A synergistic assembly complex of oyster peptide and anthocyanin and its application

Abstract

The present invention discloses a synergistic assembly complex of oyster peptides and anthocyanins and its application, belonging to the field of biomedicine technology. The complex is composed of oyster DPP-IV inhibitory peptides and anthocyanins; the oyster DPP-IV inhibitory peptide is a polypeptide shown in an amino acid sequence such as FPVGR, APPNIT, IAPPERK, LDFYLDIP or LAIL; the anthocyanin is paeoniflorin-3-O-glucoside, cyanidin-3-O-glucoside or malvidin-3-O-glucoside. The present invention analyzes the synergistic effect of oyster DPP-IV inhibitory peptides and anthocyanins in synergistic activity, and finds that the DPP-IV inhibitory activity of the FR-Pn3G complex is the strongest; optimizes the preparation conditions of the synthetic peptide FR-5 and Pn3G synergistic assembly complex, and obtains the oyster peptide and anthocyanin synergistic assembly complex with the most significant DPP-IV inhibitory activity synergistic effect, which provides a new direction for hypoglycemic and diabetes prevention and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an oyster peptide and anthocyanin synergistic assembly complex and application thereof. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by hyperglycemia and insulin resistance. It is the third most dangerous disease for human life and health after cardiovascular and cerebrovascular diseases and cancer. According to the International Diabetes Federation (IDF), the number of diabetes patients in the world in 2021 is about 537 million. It is predicted that the number of diabetes patients in the world will reach 643 million in 2030 and exceed 783 million in 2045. It can be seen that the large number of diabetes patients and the increasing number of patients have seriously endangered human life and health. Among them, type II diabetes is the main type of diabetes, accounting for about 90% of the number of diabetes patients. Therefore, the key to controlling diabetes is to effectively reduce the prevalence of type II diabetes. Dipeptidyl peptidase-IV (DPP-IV) has been confirmed to be one of the important targets for the treatment of type II diabetes. Glucagon-like peptide-1 (GLP-1) can promote insulin secretion and inhibit glucagon secretion under glucose stimulation, thereby playing a role in blood sugar regulation. DPP-IV cuts the N-terminal dipeptide residue of GLP-1 to make GLP-1 lose its insulinotropic activity, resulting in imbalance in blood sugar regulation. Using DPP-IV as a therapeutic target can effectively prolong the half-life of GLP-1, promote glucose-induced insulin secretion, and regulate blood sugar without the risk of hypoglycemia. Therefore, finding DPP-IV inhibitors has become a hot topic in the research of new drugs for type 2 diabetes.

[0003] At present, most of the drugs that use DPP-IV as a target to treat type II diabetes are chemically synthesized drugs, such as Saxagliptin, Sitagliptin and Linagliptin, but these drugs will produce certain side effects when taken for a long time, such as severe allergic reactions, gastrointestinal adverse reactions, etc. Existing studies have shown that DPP-IV inhibitors derived from natural foods are highly safe and easily absorbed, and have the potential to prevent and treat type II diabetes.

[0004] Oysters, commonly known as "raw oysters", are a high-protein, low-fat marine food known as "undersea milk". Oyster active peptides have good biological activities, such as anti-oxidation, immunomodulation, lowering blood pressure, anticoagulation, lowering blood sugar, etc. In addition, the special marine environment (such as high salt, low oxygen and low temperature, etc.) makes oyster protein have a unique amino acid composition and sequence structure, which is an advantageous resource for the preparation of DPP-IV inhibitory peptides. However, oyster peptides have poor stability during digestion, resulting in low bioavailability and limited activity. Anthocyanins are a class of water-soluble phenolic compounds widely found in plants, with significant DPP-IV inhibitory activity, but anthocyanins are sensitive to environmental factors such as temperature, light, and pH, and have poor stability, which limits their activity.

[0005] Although a large number of literatures have reported that individual oyster peptides and anthocyanins have the effect of inhibiting DPP-IV activity, individual oyster peptides and anthocyanins have poor digestion stability, low bioavailability, and poor DPP-IV inhibitory activity. Studies have shown that the assembly of active peptides and polyphenols into a complex can not only improve the physicochemical stability of polyphenols, but also improve the antioxidant activity of the two after assembly. Therefore, assembling oyster peptides and anthocyanins into a complex has the potential to protect the active structures of both and synergistically enhance the DPP-IV inhibitory activity. However, there are few reports on the preparation of oyster peptide and anthocyanin synergistic assembly complexes, the structural characteristics of oyster DPP-IV inhibitory peptides in the complex, the types of anthocyanins, and other factors that affect the assembly effect of the two, as well as their synergistic effect in inhibiting DPP-IV activity. Summary of the invention

[0006] The purpose of the present invention is to provide an oyster peptide and anthocyanin synergistic assembly complex and its application to solve the problems existing in the above-mentioned prior art. The present invention provides an oyster peptide and anthocyanin synergistic assembly complex with the most significant DPP-IV inhibitory activity enhancement effect, which provides a new direction for lowering blood sugar and treating and preventing Type II diabetes.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an oyster peptide and anthocyanin synergistic assembly complex, wherein the complex is composed of an oyster DPP-IV inhibitory peptide and anthocyanin; wherein the oyster DPP-IV inhibitory peptide comprises any one or more of the polypeptides represented by amino acid sequences such as FPVGR, APPNIT, IAPPERK, LDFYLDIP, LAIL, TVPIYEG, TVPIYE, APPERKY, PYFPG, APPER, HAPDHANPGDLG, WDPNDLNPK, PGNKLTP, LPVLA, LPR and PPNIT.

