Astaxanthin carrier with liver targeting and intestinal slow release effects for improving acute alcoholic liver injury and preparation method of astaxanthin carrier

Abstract

The invention discloses an astaxanthin carrier with liver targeting and intestinal slow release effects for improving acute alcoholic liver injury and a preparation method of the astaxanthin carrier, and belongs to the field of food science and biological medicine. The astaxanthin in the astaxanthin carrier is loaded by sea cucumber peptide (SCP) and proanthocyanidins (PCs) and encapsulated in hyaluronic acid and the sea cucumber peptide carrier, so that liver enrichment of stable and high-expression astaxanthin is realized, and the effect of improving acute alcoholic liver injury is realized in animal experiments. Therefore, the problems that the astaxanthin is poor in stability and low in availability when being directly applied to living bodies are effectively solved. The carrier has high loading capacity, intestinal slow release and liver targeting capacity, can effectively relieve symptoms of the acute alcoholic liver injury by repairing liver tissue injury, reduces oxidative stress and expression level of inflammatory factors, and lays a foundation for relieving and treating the acute alcoholic liver injury by accurately delivering AXT.

Description

Technical Field

[0001] The present invention belongs to the fields of food science and biomedicine, and particularly relates to an astaxanthin carrier with liver targeting and intestinal sustained release effects for improving acute alcoholic liver injury and a preparation method thereof. Background Art

[0002] Alcohol metabolism mainly occurs in the liver. Excessive alcohol consumption can lead to a series of liver diseases, collectively referred to as acute alcoholic liver damage (AALD). This damage develops through several stages, including alcoholic fatty liver, alcoholic steatohepatitis, liver fibrosis, cirrhosis, and in severe cases, hepatocellular carcinoma. AALD is associated with multiple molecular and cellular processes, such as oxidative stress caused by reactive oxygen species (ROS) generated during hepatic ethanol metabolism, activation of the NF-kB pathway and inflammatory cytokines such as TNF-α, IL-6, and IL-1β by chronic alcohol consumption, accumulation of unfolded or misfolded proteins in endoplasmic reticulum stress, and lipid metabolism disorders. Excessive ROS production can lead to hepatocyte mitochondrial dysfunction and lipid accumulation, resulting in significant hepatocyte damage. Therefore, the timely clearance of ROS in the liver during alcohol metabolism is crucial for the effective treatment of AALD.

[0003] Astaxanthin (AXT) is a lipophilic carotenoid widely distributed in phytoplankton, algae, crustaceans, and other marine organisms. Its molecular structure has conjugated double bonds and functional groups such as hydroxyl and keto groups, which can react with oxygen free radicals to neutralize, thus showing strong antioxidant properties. Studies have shown that AXT can activate the Nrf2 signaling pathway and upregulate the expression of genes such as heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), superoxide dismutase 2 (SOD2), and heat shock protein 70 (HSP70), enhancing the body's ability to resist oxidative stress. However, the development of oral AXT therapy is significantly hindered by its structural degradation, short intestinal residence time, and low gastrointestinal bioavailability. The acidic conditions in the stomach, together with bile salts, digestive enzymes, and the mucus / mucosal barrier of the intestine, easily destroy unprotected AXT molecules after oral administration. Therefore, preventing the degradation and loss of function of astaxanthin is a challenging problem.

[0004] The nano-bioactive compound delivery system is a method to solve the above problems because it can address several key challenges, including low bioavailability, adverse pharmacokinetics, and persistent toxic side effects. Ideally, a nano-sized delivery system can maintain colloidal stability while enabling the target to reach the designated location. This design can keep bioactive compounds encapsulated during circulation, promote targeted delivery through enhanced permeability and retention effects, or achieve specific targeting through ligands. Therefore, the nano-bioactive compound delivery system opens up new possibilities for precise AALD treatment.

