Astaxanthin composite emulsion for resisting ionizing radiation intestinal injury and preparation method of astaxanthin composite emulsion

Abstract

The invention provides an astaxanthin composite emulsion for resisting ionizing radiation intestinal injury and a preparation method thereof.The preparation method comprises the steps that firstly, sea cucumber peptide and fucoidin are mixed and stirred in water, the obtained mixture is placed in a water bath kettle to react, dialysis and freeze drying are conducted after the reaction is finished, and a Maillard reaction compound is obtained; the preparation method comprises the following steps: mixing astaxanthin and DHA algal oil, stirring in a dark place overnight, centrifuging to remove insoluble substances, and collecting supernate to obtain an astaxanthin DHA algal oil solution; and redissolving the obtained Maillard reaction compound, mixing the redissolved Maillard reaction compound with an astaxanthin DHA algae oil solution, carrying out full vortex treatment, and then carrying out high-speed homogenization treatment to obtain the astaxanthin composite emulsion. According to the method, an astaxanthin-loaded Pickering emulsion delivery system is prepared by utilizing the interfacial activity of a Maillard reaction product of sea cucumber peptide and fucoidin, the system realizes covalent binding of the sea cucumber peptide and the fucoidin, the water solubility and the stability of the astaxanthin are improved, and meanwhile, the astaxanthin-loaded Pickering emulsion is prepared. The application of astaxanthin in health food and synthetic drugs for preventing ionizing radiation damage is widened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food processing and radiation protection, and particularly relates to astaxanthin composite emulsion for resisting ionizing radiation intestinal injury and a preparation method thereof. BACKGROUND

[0002] Radiation is considered as the fourth pollution after water, air and noise pollution. From the perspective of energy size, radiation can be divided into ionizing radiation and non-ionizing radiation. Ionizing radiation has high energy and can ionize neutral atoms, thereby changing the structure and physical and chemical properties of atoms. When ionizing radiation acts on the human body, biological macromolecules such as nucleic acids and proteins in the human body will be ionized or excited, the structure and function of cells are destroyed, and long-term health effects such as malignant tumors and cardiovascular diseases are induced. At present, most special workers rely on taking drugs before radiation exposure to achieve the purpose of reducing damage. However, most synthetic drugs have toxic side effects and cannot achieve long-term prevention effect.

[0003] Astaxanthin, also known as astaxanthin and astaxanthin, is a natural pigment extracted from Chlorella vulgaris, which is a ketone type carotenoid. The conjugated double bond, hydroxyl group and ketone group at the end of the conjugated double bond chain in the astaxanthin molecule form α-hydroxy ketone, and these structures have active electron effect, which can provide electrons or attract unpaired electrons of free radicals, effectively quenching singlet active oxygen and other free radicals. The excellent antioxidant and anti-inflammatory activity of astaxanthin makes it a natural compound with auxiliary protection against radiation hazards, which has been confirmed in vitro cell, molecular level and in vivo animal experiments. However, in practical application, it is found that astaxanthin has poor water solubility, is easy to decompose under light, has low absorption rate in the gastrointestinal tract, and has fast metabolism, etc. These problems lead to low bioavailability, which limits its application in the field of food.

[0004] Therefore, around the specific demand of "preventing ionizing radiation intestinal injury", exploring the breakthrough of the application of astaxanthin in radiation protection has become a key scientific problem and a research direction to be broken through. SUMMARY

[0005] In view of the technical bottleneck of poor solubility and low oral bioavailability of astaxanthin in the prior art, the purpose of the present application is to reduce the degradation of astaxanthin by gastric acid, promote slow release in the intestinal tract, improve the antioxidant activity and bioavailability of astaxanthin, and provide an astaxanthin composite emulsion for preventing ionizing radiation intestinal injury and a preparation method thereof. The method utilizes the interfacial activity of the Maillard reaction product of sea cucumber peptide and fucoidan to prepare a Pickering emulsion delivery system loaded with astaxanthin. The system realizes the covalent combination of sea cucumber peptide and fucoidan, improves the water solubility and stability of astaxanthin, and widens the application of astaxanthin in health foods and synthetic drugs for preventing ionizing radiation damage.

[0006] It has been found in existing research that the construction of delivery carriers such as emulsions, nanoparticles, liposomes and microcapsules can achieve the purpose of encapsulating astaxanthin, resist the erosion of gastric acid environment, slowly release in the intestinal tract, and improve the bioavailability. However, due to the complex types of encapsulating materials and various encapsulating methods, the existing delivery carriers generally have problems such as uneven astaxanthin loading efficiency, insufficient emulsion stability, and poor targeting suitability for ionizing radiation intestinal injury prevention, which makes it difficult to fully exert the anti-radiation activity of astaxanthin. Therefore, the inventors have conducted research and found that under the condition of using the covalent complex formed by the Maillard reaction of sea cucumber peptide and fucoidan as an emulsifier, the prepared astaxanthin delivery system can significantly improve the water solubility and stability of astaxanthin, reduce gastric acid degradation and achieve slow release in the intestinal tract, and its anti-radiation intestinal injury effect is better than that of free astaxanthin. Based on this, in the further research on the optimal ratio and process parameters of sea cucumber peptide and fucoidan, the inventors found that the Pickering emulsion prepared by mixing the Maillard reaction product of sea cucumber peptide and fucoidan with a mass ratio of 1:1 and astaxanthin-DHA algal oil solution with a certain mass ratio has a significantly improved encapsulation rate of astaxanthin and exhibits better bioavailability and anti-inflammatory and antioxidant activity in anti-radiation protection. In view of this, the inventors provide the following scheme of the present application.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] The present application provides a preparation method of an astaxanthin composite emulsion for preventing ionizing radiation intestinal injury, comprising the following steps:

[0009] Step (1): Mix sea cucumber peptide and fucoidan in water and stir to obtain a mixture, which is then placed in a water bath for reaction. After the reaction is completed, the mixture is dialyzed and freeze-dried to obtain a Maillard reaction complex;

[0010] Step (2): Mix astaxanthin and DHA algal oil, stir in the dark overnight, centrifuge to remove insoluble substances, and collect the supernatant to obtain an astaxanthin DHA algal oil solution;

[0011] Step (3): the Maillard reaction complex obtained in step (1) is redissolved, mixed with the astaxanthin DHA algal oil solution in step (2), vortexed thoroughly, and then subjected to high-speed homogenization treatment to obtain the astaxanthin composite emulsion.

[0012] Further, in step (1), the mass ratio of the sea cucumber peptide and fucoidan is 4-1:1; preferably, the mass ratio of the two is 1:1.

[0013] Further, in step (1), the water bath reaction is carried out at 90-95 ℃ for 4-10 h; preferably, the reaction is carried out at 95 ℃ for 6 h.

[0014] Further, in step (1), the dialysis is carried out using a dialysis bag with a molecular weight cutoff of 1000-3500 Da; the freeze-drying time is 24-72 h; preferably, the freeze-drying time is 72 h.

[0015] Further, in step (2), the amount of astaxanthin added to the DHA algal oil is 0.1 mg / mL.

[0016] Further, in step (2), the centrifugation is carried out at 10000 rpm for 5-15 min; preferably, the centrifugation is carried out for 10 min.

[0017] Further, in step (3), the mass ratio of the Maillard reaction complex and the astaxanthin DHA algal oil solution is 2-3:3; preferably, the mass ratio of the two is 2:3.

[0018] Further, in step (3), the homogenization is carried out at a speed of 12000 rpm for 2-5 min; preferably, the homogenization is carried out for 3 min.

[0019] The second aspect of the present application provides an astaxanthin composite emulsion for preventing ionizing radiation damage prepared by the method of the first aspect.

[0020] The third aspect of the present application provides the use of the preparation method of the first aspect or the astaxanthin composite emulsion for preventing ionizing radiation damage of the second aspect in the preparation of health food and / or drugs for preventing ionizing radiation damage.

[0021] The present application has the following advantages compared with the prior art:

[0022] (1) In the preparation method of the present application, the sea cucumber peptide-fucoidan covalent combination product is used as an emulsifier to regulate the properties of the water-oil interface, so that the system has excellent physicochemical stability, can efficiently load astaxanthin, and significantly improves the water solubility of astaxanthin.

[0023] (2) The astaxanthin composite emulsion prepared by the application can reduce the degradation of astaxanthin by gastric acid, realize the slow release of astaxanthin in the intestinal tract, and improve the antioxidant and anti-inflammatory activity of astaxanthin; in terms of preventing intestinal damage caused by ionizing radiation, the astaxanthin composite emulsion system has a more significant effect than free astaxanthin, and the bioavailability of astaxanthin is improved. The application can provide a new idea for developing a fat-soluble active factor carrying system, and broaden the application of the active factor carrying system in the development of health foods or drugs for preventing ionizing radiation damage. BRIEF DESCRIPTION OF DRAWINGS

[0024] The application will be further described below in combination with the drawings and examples:

[0025] Figure 1 (A) apparent diagram, (B) cold field scanning electron microscope diagram, (C) average particle size, (D) Fourier transform infrared spectrogram, (E) fluorescence spectrogram, (F) circular dichroism spectrogram, (G) secondary structure distribution and (H-I) three-phase contact angle of the sea cucumber peptide and fucoidan Maillard compound in Example 1 and Comparative Example 1 of the application;

[0026] Figure 2 (A-B) bright field structure diagram, (C) average particle size, (D) Zeta potential absolute value, (E) bright field microscope diagram, (F) particle size distribution diagram of the astaxanthin composite emulsion in Example 1 and Comparative Example 1 of the application;

[0027] Figure 3 (A-D) cold field scanning electron microscope diagram, (E-G) laser confocal microscope diagram of the astaxanthin composite emulsion in Example 1 and Comparative Example 1 of the application;

[0028] Figure 4 Modulus of the astaxanthin composite emulsion in Example 1 and Comparative Example 1 of the application changes with (A-B) strain scanning and (C) frequency; (D) thixotropic recovery characteristics of the emulsion and changes of the viscosity of the emulsion with (E) shear rate and (F) temperature scanning;

[0029] Figure 5 (A) encapsulation rate and retention rate, (B) ultraviolet irradiation stability and (C) thermal stability of astaxanthin in the Pickering emulsion prepared in Example 1 and Comparative Example 1 of the application;