[0009] Furthermore, the anthocyanin comprises paeoniflorin-3-O-glucoside (Pn3G), cyanidin-3-O-glucoside (C3G), malvidin-3-O-glucoside (Mv3G), delphinidin-3-O-glucoside (D3G), pelargonidin-3-O-glucoside (Pg3G) or petunidin-3-O-glucoside (Pt3G).

[0010] Furthermore, the oyster DPP-IV inhibitory peptide is any one of the polypeptides represented by the amino acid sequences such as FPVGR, APPNIT, IAPPERK, LDFYLDIP, and LAIL; the anthocyanin is paeoniflorin-3-O-glucoside, cyanidin-3-O-glucoside or malvidin-3-O-glucoside.

[0011] Furthermore, the concentration ratio of the oyster DPP-IV inhibitory peptide to the anthocyanin is 1:6 to 9:1.

[0012] The present invention also provides a method for preparing the above-mentioned complex, comprising the steps of mixing the oyster DPP-IV inhibitory peptide and anthocyanin for reaction.

[0013] Furthermore, the reaction conditions are pH 3-7, the reaction time is 0.5-5h, and the reaction temperature is 25-50°C; the mixed concentration ratio of the oyster DPP-IV inhibitory peptide and the anthocyanin is 1:6 to 9:1.

[0014] The present invention also provides the use of the above-mentioned complex in preparing medicine for preventing and / or treating diabetes.

[0015] Furthermore, the diabetes is type II diabetes.

[0016] The present invention also provides a medicine for preventing and treating type II diabetes, comprising the above-mentioned compound.

[0017] The present invention also provides a blood sugar lowering food comprising the above compound.

[0018] The present invention discloses the following technical effects:

[0019] The present invention prepares oyster peptides by enzymatic hydrolysis, determines the molecular weight distribution by gel permeation chromatography, and analyzes the amino acid composition and peptide spectrum sequence of oyster peptides by amino acid analyzer and LC-MS / MS technology. The in vitro DPP-IV activity inhibition model and Compusyn synergistic calculation model are used to analyze the activity enhancement effect of oyster DPP-IV inhibitory peptides and anthocyanins. The results show that the molecular weight of oyster peptides is mainly concentrated below 3kDa (55.44%), and branched-chain amino acids (BCAAs) and proline (Pro) account for 60.84% ​​of the total hydrophobic amino acids in its amino acid composition. Peptide spectrum sequence analysis shows that the main 17 peptides in oyster peptides are rich in BCAAs and Pro at the nitrogen end (N end), and have significant DPP-IV inhibitory peptide structural characteristics. Among the 5 peptides (FPVGR, APPNIT, IAPPERK, LDFYLDIP, LAIL) screened and synthesized, FPVGR has the strongest DPP-IV inhibitory activity (P<0.05). Among the six anthocyanins, anthocyanins Pn3G, C3G, and Mv3G have stronger DPP-Ⅳ inhibitory activity. After being assembled into a complex with the synthetic peptide FPVGR, synergistic studies showed that FPVGR and Pn3G, C3G, and Mv3G all have synergistic inhibitory effects on DPP-Ⅳ, among which the DPP-Ⅳ inhibitory activity of the FR-Pn3G complex is the strongest (P<0.05). The preparation conditions (concentration ratio, pH, time, temperature) of the synergistic assembly complex of the synthetic peptide FR-5 and Pn3G were optimized, and the CI values ​​were all less than 1, so the oyster peptide and anthocyanin synergistic assembly complex with the most significant DPP-Ⅳ inhibitory activity was obtained. The present invention provides a new direction for the treatment and prevention of hypoglycemia and type II diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is a relative molecular weight distribution diagram of the oyster peptide of the present invention;

[0022] Figure 2 This is a graph showing the DPP-IV inhibitory activity results of the oyster peptide of the present invention;

[0023] Figure 3 This is a graph showing the DPP-Ⅳ inhibitory effect of the synthetic peptide of the present invention. Different letters indicate statistical significance after variance analysis, P < 0.05;

[0024] Figure 4This is a graph showing the DPP-Ⅳ inhibitory effect of anthocyanins of the present invention. Different letters indicate statistical significance after variance analysis, P<0.05;

[0025] Figure 5 This is a graph showing the DPP-Ⅳ inhibitory effect of the synthetic peptide-anthocyanin complex of the present invention. Different letters indicate statistical significance after variance analysis, P < 0.05;

[0026] Figure 6 The synergistic inhibitory effect diagram of FR-5 and Pn3G on DPP-Ⅳ of the present invention; a-dose-effect diagram; b-combination index diagram; c-dose-effect reduction diagram; d-contour line diagram;

[0027] Figure 7 The synergistic inhibitory effect diagram of FR-5 and C3G on DPP-Ⅳ of the present invention; a-dose-effect diagram; b-combination index diagram; c-dose-effect reduction diagram; d-contour line diagram;

[0028] Figure 8 The synergistic inhibitory effect diagram of FR-5 and Mv3G on DPP-Ⅳ of the present invention; a-dose-effect diagram; b-combination index diagram; c-dose-effect reduction diagram; d-contour line diagram;

[0029] Fig. 9 Figure 2 is a graph of the combined index of the FR and Pn3G synergistic complexes of the present invention at different ratios; a-FR:Pn3G=1:6; b-FR:Pn3G=1:3; c-FR:Pn3G=1:1; d-FR:Pn3G=3:1; e-FR:Pn3G=6:1; f-FR:Pn3G=9:1;