[0005] Currently, in the prior art, a technology similar to the use of a nano-bioactive compound drug delivery system is CN 118236349A (a nano-dry powder of astaxanthin sea cucumber peptide with liver fibrosis alleviating effect and its preparation method). In this patent, sea cucumber peptide, procyanidins, and vanillin are used as raw materials, and a sea cucumber peptide nano-carrier is prepared based on the Mannich reaction. However, there is still room for improvement in the astaxanthin sustained-release performance and targeted therapeutic effect of this sea cucumber peptide nano-carrier. Summary of the Invention

[0006] Technical Problem

[0007] Astaxanthin needs to be encapsulated to ensure that it is not destroyed by the digestive system after oral administration, so as to achieve sustained release and exert a therapeutic effect. However, the sustained-release performance and therapeutic effect of the current astaxanthin nano-carriers still need to be improved.

[0008] Technical Content

[0009] In view of the above problems and deficiencies, the present invention provides an astaxanthin drug delivery system based on liver targeting and intestinal sustained release. The hydrophobic food functional factor astaxanthin is loaded and encapsulated in a hyaluronic acid and sea cucumber peptide carrier by sea cucumber peptide (SCP) and procyanidins (PCs), realizing the liver enrichment of astaxanthin, and achieving an improvement effect on acute alcoholic liver injury in animal experiments, thus effectively breaking through problems such as poor stability of astaxanthin and low bioavailability when directly applied in vivo.

[0010] The present invention provides an astaxanthin carrier with liver targeting and intestinal sustained release for intervening and / or improving acute alcoholic liver injury. The astaxanthin carrier is formed by loading and encapsulating astaxanthin in a hyaluronic acid and sea cucumber peptide hydrogel by sea cucumber peptide and procyanidin nanoparticles.

[0011] The present invention provides a preparation method of an astaxanthin carrier, and the preparation method includes the following steps:

[0012] (1) Preparation of the HA-SCP carrier: Add hyaluronic acid, EDC, NHS, and sea cucumber peptide to water to obtain a reaction solution, then stir to obtain a hydrogel, dialyze the hydrogel, and then perform freeze-drying to obtain the HA-SCP carrier;

[0013] (2) Preparation of SPNs nanoparticles: Dissolve sea cucumber peptide and vanillin in water, stir to obtain reaction solution 1, then add a procyanidin solution to reaction solution 1 and stir to obtain reaction solution 2, and then add vanillin and continue stirring to obtain SPNs nanoparticles;

[0014] (3) Preparation of the AXT@SPNs carrier: Mix an astaxanthin ethanol solution with an SPNs nanoparticle solution to form a coarse emulsion, then ultrasonicate the coarse emulsion, and then rotary evaporate to remove ethanol to obtain the AXT@SPNs carrier;

[0015] (4) Preparation of AXT@SPNs / HS carrier: Stir and mix the HA-SCP carrier solution and the AXT@SPNs solution, and then perform rotary evaporation on the mixture and freeze-dry it to obtain the AXT@SPNs / HS carrier, that is, the astaxanthin carrier.

[0016] Furthermore, the mass ratio of the hyaluronic acid to the EDC in step (1) is 20-30:1.

[0017] Furthermore, the mass ratio of the hyaluronic acid to the NHS in step (1) is 40-45:1.

[0018] Furthermore, the mass ratio of the hyaluronic acid to the SCP in step (1) is 0.5-3:1.

[0019] Preferably, the mass ratio of the hyaluronic acid to the SCP in step (1) is 0.5-1.5:1.

[0020] Furthermore, the concentration of the hyaluronic acid in the reaction solution in step (1) is 1-5 mg / mL.

[0021] Furthermore, the stirring time in step (1) is 10-20 hours.

[0022] Furthermore, in step (1), dialysis is performed using a dialysis bag with a molecular weight cut-off of 500-2000 Da for 24-48 hours.

[0023] Furthermore, the mass ratio of the sea cucumber peptide to the vanillin in step (2) is 30-50:1.

[0024] Furthermore, the concentration of the sea cucumber peptide in the reaction solution 1 in step (2) is 1-5 mg / mL.

[0025] Furthermore, the concentration of the procyanidin in the procyanidin solution in step (2) is 4-8 mg / mL.

[0026] Furthermore, the volume ratio of the procyanidin solution to the reaction solution 1 in step (2) is 0.5-1.5:1.

[0027] Furthermore, the concentration of the vanillin in the reaction solution 2 in step (2) is 0.03-0.05 mg / mL.

[0028] Furthermore, the stirring temperature in step (2) is 40-60 °C, and the time is 0.5-3 hours.

