Antarctic krill peptide-polyphenol complex and application thereof

Abstract

The application discloses a euphausia superba peptide-polyphenol compound and application, and belongs to the field of biotechnology / food technology. The euphausia superba peptide-polyphenol compound is prepared by dissolving euphausia superba peptide and polyphenol in a phosphate buffer, and stirring and standing. The high-concentration euphausia superba blood sugar-reducing peptide is compounded with low-concentration polyphenol, the bad smell of the active peptide is covered through sensory cognition, the two active substances are synergized in the blood sugar-reducing effect, the inhibitory effect of the compound on alpha-glucosidase, alpha-amylase and DPP-IV is better than that of single active polypeptide / polyphenol, the synergistic effect of the compound combination makes the functional activity of the compound enhanced, and the experimental results show that the compound also has the effect of delaying drug resistance.

Description

Technical Field

[0001] This invention relates to an Antarctic krill peptide-polyphenol complex, belonging to the field of biotechnology / food technology. Background Technology

[0002] With irregular changes in lifestyle and dietary structure, the prevalence of diabetes mellitus (DM) is increasing, making it the third most common chronic non-communicable disease after cancer and cardiovascular disease, and seriously threatening human health. This places a heavy burden on patients, their families, and society. Currently available diabetes medications have side effects, making the development of natural, safe, and medicinally valuable hypoglycemic substances of practical significance.

[0003] Studies have identified many natural anti-diabetic active ingredients, such as ginkgo leaf extract and plant polysaccharides. Research on the blood sugar-lowering effects of bioactive peptides is relatively limited. Compared to proteins, bioactive peptides possess superior physicochemical properties and processing performance, as well as important physiological functions, such as providing nutrients necessary for human growth and development, promoting mineral absorption, regulating the nervous system, and exhibiting antioxidant, blood sugar-lowering, blood pressure-lowering, blood lipid-lowering, cholesterol-lowering, liver-protecting, and antibacterial effects. Therefore, they have become a focus of research both domestically and internationally.

[0004] Polyphenols have also been proven to be a class of natural substances with hypoglycemic activity. Studies have shown that polyphenols can be combined with other active substances to achieve the advantages of multiple targets and multiple mechanisms of action. At the same time, the complex exhibits enhanced functional activity and delayed drug resistance through synergistic effects.

[0005] The digestion and absorption of carbohydrates such as starch in the human body relies on two key enzymes: α-glucosidase and α-amylase. Therefore, inhibiting the activity of these two key enzymes can slow down the rate at which carbohydrates are broken down into monosaccharides, thereby controlling the rapid rise in postprandial blood glucose. High expression of dpp-4 in the intestine breaks down the protein glp-1, which (stimulates insulin, inhibits glucagon, inhibits gastric emptying, and promotes pancreatic islet cell regeneration) and has a blood glucose-lowering effect. Inactivating dpp-4 to prevent the breakdown of glp-1 is one of the main approaches to treating diabetes.

[0006] Antarctic krill peptide-polyphenol complex controls blood glucose through multiple mechanisms by inhibiting α-glucosidase, α-amylase, and DPP-IV. It is a hypoglycemic complex with high economic value and market prospects.

[0007] Existing studies have shown that bioactive peptides can effectively improve the effects of diabetes. For example, in the study by Wang Junbo et al., marine collagen peptides can alleviate the structural damage of pancreatic β-cells in rats with hyperinsulinemia, increase granule secretion, reduce lipid droplet formation, and significantly improve the biological activity of insulin; they also significantly reduce fasting insulin levels and have a certain improving effect on fasting blood glucose and oral glucose tolerance. In the study by Huang Fengjie et al., shark liver bioactive peptide S-8300 has antioxidant effects, protecting pancreatic β-cells by scavenging free radicals, regulating glucose and lipid metabolism, and delaying the depletion of pancreatic β-cells, thus potentially treating diabetes to some extent.