[0030] Fig.10 The results of the DPP-Ⅳ inhibition of the Pn3G-FR complex of the present invention are shown in Figure 1, including FR:Pn3G=6:1; FR:Pn3G=9:1, and different letters indicate statistical significance after variance analysis, P<0.05;

[0031] Fig.11 The DPP-Ⅳ inhibitory effect of the Pn3G-FR complex prepared under different pH conditions of the present invention is shown in the figure. Different letters indicate statistical significance after variance analysis, P<0.05;

[0032] Fig.12 The particle size and Zeta potential of the Pn3G-FR complex prepared under different pH conditions of the present invention, different letters indicate statistical significance after variance analysis, P < 0.05;

[0033] Fig.13 This is a graph showing the DPP-Ⅳ inhibitory effect of the Pn3G-FR complex prepared at different times according to the present invention;

[0034] Fig.14 The particle size and Zeta potential of the Pn3G-FR complex prepared at different times in the present invention are shown in Figure 1. Different letters indicate statistical significance after variance analysis, P < 0.05;

[0035] Fig.15 The DPP-Ⅳ inhibitory effect of the Pn3G-FR complex prepared under different temperature conditions of the present invention is shown in the figure. Different letters indicate statistical significance after variance analysis, P<0.05;

[0036] Fig.16 The particle size and Zeta potential of the Pn3G-FR complex prepared under different temperature conditions of the present invention are shown in Figure 2. Different letters indicate statistical significance after variance analysis, P < 0.05;

[0037] Fig.17 The diagram is a diagram of the synergistic inhibitory effect of FR-5 and Pn3G on DPP-Ⅳ under the optimal preparation conditions of the present invention; a-dose-effect diagram; b-combination index diagram; c-dose-effect reduction diagram; d-iso-effect diagram. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The main experimental materials, reagents and equipment involved in the following examples are:

[0044] (1) Oyster meat: purchased from Dongfeng Market, Zhanjiang City, Guangdong Province; peony cyanidin-3-O-glucoside (Pn3G), cyanidin-3-O-glucoside (C3G), malvidin-3-O-glucoside (Mv3G), delphinidin-3-O-glucoside (D3G), pelargonidin-3-O-glucoside (Pg3G) and petunidin-3-O-glucoside (Pt3G) were all purchased from Chengdu Caoyuankang Biotechnology Co., Ltd. (purity>98.0%); synthetic peptides: commissioned by Wuxi Maimotop Biotechnology Co., Ltd. for synthesis. Alkaline protease (200000 U / g): purchased from Beijing Solebao Biotechnology Co., Ltd.; DPP-IV inhibitor screening kit: purchased from Cayman Chemical Company, USA; formic acid and acetonitrile were chromatographic grade, and other reagents were analytical grade.

[0045] (2) PHS-2F pH meter, Shanghai Yidian Scientific Instrument Co., Ltd.; Varioskan LUX fully automatic microplate reader, Easy-nLC 1200 liquid chromatograph, Lynx6000 high-speed floor-standing centrifuge and electrospray-combined ion trap Orbitrap mass spectrometer, Thermo Fisher Scientific, USA; LC-JSY-3 constant temperature metal bath, Shanghai Lichen Instrument Technology Co., Ltd.

[0046] The specific experimental methods are as follows:

[0047] Example 1

[0048] 1. Preparation of oyster DPP-Ⅳ inhibitory peptide

[0049] 1.1 Preparation of oyster peptides

[0050] The viscera-free Hong Kong oyster meat was thawed in running water, homogenized with water at a solid-liquid ratio of 1:3 (g:mL), the pH was adjusted to 8, alkaline protease was added (4000U of enzyme was added per g of oyster meat), and then enzymatic hydrolysis was carried out in a 50°C water bath for 3 h, the enzyme was inactivated in a boiling water bath for 15 min, cooled to room temperature in an ice water bath, and centrifuged (4°C, 8000r / min) for 15 min. The supernatant was collected by filtration with degreased gauze, and freeze-dried for later use.

[0051] 1.2 Structural characterization of oyster peptides

[0052] 1.2.1 Determination of relative molecular weight distribution of oyster peptides

[0053] The molecular weight distribution of oyster peptides was determined by gel permeation chromatography. An Agilent 1260 instrument was used, equipped with a Waters Ultrahydrogel (300mm×7.8mm) column and an Agilent RID G1362A detector. Chromatographic analysis conditions: mobile phase: 0.1mol / L sodium nitrate aqueous solution; flow rate: 1.0mL / min; standard: polypropylene glycol; column temperature: 45℃; injection volume: 40μL. The peak time (X) was linearly fitted with the logarithm of the relative molecular mass (lgM), and the standard curve was obtained: lgM=18.94-1.21X 1 +0.02986X 2 -0.0002655X 3 , R 2 =0.9996.

[0054] 1.2.2 Analysis of amino acid composition of oyster peptides

[0055] The amino acid content was determined using a fully automatic amino acid analyzer with reference to GB 5009.124-2016 “National Food Safety Standard - Determination of Amino Acids in Foods”.