[0029] Furthermore, the purity of the astaxanthin in the astaxanthin ethanol solution in step (3) is 5-20%.

[0030] Further, the concentration of astaxanthin in the astaxanthin ethanol solution in step (3) is 5 - 15 mg / mL.

[0031] Further, the concentration of SPNs nanoparticles in the SPNs nanoparticle solution in step (3) is 3 - 7 mg / mL.

[0032] Further, the volume ratio of the astaxanthin ethanol solution to the SPNs nanoparticle solution in step (3) is 1:7 - 10.

[0033] Further, the temperature of the ultrasound in step (3) is 0 - 5 °C, and the time is 10 - 20 minutes.

[0034] Further, the concentration of the HA-SCP carrier in the HA-SCP carrier solution in step (4) is 3 - 7 mg / mL.

[0035] Further, the concentration of AXT@SPNs in the AXT@SPNs solution in step (4) is 8 - 12 mg / mL.

[0036] Further, the volume ratio of the HA-SCP carrier solution to the AXT@SPNs solution in step (4) is 1:5 - 10.

[0037] Further, the solvent in the AXT@SPNs solution in step (4) is an ethanol aqueous solution with a concentration of 5 - 15%.

[0038] The present invention provides an astaxanthin carrier prepared according to the above method.

[0039] The present invention provides the application of the astaxanthin carrier in the field of drug preparation or health products.

[0040] Further, the drug in the drug preparation is a drug for treating acute alcoholic liver injury.

[0041] Further, the health product is a health product with an auxiliary protective effect on chemical liver injury.

[0042] The present invention has the following beneficial effects compared with the prior art:

[0043] (1) The present invention prepares a liver-targeted AXT@SPNs / HS carrier, which solves the problems of poor water solubility and poor stability of astaxanthin, greatly improves the bioavailability of astaxanthin, and makes the application of the targeted AXT carrier in the oral nutritional delivery system more mature and reliable.

[0044] (2) The AXT@SPNs / HS carrier of the present invention exhibits a good dispersion size (1 μm), has a high loading capacity (6.56 ± 0.19 μg / mg), intestinal sustained release and liver targeting capabilities. It can not only effectively relieve the symptoms of AALD by repairing liver tissue damage, but also down-regulate the expression levels of oxidative stress and inflammatory factors to 3.67%, laying a foundation for the precise delivery of AXT to relieve and treat acute alcoholic liver damage. Description of the Drawings

[0045] Figure 1 It is the yield of the HA-SCP carrier.

[0046] Figure 2 It is the cold field scanning electron microscope of the HA-SCP carrier.

[0047] Figure 3 It is the cold field electron microscope of AXT@SPNs / HS.

[0048] Figure 4 It is the particle size of AXT@SPNs / HS.

[0049] Figure 5 It is the drug loading of astaxanthin in AXT@SPNs / HS.

[0050] Figure 6 It is the in vitro digestion and release analysis of astaxanthin in AXT@SPNs / HS.

[0051] Figure 7 It is the in vivo biodistribution of free AXT, AXT@SPNs and AXT@SPNs / HS.

[0052] Figure 8 It is the macroscopic images of liver tissues of the acute alcoholic liver injury mouse model intervened by free SCP, AXT, SPNs, AXT@SPNs and AXT@SPNs / HS.

[0053] Figure 9 It is the H&E staining of the acute alcoholic liver injury mouse model intervened by free SCP, AXT, SPNs, AXT@SPNs and AXT@SPNs / HS.

[0054] Figure 10 It is the immunohistochemical staining and semi-quantitative analysis of HMGB1 in the acute alcoholic liver injury mouse model intervened by free SCP, AXT, SPNs, AXT@SPNs and AXT@SPNs / HS. Detailed Embodiments

[0055] To make the objectives, technical solutions and effects of the present invention clearer and more explicit, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0056] Source of raw materials

[0057] Hyaluronic acid was purchased from Macklin with a molecular weight of 60 kDa; sea cucumber peptide was purchased from Dalian Deep Blue Peptide; other reagents and raw materials used in the present invention are all commercially available or can be prepared according to common knowledge.

