more than or equal to 200U/mg.

Optionally, in the step (2), the enzyme activity of the acid protease is more than or equal to 200U/mg.

Optionally, in step (3), ultrafiltration is performed by collecting the enzymatic hydrolysate having a molecular weight of less than 3kD using an ultrafiltration tube having a molecular weight cut-off of 3 kD.

Optionally, in the step (3), the chromatography is column chromatography of the ultrafiltered enzymolysis liquid by using a Sephadex G-15 gel chromatographic column: the loading concentration is 20-40 mg/mL, the loading amount is 2-4 mL, the flow rate is 980 mu L/min, one tube is collected every 10min, and the absorbance curve is detected and drawn at 220 nm.

Optionally, the reversed-phase high performance liquid chromatography is carried out on the components after chromatography, and the conditions of the reversed-phase high performance liquid chromatography are as follows: the sample injection amount is 10-20 mu L; using a C18 reverse chromatography column at a column temperature of 25-30 ℃, mobile phase: a is water containing 0.1% trifluoroacetic acid, B is acetonitrile, gradient elution: 0-40 min, acetonitrile concentration from 0-50%, and eluting speed of 1.0mL/min; the ultraviolet detection wavelength is 280nm.

According to the embodiment of the invention, the invention also provides the application of the rana spinosa polypeptide in preparing the antihypertensive drug, so as to realize the comprehensive utilization of the high added value of the rana spinosa.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a column chromatography elution diagram of an enzymatic ultrafiltrate in accordance with an embodiment of the present invention;

FIG. 2 is an inverse high performance liquid chromatogram according to an embodiment of the invention;

FIG. 3 is a mass spectrum of a Rana spinosa polypeptide according to an embodiment of the invention;

FIG. 4 is a plan view of the conformation of the Rana spinosa polypeptide in the ACE pocket, binding pattern in the ACE active site, interaction according to an embodiment of the present invention;

FIG. 5 shows the angiotensin converting enzyme inhibitory activity of the ultrafiltration fraction according to the examples of the present invention, a-d showing the difference between groups was very significant (P < 0.05);

fig. 6 is a diagram of the cytotoxicity of the rana spinosa polypeptide against LO2 according to an embodiment of the invention.

Detailed Description

The technical scheme of the invention is described below through specific examples. It is to be understood that the mention of one or more method steps of the present invention does not exclude the presence of other method steps before and after the combination step or that other method steps may be interposed between these explicitly mentioned steps; it should also be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.

In order to better understand the above technical solution, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not limiting in any way.

Example 1

(1) Adding distilled water into Rana spinosa skin according to the ratio of 1g to 15mL, homogenizing, adjusting pH to 7.0, adding 4% papain (enzyme activity not less than 200U/mg) according to Rana spinosa skin amount, adjusting enzymolysis temperature to 50deg.C, and inactivating enzyme with boiling water for 10min after enzymolysis treatment for 4h.

(2) Adjusting the pH value of the enzyme solution after enzyme deactivation in the step (1) to 2.0, adding 6% of acid protease (the enzyme activity is more than or equal to 200U/mg) according to the quantity of frog cortex, adjusting the enzymolysis temperature to 45 ℃, and inactivating enzyme by boiling water for 10min after 4h of enzymolysis treatment.

(3) Cooling the enzyme-deactivated enzymolysis liquid in the step (2) to room temperature, centrifuging at 10000r/min for 10min at 4 ℃, and taking supernatant after centrifuging.

(4) Separating and classifying the supernatant in the step (3) by using an ultrafiltration tube with the molecular weight cut-off of 3kD and 10kD to obtain three components: QSPH-I (< 3 kD), QSPH-II (3-10 kD), QSPH-III (> 10 kD).

(5) Purifying the QSPH-I enzymolysis liquid obtained after the ultrafiltration in the step (4) by using a Sephadex G-15 gel chromatographic column (with the specification of phi 1.6X60 cm). The loading concentration is 40mg/mL, the loading amount is 4mL, the flow rate is 980 mu L/min, a tube is collected every 10min, the absorbance curve is detected and drawn at 220nm, the results are shown in figure 1, the components 1-4 are respectively collected, the angiotensin converting enzyme inhibitory activity is detected after freeze drying, wherein the 11 th-13 th tube is the component with the highest angiotensin converting enzyme inhibitory activity, and the gel chromatography enzymatic hydrolysate is obtained.