[0008] Li Mingxi confirmed that Phyllanthus emblica extract can exert a hypoglycemic effect by increasing the expression of GLUT-2 and PPARγ and inhibiting related inflammatory pathways. The main active ingredient of the extract was identified as polyphenols. Huang et al. found that in the oral glucose tolerance test, polyphenols greatly alleviated the hyperglycemic symptoms of high fructose diet (HFD) induced diabetic mice.

[0009] Li Xingya et al. confirmed at both cellular and animal levels that a certain ratio of tea polysaccharides and tea polyphenols in combination has a good synergistic preventive and therapeutic effect on SD diabetic rats. The ratio TP / TPS=5 (tea polyphenols / tea polysaccharides=5) showed a significant effect in assisting in lowering blood glucose in DM rats. Wang Lingge used radioimmunoassay and other methods to detect the effects of the combination of green tea polysaccharides and tea polyphenols on insulin secretion function in MIN6 cells. The study showed that its hypoglycemic mechanism is closely related to mitochondrial function. It reduces oxidative stress damage to cells by scavenging ROS, protecting DNA and mitochondrial function, and increasing ATP levels, while simultaneously inhibiting Caspase-3 activity to prevent apoptosis, thus achieving a protective effect on MIN6 cells. The synergistic hypoglycemic mechanism of polyphenolic compounds and other active substances is achieved by inhibiting key enzymes in diabetes.

[0010] Currently, most bioactive peptides achieve blood sugar lowering effects only as independent components. Research on the synergistic effect of polyphenols and bioactive peptide complexes in lowering blood sugar is also rare in existing technologies. Furthermore, no research has been reported on the synergistic effect of polypeptide-polyphenol complexes in regulating blood sugar. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide an Antarctic krill peptide-polyphenol complex and its application, so as to improve the bioavailability of active peptides and realize the enhancement effect of Antarctic krill peptide / polyphenol in regulating blood sugar through multiple mechanisms.

[0012] The technical problem to be solved by this invention is achieved by the following technical solution:

[0013] An Antarctic krill peptide-polyphenol complex is prepared by mixing and dissolving Antarctic krill peptides and polyphenols at pH 6.5-7, followed by stirring and standing.

[0014] The weight ratio of Antarctic krill peptides to polyphenols is 10:0.1-5, preferably 10:0.25-2, and more preferably 10:0.5-2. In a preferred embodiment of the present invention, the ratio is 10:1.

[0015] Preferably, the amino acid sequence of the Antarctic krill peptide is FAGDADAPR.

[0016] Preferably, Antarctic krill peptides and polyphenols are dissolved in a phosphate buffer solution with a pH of 6.5-7.

[0017] Preferably, the preparation method of the Antarctic krill peptide includes the following steps:

[0018] S1. Preparation of Antarctic krill enzymatic hydrolysate: Defatted Antarctic krill powder is mixed with acidic phosphate buffer, enzymatic hydrolysate is added, and enzymes are inactivated after enzymatic hydrolysis to obtain Antarctic krill enzymatic hydrolysate;

[0019] S2. Isolation and purification of Antarctic krill peptides: After centrifugation of the cooled Antarctic krill enzymatic hydrolysate, the supernatant was collected and then filtered and ultrafiltered to obtain the filtrate.

[0020] Preferably, the filtrate after vacuum filtration and ultrafiltration in S2 is concentrated and dried to obtain Antarctic krill peptides.

[0021] Furthermore, the drying time is 1-2 days.

[0022] Preferably, in S1, the pH of the phosphate buffer is 6.5-7, the ratio of defatted Antarctic krill powder to phosphate buffer is 1g:4-10mL, and enzymatic hydrolysis is performed at 50-60℃ for 3-5h; the enzymes include neutral protease, alkaline protease, trypsin and lipase, and the amounts used are 1%, 1%, 1% and 0.3% respectively based on the weight of defatted Antarctic krill powder, the enzyme inactivation temperature is 100℃ and the enzyme inactivation time is 8-10min.

[0023] Preferably, the mixed solution is dried to obtain Antarctic krill peptide-polyphenol complex dry powder.