[0030] Figure 6 (A) bright field microscope change diagram (the scale is 100 μm), (B) free fatty acid release rate and (C) bioaccessibility of the astaxanthin composite emulsion prepared in Example 1 and Comparative Example 1 of the application in the in vitro simulated digestion experiment;

[0031] Figure 7Effect of the astaxanthin complex emulsion micelles prepared in Example 2 of the present application on (A) the viability of small intestinal crypt epithelial cells (IEC-6), (B) the viability of macrophages (RAW 264.7), and (C-D) hemolytic experiments; effect of free astaxanthin in Comparative Example 3 on (E) the viability of IEC-6 cells, (F) the viability of RAW 264.7 cells, and (G-H) hemolytic experiments;

[0032] Figure 8 H&E staining sections of (A-D) heart, liver, kidney, lung, spleen, and stomach after oral gavage of the samples prepared in Example 3, Comparative Example 2, and Comparative Example 3 of the present application for 15 days;

[0033] Figure 9 RAW 264.7 cell reactive oxygen species analysis (scale bar: 100 μm) after hydrogen peroxide induction of the samples prepared in Comparative Example 3 and Example 2 of the present application;

[0034] Figure 10 (A) Changes in the body weight of mice and (B) survival rate of mice in a model for preventing intestinal damage caused by ionizing radiation using the samples prepared in Example 3, Comparative Example 2, and Comparative Example 3 of the present application;

[0035] Figure 11 (A) White blood cell count, (B) red blood cell count, and (C) platelet count in mice and (D) expression levels of tumor necrosis factor alpha (TNF-α), (E) myeloperoxidase (MPO), and (F) inducible nitric oxide synthase (iNOS) factors in the colon tissue of mice in a model for preventing intestinal damage caused by ionizing radiation using the samples prepared in Example 3, Comparative Example 2, and Comparative Example 3 of the present application;

[0036] Figure 12 H&E staining sections of the colon tissue of mice in a model for preventing intestinal damage caused by ionizing radiation using the samples prepared in Example 3, Comparative Example 2, and Comparative Example 3 of the present application (A-E). DETAILED DESCRIPTION

[0037] The examples are provided to better illustrate the present application and should not be construed as limiting the present application to their details. Substantial modifications of the described embodiments are considered to fall within the scope of the present application.

[0038] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the properties. The endpoints of the ranges and any numerical values are to be understood to be approximate such that slight variations are to be expected. For numeric ranges, the endpoints are provided as a separate point.

[0039] The present application will be described in detail below by way of examples. It should be understood that the following examples are only used to illustrate and explain the present application in further detail, and are not intended to limit the present application.

[0040] In the following examples, astaxanthin (96%) was purchased from Macleod Biochemicals. Sea cucumber peptide and fucoidan were provided by Weihai Yulong Group Co., Ltd. Unless otherwise specified, other reagents were purchased from Damo Chemical Reagent Co., Ltd.

[0041] In the following examples, the detection methods and detection devices involved include:

[0042] 1. Characterization of the micro-morphology of the complex: Cryo-SEM (Cryo-SEM, SU8010, Hitachi, Japan) was used to observe the micro-morphology of the complex and the emulsion.

[0043] 2. Determination of the particle size of the complex: After diluting the sample 100 times, the average particle size was determined using a dynamic light scattering instrument (LS Instruments AG, Switzerland).

[0044] 3. Fourier transform infrared spectroscopy experiment: The freeze-dried sample was mixed with dry potassium bromide at a mass ratio of 1:100, and ground into powder under infrared lamp irradiation. Fourier transform infrared spectroscopy (FTIR, PerkinElmer, Norwalk, CT) was used to detect the spectrum in the range of 4000-400 cm-1, with a resolution of 4 cm-1.

[0045] 4. Fluorescence spectroscopy experiment: The sample was diluted 10 times, and the endogenous fluorescence of the sample was detected using a fluorescence spectrophotometer (F-2700, Hitachi, Japan) with an excitation wavelength of 280 nm, a scanning speed of 60 nm / min, and a response time of 2 s. The emission spectrum of 3000-500 nm was recorded.

[0046] 5. Circular dichroism and secondary structure determination experiment: The sample was diluted to a concentration of 1 mg / mL, and the secondary structure of the sample was determined using a circular dichroism polarimeter (J-1500, JASCO, Japan). The scanning wavelength range was set to 190-260 nm, the bandwidth was 1 nm, the response time was 1 s, and the scanning speed was 50 nm / min.

[0047] 6. Three-phase contact angle (θ) determination experiment: The freeze-dried complex powder was pressed into a thin sheet and placed on a glass sheet. A high-precision syringe was used to add a drop of deionized water to the surface of the sheet. The camera of a video optical contact angle measuring instrument (DSA25, KRUSS, Germany) was used to capture the droplet morphology image, and the contact angle was calculated after baseline fitting.

[0048] 7. Pickering emulsion particle size distribution and potential value determination experiment: The sample was diluted 100 times, and the dynamic light scattering instrument (LS Instruments AG, Switzerland) was used to determine the emulsion particle size distribution and Zeta potential value.

