Schiff base mediated peptide self-assembly as well as preparation method and application thereof

Abstract

The invention belongs to the field of bioactive peptides, and relates to a Schiff base mediated peptide self-assembly body as well as a preparation method and application thereof, in particular to a supramolecular self-assembly body of an egg white source bioactive peptide with an amino acid sequence of Gln-Ile-Gly-Leu-Phe (QIGLF) and application of the supramolecular self-assembly body. The self-assembly body is assembled by a group of mixed construction modules (2QIGLF-3GA) which are generated after the QIGLF and glutaraldehyde (GA) are subjected to Schiff base reaction, and the molecular formula of the mixed construction modules (2QIGLF-3GA) is C71H104N12O16). The peptide self-assembly provided by the invention can be effectively combined with a hydrophobic substance curcumin, and has excellent encapsulation capability (gt; the peptide-based supramolecular biomaterial has the advantages that the antioxidant capacity of curcumin and the transport level of curcumin on a Caco-2 cell monolayer membrane can be remarkably improved, meanwhile, the peptide-based supramolecular biomaterial is safe, free of toxic and side effects and the like, can be used as a high-load-rate delivery carrier of hydrophobic food and medicine components, and has important significance in the field of peptide-based supramolecular biomaterials.

Description

Technical Field

[0001] The invention belongs to the field of bioactive peptides, and specifically relates to a supramolecular self-assembly of an egg white-derived active peptide with an amino acid sequence of Gln-Ile-Gly-Leu-Phe (QIGLF), a preparation method and an application thereof. Background Art

[0002] Inspired by the beauty of natural protein and peptide structure harmony and function, researchers pursue the supreme wisdom of nature to simulate and manufacture complex and highly ordered superstructures, thereby producing new biomaterials with multiple functions. Peptides released after the degradation of food proteins are not only the basic materials for building and repairing organisms, but also undertake the tasks of human growth and the repair and renewal of damaged cells. They also provide an important treasure for obtaining self-assembling peptide building blocks to organize higher-order structures. Although some food-derived peptides with clear structural characteristics can self-assemble to form diverse fine structures based on non-covalent interactions, self-assembly with the help of covalent chemistry is an important way to help food-derived peptides that lack secondary structures (such as β-folds), intrinsic disorder or amphiphilic properties develop superstructures with subtle architectures, dynamic interactions and multifunctionality to perform biological functions. At present, a series of dynamic reactions with reversible covalent bonding characteristics have been successfully used to design functional materials, bottom-up assembly processes and molecular machines.

[0003] Schiff base bonds (imine bonds - N = CH-) are dynamic covalent bonds formed between aldehyde (or ketone) groups and amino groups, which can synergistically regulate the structure and function of the assembly with non-covalent interactions. This bond gives the assembly autofluorescence properties, which facilitates biological tracking in vivo without the need for external fluorescent dyes. More importantly, the regulation of dynamic covalent interactions gives the assembly unique adaptability, dynamic properties and relatively stable networks in biological systems. This Schiff base-induced dynamic covalent assembly has become a powerful and effective strategy for constructing peptide-based nanocarriers to encapsulate and deliver food and drug ingredients. It also provides a new idea to solve the problem of limited bioavailability of hydrophobic components due to extremely low water solubility.

[0004] However, the practical application of supramolecular nano-delivery carriers still faces many challenges, especially in the encapsulation of hydrophobic compounds. A significant problem is that it is difficult to effectively improve the encapsulation capacity, and the encapsulation rate is usually less than 10%. In addition, the efficiency of the assembly penetrating intestinal epithelial cells into the blood circulation is extremely low, which significantly limits the number of loaded effective ingredients reaching the target site and its subsequent ability to exert physiological regulatory functions. Therefore, there is an urgent need to construct high-quality nano-carriers with excellent encapsulation ability and effective overcoming of the intestinal absorption barrier. Based on the dynamic covalent bond of Schiff base to give food-borne bioactive peptides supramolecular structure, it is of great significance and prospect to design hydrophobic delivery carriers with efficient drug encapsulation ability, high biocompatibility and effective penetration of the intestinal epithelial absorption barrier.