[0024] Furthermore, the drying time is 1-2 days.

[0025] Preferably, the polyphenol is gallic acid.

[0026] The above-described application of Antarctic krill peptide-polyphenol complex in the preparation of drugs for treating diabetes or hypoglycemic agents.

[0027] The above-described Antarctic krill peptide-polyphenol complex is used in the preparation of drugs that inhibit α-amylase, α-glucosidase, or DPP-IV.

[0028] Preferably, in S2, the centrifuge speed is 8000-12000 r / min, the centrifugation temperature is 3-5℃, the vacuum filtration uses a 0.45-0.5μm membrane, the ultrafiltration uses a 3k molecular weight membrane, and the molecular weight of the purified Antarctic krill active peptide solution is ≤3000 Da.

[0029] The beneficial effects of this invention are as follows: This invention combines a high concentration of Antarctic krill hypoglycemic peptides with a low concentration of polyphenols. Through sensory perception, it is believed that the unpleasant odor of the active peptides is masked, and the hypoglycemic effect of the two active substances is enhanced. The inhibitory effect of the complex on α-glucosidase, α-amylase and DPP-IV is better than that of the single active peptide / polyphenol, indicating that the synergistic effect of the complex enhances the functional activity of the complex. At the same time, the experimental results show that the complex also plays a role in delaying drug resistance. Attached Figure Description

[0030] Figure 1 The UV spectra of the peptides and polyphenols of the present invention in phosphate buffer solution at different ratios are shown below.

[0031] Figure 2 The graph shows the inhibition rate of the complex in Example 2 against α-amylase.

[0032] Figure 3 The graph shows the inhibition rate of the complex in Example 3 against α-glucosidase.

[0033] Figure 4 The graph shows the inhibition rate of the complex in Example 4 against DPP-Ⅳ.

[0034] Figure 5 Fourier transform infrared spectrum of Antarctic krill peptide in this invention;

[0035] Figure 6 Infrared spectrum of the Antarctic krill peptide / polyphenol complex in this invention; Detailed Implementation

[0036] To facilitate a clear understanding of the technical means, creative features, objectives, and effects of this invention, the invention will be further described below in conjunction with specific embodiments.

[0037] Example 1

[0038] Antarctic krill peptide and gallic acid were mixed and dissolved in phosphate buffer at a weight ratio of 10:1, and the mixture was stirred and allowed to stand to obtain the Antarctic krill peptide-gallic acid complex. The amino acid sequence of Antarctic krill peptide is FAGDADAPR.

[0039] The preparation method of the Antarctic krill peptide includes the following steps:

[0040] S1. Preparation of Antarctic krill enzymatic hydrolysate: 20g of defatted Antarctic krill powder was mixed with 200mL of pH=6.8 phosphate buffer. The hydrolysate consisted of 1% neutral protease, 1% alkaline protease, 1% trypsin and 0.3% lipase, respectively, based on the weight of the defatted Antarctic krill powder. The enzymatic hydrolysis time was 3h, the enzyme inactivation temperature was 100℃ and the enzyme inactivation time was 8min. After enzymatic hydrolysis, the enzyme was inactivated to obtain Antarctic krill enzymatic hydrolysate.

[0041] S2. Isolation and purification of Antarctic krill peptides: After cooling, the enzymatic hydrolysate of Antarctic krill was centrifuged at 10,000 r / min and 4℃. The supernatant was then collected, filtered using a 0.45 μm membrane, and ultrafiltered using a 3k molecular weight membrane to obtain the filtrate. The molecular weight of the obtained filtrate was ≤3000 Da.

[0042] After filtration and ultrafiltration were completed in S2, the filtrate was concentrated and dried for 1.5 days to obtain Antarctic krill peptides. Its amino acid sequence is FAGDADAPR.

[0043] The mixed solution was dried for 2 days to obtain Antarctic krill peptide-gallic acid complex dry powder.