[0049] 8. Microscopic morphology characterization of Pickering emulsion: 0.5 mg / mL fluorescein isothiocyanate (FITC) and 0.1 mg / mL Nile red were used to stain the sea cucumber peptide-fucoidan Maillard reaction complex and DHA algal oil, respectively. Then, the two were mixed in a mass ratio of 2:3, vortexed thoroughly, and homogenized at a speed of 12,000 rpm for 3 min to obtain a Pickering emulsion loaded with astaxanthin. Leica laser confocal microscope was used for observation, and the excitation wavelengths of FITC and Nile red were set to 525 nm and 488 nm, respectively, to show the distribution of the water phase and the oil phase.

[0050] 9. Rheological experiment of Pickering emulsion: The Discovery HR-2 rheometer was used to evaluate the rheological properties of the emulsion. The Pickering emulsion was applied to a 40 mm parallel plate, and the surrounding excess sample was removed by a scraper. The gap height was set to 1000 μm. The storage modulus (G') and loss modulus (G'') of the sample were monitored under the conditions of strain range of 0.01%-1000% and frequency range of 0.1-10 Hz, respectively. The viscosity change curve was determined at a temperature of 25 ℃ and a shear rate range of 0.1-100 s -1 ​​​Each stage shear rate was maintained for 120 s. The temperature sweep test was performed at a constant angular frequency of 4 rad / s to determine the viscosity curve in the temperature range of 20-90 °C.

[0051] 10. Astaxanthin content determination experiment: 0.2 g of emulsion sample was dissolved in 4 mL of dichloromethane and methanol mixed reagent (2:1 / v:v), centrifuged at 8,000 rpm for 10 min, the supernatant was taken and the absorbance was determined at 485 nm by ultraviolet spectrophotometer to obtain the total astaxanthin content in the emulsion. 0.2 g of emulsion sample was dissolved in 4 mL of dichloromethane and centrifuged at 8,000 rpm for 10 min, the organic phase was separated by filtration, and then the absorbance was determined at 485 nm by ultraviolet spectrophotometer to obtain the unencapsulated astaxanthin content in the emulsion. Finally, the encapsulation efficiency of astaxanthin was calculated according to the standard curve.

[0052] 11. Cytotoxicity determination experiment: IEC-6 cells and RAW 264.7 cells were inoculated in 96-well plates (5000 cells per well) respectively, and cultured for 24 h to wait for cell adhesion. Astaxanthin and astaxanthin complex emulsion micelles layer were dissolved in culture medium respectively, and gradiently diluted to concentrations of 2.5, 5, 10, 20 and 40 μg / ml. Then all the culture medium was discarded, and incubated with cells for 24 h. 10 μL of 5 mg / mL 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) was added to each well, and incubated for 4 h. The culture medium was discarded under light shielding condition, 200 μL of dimethyl sulfoxide (DMSO) was added to each well, and placed on a shaker for 10 min. The wavelength of the enzyme marker was set to 570 nm, and the absorbance was determined under this condition.

[0053] 12、Hemolysis experiment: All animal experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and relevant guidelines, EU Directive 2010 / 63 / EU, and the guidelines and animal protocol (DLPU2024PK019) were approved by the Ethics Committee of Dalian Polytechnic University. Blood was collected from the eyeball of mice, and 500 μL of whole blood was added to 5 mL of PBS solution and centrifuged at 10,000 rpm for 5 min at 4 ℃ to collect red blood cells. The obtained red blood cell pellet was washed twice with PBS and resuspended in 10 mL of PBS to obtain a red blood cell suspension. Each 200 μL of the red blood cell suspension was mixed with 800 μL of astaxanthin and astaxanthin complex emulsion micellar layer solution at different concentrations (0.25, 0.5, 1, 1.5, and 2 mg / mL), respectively, and incubated for 1-3 h. Deionized water and PBS solution groups were set as positive and negative controls, respectively. After incubation, centrifugation was performed at 10,000 rpm for 5 min, and the supernatant was used to record the absorbance at a wavelength of 570 nm using a microplate reader.

[0054] 13、Cellular reactive oxygen species analysis experiment: RAW264.7 cells were seeded in a 12-well plate (5000 cells per well) and cultured for 24 h to allow cell adhesion. The control group, hydrogen peroxide (H2O2) group, Comparative Example 3 group, and Example 3 group were set up, with 3 replicates for each group. The cell culture medium was discarded, and 1 mL of RAW264.7 cell-specific culture medium was added to the control group,

[0055] ​​​​​​14. Mouse weight monitoring experiment: All groups of mice were given free access to water from day 1 to day 16, in addition, the sea cucumber peptide-fucoidan complex (SFE) group, the free astaxanthin (AXT) group and the astaxanthin complex emulsion (AXT-SFE) group were given 200 μL (astaxanthin concentration 20 mg / kg / d) samples by gavage every day. On day 15, the mice were irradiated at 1.5 Gy / min and a total dose of 6 Gy using an X-ray device (except for the blank control group), and the mice were deprived of food for 24 h after irradiation. The mice were sacrificed on day 16, and the body weight of the mice was measured at a fixed time every day. Mouse survival rate monitoring experiment: 30 six-week-old BALB / C mice were randomly divided into 5 groups, and given free access to water from day 1 to day 16, in addition, the SFE group, the AXT group and the AXT-SFE group were given 200 μL (astaxanthin concentration 20 mg / kg / d) samples by gavage every day. On day 15, the mice were irradiated at 1.5 Gy / min and a total dose of 6 Gy using an X-ray device (except for the blank control group), and the survival of the mice was recorded every day for a total of 30 days.

