CaPhy-NAD nanoscale enzyme, preparation method, application and alcoholism preparation thereof

Abstract

The application belongs to the technical field of nanomaterials, and provides a CaPhy-NAD nanoenzyme, a preparation method, application and an alcoholism treatment preparation thereof. The CaPhy-NAD nanoenzyme is prepared by using calcium phytate nanoparticles as a carrier and in-situ loading of ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase and coenzyme NAD. 2+ The CaPhy-NAD nanoenzyme can effectively activate the catalytic activity of acetaldehyde dehydrogenase, and the three enzymes can have a high-efficiency confined cascade reaction in the pore channel of the calcium phytate nanoparticles, so that the recycling of the coenzyme NAD is realized, and the three types of alcohol metabolism-related toxic substances, i.e., ethanol, acetaldehyde and hydrogen peroxide, can be simultaneously removed. The CaPhy-NAD nanoenzyme prepared by the application has excellent catalytic activity and good biological safety, and can be orally administered, thereby providing a new technical idea and solution for the prevention and treatment of alcoholism.

Description

TECHNICAL FIELD

[0001] This application belongs to the field of nanomaterials technology, and in particular relates to a CaPhy-NAD nanozyme, its preparation method, application and hangover remedy. BACKGROUND

[0002] Alcohol is a widely consumed beverage globally, and excessive consumption leading to alcohol poisoning has become a significant public health issue. According to the World Health Organization's (WHO) 2022 Global Alcohol and Health Report, there are 2.3 billion alcohol drinkers worldwide, and approximately 3 million people die annually from alcohol-related illnesses, accounting for 5.3% of all deaths globally. Long-term alcohol abuse can easily lead to alcoholic liver disease (ALD), neurodegeneration, cardiovascular disease, and cancer, while acute alcohol poisoning can cause respiratory depression, coma, and even death.

[0003] The metabolism of alcohol in the body mainly relies on the cascade catalytic action of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH): ethanol is first oxidized by ADH to highly toxic acetaldehyde, which is then further oxidized by ALDH to non-toxic acetic acid. Accumulation of acetaldehyde in the body can trigger DNA damage, oxidative stress, mitochondrial dysfunction, and inflammatory responses, leading to multi-organ damage. Simultaneously, reactive oxygen species (ROS) produced during alcohol metabolism exacerbate hepatocyte apoptosis and fibrosis. Long-term alcohol consumption also disrupts the body's neurotransmitter balance, further damaging the central nervous system.

[0004] Currently, commonly used clinical strategies for alcohol detoxification are mainly divided into three categories: metabolic regulators (such as metadoxine, which accelerates ethanol clearance), acetaldehyde scavengers (such as N-acetylcysteine, NAC), and opioid receptor antagonists (such as naloxone, used for the emergency treatment of acute poisoning). However, existing drugs have significant drawbacks: they can only partially relieve the symptoms of alcohol poisoning, cannot reverse alcohol-induced cell damage, and have low bioavailability and significant side effects; for example, although disulfiram can inhibit ALDH2 to reduce the desire to drink, it may cause serious cardiovascular adverse reactions.

[0005] In recent years, the research and development of novel alcohol detoxifiers has gradually shifted towards areas such as targeted regulation of metabolic enzymes, mitochondrial protection, intestinal microbiome regulation, and nano-drug delivery systems. However, current technologies still cannot simultaneously achieve efficient removal of multiple toxic intermediates of alcohol poisoning, such as ethanol, acetaldehyde, and hydrogen peroxide. Furthermore, when free enzymes are used as the core detoxification component, they exhibit poor stability and are easily inactivated under the combined action of digestive enzymes and gastric acid, making oral administration difficult—while oral administration, being unrestricted by time and space, represents the mainstream development direction for alcohol detoxification agents.

[0006] Therefore, developing a compound hangover remedy that combines high catalytic activity and high stability, can be administered orally, and can simultaneously eliminate multiple toxic intermediates of alcohol poisoning through multiple pathways has become an urgent technical problem to be solved in this field. SUMMARY

[0007] The purpose of this application is to provide a CaPhy-NAD nanozyme, which aims to solve the technical problems of existing alcohol detoxification drugs having limited efficacy, poor stability of free enzymes that are easily inactivated, and inability to simultaneously remove multiple toxic intermediate products of alcohol poisoning.