(6) Preparing the gel chromatography zymolyte into 50 mug/mL solution by double distilled water, purifying by reverse phase high performance liquid chromatography, and obtaining 1 polypeptide Gly-Phe-Gly-Phe-Gly-Gly-Phe with inhibiting activity on angiotensin converting enzyme according to the inhibiting activity of angiotensin converting enzyme; the reversed phase high performance liquid chromatography conditions are as follows: the sample injection amount is 10 mu L; using a C18 reverse phase chromatography column (Acclaim PepMap RSLC,75 μm X25 cm C18-2 μm)

) The method comprises the steps of carrying out a first treatment on the surface of the The column temperature is 30 ℃; mobile phase: a is water containing 0.1% trifluoroacetic acid, B is acetonitrile; gradient elution: acetonitrile concentration from 0 to 50% in 0-40 min; the elution speed is 1.0mL/min, and the ultraviolet detection wavelength is 280nm. The reverse high performance liquid phase result diagram is shown in fig. 2, and the single peak with the retention time of 20.812min is the peptide fragment with the strongest activity.

Example 2

Using a C18 reverse phase chromatography column (Acclaim PepMap RSLC,75 μm X25 cm C18-2 μm)

) Mobile phase was acetonitrile/water/formic acid=5/95/0.1 (v/v), mobile phase a decreased from 95% to 62% over 60 min. The mass spectrum used ThermoFisher Q Exactive system (sammer fly in united states) in combination with nanoliter spray Nano Flex ion source, spray voltage of 1.9kV and ion transport tube heating temperature of 275 ℃. The mass spectrum scanning mode is an information-dependent acquisition working mode (Data Dependent Analysis, DDA), the primary mass spectrum scanning range is 100-1500m/z, the scanning resolution is 70000, and the maximum injection time is 100ms. At most 20 secondary maps with charges of 1+ to 3+ are acquired under each DDA cycle, and the maximum injection time of the secondary mass spectrum ions is 50ms. The collision cell energy (high energy collision induced dissociation, HCD) was set at 28eV, for all precursor ions, and the dynamic exclusion was set at 6 seconds.

And (3) obtaining a mass spectrum shown in figure 3, selecting ions of the target peptide as parent ions to collide with inert gas, breaking peptide bonds in a peptide chain to form a series of ions, marking an N-terminal fragment ion series as a b series and a C-terminal fragment ion series as a y series, and comprehensively analyzing the fragment ion series to obtain the amino acid sequence of the peptide fragment as Gly-Phe-Gly-Phe-Gly-Gly-Phe.

Example 3

Screening for biological Activity on peptides identified by Mass Spectrometry was performed using software Autodock (version 4.2) based on molecular docking, the spatial structure of the peptides was mapped by ChemDraw 11.0 (Cambridge Soft Corporation, cambridge, massachusetts, USA) software. The Open Babel GUI is used to save the polypeptide structure as a mol2 format, and the Tripos force field energy minimization principle is used to construct the polypeptide molecular structure. The optimization of the protonation state and hydrogen orientation was performed in Autodock and saved as pdbqt file as ligand file. Calculation of docking was performed using Lamarkian Genetic Algorithm (LGA) and set with energy range=4.0, output=9.0, num modes=30.0

The 3D structure of angiotensin converting enzyme (PDB ID:1O 86) was obtained from the PDB database and the incomplete side chains were replaced with Dunback 2010 rotamer library. The coordinates of the butt joint in the ACE structure are x:40.6559, y:37.3827, z:43.3401 radius of

As a result, as shown in FIG. 4, A is the conformation of Gly-Phe-Gly-Phe-Gly-Gly-Phe in the ACE pocket; B. binding patterns in ACE active sites; C. a plan view of the interaction. Three pockets forming angiotensin converting enzyme active sites have been reported in studies: s1 (Ala 354, glu384 and Tyr 523), S2 (Gln 281, his353, lys511, his513 and Tyr 520) and S1' (Gln 162). Ligand molecules that interact with these residues may achieve a reduction in enzyme activity through competitive inhibition. Gly-Phe-Gly-Phe forms pi-pi T-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 Tyr 523. Therefore, gly-Phe-Gly-Phe-Gly-Gly-Phe has stronger binding capacity in the S1 and S2 regions and can competitively inhibit the activity of angiotensin converting enzyme.

Example 4

In vitro ACE inhibition experiments were performed on QSPH-I, QSPH-II, QSPH-III prepared in example 1: the sample was dissolved in borate buffer (0.1M boric acid mixed with 0.3M sodium chloride and pH adjusted to 8.3 with NaOH). mu.L of the sample was mixed with 100. Mu.L of 5mM HHI and then subjected to a water bath at 37℃for 10 minutes. Then 50. Mu.L of 0.1U/mL ACE solution was added and mixed and then water was put in a 37℃water bath for 10min. Finally, 150 mu L of 1M hydrochloric acid is added to terminate the reaction, and 1.5mL of ethyl acetate is added to be mixed evenly under shaking. After centrifugation (4000 r/min,10 min) 0.8mL of ethyl acetate layer solution was taken in a test tube, dried in an oven at 95℃and mixed well with 4mL of deionized water, and the absorbance at 228nm was measured. The control group had no ACE inhibitor added during the reaction but after the reaction. The blank was quenched by the addition of 150. Mu.L of 1M hydrochloric acid prior to the reaction. Captopril was used as a positive control.