[0056] 15. Mouse colon H&E staining experiment: The mice were given free access to water from day 1 to day 16, in addition, the Comparative Example 2 group, the Comparative Example 3 group and the Example 3 group were given 200 μL (astaxanthin concentration 20 mg / kg / d) samples by gavage every day. On day 15, the mice were irradiated at 1.5 Gy / min and a total dose of 6 Gy using an X-ray device (except for the blank control group), and the mice were sacrificed on day 16. The collected colon was fixed with 4% (w / v) paraformaldehyde, stained with hematoxylin and eosin (H&E), and the histopathological changes were observed.

[0057] Example 1 - Preparation of astaxanthin complex emulsion

[0058] The present example provides a preparation method of an astaxanthin complex emulsion for resisting ionizing radiation-induced intestinal injury, comprising the following steps:

[0059] Step (1): 1 g of sea cucumber peptide and 1 g of fucoidan were weighed and mixed and stirred in 10 mL of deionized water. The obtained mixture was placed in a 95 °C water bath and uniformly heated for 6 h. After the reaction was completed, the mixture was dialyzed using a 3500 Da dialysis bag for 24 h, and then freeze-dried for 72 h to obtain a Maillard reaction complex of sea cucumber peptide and fucoidan.

[0060] Step (2): 5 mg of astaxanthin was weighed and placed in 50 mL of DHA algal oil. The mixture was stirred overnight in the dark, centrifuged at 10,000 rpm for 10 min to remove insoluble substances, and the supernatant was collected to obtain an astaxanthin DHA algal oil solution.

[0061] Step (3): 1.5 g of the Maillard reaction complex obtained in step (1) was weighed into 10 mL of deionized water and dissolved, mixed with the astaxanthin DHA algal oil solution in step (2) at a mass ratio of 2:3, vortexed thoroughly, and homogenized at a speed of 12,000 rpm for 3 min to obtain the astaxanthin composite emulsion.

[0062] Example 2 - In vitro simulation of digestion of astaxanthin composite emulsion

[0063] This example extracts the micellar layer after in vitro simulation of digestion of the astaxanthin composite emulsion described in Example 1 by simulating in vitro digestion experiments, specifically including:

[0064] The simulated saliva (SSF), simulated gastric juice (SGF) and simulated intestinal juice (SIF) were preheated in a 37 °C water bath for 30 min. In the saliva digestion stage, 5 g of astaxanthin composite emulsion was mixed thoroughly in 5 mL of SSF, and the mixture was placed in a 37 °C shaking bed and incubated at 100 rpm for 2 min. In the gastric digestion stage, 32 mg of pepsin was dissolved in 10 mL of SGF, and the saliva digestion mixture (1:1, v:v) was added to the freshly prepared simulated gastric juice. The mixture was placed in a 37 °C shaking bed and incubated at 100 rpm for 2 h. In the intestinal digestion stage, 120 mg of porcine lipase, 120 mg of porcine trypsin and 120 mg of bovine bile were respectively dissolved in 20 mL of SIF, and the gastric juice digestion mixture (1:1, v:v) was added to the freshly prepared simulated intestinal juice. The mixture was placed in a 37 °C shaking bed and incubated at 100 rpm for 2 h. The reaction mixture was centrifuged at 10,000 rpm for 1 h, and the micellar layer in the middle was aspirated, dialyzed for 24 h using a 500 Da dialysis bag, and freeze-dried. The content of astaxanthin was calculated according to the standard curve of astaxanthin.

[0065] Example 3 - Preventive effect of astaxanthin composite emulsion on intestinal injury caused by electric radiation

[0066] This example verifies the actual protective effect of the astaxanthin composite emulsion described in Example 1 by constructing a mouse ionizing radiation model, specifically including:

[0067] Sixty 6-week-old BALB / C mice were randomly divided into five groups, and all the mice in the groups were given water without restriction from day 1 to day 16. In addition, the sea cucumber peptide-fucoidan Maillard reaction complex (SFE) group described in Comparative Example 2, the free astaxanthin (AXT) group of Comparative Example 3, and the astaxanthin complex emulsion (AXT-SFE) group of Example 1 were given 200 μL (astaxanthin concentration 20 mg / kg / d) of the sample by gavage every day. On the 15th day, the mice were irradiated with X-ray equipment at a dose rate of 1.5 Gy / min and a total dose of 6 Gy, except for the blank control group.

[0068] All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and relevant guidelines, EU Directive 2010 / 63 / EU, and the guidelines and animal program (DLPU2024PK019) have been approved by the Ethics Committee of Dalian Polytechnic University.