[0058] The preparation method of SPNs nanoparticles is as follows:

[0059] Dissolve 40 mg of SCP and 1 mg of vanillin in 11 mL of water, and then stir with a magnetic stirrer at room temperature for 30 minutes. Then, the self-assembly of SCP nanoparticles (SPNs) was initiated by adding 15 mL of a 6 mg / mL procyanidin (PCs) solution, and after continuing to stir at 55 °C for 2 h, vanillin was added, and the concentration of vanillin in the reaction solution was 0.038 mg / mL to promote the cross-linking of the Mannich reaction and the formation of SPNs nanoparticles.

[0060] Example 1 Preparation of HA-SCP carrier

[0061] Using EDC / NHS as a cross-linking agent and catalyst, the amide reaction was carried out using the carboxyl group of hyaluronic acid (HA) and the amino group of sea cucumber peptide (SCP) to synthesize the HA-SCP carrier. The specific method is as follows: Add HA (200 mg), EDC (7.75 mg, 50.0 μmol), NHS (4.6 mg, 40.0 μmol) and different amounts of SCP (150, 300, 600 mg) to 60 mL of water in sequence, stir overnight, and the obtained HA-SCP hydrogels were named HS 2.5 , HS5 and HS 10 . Then, the hydrogel was dialyzed (MW = 500 Da) for 36 hours to remove unreacted SCP, and then freeze-dried to obtain the HA-SCP carrier.

[0062] The yield was calculated according to the following formula (1):

[0063] Yield (%) = T i / T0 (1)

[0064] T i Is the content (g) of the HA-SCP carrier, and T0 is the total input amount (g) of HA and SCP.

[0065] The results are as Figure 1As shown, when HA (200 mg) was mixed with SCP at final concentrations of 2.5, 5, and 10 mg / mL, the yields (grafting rates) of the HA-SCP carriers were 72.67%, 37.06%, and 22.29%, respectively. This indicates that the HA-SCP carriers were successfully synthesized and had a relatively high yield.

[0066] The microscopic morphology was observed using a cold field scanning electron microscope, as Figure 2 shown. It transitioned from the smooth layered structure of pure hyaluronic acid to the porous interconnected structure in the HA-SCP carrier, indicating that the HA-SCP carrier of the present invention can create a more stable and functional delivery system.

[0067] Example 2 Preparation and Characterization of AXT@SPNs / HS Carriers

[0068] The AXT ethanol solution (10 mg / mL, AXT purity was 10%) was mixed with the SPNs nanoparticle solution (5 mg / mL) at a ratio of 1:9 (v / v), and a coarse emulsion was formed using an Ultra-Turrax T25 high-speed vortex. Then, the mixture was homogenized in an ice bath at 50% amplitude for 15 minutes (on for 5 s / off for 5 s) under ultrasonic conditions to facilitate the incorporation of AXT. Ethanol was removed by rotary evaporation at 45°C to obtain AXT@SPNs.

[0069] 2 mL of the HA-SCP carrier (5 mg / mL) was added to 16 mL of an ethanol aqueous solution (ethanol concentration was 10%) containing AXT@SPNs (10 mg / mL), and the mixture was thoroughly mixed using an IKA stirrer at 10000 rpm for 2 minutes. Then, the mixture was rotary evaporated and freeze-dried to obtain the astaxanthin carrier (AXT@SPNs / HS).

[0070] The particle size and potential were observed using a scanning electron microscope and measured using a laser particle size analyzer, and the results are as Figure 3 shown. The cryo-SEM images showed that AXT@SPNs presented a spherical morphology and were embedded in the network structure of the HA-SCP carrier, which was a covalent crosslink formed by the amide reaction between the HA and SCP solutions. Figure 4 As shown in, due to encapsulation in the HA-SCP carrier, the average size of the balloon-like AXT@SPNs / HS carrier was approximately 1281 nm, which was larger than that of AXT@SPNs with a size of approximately 510 nm.

[0071] Figure 5 The absorbance value was detected at 480 nm using a UV spectrophotometer, and the drug loading of the sample was calculated based on the astaxanthin standard curve. The drug loading calculation formula was: Drug loading = [(the content of astaxanthin added to the sample - the content of astaxanthin on the surface of the sample) / total sample mass] * 100%.

[0072] It was calculated that the drug loading of AXT in AXT@SPNs and AXT@SPNs / HS was 6.81±0.10 and 6.56±0.19 μg / mg, respectively.