[0044] Prepare 10 mg / mL Antarctic krill peptide and 5 mg / mL gallic acid using pH 6.8 phosphate buffer. Add 5 mL of Antarctic krill peptide and 0, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, 1, and 1.125 mL of gallic acid to test tubes 1-10, respectively. Make up to 10 mL, stir magnetically at 30℃ for 5 min, and measure the UV absorbance.

[0045] like Figure 1 As shown, when the concentration of gallic acid is 0-0.25 mg / mL, the rate of increase in absorbance is faster when λ = 260 nm. When the concentration is 0.25-0.5 mg / mL, the rate of increase in absorbance is slower when λ = 260 nm. When the concentration is 0.5 mg / mL, the rate of increase in absorbance tends to be stable when λ = 260 nm.

[0046] from Figures 5-6 It is evident that a chemical reaction occurred between Antarctic krill peptides and gallic acid, resulting in the formation of chemical bonds. This indicates that the synergistic effect of the complex enhances its functional activity.

[0047] Example 2: Inhibitory effect on α-amylase

[0048] (I) Experimental Methods

[0049] Step 1: Preparation of Inhibitors

[0050] Prepare 10 mg / mL Antarctic krill peptide and 5 mg / mL gallic acid using pH 6.8 phosphate buffer. Add 5 mL of Antarctic krill peptide and 0, 0.25, 0.5, 0.75 and 1 mL of gallic acid to test tubes 1, 2, 3, 4 and 5 respectively, and bring the volume to 10 mL. Stir magnetically at 30°C for 5 min.

[0051] Step 2: Preparation of starch solution

[0052] Prepare a starch solution by dissolving 1g of soluble starch in 100mL of distilled water;

[0053] Step 3: Determination of the inhibitory effect on α-amylase

[0054] Add 3 mL of α-amylase (100 U activity units, 0.5 g to bring the volume to 50 mL) to each test tube, incubate at 37 ℃ for 5 min, then add 4 mL of α-starch solution, incubate at 37 ℃, take 2 mL of the mixture every 15 minutes, add 1 mL of DNS reagent, boil the mixture in water for 5 min, cool to room temperature, bring the volume to 10 mL with distilled water, and measure the absorbance at 540 nm for 1 h.

[0055] Phosphate buffer was used as a substitute for the inhibitor (A1) and as a substitute for α-amylase as a blank control (A2); phosphate buffer solution was used as a substitute for α-amylase as a background control (A4); phosphate buffer was used as a substitute for the inhibitor as a blank control (A1), and five different ratios of inhibitors were used as experimental groups (A3).

[0056]

[0057]

[0058] The test results are attached. Figure 2 As shown.

[0059] Example 3: Detection method for inhibition of α-glucosidase

[0060] Step 1: Preparation of the inhibitor;

[0061] Prepare 10 mg / mL Antarctic krill peptide and 5 mg / mL gallic acid using 6.8 phosphate buffer. Add 5 mL of Antarctic krill peptide and 0, 0.25, 0.5, 0.75, and 1 mL of gallic acid to test tubes 1, 2, 3, 4, and 5 respectively, and bring the volume to 10 mL. Stir magnetically at 30 °C for 5 min.

[0062] Step 2: Determination of the inhibitory effect on α-glucosidase

[0063] 200 μL of sample extract and 200 μL of 0.02 mg / mL α-glucosidase (33 μL / mg) (dissolved in 0.01 mol / L, pH 6.8 sodium phosphate buffer) were reacted at 37 °C for 15 min. Then, 200 μL of 2.5 mmol / L p-nitrophenyl-α-D-pyranose (pNPG) solution was added, and the reaction was carried out at 37 °C for 10 min. Finally, 5 mL of 0.10 mol / L Na₂CO₃ solution was added to terminate the reaction. The absorbance A was measured at 400 nm. The blank was replaced with buffer solution, and the control was replaced with phosphate buffer solution.

[0064]

[0065] The test results are attached. Figure 3 As shown.