[0008] This application is implemented as follows: a CaPhy-NAD nanozyme, wherein the CaPhy-NAD nanozyme is composed of a calcium phytate nanoparticle carrier, and ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase, and coenzyme loaded on the calcium phytate nanoparticle carrier; the calcium phytate nanoparticles release a small amount of CaPhy-NAD nanoparticles under acidic conditions. 2+ It can activate the catalytic activity of acetaldehyde dehydrogenase. Ethanol oxidase, acetaldehyde dehydrogenase, and horseradish peroxidase undergo a confined cascade reaction within the pores of calcium phytate nanoparticles, achieving the cyclic regeneration of coenzymes and simultaneously removing three toxic substances: ethanol, acetaldehyde, and hydrogen peroxide.

[0009] Another object of this application is a method for preparing the above-mentioned CaPhy-NAD nanozyme, comprising: Using deionized water as a solvent, prepare the following solutions: A) a mixed solution containing ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase, and coenzyme; B) an aqueous solution containing a dispersant; C) an aqueous solution containing a phosphorus source; and D) an aqueous solution containing a calcium source. Solutions A, B, C, and D are simultaneously fed into a vortex mixer and mixed under ultrasonic assistance. After the reaction is completed, the mixture is centrifuged, washed, and lyophilized to obtain the CaPhy-NAD nanozyme.

[0010] Another objective of this application is the application of the aforementioned CaPhy-NAD nanozyme in the preparation of alcohol detoxification drugs.

[0011] Another object of this application is a hangover remedy comprising a pharmaceutically effective amount of CaPhy-NAD nanozyme as described above, and pharmaceutically acceptable excipients.

[0012] This application innovatively constructs an enzyme cascade system of ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase, and coenzyme NAD. CaPhy-NAD nanozymes are prepared by co-loading with calcium phytate, using a dispersant, and ultrasound-assisted in-situ encapsulation. This preparation process achieves in-situ loading of the enzyme and coenzyme, allowing the nanozyme to possess both high enzyme activity and high stability. It not only exhibits excellent catalytic degradation activity against ethanol but also effectively removes toxic intermediates in alcohol metabolism, such as H2O2 and CH3CHO. Simultaneously, the calcium phytate protective layer endows the nanozyme with extremely strong acid and digestive enzyme resistance. Its catalytic activity under extreme pH, high temperature, and trypsin action is far superior to that of the free enzyme and can maintain more than 70%, fully verifying the feasibility of oral administration. Cell and in vivo experiments further confirm the excellent biocompatibility of this nanozyme. This CaPhy-NAD nanozyme, which combines high catalytic activity, high stability, good biocompatibility, and the advantage of oral administration, provides a novel technical approach for the prevention and treatment of alcohol poisoning. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 shows the material preparation and characterization of the CaPhy-NAD nanozyme provided in the embodiments of this application; where (a) is the SEM image of CaPhy-NAD nanoparticles, (b) is the elemental mapping diagram, (c) is the DLS dynamic scattering spectrum, (d) is the XPS fine spectrum, (e) is the XRD spectrum, (f) is the adsorption equilibrium isotherm, (g) is the pore size distribution diagram, and (h) is the detection diagram of enzyme and coenzyme loading. Figure 2 shows the substrate degradation catalyzed by CaPhy-NAD nanozymes provided in the embodiments of this application; each group is (1) CaPhy-NAD, (2) AOx@CaPhy-NAD, (3) AOx / HRP@CaPhy-NAD, and (4) AOx / HRP / ALDH@CaPhy-NAD; where (a) is the change in ethanol concentration catalyzed by different nanozymes, (b) is the amount of acetaldehyde generated by different nanozymes catalyzed by ethanol degradation, and (c) is the amount of hydrogen peroxide generated by different nanozymes catalyzed by ethanol degradation. Figure 3 shows the stability test diagram of CaPhy-NAD nanozyme provided in the embodiments of this application; where (a) is a comparison of the enzyme activity of CaPhy-NAD nanozyme (red) and natural enzyme (blue) at pH=1~11, and (b) and (c) are a comparison of the enzyme activity of CaPhy-NAD nanozyme and natural enzyme after treatment at 37~67℃ for 5 min. Figure 4 shows the biocompatibility test results of the CaPhy-NAD nanozyme provided in the embodiments of this application; each group is (1) PBS+EtOH, (2) AOx+EtOH, (3) AOx / HRP+EtOH, (4) AOx / HRP / ALDH+EtOH, (5) PBS, (6) AOx, (7) AOx / HRP, (8) AOx / HRP / ALDH, (9) AOx@CaPhy-NAD+EtOH, (10) AOx / HRP@CaPhy-NAD+EtOH, (11) AOx / HRP / ALDH@CaPhy-NAD+EtOH, (12) CaPhy-NAD+EtOH, (13) AOx@CaPhy-NAD, (14) AOx / HRP@CaPhy-NAD, (15) AOx / HRP / ALDH@CaPhy-NAD, (16) CaPhy-NAD nanozyme; Figure 5 shows the activity test diagram of the freeze-dried CaPhy-NAD nanozyme provided in the embodiments of this application; where (a) is a schematic diagram of the freeze-drying of CaPhy-NAD nanozyme, (b) is the particle size change of CaPhy-NAD nanozyme before and after freeze-drying, and (c) is the enzyme activity of CaPhy-NAD nanozyme before and after freeze-drying with or without BSA. Figure 6 is a schematic diagram of the CaPhy-NAD nanozyme provided in the embodiments of this application for the treatment of acute alcohol poisoning; Figure 7 shows the detection of ethanol and acetaldehyde content in the blood of mice at different times after drinking alcohol, as provided in the embodiments of this application; each group (1) Blank, (2) PBS, (3) AOx@CaPhy-NAD, (4) AOx / HRP@CaPhy-NAD, (5) AOx / HRP / ALDH@CaPhy-NAD; where (a) is the change in ethanol content and (b) is the change in acetaldehyde content; Figure 8 is a graph showing the detection of oxidative stress levels in mouse livers provided in the embodiments of this application; where (a) is the liver weight, (b) is the standard curve of protein content in the liver, (c) is the MDA content in the liver, and (d) is the ALT level in the liver. Figure 9 shows the detection of biochemical levels in mouse blood provided in the embodiments of this application; each group (1) Blank, (2) PBS, (3) AOx@CaPhy-NAD, (4) AOx / HRP@CaPhy-NAD, (5) AOx / HRP / ALDH@CaPhy-NAD; where (a) is ALT content, (b) is AST content, (c) is BUN content, and (d) is CRE content; Figure 10 shows the mouse major organ sections and H&E staining images provided in the embodiments of this application; each group (1) Blank, (2) PBS, (3) AOx@CaPhy-NAD, (4) AOx / HRP@CaPhy-NAD, (5) AOx / HRP / ALDH@CaPhy-NAD; Figure 11 is a hemolysis assay of the CaPhy-NAD nanozyme provided in the embodiments of this application. DETAILED DESCRIPTION