[0073] In vitro simulated digestion cumulative release rate of AXT@SPNs / HS in Example 3

[0074] During the simulated gastrointestinal digestion process, the cumulative release rate of AXT in AXT@SPNs and AXT@SPNs / HS was measured to further study the pH response.

[0075] The experimental procedure was as follows: Equal amounts (13 mg) of AXT@SPNs and AXT@SPNs / HS were resuspended in 20 mL of simulated gastric juice (SG, pH = 2.0) and 25 mL of simulated intestinal fluid (SIF, pH = 6.8), respectively, and then further incubated at 37 °C with gentle shaking at 100 rpm. At specific time intervals (0, 1, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8 h), 1 mL of each sample was taken and an equal amount of fresh liquid was added. 200 μL of absolute ethanol was added to the sample, and after centrifugation at 5000 rpm for 10 minutes, 100 μL of the supernatant was added to a 96-well microplate to measure the absorbance. The content of AXT in the sample was quantified according to the standard curve.

[0076] Figure 6 The results showed that very little AXT was released from the carrier after 5 minutes of digestion in saliva. However, when acting in gastric juice for 2 h, the released AXT in AXT@SPNs reached 39.05%, but the released AXT in AXT@SPNs / HS was only 26.55%, and with the extension of digestion time, the release rate of AXT in simulated intestinal fluid continued to increase. After 4 h of digestion, the release rate of AXT in AXT@SPNs rose to 67.51%, which was 71.21% higher than the release rate of AXT in AXT@SPNs / HS. This indicates that AXT@SPNs / HS has a sustained-release property in the gastrointestinal environment, which is attributed to the coating of the HA-SCP carrier, which can effectively improve the drug efficacy, reduce gastrointestinal irritation, improve compliance, and optimize the release and absorption of the drug.

[0077] Example 4 In vivo biodistribution

[0078] NR@SPNs and NR@SPNs / HS were used to evaluate the distribution of AXT@SPNs and AXT@SPNs / HS in AALD mice during 12 h of digestion. The preparation processes of the NR@SPNs and NR@SPNs / HS were carried out with reference to Example 1 and Example 2, where only AXT was replaced with an equal amount of Nile red (NR).

[0079] The experimental procedure was as follows: KM mice at 4 - 5 weeks of age (body weight approximately 20 ± 0.75 g) were taken. After 3 weeks of adaptation, 3 mice per cage were intragastrically administered 56° white liquor at a dosage of 7 mL / kg to obtain AALD mice. Then, the free NR group, NR@SPNs group, and NR@SPNs / HS group were selected, and AALD mice were fed with an equal amount of 10 mg / kg body weight. After 4, 8, and 12 hours of digestion, the mice were dissected and euthanized with CO2. The fluorescence images of the mouse organs were provided by the MIIS XFP - BIX in - vivo imaging system of Molecular Devices Corporation in Sunnyvale, USA.

[0080] Figure 7 The results showed that 4 hours after intragastric administration of NR@SPNs / HS, compared with the NR@SPNs group and the NR group, stronger fluorescence signals were observed in the livers of alcohol - treated mice. The fluorescence signal of NR in the liver reached the highest value 8 h after administration. The increased liver uptake may be attributed to the over - expression of hyaluronic acid receptors in damaged hepatocytes. In addition, 12 h after administration, fluorescence signals could still be detected in the liver, indicating an extended retention time of the nanoparticles in vivo.

[0081] Establishment and intervention of a mouse model of acute alcoholic liver injury in Example 5

[0082] KM mice at 4 - 5 weeks of age were divided into 7 groups (n = 6). Two of the groups were intragastrically administered water, and the remaining groups were intragastrically administered free SCP (67 mg / kg / day), 10% purity AXT (15 mg / kg / day, equivalent to AXT 30 μg / 20 g / day), SPNs nanoparticles (220 mg / kg / day), AXT@SPNs (220 mg / kg / day, equivalent to AXT 30 μg / 20 g / day), and AXT@SPNs / HS (228 mg / kg / day, equivalent to AXT 30 μg / 20 g / day) for 14 consecutive days. After fasting overnight, 56 - degree white wine (10 mL / kg body weight) was intragastrically administered to induce acute liver injury. One of the groups was used as a control group and only received water, while the other group was designated as the alcohol group, and the mice were sacrificed with CO2 8 h later.