[0056] 1.2.3 Peptide spectrum sequence identification of oyster peptides

[0057] The peptide spectrum sequence of oyster peptides was analyzed by LC-MS / MS technology. A C18 column (100μm×180mm) was used, the injection volume was 5μL, the flow rate was 600nL / min, the mobile phase A was an aqueous solution containing 0.1% formic acid, and the composition of mobile phase B was an aqueous solution containing 0.1% formic acid: acetonitrile = 2:8 (v / v). The linear gradient elution mode was adopted: 0-2min, B phase 4%-8%; 2-45min, B phase 8%-40%; 45-55min, B phase 40%-60%; 55-66min, B phase 60%-95%. The scanning range of the mass spectrometer was 100-1500m / z, scanning mode: DDA; capillary voltage: 2200V; capillary temperature: 320℃.

[0058] 1.3 Determination of DPP-IV inhibitory activity of oyster peptides

[0059] The DPP-Ⅳ inhibitor screening kit was used to determine the DPP-Ⅳ inhibitory activity of oyster peptides by the fluorescent substrate method. Sitagliptin was used as a positive control, 30 μL of buffer was pipetted into the microwells of a 96-well plate, 10 μL of DPP-Ⅳ solution and 10 μL of sample solution were added, and then 50 μL of substrate (Gly-Pro-AMC) was added to react at 37°C for 30 minutes, and the fluorescence value was determined by a multifunctional microplate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The inhibition rate was calculated according to the following formula.

[0060]

[0061] Where: A and B are the fluorescence values ​​of the control group and the sample group after deducting the blank.

[0062] 1.4 Artificial synthesis of oyster DPP-IV inhibitory peptide

[0063] The DPP-Ⅳ inhibitory peptide was commissioned to Wuxi Maimotop Biotechnology Co., Ltd. to synthesize using the solid phase synthesis method, and the synthetic peptide was identified and purified using high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS). The purity of the final synthetic peptide was greater than 98%.

[0064] 1.5 Synthesis of DPP-Ⅳ inhibitory peptides and determination of DPP-Ⅳ inhibitory activity of anthocyanins

[0065] The method for determining the DPP-Ⅳ inhibitory activity of synthetic DPP-Ⅳ inhibitory peptides and anthocyanins is the same as 1.3.

[0066] 2. Preparation of oyster peptide and anthocyanin synergistic assembly complex with enhanced DPP-IV inhibitory activity

[0067] Ultrapure water was used to prepare synthetic peptide and anthocyanin solutions with a concentration of 1 mg / mL, and the sample solutions were respectively pipetted into 2 mL centrifuge tubes to adjust the concentration ratio (c / c) of synthetic peptide: anthocyanin in the mixed solution to 6:1. After sufficient oscillation and mixing, the mixture was reacted at 25°C and 1000 r / min for 2 h to obtain the oyster peptide and anthocyanin synergistic assembly complex (synthetic peptide-anthocyanin complex).

[0068] 3. Analysis of the synergistic effect of synthetic peptides and anthocyanins in inhibiting DPP-Ⅳ activity

[0069] 3.1 Determination of DPP-Ⅳ inhibitory activity of synthetic peptide-anthocyanin complex

[0070] The method for determining the DPP-Ⅳ inhibitory activity of synthetic peptide-anthocyanin complex is the same as 1.3.

[0071] 3.2 Synergistic effects of synthetic peptides and anthocyanins

[0072] The constant ratio combination design method in the Compusyn synergistic calculation model was used to input the DPP-Ⅳ inhibitory activity test results of synthetic peptides, anthocyanins and synthetic peptide-anthocyanin complexes (synthetic peptide: anthocyanin = 6:1, c / c) into the software respectively. The synergistic effect of oyster peptides and anthocyanins was evaluated by calculating the generated dose-effect graphs, dose-effect reduction graphs, combination index graphs and contour graphs.

[0073] 4. Optimization of the concentration ratio conditions for preparing the synergistic assembly complex of oyster peptides and anthocyanins

[0074] The difference from the above-mentioned second step of preparing a synergistic assembly complex of oyster peptides and anthocyanins with enhanced DPP-Ⅳ inhibitory activity is that the sample solution is prepared with ultrapure water and the pH is adjusted to 5.0, and the concentration ratios of synthetic peptides and anthocyanins are 1:6, 1:3, 1:1, 3:1, 6:1 and 9:1 respectively; the other steps are the same. The optimal complex is screened based on the synergistic strength of the prepared complex combined with its DPP-Ⅳ inhibitory activity.

[0075] 5. Optimization of pH conditions for the preparation of oyster peptide and anthocyanin synergistic assembly complexes

[0076] The difference from the above two, preparation of oyster peptide and anthocyanin synergistic assembly complex with enhanced DPP-Ⅳ inhibitory activity, is that the pH of the sample solution is adjusted to 3.0, 4.0, 5.0, 6.0 and 7.0 respectively with ultrapure water; the other steps are the same. The optimal complex is screened based on the strength of the DPP-Ⅳ inhibitory activity of the prepared complex combined with its particle size and potential size.

[0077] 6. Optimization of time conditions for preparing oyster peptide and anthocyanin synergistic assembly complexes

[0078] The difference from the above two, preparation of oyster peptide and anthocyanin synergistic assembly complex with enhanced DPP-Ⅳ inhibitory activity, is that the sample solution is prepared with ultrapure water and the pH is adjusted to 5.0, and the reaction time is 0.5, 1.0, 2.0, 3.0 and 5.0h respectively; the other steps are the same. The optimal complex is screened based on the strength of the DPP-Ⅳ inhibitory activity of the prepared complex combined with its particle size and potential size.