[0069] Comparative Example 1

[0070] To explore the effects of different mass ratios of sea cucumber peptide and fucoidan on the structure and function of astaxanthin complex emulsion, the emulsion was prepared according to the method described in Example 1, with the difference being that:

[0071] In step (1), the amounts of sea cucumber peptide and fucoidan were adjusted so that the mass ratio of the two was 4:1, 3:1 and 2:1, respectively, to obtain three different ratios of sea cucumber peptide and fucoidan Maillard reaction complex. Finally, three different ratios of astaxanthin complex emulsion were prepared.

[0072] Comparative Example 2

[0073] 1 g of sea cucumber peptide and 1 g of fucoidan were weighed and stirred thoroughly in 10 mL of deionized water. The mixture was placed in a 95 °C water bath and heated evenly for 6 h. After the reaction, the mixture was dialyzed for 24 h using a 3500 Da dialysis bag and freeze-dried to obtain the Maillard reaction complex of sea cucumber peptide and fucoidan.

[0074] Comparative Example 3

[0075] 20 mg of astaxanthin was weighed and placed in 10 mL of deionized water. The mixture was stirred overnight in the dark to obtain an aqueous solution of free astaxanthin.

[0076] Test Example:

[0077] In this test example, the structure, stability and functional activity of the sea cucumber peptide and fucoidan Maillard reaction complex and astaxanthin complex emulsion prepared in the above examples and comparative examples were analyzed.

[0078] Figure 1The macroscopic images showed that the complex solutions were well dispersed, and the solution color changed with different mass ratios of sea cucumber peptide and fucoidan. Cryo-SEM images showed that the complexes prepared in Example 1 and Comparative Example 1 were spherical in structure, and the complex prepared in Example 1 had the smallest particle size (398.53 ± 3.84 nm).

[0079] Figure 1 The chemical structures of the sea cucumber peptide and fucoidan Maillard reaction complexes prepared in Example 1 and Comparative Example 1 were characterized in D-G. Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy and circular dichroism (CD) and secondary structure analysis showed that the complex prepared in Example 1 exhibited characteristic peaks of each individual component, indicating the presence of functional group interactions therebetween. In addition, the complex exhibited the strongest fluorescence quenching effect, suggesting that the intermolecular interactions caused changes in the structure of endogenous amino acids. Further analysis showed that the proportion of random coil structure in the complex increased from 60.9% to 74.3%, and the β-turn increased from 3.4% to 10.4%, which confirmed that the sea cucumber peptide and fucoidan had been successfully complexed. Figure 1 The interfacial wettability of the sea cucumber peptide and fucoidan Maillard reaction complexes prepared in Example 1 and Comparative Example 1 was characterized in H-I. The results showed that the three-phase contact angle (θ) was less than 90°, indicating that the hydrophilicity was dominant. Among them, the θ value of the complex prepared in Example 1 was 75.43 ± 0.06°, which was not significantly different from the θ value of fucoidan alone (76.87 ± 0.86°).

[0080] Figure 2 A-F shows the morphological characteristics of the astaxanthin complex emulsion prepared in Example 1 and Comparative Example 1. The results showed that the sample prepared in Example 1 remained in a solid state without flowing after being inverted 180°, and the emulsion droplets were closely arranged. In addition, the emulsion prepared in Example 1 had the smallest average particle size (9.23 ± 1.03 μm) and the most concentrated distribution, indicating that the emulsion structure was the most stable under this condition. This result can be attributed to the covalent binding of sea cucumber peptide and fucoidan, which promotes the exposure of hydrophobic residues inside the sea cucumber peptide, thereby enhancing the surface wettability and interfacial adsorption capacity of the complex.

[0081] Figure 3The microstructure of astaxanthin Pickering emulsion prepared in Example 1 and Comparative Example 1 was presented in Figure A-G. The results showed that the emulsion droplets were crosslinked by the complex of sea cucumber peptide and fucoidan through the interface, forming a stable honeycomb network structure. This structure provides an important physical barrier, not only effectively preventing the coalescence and rupture of droplets, but also helping to maintain the good dispersibility and long-term stability of the emulsion. Nile blue staining makes the water phase present green fluorescence, while Nile red staining makes the oil phase present red fluorescence. The fluorescence microscopic image shows that the oil droplets are red spherical droplets, and the outer layer of the oil droplets is covered with a ring of dense green fluorescence, indicating that the complex of sea cucumber peptide and fucoidan Maillard reaction firmly adsorbs to the oil-water interface, and builds a typical O / W type emulsion structure.

[0082] Figure 4 The rheological behavior of astaxanthin Pickering emulsion prepared in Example 1 and Comparative Example 1 was evaluated. In the frequency scanning test range of 0.1-10 Hz, the storage modulus (G') of all samples was always higher than the loss modulus (G"), indicating that the emulsion showed a dominant gel property of elasticity. In the shear rate range of 0.1-100 s -1 , the apparent viscosity of all samples gradually decreased, showing shear thinning characteristics. Further experiments found that the astaxanthin Pickering emulsion could maintain the integrity of the three-dimensional gel network structure during dynamic shear and temperature scanning, significantly improving its applicability in high-temperature intensive food manufacturing processes.