[0005] The present invention aims to provide a Schiff base dynamic covalent bond-mediated peptide-based supramolecular self-assembly, which can be used to effectively encapsulate hydrophobic curcumin and improve its bioaccessibility. Summary of the invention

[0006] The present invention discloses a peptide-based supramolecular self-assembly (QIGLF-GA SA) which is assembled from a group of mixed building blocks based on the amino acid sequence of Gln-Ile-Gly-Leu-Phe and mediated by a Schiff base dynamic covalent bond. The self-assembly can be used for the efficient encapsulation, antioxidant activity and bioaccessibility gain of the hydrophobic component curcumin (Cur).

[0007] The technical solution of the present invention is:

[0008] Step 1: Construction and identification of peptide self-assemblies

[0009] 1.0-4.0 mg / mL of QIGLF was dissolved in ultrapure water, and 1.0-3.0% (v / v) glutaraldehyde (GA, 50%) solution was added under continuous magnetic stirring. The pH of the system was adjusted to 5.0-7.0, and the reaction was carried out in a water bath at 30-45°C for 18-36 hours. After the reaction was completed, the system was dialyzed through a 3.5 kDa dialysis bag for 24-36 hours, and the dialysis fluid was water. During this period, the dialysis fluid was replaced twice at equal intervals to remove GA that did not participate in the reaction. Thereafter, the reaction system was freeze-dried to obtain the peptide self-assembly reported in the present invention. Thereafter, the self-assembly was measured by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) using water as solvent and α-cyano-4-hydroxycinnamic acid (CHCA) as matrix, and the molecular weight range of the test was 1000-10000Da, and positive ion detection was performed.

[0010] The reagent for adjusting the pH of the solution is preferably NaOH, more preferably 1.0M NaOH; the water is preferably deionized water; the reaction temperature is preferably 37°C, and the reaction time is preferably 24h. The mixed building block is identified by MALDI-TOF-MS method, including 2QIGLF-3GA (molecular formula: C 71 H 104 N 12 O 16 ), 2QIGLF-2GA, 2QIGLF-4GA, and 2QIGLF-5GA, the content ratio of the four is approximately 11:3:6:2.

[0011] Step 2: Centrifugation method to determine the encapsulation capacity of the pH-driven self-assembly for hydrophobic curcumin

[0012] The aqueous solution of the self-assembly (0.25-1.0 mg / mL) was mixed with an equal volume of curcumin solution (0.1 mg / mL, pre-dissolved in water at pH = 12.0), and then the pH of the mixture was adjusted to 12.0 with NaOH solution, and stirred continuously. Subsequently, the pH of the system was quickly adjusted to 7.0 with HCl solution to obtain a supramolecular co-assembly loaded with curcumin. After that, the sample was centrifuged to remove the unencapsulated curcumin, and a standard curve (R 2 >0.999) method was used to quantify the concentration of curcumin in the supernatant, and the encapsulation capacity (EC) of the self-assembly for curcumin was calculated according to formula (1).

[0013]

[0014] The NaOH used to adjust the pH is preferably 6M, and the HCl is preferably 1M; the centrifugation condition of the sample is 8000-12000 rpm / min, 10 min, preferably 10000 rpm / min, 10 min.

[0015] Step 3: DPPH and ABTS determination of antioxidant activity gain

[0016] (1) DPPH free radical scavenging activity: First, prepare a 0.2 mM DPPH solution with 75% ethanol and dilute it appropriately so that its ultraviolet absorption at 517 nm is 0.8 ± 0.02. After the sample to be tested is prepared, react in the dark for 1.5 to 2 hours. After the reaction is completed, the sample is centrifuged to obtain the supernatant. Thereafter, the sample (curcumin concentration 10 ug / ml) is mixed with the DPPH solution in a volume ratio of 1:2 and incubated in the dark for 0.5 hours. DPPH is mixed with pure water as a control. After the reaction is completed, the absorbance value of the sample at 517 nm is measured using a UV spectrophotometer. The calculation formula for DPPH free radical scavenging activity is as follows:

[0017]

[0018] In the formula, A C and A S are the absorbance values ​​of the control group and the experimental group, respectively.