[0079] 7. Optimization of temperature conditions for the preparation of oyster peptide and anthocyanin synergistic assembly complexes

[0080] The difference from the above two, preparation of oyster peptide and anthocyanin synergistic assembly complex with enhanced DPP-Ⅳ inhibitory activity, is that the sample solution is prepared with ultrapure water and the pH is adjusted to 5.0, and the reaction temperature is set to 25, 30, 37, 45, and 50°C respectively; the other steps are the same. The optimal complex is screened based on the strength of the DPP-Ⅳ inhibitory activity of the prepared complex combined with its particle size and potential size.

[0081] 8. Analysis of the synergistic effect of oyster peptide-anthocyanin complexes under optimal preparation conditions

[0082] Same as method 3.2.

[0083] IX. Statistical Analysis

[0084] All experiments were repeated 3 times, and the experimental results were expressed as mean ± standard deviation. SPSS27.0 software was used to process the experimental data and perform one-way ANOVA. Duncan analysis was used for multiple comparisons, and P < 0.05 indicated significant differences between groups. Origin 2021, GraphPad Prism 9 and other software were used to draw images.

[0085] 10. Results and Analysis

[0086] 10.1 Structural Characterization of Oyster Peptides

[0087] 10.1.1 Relative molecular weight distribution of oyster peptides

[0088] After the oyster peptide prepared by alkaline protease hydrolysis was eluted through the chromatographic column, four chromatographic peaks were separated and the molecular weight distribution was calculated by peak area normalization method. The results are as follows: Figure 1 As shown in the figure. The molecular weight of oyster peptides is mainly concentrated below 3kDa, accounting for 55.44% of the total. The molecular weight of active peptides is closely related to their DPP-Ⅳ inhibitory activity, and active peptides with lower molecular weight tend to have stronger DPP-Ⅳ inhibitory activity.

[0089] 10.1.2 Analysis of Amino Acid Composition of Oyster Peptides

[0090] The DPP-IV inhibitory activity of marine bioactive peptides is closely related to their structure, such as amino acid composition, molecular weight, peptide sequence, etc. As shown in Table 1, the oyster peptides prepared by the present invention are mainly composed of glutamic acid, aspartic acid, threonine, histidine, glycine and lysine, accounting for 55.88% of the total amino acids, and the essential amino acid content of oyster peptides is relatively rich (35.30%). Hydrophobic amino acids, especially branched-chain amino acids (BCAAs, including valine, leucine and isoleucine) and proline are important composition characteristics of DPP-IV inhibitory peptides. The hydrophobic amino acid content of the oyster peptides prepared in this study is 27.39%, of which branched-chain amino acids and proline account for a high proportion, accounting for 60.84% ​​of the total hydrophobic amino acids, indicating that the prepared oyster peptides have the amino acid composition characteristics of DPP-IV inhibitory peptides.

[0091] Table 1 Amino acid composition of oyster peptides

[0092]

[0093] Note: TAA (total amino acids); EAA (essential amino acids); NEAA (non-essential amino acids); HAA (hydrophobic amino acids); BCAAs (branched-chain amino acids).

[0094] 10.1.3 Peptide sequence analysis of oyster peptides

[0095] The peptide spectrum sequence of oyster peptides was analyzed by LC-MS / MS technology, and a total of 100 peptide chains were detected. First, the oyster peptides were evaluated by literature comparison to see if they had typical DPP-IV inhibitory peptide characteristics. DPP-IV inhibitory peptides have the characteristics of high hydrophobic amino acid content and relatively short peptide chains (generally less than 15 amino acid residues), and when proline is located in the first four positions of the N-terminus of the peptide, the active peptide has a higher DPP-IV inhibitory activity. In addition, peptides rich in BCAAs in the peptide sequence structure or BCAAs located in specific positions of the peptide sequence have the potential to become DPP-IV inhibitory peptides, such as BCAAs located in the first three positions of the N-terminus of the peptide segment, showing significant DPP-IV inhibitory activity. In this study, a total of 42 peptides met the structural characteristics of DPP-IV inhibitory peptides, of which the main 17 peptides are shown in Table 2. These peptides are hydrophobic, rich in branched-chain amino acids and proline, and are mainly located in the first four positions of the N-terminus of the peptide chain. They have significant DPP-IV inhibitory peptide structural characteristics, indicating that they have potential DPP-IV inhibitory activity.

[0096] GLP-1 can regulate the body's blood sugar balance. DPP-IV inhibitory peptides can inhibit DPP-IV activity, slow down the decomposition of DPP-IV on GLP-1, and help regulate blood sugar balance. Among them, the peptides with Pro or Ala at the second position of the N-terminus are more likely to inhibit DPP-IV activity through competitive inhibition and play a hypoglycemic effect. In addition, considering that anthocyanins are highly hydrophilic, and the key forces for the interaction between active peptides and anthocyanins are generally hydrophobic forces and hydrogen bonds, the active peptides selected for assembly with anthocyanins should have both certain hydrophilicity and hydrophobicity. Based on the characteristic structure of DPP-IV inhibitory peptides and the conditions for the interaction between active peptides and anthocyanins, 5 peptides (FPVGR, APPNIT, IAPPERK, LDFYLDIP, LAIL) were comprehensively screened from the 17 DPP-IV inhibitory peptide sequences in Table 2 for synthesis and subsequent experiments.

[0097] Table 2 Main DPP-Ⅳ inhibitory peptide sequences of oyster peptides

[0098]

[0099] Note: [+42.011]: Acetyl (Protein NTerm) acetylation modification occurs at the nitrogen terminus of the protein, and the relative molecular mass changes to +42.011Da.