[0083] Figure 5 The stability of astaxanthin in the Pickering emulsion prepared in Example 1 and Comparative Example 1 was evaluated. The encapsulation efficiency of astaxanthin in the emulsion of Example 1 reached 92.27%, showing excellent encapsulation capacity. In the storage stability test, the retention rate of astaxanthin gradually decreased over time, and the final retention rate after 10 days of storage was 79.44 ± 0.08%. After 12 h of ultraviolet irradiation, the retention rate of astaxanthin in Example 1 was 79.13 ± 0.16%, indicating that the emulsion maintained a strong three-dimensional crosslinked framework, thereby enhancing the resistance to ultraviolet-induced structural degradation. The thermal stability test further showed that after heating at 90 °C water bath for 60 min, the retention rate of astaxanthin could still be maintained at more than 80%. This may be because the complex of sea cucumber peptide and fucoidan Maillard reaction assembled at the oil-water interface to form a high-efficiency interface barrier, effectively blocking the transfer of water vapor molecules to astaxanthin, maintaining the stability of astaxanthin.

[0084] Figure 6In vitro simulated digestion of astaxanthin complex emulsions prepared in Example 1 and Comparative Example 1. The microstructure of all samples did not change significantly during the oral digestion phase. After the gastric digestion phase, the droplet size of the emulsions in Example 1 and Comparative Example 1 increased, which can be attributed to the weakening of the repulsive forces between the droplets due to the acidic environment and ionic strength, leading to flocculation. Finally, the droplet size decreased during the intestinal digestion phase, and the emulsions were almost completely digested, which can be attributed to the adsorption of bile salts and phospholipids at the oil-water interface, and the subsequent hydrolysis of triglycerides mediated by lipase, promoting the breakdown and size reduction of oil droplets. The release of free fatty acids (FFA) from the emulsions prepared in Example 1 and Comparative Example 1 showed a gradual increase followed by a plateau. The maximum FFA release rate was achieved at 80 min during the intestinal digestion phase, and the FFA release rates were as follows: Example 1 (60.39%) > Comparative Example 1 (sea cucumber peptide: fucoidan = 2:1, m:m) (52.45%) > Comparative Example 1 (sea cucumber peptide: fucoidan = 3:1, m:m) (31.04%) > Comparative Example 1 (sea cucumber peptide: fucoidan = 4:1, m:m) (30.63%) > algal oil (23.49%). The emulsions significantly improved the FFA release rate during the intestinal digestion phase, which was beneficial for the arrangement and aggregation of bile salts and phospholipids to form a micellar layer, thereby increasing the solubility of astaxanthin. The results showed that the emulsions stabilized by the Maillard reaction complex of sea cucumber peptide and fucoidan significantly improved the bioavailability of astaxanthin compared to free astaxanthin, promoted the absorption of astaxanthin, and provided an effective system for the transport of fat-soluble functional factors.

[0085] Figure 7 Toxicity determination experiments for the samples in Example 2 and Comparative Example 3. After co-incubation of astaxanthin and the micellar layer of astaxanthin complex emulsion with IEC-6 cells and RAW 264.7 cells, the cell viability increased in a dose-dependent manner within a concentration range of 0-20 μg / mL. When the concentration increased to 40 μg / mL, the cell viability decreased slightly. The results showed that the astaxanthin complex emulsion promoted cell proliferation and had good biocompatibility. Blood compatibility analysis is a key factor for the effective application of emulsions in vivo. Within a concentration range of 0.25-2 mg / mL, the hemolysis rate of the Pickering emulsion carrier system was less than 5% within 1 or 3 h, and no obvious hemolysis was observed when red blood cells were in contact with the sample. The results showed that the Pickering emulsion carrier system had good blood compatibility and could be used for subsequent in vivo experiments.

[0086] Figure 8 Biological safety of the samples prepared in Example 3, Comparative Example 2, and Comparative Example 3 in vivo. No pathological changes were found in the heart, liver, kidney, lung, spleen, and stomach by observing H&E stained sections. The results showed that the samples prepared in Example 3, Comparative Example 2, and Comparative Example 3 had good biological safety.

[0087] Figure 9AD is an assay for reactive oxygen species (ROS) in cells of the samples prepared in Comparative Example 3 and Example 2. Hydrogen peroxide ( This leads to an increase in intracellular reactive oxygen species (ROS). DCFH-DA is commonly used as a fluorescent probe to quantify intracellular ROS levels; the intensity of green fluorescence is positively correlated with ROS content. Figure 9 As shown in Figure B, RAW 264.7 cells in After induction, the intensity of green fluorescence within the cells was significantly enhanced. After treatment with Comparative Example 3 and Example 2, the fluorescence intensity was significantly reduced, with Example 2 showing the lowest ROS level. Astaxanthin, as a natural antioxidant, has the potential to quench ROS, but its poor water solubility limits its effective absorption and accumulation within cells, thus affecting its antioxidant effect. The Maillard reaction complex of sea cucumber peptide and fucoidan possesses a certain antioxidant capacity. Example 2 not only improved the solubility and cellular uptake of astaxanthin but may also further enhance the antioxidant effect through the active ingredients carried by the complex, thereby better exerting its ROS scavenging effect.