[0019] The centrifugal conditions are 4°C, 9000-12000 r / min, 5-15 min, preferably 10000 r / min, 10 min.

[0020] (2) ABTS free radical scavenging activity: ABTS reagent with a concentration of 7 mM and potassium persulfate with a volume ratio of 1:1 were mixed, incubated in the dark at room temperature for 12 to 16 h, and diluted with PBS buffer before use to obtain an absorbance of 0.70 ± 0.02 at 734 nm to obtain ABTS working solution. 20 μl of sample (curcumin concentration 10 μg / mL) was added to each well of a 96-well plate, followed by 180 μl of ABTS working solution. After shaking for 10 s, the mixture was allowed to stand at room temperature for 5 to 10 min, and the absorbance of the reaction system at 734 nm was measured using an enzyme-labeled instrument. The formula for calculating ABTS free radical scavenging activity is:

[0021]

[0022] In the formula, A C , A S and A B The absorbance values ​​of the control group, experimental group and blank group are shown in Figure 2.

[0023] Step 4: MTS assay to determine cytotoxicity and hemolysis assay to evaluate biocompatibility

[0024] (1) MTS method for cytotoxicity determination: Caco-2 or L02 cells were inoculated in a 96-well plate (90 μL), cultured for 12 to 16 hours, and then 10 μL of sample was added to each well for further incubation for 24 to 36 hours. 20 μL MTS was added and treated for 2 to 4 hours. The absorbance at 490 nm was recorded using an ELISA reader. The blank group used 90 μL DMEM instead of the cell suspension, and the control group used 10 μL DMEM instead of the sample. Cell viability was calculated according to formula (4):

[0025]

[0026] Among them, A S , A B and A C Represent the absorbance values ​​of the sample group, blank group and control group, respectively.

[0027] (2) Hemolysis experiment: Fresh sterile defibrotized rabbit blood was washed several times with physiological saline and diluted to obtain a 4% red blood cell suspension. An appropriate amount of sample was mixed with an equal volume of red blood cell suspension and shaken at 37°C for 3 to 6 hours. Ultrapure water and physiological saline were used as positive and negative control groups, respectively. After incubation, the samples were centrifuged. The absorbance of the supernatant at 540 nm was detected using an ELISA instrument, and the hemolysis rate was calculated using formula (5):

[0028]

[0029] Among them, A S , A B and A C Represent the absorbance values ​​of the sample, negative control group and positive control group, respectively.

[0030] The sample centrifugation condition is 2000-4000 r / min, 10 min, preferably 3000 r / min, 10 min.

[0031] Step 5: Caco-2 Cell Monolayer Transport Assessment Experiment Bioaccessibility Gain

[0032] Caco-2 cells were seeded in 12-well Transwell plates and cultured for more than 21 days until the transmembrane electrical resistance (TEER) value reached 400-600 Ω / cm 2 Different samples with the same curcumin content were added to the top of the monolayer membrane, incubated for 2 to 3 hours, and then samples were taken from the other side. After the curcumin content in the sample was quantified using an ELISA instrument, the apparent permeability coefficient (P app ).

[0033]

[0034] Where Q is the total amount of sample permeated (μg), A is the membrane area (cm 2 ), C is the initial concentration of the donor zone sample (μg / mL), and t is the total time of the experiment (s).

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention is based on the self-assembly of food-derived peptides mediated by Schiff base dynamic covalent bonds, and the reported self-assembly has high biocompatibility.

[0037] (2) The present invention significantly improves the encapsulation capacity of curcumin (>22%), which is much higher than the current actual level of nanocarrier loading rate generally less than 10%.

[0038] (3) The present invention significantly increases the antioxidant activity of curcumin. After the self-assembly encapsulated curcumin, the DPPH and ABTS free radical scavenging activities can reach 35.4% and 19.4%, respectively, which are approximately 11.8 times and 2.6 times that of free curcumin under the same experimental conditions.