ACE inhibition ratio (%) = [ (A1-A0)/(A1-A2) ]x100%;

wherein: a0 is a sample group; a1 is a control group; a2 is a blank group.

As shown in FIG. 5, gly-Phe-Gly-Phe-Gly-Gly-Phe has good inhibitory effect on angiotensin converting enzyme.

Cytotoxicity experiments were performed on QSPH-I prepared in example 1: preparing LO2 cells in logarithmic growth phase into uniform single cell suspension, diluting to 2 ten thousand/100 mu L with culture medium, spreading to 96-well plate with 100 mu L per well, culturing for 12-16 hr, removing old culture medium, adding culture medium containing polypeptide at different concentrations (culture medium: polypeptide solution=99:1), and culturing for 24 hr. Finally, 100 mu L of new culture medium and 15 mu L of 0.5mg/mL of thiazole blue solution are added to each well under the light-shielding condition, the supernatant is sucked and removed after incubation for 4 hours in an incubator, 150 mu L of DMSO is added to each well, the wells are vibrated for 10 minutes under the light-shielding condition, and the light absorption value is detected at 490 nm. Setting 6 parallel holes for each group of samples, and setting a PBS control group; the cell viability was calculated as follows:

cell viability (%) = [ (a) ₁ -A ₀ )/(A ₂ -A ₀ )]×100％；

Wherein: a is that ₀ Is a blank group; a is that ₁ Is a sample group; a is that ₂ Is a control group.

As shown in FIG. 6, the polypeptide Gly-Phe-Gly-Phe-Gly-Gly-Phe had growth promoting effect on LO2 cells at a concentration of 0.1-10. Mu.g/mL, and the cell survival rate was 98-120%.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (8)

1. A rana spinosa polypeptide for inhibiting the activity of angiotensin converting enzyme, which is characterized in that the amino acid sequence is shown in SEQ ID NO: 1.

2. A method for producing the xanthine oxidase activity-inhibiting polypeptide of the thorn frog of claim 1, comprising the steps of:

(1) Adding distilled water into the Rana spinosa skin according to the feed liquid ratio of 1g to 15-25 mL for homogenization, adjusting the pH value to 6.5-7.5, adding 4-6% papain according to the leather quality of the Rana spinosa, adjusting the enzymolysis temperature to 40-50 ℃, and inactivating enzyme in boiling water for 10min after enzymolysis for 4-6 h;

(2) Adjusting the pH value of the enzyme-deactivated enzymolysis solution in the step (1) to 1.0-3.0, adding 4-6% of acid protease according to the leather quality of the frog, adjusting the enzymolysis temperature to 45-55 ℃, and inactivating the enzyme by boiling water for 10min after the enzymolysis treatment for 4-6 h;

(3) Centrifuging the enzyme-inactivated enzymolysis solution obtained in the step (2), taking supernatant, and performing ultrafiltration and chromatography to obtain the rana spinosa polypeptide for inhibiting the activity of angiotensin converting enzyme.

3. The process according to claim 2, wherein in the step (1), the papain has an enzyme activity of 200U/mg or more.

4. The process according to claim 2, wherein in the step (2), the enzyme activity of the acid protease is not less than 200U/mg.

5. The method of claim 2, wherein in step (3), ultrafiltration is performed by collecting the enzymatic hydrolysate having a molecular weight of less than 3kD using an ultrafiltration tube having a molecular weight cut-off of 3 kD.

6. The method according to claim 2, wherein in the step (3), the chromatography is column chromatography of the ultrafiltered enzymatic hydrolysate using a Sephadex G-15 gel column: the loading concentration is 20-40 mg/mL, the loading amount is 2-4 mL, the flow rate is 980 mu L/min, one tube is collected every 10min, and the absorbance curve is detected and drawn at 220 nm.

7. The method of claim 6, wherein the reversed-phase hplc purification is performed on the chromatographed components under the following conditions: the sample injection amount is 10-20 mu L; using a C18 reverse chromatography column at a column temperature of 25-30 ℃, mobile phase: a is water containing 0.1% trifluoroacetic acid, B is acetonitrile, gradient elution: 0-40 min, acetonitrile concentration from 0-50%, and eluting speed of 1.0mL/min; the ultraviolet detection wavelength is 280nm.

8. The use of the acantha fortuneana polypeptide according to claim 1 for the preparation of a medicament for lowering blood pressure.

Images