[0100] 10.2 Analysis of DPP-IV inhibitory activity of oyster peptides

[0101] DPP-IV can promote the rapid degradation of GLP-1. By inhibiting the activity of DPP-IV, GLP-1 inactivation can be prevented, thereby promoting the production of insulin and achieving the purpose of lowering blood sugar. Figure 2 As shown in Figure 2, oyster peptide has good DPP-Ⅳ inhibitory activity, and its half inhibitory concentration (IC 50 ) was 0.60±0.06mg / mL, and its inhibition on DPP-Ⅳ activity showed a dose-dependent trend, and the inhibitory effect increased with the increase of oyster peptide mass concentration.

[0102] 10.3 Analysis of DPP-Ⅳ inhibitory activity of synthetic peptides and anthocyanins

[0103] Depend on Figure 3 It can be seen that the peptides FPVGR (FR-5), APPNIT (AT-6), IAPPERK (IK-7), LDFYLDIP (LP-8), and LAIL (LL-4) screened and synthesized based on the peptide sequence analysis results all have good DPP-Ⅳ inhibitory activity, and their IC 50The DPP-IV inhibitory activity of active peptides was 0.06-2.15 mg / mL. Aromatic amino acids (including Trp, Tyr, Phe) and proline (Pro) made important contributions to the DPP-IV inhibitory activity of active peptides, especially when aromatic amino acids and Pro were located at positions 1 to 4 of the N-terminus of the peptide chain, the DPP-IV inhibitory activity of active peptides was stronger. Among the 5 synthetic peptides, FR-5 had the strongest DPP-IV inhibitory activity (P<0.05), and its IC 50 The value was 0.06357±0.003mg / mL, which may be due to the fact that Phe and Pro are located at the 1st and 2nd positions of the N-terminus of the peptide chain, respectively. The active peptides with Pro or Ala at the second position of the N-terminus are more likely to interact with DPP-IV and inhibit DPP-IV activity, but the DPP-IV inhibitory activity of LP-8 is significantly higher than that of AT-6, IK-7 and LL-4. This may be due to the fact that the 3rd and 4th positions of the N-terminus of the LP-8 peptide chain are both aromatic amino acids, while there are no aromatic amino acids in the amino acid composition of the other three peptide chains. Since FR-5 has the strongest DPP-IV inhibitory activity, FR-5 was selected for subsequent synergistic assembly with anthocyanins.

[0104] like Figure 4 As shown in Figure 3, the IC values ​​of six common anthocyanins: peonydin-3-O-glucoside (Pn3G), cyanidin-3-O-glucoside (C3G), malvidin-3-O-glucoside (Mv3G), delphinidin-3-O-glucoside (D3G), pelargonidin-3-O-glucoside (Pg3G) and petunidin-3-O-glucoside (Pt3G) for DPP-Ⅳ. 50 The concentrations of anthocyanins in the anthocyanins were 6.99-23.62 μg / mL, and all of them had strong DPP-Ⅳ inhibitory activity. The order of DPP-Ⅳ inhibitory activity of the six anthocyanins was Pn3G = C3G = Mv3G > Pg3G > D3G > Pt3G. This difference may be due to the different binding abilities of different groups at the 3' and 5' positions on the B ring of anthocyanins with DPP-Ⅳ. In this study, the three anthocyanins with the best DPP-Ⅳ inhibitory activity (Pn3G, C3G, Mv3G) were selected for subsequent synergistic inhibition of DPP-Ⅳ experiments with the synthetic peptide FR-5.

[0105] 10.4 Analysis of DPP-Ⅳ inhibitory activity of synthetic peptide FR-5-anthocyanin complex

[0106] In order to explore whether the synthetic peptide FR-5 and anthocyanins have a synergistic effect on DPP-Ⅳ inhibitory activity, the DPP-Ⅳ inhibitory activities of FR-Pn3G, FR-C3G and FR-Mv3G complexes were measured respectively. Figure 5 It can be seen that the three synergistic complexes of FR-Pn3G, FR-C3G and FR-Mv3G have strong inhibitory effects on DPP-Ⅳ, and their IC 50The values ​​were 26.70±0.26μg / mL, 30.89±0.81μg / mL and 28.68±0.87μg / mL, respectively, which were significantly lower than the IC of FR-5 alone. 50 value (63.57±2.73μg / mL), but higher than the IC value of anthocyanins alone 50 However, the complex is composed of FR-5 and anthocyanin in a ratio of 6:1 (c / c). Although the complex inhibits DPP-Ⅳ with an IC 50 The value was larger than that of anthocyanin alone, but the amount of anthocyanin in the complex was less than that of anthocyanin alone. In addition, among the three synthetic peptide-anthocyanin complexes, FR-Pn3G had the strongest DPP-Ⅳ inhibitory activity (P<0.05), which may be due to the fact that the structure of FR-Pn3G complex is more likely to interact with DPP-Ⅳ and inhibit its activity compared with FR-C3G and FR-Mv3G complexes.