[0039] (4) After the self-assembly encapsulated 100 μg / mL of curcumin (corresponding to a self-assembly concentration of 250 μg / mL), the results of the cytotoxicity experiment were: Caco-2 cell survival rate was 83.4% to 88.2%, L02 cell survival rate was 89.2% to 91.5%; the hemolysis rate was 0.414% to 0.307%, which confirmed the high biocompatibility of the self-assembly.

[0040] (5) The self-assembly can significantly improve the bioaccessibility of curcumin. After encapsulating curcumin in the self-assembly, the apparent permeability coefficient of curcumin on the Caco-2 cell monolayer membrane can be increased. aap The value increased to 3.996×10 -7 ~4.223×10 -7 cm / s, which is 11.23 to 12.68 times that of free curcumin under the same experimental conditions.

[0041] The supramolecular self-assembly reported in the present invention has important practical significance and broad application prospects in the encapsulation and delivery of hydrophobic components. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present invention is attached Figure 5 width, of which:

[0043] Figure 1 Identification of chemical composition of self-assemblies (MALDI-TOF-MS);

[0044] Figure 2 The encapsulation ability of curcumin by self-assemblies;

[0045] Figure 3 The self-assembly gained activity on scavenging DPPH and ABTS free radicals of curcumin;

[0046] Figure 4 Evaluation of biocompatibility of self-assemblies;

[0047] Figure 5 Gained bioaccessibility of curcumin by self-assembly. DETAILED DESCRIPTION

[0048] The present invention is further described below by way of specific embodiments.

[0049] Example 1. Construction and identification of peptide self-assemblies

[0050] QIGLF was dissolved in ultrapure water at a concentration of 2.0 mg / mL, and 2.5% (v / v) GA solution (50%) was added. The pH of the system was adjusted to 7.0 with 1M NaOH, and the reaction was carried out in 37°C water for 24 hours. After the reaction was completed, the system was dialyzed through a 3.5 kDa dialysis bag for 36 hours, and the dialysis fluid was water. The dialysis fluid was replaced twice during the reaction. Thereafter, the reaction system was freeze-dried to obtain the peptide self-assembly reported in the present invention. Thereafter, the self-assembly was measured by MALDI-TOF-MS using water as solvent and CHCA as matrix. The molecular weight range of the test was 1000-10000 Da, and positive ion detection was performed. The results are shown in FIG. Figure 1 As shown, it is demonstrated that the reported self-assembly is assembled via a set of mixed basic building blocks dominated by 2QIGLF-3GA.

[0051] Example 2. Determination of the encapsulation ability of the self-assembly for hydrophobic curcumin

[0052] The prepared self-assembly aqueous solution (0.25 mg / mL) was mixed with an equal volume of curcumin solution (0.1 mg / mL, pre-dissolved in water at pH = 12.0), and then the pH of the mixture was adjusted to 12.0 with 6M NaOH, and stirred continuously. Subsequently, the pH of the solution was quickly reduced to 7.0 with 1M HCl to obtain a supramolecular co-assembly loaded with curcumin. The encapsulation capacity of the self-assembly for curcumin was established by a UV-visible spectrophotometer (UV-2250, Shimadzu, Japan) at 425 nm to establish a standard curve (R 2 >0.999) for quantitative determination. Specifically, the freshly prepared samples were centrifuged at 10000rpm / min for 10min at 4°C to remove any unreacted or unencapsulated curcumin. Then, 0.5mL of the supernatant was diluted with 3.5mL of 75% ethanol and mixed evenly for determination. The encapsulation capacity of the self-assembly for curcumin was calculated according to the following formula.

[0053]

[0054] Figure 2 The results shown show that after the self-assembly is formed at physiological pH, its encapsulation capacity for curcumin is as high as more than 22%, which is about 23 times that of the original peptide chain QIGLF. The reported self-assembly has extremely excellent curcumin encapsulation capacity.

[0055] Example 3. Evaluation of antioxidant activity

[0056] The pre-dissolved DPPH solution (0.2 mM, 75% ethanol) was diluted to a UV absorbance of 0.800 at 517 nm. Subsequently, the sample was centrifuged at 4°C, 10000 r / min for 10 min after standing for 2 h, and the supernatant (Cur 10 μg / mL) was mixed with the DPPH solution at a volume ratio of 1:2 and incubated in the dark for 30 min. DPPH was mixed with pure water as a control. The DPPH free radical scavenging activity was calculated according to the following formula:

[0057]

[0058] In the formula, A C and A S are the absorbance values ​​of the control group and the experimental group, respectively.