[0083] (1) Macro - morphological analysis of liver tissue: The morphological features of the liver tissue of each mouse were examined and photographed.

[0084] Figure 8The experimental results showed that the liver in the control group was rosy, bright, with a shiny surface, sharp edges, and no lesions. In contrast, the liver in the alcohol group had dulled edges, characterized by white punctate granules, tissue enlargement, and a dull appearance. Although the liver color was slightly improved in the groups exposed to free SCP and AXT, traces of tissue necrosis were still obvious. On the contrary, obvious tissue necrosis occasionally occurred in the livers of the SPNs group and the AXT-loaded SPNs group, although their color was slightly lighter. Notably, the livers in the AXT@SPNs / HS group showed a bright red hue without any signs of tissue necrosis.

[0085] (2) H&E staining analysis of liver tissue: The liver tissue was fixed in 4% (w / v) formalin solution, and the tissue was subsequently wrapped in paraffin blocks. The tissue was sectioned with a thickness of 5 μm and stained with H&E.

[0086] Figure 9 The results showed that the histological appearance of the liver tissue in the control group was normal, with round cell nuclei and clear cell spaces around the central vein. In the alcohol group, however, the hepatocytes were severely damaged, with disrupted structures, cell swelling and necrosis, disappearance or fusion of cell boundaries, and infiltration of inflammatory cells. After treatment with free SCP and AXT, the liver damage was alleviated, but the hepatocytes were still disordered, the inflammatory cells continued to infiltrate, the cell boundaries were blurred, and obvious vacuolization occurred. In the SPNs carrier group, the hepatocytes still had swelling and vacuolization, the cell boundaries were slightly blurred, but no fusion was seen, which was an improvement compared to the SCP group and the AXT group. In the AXT@SPNs group, the cell structure and arrangement were relatively complete and orderly, the cell spaces were clear, and there was less infiltration of inflammatory cells near the central vein. Notably, the liver tissue morphology in the AXT@SPNs / HS group was very similar to that in the control group. As shown above, AXT@SPNs / HS can effectively reduce the damage to the liver tissue structure caused by acute alcohol in mice.

[0087] Thus, it can be seen that the astaxanthin carrier provided by the present invention with the characteristics of liver targeting and intestinal sustained release can significantly improve acute alcohol-induced liver injury. After loading astaxanthin with sea cucumber peptide-proanthocyanidin nanoparticles and encapsulating it in an amide reaction to construct a hyaluronic acid-sea cucumber peptide hydrogel, it shows liver targeting and intestinal sustained release characteristics, solves many limitations such as the poor water solubility, poor stability, and low in vivo bioavailability of astaxanthin, can significantly improve acute alcoholic liver injury, and has a wider application range.

[0088] (3) Immunohistochemical staining analysis of high mobility group box 1 (HMGB1) in liver tissue: The liver tissue was fixed in 4% (w / v) formalin solution, and the tissue was subsequently wrapped in paraffin blocks. The primary antibody against HMGB1 was incubated at 1:3000 at 4 °C overnight. Then, the secondary antibody HRP goat anti-rabbit IgG was incubated at 1:200 at room temperature for 1 hour.

[0089] HMGB1 is mainly secreted by inflammatory cells and is a significant extracellular damp substance involved in the progression of various liver diseases. Figure 10 Show the results of immunohistochemical staining of HMGB1 in liver tissue sections and semi-quantitative analysis by Image J software. The expression area of HMGB1 (brown) was observed to be 19.94% in the alcohol group. This represented a significant 8.97-fold increase compared with the control group. Both the free SCP group (13.09%) and the AXT group (16.56%) showed large HMGB1 expression areas. In contrast, after AXT@SPNs / HS intervention, the expression area of HMGB1 decreased to 3.67%, which was not much different from the control group and was more effective in decreasing the expression area than the carrier SPNs group (11.29%) and the AXT@SPNs group (7.63%).