[0107] 10.5 Analysis of the synergistic effect of synthetic peptide FR-5 and anthocyanins

[0108] In order to further explore the synergistic effect of synthetic peptides and anthocyanins, the Compusyn synergistic computational model was used to study the results. Figure 7 , Figure 8 and Fig. 9 As shown in the dose-effect Figure 6 Middle a, Figure 7 Middle a, Figure 8 As shown in Figure a, FR-5, Pn3G, C3G, Mv3G and FR-Pn3G / C3G / Mv3G complex have a good dose-effect relationship in inhibiting DPP-Ⅳ activity, and Pn3G, C3G, and Mv3G have stronger inhibitory effects on DPP-Ⅳ than FR-5, indicating that the synergistic activity of FR-5 and anthocyanins mainly depends on anthocyanins. The combination index (CI) <1, CI = 1, and CI > 1 represent synergistic, additive, and antagonistic effects, respectively. Figure 6 Middle b, Figure 7 Middle b, Figure 8 As shown in (b), the combined inhibitory effect of FR-5 and Pn3G / C3G / Mv3G on DPP-Ⅳ is concentration-dependent, and the synergistic effect of FR-5 and the three anthocyanins is similar. After Fa≥0.45, Fa≥0.5 and Fa≥0.35, the corresponding CI<1, indicating that FR-5 and Pn3G / C3G / Mv3G have a synergistic inhibitory effect on DPP-Ⅳ. Figure 6 Middle c, Figure 7 Middle c, Figure 8As shown in c, the dose reduction index (DRI) of FR-5 and Pn3G / C3G / Mv3G is greater than 1, and increases with the increase of the inhibitory effect (Fa), indicating that the combined use can significantly reduce the dosage of FR-5 and anthocyanins. The contour map refers to the equivalent dose map of the combination of two drugs. The combination data points falling on the contour line, the lower left, and the upper right represent additive effects, synergistic effects, and antagonistic effects, respectively. The horizontal and vertical axes represent the doses required for the inhibition rates of FR-5 and anthocyanins to reach 90% (Fa=0.9, green line), 75% (Fa=0.75, red line), and 50% (Fa=0.5, blue line), respectively. Figure 6 Middle d, Figure 7 Medium D and Figure 8 As can be seen in figure d, when FR-5 and Pn3G / C3G / Mv3G were added simultaneously, the points at which the inhibition rates were 90%, 75% and 50% were at the lower left of the corresponding contour lines, indicating that there was a significant synergistic inhibitory effect of DPP-Ⅳ between FR-5 and Pn3G / C3G / Mv3G.

[0109] Based on the DPP-IV inhibitory activity results and synergistic effect analysis of FR-5 synergistic with Pn3G / C3G / Mv3G, FR-5 and Pn3G were selected for subsequent condition optimization experiments for synergistic assembly of complexes.

[0110] 10.6 Analysis of the results of optimization of preparation conditions for the synergistic assembly complex of synthetic peptide FR-5 and anthocyanins

[0111] 10.6.1 Analysis of the results of concentration ratio optimization

[0112] Fig. 9 is the joint index diagram of the FR-5 and Pn3G assembly complex at different concentration ratios. Fig. 9 As shown in a, b, and c, when the concentration ratio of FR-5 and Pn3G is 1:6, 1:3, and 1:1, the corresponding CI is <1 after Fa≥0.95, Fa≥0.90, and Fa≥0.85, respectively, indicating that at these three concentration ratios, the assembled complex has the synergistic inhibitory effect of FR-5 and Pn3G at high concentrations of DPP-Ⅳ. Fig. 9 As shown in Figure d, when the concentration ratio of FR-5 and Pn3G is 3:1, when Fa≥0.5, the corresponding CI<1, indicating that at this ratio, FR-5 and Pn3G have a synergistic inhibitory effect on DPP-Ⅳ. Fig. 9 As shown in Figures e and f, when the concentration ratio of FR-5 and Pn3G is 6:1 and 9:1, the CI is less than 1, indicating that at these two concentration ratios, FR-5 and Pn3G can synergistically inhibit DPP-Ⅳ activity. Therefore, when the concentration ratio of FR-5 and Pn3G is 6:1 and 9:1, the two substances have the best synergistic effect in inhibiting DPP-Ⅳ activity.

[0113] To further screen the optimal concentration ratio conditions, the inhibitory activity of the complex assembled at a concentration ratio of FR-5 to Pn3G of 6:1 and 9:1 against DPP-Ⅳ was measured. Fig.10 When the concentration ratio of FR-5 and Pn3G is 6:1, the IC value of the assembled complex for DPP-Ⅳ is 50 The concentration of FR-5 in the complex was 1:1 (13.50±0.87μg / mL), which was significantly lower than that of the assembled complex when the concentration ratio of FR-5 and Pn3G was 9:1 (26.06±0.51μg / mL), indicating that the assembled complex had stronger inhibitory activity against DPP-Ⅳ when the concentration ratio of FR-5 and Pn3G was 6:1. Therefore, the concentration ratio of FR-5:Pn3G=6:1 was selected for subsequent experiments on optimizing the preparation conditions of the complex.

[0114] 10.6.2 Analysis of pH Condition Optimization Results

[0115] When the concentration of Pn3G-FR complex was 0.4 mg / mL under different pH conditions, the inhibitory activity against DPP-Ⅳ was as follows: Fig.11 The inhibition rate of the complex prepared under pH 3 to 7 conditions on DPP-Ⅳ was 49.18% to 70.25%, among which the complex prepared under pH 5 conditions had the strongest DPP-Ⅳ inhibition activity (P<0.05, inhibition rate was 70.25±0.91%).