[0059] ABTS free radicals were prepared by stirring a mixture of 7.0 mM ABTS aqueous solution and 2.45 mM potassium persulfate in the dark at room temperature for 12 h. Then, the ABTS working solution was prepared by diluting the mixture with PBS (0.2 M, pH 7.4) until the absorbance at 734 nm reached 0.700. Next, 180 μL of the working solution was added to the wells in a 96-well microplate, and 20 μL of the sample (Cur 10 μg / mL) was added, incubated in the dark for 5 min, and then the absorbance at 734 nm was measured using a microplate reader (Spark TECAN, Switzerland). The ABTS free radical scavenging activity was calculated using the following formula:

[0060]

[0061] In the formula, A C , A S and A B The absorbance values ​​of the control group, experimental group and blank group are shown in Figure 2.

[0062] Figure 3 The results shown indicate that the DPPH and ABTS free radical scavenging activities of curcumin encapsulated in the self-assembly can reach more than 35.4% and 19.4%, respectively, which are approximately 11.8 times and 2.6 times that of free curcumin under the same experimental conditions.

[0063] Example 4. Biocompatibility evaluation

[0064] Hemolysis analysis: Fresh sterile defibrotized rabbit blood was washed several times with saline and diluted to obtain a 4% red blood cell suspension. Based on the highest curcumin concentration of 100 μg / mL, the sample was mixed with an equal volume of red blood cell suspension and shaken at 37°C for 3 h. Deionized water and saline were used as positive and negative control groups, respectively. After incubation, the samples were centrifuged at 3000 r / min for 10 min. The absorbance of the supernatant at 540 nm was detected using a multi-mode microplate reader (Spark, TECAN, Switzerland), and the hemolysis rate was calculated using the following formula:

[0065]

[0066] Among them, A S , A B and A C Represent the absorbance values ​​of the sample, negative control group and positive control group, respectively.

[0067] Cytotoxicity analysis: Caco-2 (human colon cancer cells) and L02 (human liver cells) were inoculated in a 96-well plate (90 μL) and cultured for 12 hours, and 10 μL of sample was added to each well. After incubation for 24 hours, 20 μL of MTS was added for treatment for 2 hours. The absorbance value at 490 nm was recorded using a multi-mode microplate reader (Spark, TECAN, Switzerland). The blank group used 90 μL DMEM instead of the cell suspension, and the control group used 10 μL DMEM instead of the sample. Cell viability was calculated according to the following formula:

[0068]

[0069] Among them, A S , A B and A C Represent the absorbance values ​​of the sample group, blank group and control group, respectively.

[0070] Figure 4 The results of cytotoxicity and hemolysis rate fully confirmed the high biocompatibility of the assembly and its co-assembly with curcumin. Even when the content of curcumin in the sample reached 100 μg / mL, there was still no obvious cytotoxicity and hemolysis (cell viability > 85%, hemolysis rate < 1%). This provides a solid safety guarantee for the application of the reported self-assembly as a delivery carrier in vivo.

[0071] Example 5. Bioaccessibility Gain Assessment

[0072] Caco-2 cells were seeded in 12-well Transwell plates (pore size 0.4 μm, growth surface area 1.12 cm 2, Corning Incorporated) and cultured for 21 days to form a monolayer. The integrity of the monolayer was checked by measuring the transepithelial electrical resistance (TEER) value using an EVOM epithelial voltammeter / resistance meter (Millicell-ERS, Millipore, USA). Only cells with a TEER value above 600 Ω / cm 2 The transport of the cells was studied. Different samples were added to the top of the monolayer with the same curcumin content as the standard, and samples were taken from the other side after incubation for 2 hours. The curcumin content in the sample was quantified using a multi-mode microplate reader (Spark, TECAN, Switzerland, 425nm), and the apparent permeability coefficient was calculated according to the following formula.