[0066] Example 4: Detection method for DPP-Ⅳ inhibition

[0067] Step 1: Preparation of Inhibitors

[0068] Prepare 10 mg / mL Antarctic krill peptide and 5 mg / mL gallic acid using pH 6.8 phosphate buffer. Add 5 mL of Antarctic krill peptide and 0, 0.5, 0.75 and 1 mL of gallic acid to test tubes 1, 2, 3 and 4 respectively, and bring the volume to 10 mL. Stir magnetically at 30°C for 5 min.

[0069] Step 2: Determination of the inhibitory effect of DPP-IV

[0070] The inhibition rate of DPP-IV was determined using a kit method (fluorescence method), with sitagliptin as a positive control.

[0071] Add 50 μL of inhibitor (1, 2, 3, 4) and 10 μL of inhibitor sequentially to the ELISA plate, mix well, and incubate at 37°C for 10 min. Then add 25 μL of substrate and incubate at 37°C for another 15 min. Detect fluorescence (excitation wavelength 360 nm, emission wavelength 460 nm). Record the results and calculate the inhibition rate of the DPP-IV inhibitory peptide using the following formula.

[0072]

[0073] In the formula: F control — without inhibitor; F sample — containing sample or inhibitor.

[0074] The test results are attached. Figure 4 As shown.

[0075] Comprehensive Appendix Figure 2 To the attached Figure 4It was found that the Antarctic krill peptide (5 mg / mL) / gallic acid (0.5 mg / mL) complex inhibited glucosidase by 54.73%, amylase by 51.16%, and DPP-IV by 58.43%.

[0076] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and various changes and modifications can be made without departing from the spirit and scope of the invention, all of which fall within the scope of the claims. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A Euphausia superba peptide-polyphenol complex, characterized in that, The Euphausia superba peptide-polyphenol is prepared by mixing and dissolving Euphausia superba peptide and polyphenol under the condition of pH 6.5-7, and stirring and standing.

2. The Euphausiids peptide-polyphenol complex according to claim 1, characterized in that, The weight ratio of the Euphausia superba peptide to the polyphenol is 10:0.1-5.

3. The Euphausiids peptide-polyphenol complex according to claim 1, characterized in that, The Euphausia superba peptide and the polyphenol are dissolved in a phosphate buffer with pH 6.5-7.

4. The Euphausiids peptide-polyphenol complex according to claim 1, characterized in that, The preparation method of the Euphausia superba peptide comprises the following steps: S1. Preparation of Euphausia superba enzymatic hydrolysate: defatted Euphausia superba powder is mixed with an acid phosphate buffer, and an enzymatic hydrolysate is added, and after enzymolysis, the enzyme is inactivated to obtain the Euphausia superba enzymatic hydrolysate; S2. Isolation and purification of Euphausia superba peptide: after centrifugation of the cooled Euphausia superba enzymatic hydrolysate, the supernatant is taken, and the filtrate is obtained through suction filtration and ultrafiltration.

5. The Euphausiids peptide-polyphenol complex according to claim 4, characterized in that, The filtrate after suction filtration and ultrafiltration in S2 is concentrated and dried to obtain the Euphausia superba peptide.

6. The Euphausiids peptide-polyphenol complex according to claim 4, characterized in that, In S1, the pH of the phosphate buffer is 6.5-7, the solid-liquid ratio of the defatted Euphausia superba powder to the phosphate buffer is 1g:4-10mL, and the enzymolysis is carried out at 50-60℃ for 3-5h; the enzymes include neutral protease, alkaline protease, trypsin and lipase, and the dosages of the enzymes are 1%, 1%, 1% and 0.3% respectively based on the weight of the defatted Euphausia superba powder.

7. The Euphausiids peptide-polyphenol complex according to claim 1, characterized in that, The mixed solution is dried to obtain the Euphausia superba peptide-polyphenol complex dry powder.

8. Use of the Euphausia superba peptide-polyphenol complex of any one of claims 1-7 in the preparation of a drug for treating diabetes or a blood glucose-lowering drug.

9. Use of the Euphausia superba peptide-polyphenol complex of any one of claims 1-7 in the preparation of a drug for inhibiting DPP-Ⅳ.

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