[0116] In order to study the effect of different pH preparation conditions on the particle size and potential of the Pn3G-FR complex, dynamic light scattering analysis (DLS) was performed on the prepared Pn3G-FR complex. Particles with smaller particle sizes have larger surface areas, which can increase their interactions with other molecules or ions in the solution, thereby improving the stability of the particles in the system. Fig.12 It can be seen that the particle size of the complex prepared under pH 5 and pH 6 conditions is the smallest (P<0.05), which is 197.60±0.44nm and 196.80±1.20nm respectively. Zeta potential can measure the strength of mutual repulsion or attraction between particles and can evaluate the stability of particles in liquids. The larger the absolute value of Zeta potential, the more stable the solution system. The Zeta potential of the complex prepared under pH 5 and pH 7 conditions is the largest (P<0.05), which is 39.37±0.31mV and 38.67±0.98mV respectively. Therefore, the complex prepared under pH 5 conditions has the characteristics of the smallest particle size and the largest potential.

[0117] In summary, the complex prepared under pH 5 conditions has the strongest DPP-IV inhibitory activity and the solution system is relatively stable, so the solution system pH 5 was selected for subsequent complex preparation condition optimization experiments.

[0118] 10.6.3 Analysis of Preparation Time Condition Optimization Results

[0119] When the concentration of Pn3G-FR complexes prepared at different times was 0.4 mg / mL, the inhibitory activity against DPP-Ⅳ was as follows: Fig.13 The inhibition rate of the complexes prepared in 0.5-5.0h on DPP-Ⅳ was 70.07%-71.04%, and they all had good DPP-Ⅳ inhibitory activity, indicating that the preparation time of the complex had no significant effect on the DPP-Ⅳ inhibitory activity of the complex.

[0120] Fig.14 The particle size and potential value of the Pn3G-FR complex prepared at different times. Among them, the particle size of the complex prepared at 2h is the smallest (P<0.05), which is 197.60±0.44nm, followed by the complexes prepared at 1h and 3h, which are 214.55±0.92nm and 213.77±0.87nm, respectively. The Zeta potential value of the complex prepared at 0.5h and 5h is the highest (P<0.05), which is 40.37±0.25mV and 40.60±0.17mV, respectively, followed by the complex prepared at 2h (Zeta potential is 39.37±0.15mV). Therefore, the particle size of the complex prepared at 2h is the smallest, the potential value is relatively high, and the solution system is relatively stable.

[0121] In summary, the complex prepared with a preparation time of 2 h has good DPP-IV inhibitory activity and the solution system is relatively stable. Therefore, a preparation time of 2 h was selected for subsequent optimization experiments on complex preparation conditions.

[0122] 10.6.4 Analysis of Preparation Temperature Condition Optimization Results

[0123] When the concentration of Pn3G-FR complex was 0.4 mg / mL under different temperature preparation conditions, the inhibitory activity against DPP-Ⅳ was as follows: Fig.15 The inhibition rate of the complex prepared at 25-50°C on DPP-Ⅳ was 54.97%-70.25%, among which the complex prepared at 25°C had the strongest DPP-Ⅳ inhibition activity (P<0.05, inhibition rate was 70.25±0.91%). The particle size and potential values ​​of the Pn3G-FR complex prepared at different temperatures are shown in Fig.16 As shown in the figure, the particle size of the complex prepared at 25°C was the smallest and the Zeta potential value was the largest (P<0.05), which were 197.60±0.44nm and 39.37±0.15mV, respectively. Therefore, the complex prepared at 25°C had the strongest DPP-Ⅳ inhibitory activity and the solution system was relatively stable.

[0124] In summary, the optimal preparation conditions for the complex are a concentration ratio of synthetic peptide FR-5 to anthocyanin of 6:1, pH 5, time 2 h and temperature 25 °C.

[0125] 10.7 Analysis of the synergistic effect of peptide-anthocyanin complexes synthesized under optimal preparation conditions

[0126] Depend on Fig.17 As shown in Figure a, the FR-5-Pn3G complex prepared under the optimal preparation conditions has a concentration-dependent inhibition on DPP-Ⅳ, and the inhibition rate on DPP-Ⅳ gradually increases with the increase of concentration. Fig.17 In b, the CI values ​​were all less than 1, indicating that FR-5 and Pn3G could synergistically inhibit DPP-Ⅳ activity. Fig.17 Figure c shows that the dose reduction index of FR-5 and Pn3G is greater than 1, indicating that at the same level of effect, the combined use of FR-5 and Pn3G can significantly reduce the dose of both. Fig.17 As shown in Figure d, when FR-5 and Pn3G are used simultaneously, the data point of Fa=0.5 / 0.75 / 0.9 is at the lower left of the corresponding contour line, further indicating that FR-5 and Pn3G have a synergistic inhibitory effect on DPP-Ⅳ activity.

[0127] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a synergistic assembly complex of oyster peptides and anthocyanins, It is characterized in that The method comprises the steps of mixing oyster DPP-IV inhibitory peptide and anthocyanin to react; The amino acid sequence of the oyster DPP-IV inhibitory peptide is FPVGR; The anthocyanin is peony pigment-3-O-glucoside; The reaction conditions are pH 5, reaction time is 2h, and reaction temperature is 25°C; The mixed concentration ratio of the oyster DPP-IV inhibitory peptide and the anthocyanin is 6:

1.

2. A synergistic assembly complex of oyster peptides and anthocyanins, It is characterized in that The composite is prepared by the preparation method according to claim 1.

3. Use of the complex as claimed in claim 2 in the preparation of a drug for preventing and / or treating type II diabetes.

4. A drug for preventing and treating type II diabetes, It is characterized in that Comprising the complex according to claim 2.

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