[0073]

[0074] Where Q is the total amount of sample permeated (μg), A is the membrane area (cm 2 ), C is the initial concentration of the donor zone sample (μg / mL), and t is the total time of the experiment (s).

[0075] Figure 5 The experimental results showed that free curcumin crosses the Caco-2 cell monolayer membrane app The value is only 3.4×10 - 8 cm / s, and the mixture of pure peptide chain QIGLF and curcumin is only 5.0×10 -8 cm / s. By comparison, it was found that the self-assembly encapsulated curcumin could significantly improve the transport level of curcumin on the Caco-2 monolayer cell membrane. app Reached 4.1×10 -7 cm / s, which are 12 and 8.2 times of free curcumin and the mixture of QIGLF and curcumin, respectively, fully verifying the bioaccessibility gain of curcumin by the self-assembly.

[0076] Project support:

[0077] This invention is funded by the “Jilin Province Science and Technology Development Plan-Key Technology Research and Product Development for Composite Functional Egg White Peptide Manufacturing” (20240303044NC).

Claims

1. A Schiff base-mediated peptide self-assembly, characterized in that: The basic peptide sequence of the self-assembly body is Gln-Ile-Gly-Leu-Phe (QIGLF).

2. The self-assembly according to claim 1, characterized in that The preparation method is as follows: Step 1: Construction and identification of peptide self-assemblies 1.0-4.0 mg / mL of QIGLF was dissolved in ultrapure water, and 1.0-3.0% (v / v) glutaraldehyde (GA, 50%) solution was added under continuous magnetic stirring; the pH of the system was adjusted to 5.0-7.0, and the reaction was carried out in a water bath at 30-45°C for 18-36 hours; after the reaction was completed, the system was dialyzed through a 3.5 kDa dialysis bag for 24-36 hours and then freeze-dried; thereafter, the self-assembled body was measured by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) using water as solvent and α-cyano-4-hydroxycinnamic acid (CHCA) as matrix, and the test molecular weight range was 1000-10000Da, and positive ion detection was used; Step 2: Centrifugation method to determine the encapsulation capacity of the pH-driven self-assembly for hydrophobic curcumin A 0.25-1.0 mg / mL aqueous solution of the self-assembled product was mixed with an equal volume of a 0.1 mg / mL curcumin solution pre-dissolved in water at pH = 12.0, and then the pH of the mixture was adjusted to 12.0 with a NaOH solution and stirred continuously; Subsequently, the pH of the system was quickly adjusted to 7.0 with HCl solution to obtain the curcumin-loaded supramolecular co-assembly; After that, the sample was centrifuged to remove the unencapsulated curcumin, and the concentration of curcumin in the supernatant was quantified by establishing a standard curve at 425 nm using an ultraviolet spectrophotometer, and the encapsulation capacity of the self-assembly for curcumin was calculated according to formula (1); Step 3: Determination of antioxidant activity gain by DPPH and ABTS (1) DPPH free radical scavenging activity: First, prepare a 0.2 mM DPPH solution with 75% ethanol and dilute it appropriately so that its ultraviolet absorbance at 517 nm is 0.8±0.02; after the sample is prepared, react in the dark for 1.5 to 2 h. After the reaction is completed, centrifuge the sample to obtain the supernatant. Then, mix the sample with the DPPH solution in a volume ratio of 1:2 and incubate in the dark for 0.5 h; DPPH is mixed with pure water as a control. After the reaction is completed, use an ultraviolet spectrophotometer to measure the absorbance of the sample at 517 nm. The DPPH free radical scavenging activity is calculated as follows: In the formula, A C and A S are the absorbance values ​​of the control group and the experimental group, respectively; The centrifugation conditions are 4°C, 9000-12000 r / min, 5-15 min, preferably 10000 r / min, 10 min; (2) ABTS free radical scavenging activity: 7 mM ABTS reagent and 2.45 mM potassium persulfate were mixed at a volume ratio of 1:1, incubated in the dark at room temperature for 12 to 16 h, and diluted with PBS buffer before use to obtain an absorbance of 0.70 ± 0.02 at 734 nm to obtain ABTS working solution; 20 μl of sample was added to each well of a 96-well plate, followed by 180 μl of ABTS working solution, and the plate was shaken for 10 s and then allowed to stand at room temperature for 5 to 10 min. The absorbance of the reaction system at 734 nm was measured using an enzyme-labeled instrument; the ABTS free radical scavenging activity was calculated as follows: In the formula, A C , A S and A B They are the absorbance values ​​of the control group, experimental group and blank group respectively; Step 4: MTS assay for cytotoxicity and hemolysis assay for biocompatibility (1) MTS method for cytotoxicity determination: Caco-2 or L02 cells were inoculated in a 96-well plate and cultured for 12 to 16 hours. 10 μL of sample was added to each well and incubated for 24 to 36 hours. 20 μL of MTS was added and treated for 2 to 4 hours. The absorbance at 490 nm was recorded using a microplate reader. 90 μL DMEM was used instead of cell suspension in the blank group, and 10 μL DMEM was used instead of sample in the control group. Cell viability was calculated according to formula (4): Among them, A S , A B and A C Represent the absorbance values ​​of the sample group, blank group and control group, respectively; (2) Hemolysis experiment: Fresh sterile defibrotized rabbit blood was washed with physiological saline several times and diluted to obtain a 4% red blood cell suspension; an appropriate amount of the sample was mixed with an equal volume of the red blood cell suspension and shaken at 37°C for 3 to 6 hours; ultrapure water and physiological saline were used as positive and negative control groups, respectively; after the incubation, the samples were centrifuged at 3000 r / min for 10 minutes; the absorbance of the supernatant at 540 nm was detected using an ELISA instrument, and the hemolysis rate was calculated using formula (5): Among them, A S , A B and A C Represent the absorbance values ​​of the sample, negative control group, and positive control group, respectively; Step 5: Caco-2 Cell Monolayer Transport Assessment Experiment Bioaccessibility Gain Caco-2 cells were seeded in 12-well Transwell plates and cultured for more than 21 days until the transmembrane electrical resistance (TEER) value reached 400-600 Ω / cm 2 Different samples with the same curcumin content were added to the top of the monolayer membrane, incubated for 2 to 3 hours, and then samples were taken from the other side. The curcumin content in the sample was quantified using an ELISA instrument, and the apparent permeability coefficient (P app ): Where Q is the total amount of sample permeated, in μg, and A is the membrane area, in cm 2 , C is the initial concentration of the donor zone sample in μg / mL, and t is the total time of the experiment in s.