[0088] Figure 10 Figure A shows the trend of mouse body weight change in a model of intestinal damage induced by ionizing radiation in Examples 3, 2, and 3, using the astaxanthin complex emulsion, sea cucumber peptide-fucose complex, and free astaxanthin prepared in Examples 3, 2, and 3. The experiment showed that compared with the blank control group, the X-ray group mice experienced a significant decrease in body weight after modeling. After prevention with the samples prepared in Examples 2, 2, and 3, the degree of body weight loss was reduced, with the astaxanthin complex emulsion showing the most significant preventative effect on body weight. Figure 10 As shown in Figure B, except for the control group where all mice survived, mice in all other groups died successively. The X-ray treatment group showed mortality as early as day 11, and all mice died by day 13. In contrast, Comparative Example 2 showed mortality extending to day 17, Comparative Example 3 extended to day 18, and Example 3 extended to day 25. These results indicate that Example 3 has a significant preventive effect against systemic damage and even death caused by X-ray ionizing radiation.

[0089] Figure 11 The values ​​in AC represent the changes in blood routine parameters (including white blood cell count, red blood cell count, and platelet count) in mice after irradiation in different treatment groups. Compared with the control group, the X-ray treatment group significantly reduced the white blood cell count, red blood cell count, and platelet count in mice. The samples prepared in Comparative Examples 2, 3, and 3 reduced the imbalance of blood routine parameters in mice after irradiation. In the Example 3 treatment group, the white blood cell and red blood cell counts of mice recovered to near-normal levels. Figure 11As shown in D-F, compared with the control group, the expression levels of tumor necrosis factor alpha (TNF-a), myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) factors in the colon tissue after X-ray treatment were significantly up-regulated, indicating that X-ray radiation induced acute inflammatory response in the tissue and was accompanied by tissue damage. The expression levels of related inflammatory factors in the tissue were reduced in the sample treatment groups prepared by Comparative Example 2, Comparative Example 3 and Example 3, and the expression levels of TNF-a and MPO in the colon tissue of the Example 3 treatment group were close to those of normal mice.

[0090] Figure 12 A-E are the H&E staining section images of the astaxanthin complex emulsion, sea cucumber peptide-fucoidan complex and free astaxanthin prepared in Example 3, Comparative Example 2 and Comparative Example 3 in the prevention of intestinal damage model caused by ionizing radiation. The experiment showed that compared with the colon tissue section of the blank control group of mice, the colon crypt structure of the X-ray group was obviously damaged, the crypt structure was deformed, and inflammatory cells infiltrated. After prevention by Example 1, Comparative Example 2 and Comparative Example 3, the degree of inflammation of the colon tissue was significantly reduced, the damage to the crypt structure was reduced, and the infiltration of inflammatory cells was reduced. Notably, Example 1 prevention observed almost normal histological microstructure in the colon tissue, which was comparable to healthy controls, indicating that the astaxanthin complex emulsion had better prevention effect on mice. These results provide strong experimental evidence for the use of sea cucumber peptide and fucoidan Maillard reaction complex stabilized Pickering emulsion to improve the application of astaxanthin in radiation protection, especially in reducing intestinal damage.

[0091] Finally, it should be noted that the above is only used to illustrate the technical solutions of the present application and not to limit, although the present application has been described in detail with reference to the preferred arrangement, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing astaxanthin complex emulsion against ionizing radiation-induced intestinal injury, characterized in that, The preparation method comprises the following steps: Step (1): mixing sea cucumber peptide and fucoidan in water and stirring, placing the obtained mixture in a water bath for reaction, dialyzing and freeze-drying after the reaction is completed to obtain a Maillard reaction compound; Step (2): mixing astaxanthin and DHA algal oil, stirring in the dark overnight, removing insoluble substances by centrifugation, and collecting the supernatant to obtain an astaxanthin DHA algal oil solution; Step (3): resuspending the Maillard reaction compound obtained in step (1) and mixing with the astaxanthin DHA algal oil solution in step (2), vortexing thoroughly, and then high-speed homogenizing to obtain the astaxanthin composite emulsion.

2. The production method according to claim 1, characterized by, In step (1), the mass ratio of the sea cucumber peptide and fucoidan is 4-1:

1.

3. The production method according to claim 1, characterized by, In step (1), the water bath reaction conditions are 90-95℃ for 4-10 h.

4. The method of claim 1, wherein, In step (1), the dialysis uses a dialysis bag with a molecular weight cutoff of 1000-3500 Da; the freeze-drying time is 24-72 h.

5. The preparation method according to claim 1, characterized in that, In step (2), the amount of astaxanthin added to the DHA algal oil is 0.1 mg / mL.

6. The method of claim 1, wherein, In step (2), the centrifugation conditions are 10000 rpm for 5-15 min.

7. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the Maillard reaction compound to the astaxanthin DHA algal oil solution is 2-3:

3.

8. The method of claim 1, wherein, In step (3), the homogenization occurs at a speed of 12000 rpm for 2-5 min.

9. The astaxanthin composite emulsion against ionizing radiation-induced intestinal injury prepared by the method of any one of claims 1-8.

10. Use of the preparation method of any one of claims 1-8 or the astaxanthin composite emulsion against ionizing radiation-induced intestinal injury of claim 9 in the preparation of health foods and / or drugs for preventing ionizing radiation damage.

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