A kind of polypeptide for inhibiting alpha-glucosidase activity and its preparation method and application

Abstract

The present invention discloses a polypeptide of Rana spinosa for inhibiting α-glucosidase activity, 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 α-glucosidase inhibition and safety without toxic side effects, and can be used as a medicine for type 2 diabetes, thereby broadening the added value of frog skin.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a Rana spinosa polypeptide capable of inhibiting alpha-glucosidase activity, 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] Diabetes can be divided into type I, type II, special types and gestational diabetes according to different pathogenesis. Type II diabetes accounts for more than 95% of the number of diabetes patients in my country and is the main type of disease. Controlling blood sugar levels is a key factor in the treatment of type II diabetes. α-glucosidase can regulate blood sugar by regulating the metabolic process of polysaccharides. Although typical drugs currently used to treat non-insulin-dependent diabetes can better regulate blood sugar levels, most of them can cause side effects such as abdominal pain and bloating, such as acarbose and miglitol. Finding α-glucosidase inhibitors with less toxic side effects from natural products has attracted people's attention. Summary of the invention

[0004] To solve the above problems, the embodiments of the present invention provide, in a first aspect, a polypeptide of Rana spinosa that inhibits α-glucosidase activity, 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 α-glucosidase activity has a molecular weight of 685.2927 Da. The polypeptide has the advantages of strong α-glucosidase inhibition and safety without toxic side effects, and can be used as a drug for type 2 diabetes, thereby broadening 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 α-glucosidase 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 polypeptide that inhibits the activity of α-glucosidase.

[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 α-glucosidase activity.

[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 hypoglycemic 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 2is a reverse HPLC chromatogram of a Rana spinosa polypeptide 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 α-glucosidase pocket, the binding mode in the active site of α-glucosidase, and the interaction plane diagram according to the embodiments of the present invention;

[0022] Figure 5 is the α-glucosidase 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 6.7. 5% papain (enzyme activity ≥ 200 U / mg) was added based on the weight of the skin. The enzymatic hydrolysis temperature was adjusted to 45°C. After enzymatic hydrolysis for 5.3 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 2.2, 4% acidic protease (enzyme activity ≥ 200 U / mg) was added based on the weight of frog skin, the enzymatic hydrolysis temperature was adjusted to 45° C., and the enzyme was inactivated in boiling water for 10 min after the enzymatic hydrolysis treatment for 4.8 h.

[0031] (3) After the enzyme is inactivated in step (2), the enzymatic hydrolyzate is cooled to room temperature, centrifuged at 8000 r / min 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 20 mg / mL, the loading volume was 2 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 Figure 1 As shown, fractions 1 to 4 were collected and freeze-dried, and then their α-glucosidase inhibitory activities were tested. Among them, tubes 11-13 were fractions with the highest α-glucosidase 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. According to the α-glucosidase inhibitory activity, a polypeptide with inhibitory activity against α-glucosidase was obtained; the reverse phase high performance liquid chromatography conditions were: injection volume 10 μL; using a C18 reverse phase chromatography column (Acclaim PepMap RSLC, 75 μm×25 cmC18-2 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 25.539 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 working 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 Met-Pro-Gly-Pro-Met-Gly-Pro.

[0038] Example 3

[0039] Based on molecular docking, the peptides obtained by mass spectrometry identification were screened for biological activity using the software Autodock (version 4.2), and 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 α-glucosidase (PDB ID: 3A4A) was obtained from the PDB database. The binding site was determined using Discoverystudio4.5. The docking parameters conf.txt file was set. The water molecules and solvent molecules in the protein were removed. The protein was hydrogenated using Autodock tools and saved in pdbqt format for later use.

[0041] The results are as follows Figure 4 As shown, A is the conformation of Met-Pro-Gly-Pro-Met-Gly-Pro in the α-glucosidase pocket; B. Binding mode in the active site of α-glucosidase; C. Interaction plane diagram. The Met-Pro-Gly-Pro-Met-Gly-Pro peptide forms hydrogen bonds only at Arg315 and Gln353, attractive charges at Asp352 and Glu411, pi-sigma interactions at Val308, hydrophobic interactions at Lys156, and π-π stacking at Phe178, so the peptide has a certain binding ability with the receptor protein α-glucosidase.

[0042] Example 4

[0043] The QSPH-Ⅰ, QSPH-Ⅱ and QSPH-Ⅲ prepared in Example 1 were subjected to an in vitro α-glucosidase inhibition experiment: 20 μL of polypeptide solution and 20 μL of α-glucosidase were added to a 96-well plate in turn and shaken and mixed, incubated at room temperature for 20 min, then 20 μL of PNPG was added and mixed thoroughly, incubated at 37°C in the dark for 20 min, and finally 100 μL of 1M sodium carbonate solution was added to terminate the system reaction and the absorbance at 410 nm was immediately detected. The blank group was replaced with PBS buffer instead of sample, α-glucosidase and p-nitrophenyl glucose; the complete reaction group was replaced with PBS buffer instead of sample; the positive drug was replaced with acarbose instead of sample.

[0044] α-glucosidase inhibition rate (%) = [1-(D2-D1) / D3] × 100%;

[0045] Wherein: D1 is the sample control group; D2 is the sample reaction group (positive control); D3 is the complete reaction group.

[0046] The results are as follows Figure 5 As shown, Met-Pro-Gly-Pro-Met-Gly-Pro has a good inhibitory effect on α-glucosidase.

[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 6 As shown, at a concentration of 0.1 to 10 μg / mL, the Met-Pro-Gly-Pro-Met-Gly-Pro 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 α-glucosidase activity, 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 α-glucosidase activity as claimed in claim 1, characterized in that: The following steps are involved: (1) Add distilled water to the frog skin of Rana spinosa at a material-liquid ratio of 1 g:15-25 mL and homogenize, adjust the pH to 6.5-7.5, add 4%-6% papain according to the weight of frog skin, adjust the enzymatic hydrolysis temperature to 40-50℃, and after enzymatic hydrolysis for 4-6 hours, inactivate the enzyme with boiling water for 10 minutes; (2) Adjust the pH of the enzymatic solution after the enzyme inactivation in step (1) to 1.0-3.0, add 4%-6% acid protease according to the weight of frog skin, adjust the enzymatic hydrolysis temperature to 45-55°C, and inactivate the enzyme in boiling water for 10 min after the enzymatic hydrolysis for 4-6 h; (3) Centrifuging the enzymatic hydrolysate 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 α-glucosidase inhibitory activity, wherein the ultrafiltration is performed 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 plotted.

6. The preparation method according to claim 5, characterized in that: The components after chromatography were purified by reverse-phase HPLC. The conditions of reverse-phase HPLC were as follows: injection volume 10-20 μL; a C18 reverse-phase chromatographic column was used, the column temperature was 25-30°C, the mobile phase was: 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; UV detection wavelength was 280 nm.

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

Images