3. The self-assembly according to claim 1, characterized in that: The self-assembly is assembled from a set of mixed building blocks mediated by Schiff base reaction.

4. The self-assembly according to claim 1, characterized in that: The hybrid building blocks that constitute the self-assembly are 2QIGLF-3GA as the main component, with the molecular formula: C 71 H 104 N 12 O 16 In addition, 2QIGLF-2GA, 2QIGLF-4GA, and 2QIGLF-5GA participated in the formation of the self-assembly, and the content ratio of the four was approximately 11:3:6:

2.

5. The self-assembled body according to claim 1, characterized in that: The loading rate of the self-assembly for hydrophobic curcumin was 18% to 26%.

6. The self-assembled body according to claim 1, characterized in that: After the self-assembly encapsulated curcumin, the DPPH and ABTS free radical scavenging activities reached more than 35.4% and 19.4%, respectively, which were 11.8 times and 2.6 times that of free curcumin under the same experimental conditions.

7. The self-assembled body according to claim 1, characterized in that: After the self-assembly encapsulated 100 μg / mL of curcumin, the corresponding self-assembly concentration was 250 μg / mL. The results of the cytotoxicity experiment were: the survival rate of Caco-2 cells was 83.4% to 88.2%, the survival rate of L02 cells was 89.2% to 91.5%; the hemolysis rate was 0.414% to 0.307%.

8. The self-assembled body according to claim 1, characterized in that: After curcumin was encapsulated in the self-assembly, the apparent permeability coefficient of curcumin on the Caco-2 cell monolayer was P aap The value increased to 3.996×10 -7 ~4.223×10 -7 cm / s, which is 11.23 to 12.68 times that of free curcumin under the same experimental conditions.

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