[0090] Table 1

[0091] Expression area Control group 2.22％ Alcohol group 19.94％ Free SCP 13.09％ 10% purity AXT 16.56％ SPNs nanoparticles 11.29％ AXT@SPNs 7.63％ AXT@SPNs / HS 3.67％

[0092] The above specific implementation part has specifically introduced the analysis method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and idea of the present invention, rather than a limitation on the relevant content. Without departing from the principle of the present invention, those skilled in the art can also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications should also fall within the protection scope of the present invention.

Claims

1. A preparation method of an astaxanthin carrier, characterized in that, The preparation method includes the following steps: (1) Preparation of the HA-SCP carrier: Add hyaluronic acid, EDC, NHS, and sea cucumber peptides to water to obtain a reaction solution, then stir to obtain a hydrogel. Dialyze the hydrogel and then perform lyophilization to obtain the HA-SCP carrier; (2) Preparation of SPNs nanoparticles: Dissolve sea cucumber peptides and vanillin in water, stir to obtain reaction solution 1, then add the proanthocyanidin solution to reaction solution 1 and stir to obtain reaction solution 2. Then add vanillin and continue stirring to obtain SPNs nanoparticles; (3) Preparation of the AXT@SPNs carrier: Mix the astaxanthin ethanol solution with the SPNs nanoparticle solution to form a coarse emulsion, then ultrasonicate the coarse emulsion. After that, rotary evaporate to remove ethanol to obtain the AXT@SPNs carrier; (4) Preparation of the AXT@SPNs / HS carrier: Stir and mix the HA-SCP carrier solution and the AXT@SPNs solution, then perform rotary evaporation on the mixture and freeze-dry to obtain the AXT@SPNs / HS carrier, i.e., the astaxanthin carrier.

2. According to the preparation method described in claim 1, characterized in that, In step (1), the mass ratio of the hyaluronic acid to EDC is 20 - 30:1; the mass ratio of the hyaluronic acid to NHS is 40 - 45:1; the mass ratio of the hyaluronic acid to sea cucumber peptides is 0.5 - 3:1; the concentration of the hyaluronic acid in the reaction solution is 1 - 5 mg / mL.

3. According to the preparation method described in claim 1, characterized in that, In step (2), the mass ratio of the sea cucumber peptides to vanillin is 30 - 50:1; the concentration of the sea cucumber peptides in reaction solution 1 is 1 - 5 mg / mL; the concentration of the proanthocyanidins in the proanthocyanidin solution is 4 - 8 mg / mL; the volume ratio of the proanthocyanidin solution to reaction solution 1 is 0.5 - 1.5:

1.

4. According to the preparation method described in claim 1, characterized in that, In step (2), the concentration of the vanillin in reaction solution 2 is 0.03 - 0.05 mg / mL; the temperature of the stirring is 40 - 60 °C, and the time is 0.5 - 3 hours.

5. According to the preparation method described in claim 1, characterized in that, In step (3), the purity of the astaxanthin in the astaxanthin ethanol solution is 5 - 20%; the concentration of the astaxanthin in the astaxanthin ethanol solution is 5 - 15 mg / mL.

6. According to the preparation method described in claim 1, characterized in that, In step (3), the concentration of the SPNs nanoparticles in the SPNs nanoparticle solution is 3 - 7 mg / mL; the volume ratio of the astaxanthin ethanol solution to the SPNs nanoparticle solution is 1:7 - 10.

7. According to the preparation method described in claim 1, it is characterized in that, In step (4), the concentration of the HA-SCP carrier in the HA-SCP carrier solution is 3 - 7 mg / mL; the concentration of the AXT@SPNs in the AXT@SPNs solution is 8 - 12 mg / mL.

8. According to the preparation method described in claim 1, characterized in that, In step (4), the volume ratio of the HA-SCP carrier solution to the AXT@SPNs solution is 1:5 - 10; the solvent in the AXT@SPNs solution is an ethanol aqueous solution with a concentration of 5 - 15%.

9. An astaxanthin carrier, characterized in that, The astaxanthin carrier is prepared according to the preparation method described in any one of claims 1 - 8.

10. Use of the astaxanthin carrier described in claim 9 in the field of drug preparation or health products, characterized in that, The drug in the drug preparation is a drug for treating acute alcoholic liver injury, and the health product is a health product with an adjuvant protective effect on chemical liver injury.

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