[0014] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0015] The CaPhy-NAD nanozyme of this application consists of calcium phytate nanoparticles as a carrier, and ethanol oxidase (AOx), aldehyde dehydrogenase (ALDH), horseradish peroxidase (HRP), and coenzyme NAD loaded on the carrier; the calcium phytate nanoparticles release a small amount of Ca in an acidic environment. 2+ It can activate the catalytic activity of ALDH, and AOx, HRP and ALDH undergo confined cascade reactions within the pores of calcium phytate nanoparticles to achieve the recycling and regeneration of NAD, while simultaneously removing three toxic substances: ethanol, acetaldehyde and hydrogen peroxide.

[0016] The hydrated particle size of the calcium phytate nanoparticles is 380~390nm, and the polydispersity index (PDI) is ≤0.19; the pore size of the calcium phytate nanoparticles is 10~50nm, and the total loading of ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase and coenzyme NAD is 10~60wt%.

[0017] This CaPhy-NAD nanozyme exhibits excellent stability: its catalytic activity is significantly higher than that of the free enzyme under extreme pH, high temperature, and trypsin treatment; and the enzyme activity is effectively preserved after freeze-drying with the addition of a lyophilization protectant, facilitating storage and transportation. Specifically, after treatment in an acidic environment at pH=1 for 10 min, the nanozyme retains ≥93% of its catalytic activity; after heat treatment at 67℃ for 5 min, the catalytic activity retains ≥71%; after trypsin treatment for 60 min, the relative enzyme activity retains ≥92%, which is more than 1.67 times that of the free enzyme; and after freeze-drying and the addition of a lyophilization protectant, the catalytic activity of ethanol oxidase is retained ≥99%, and the catalytic activity of acetaldehyde dehydrogenase is retained ≥94%.

[0018] This application also provides a method for preparing CaPhy-NAD nanozymes, including steps such as solution preparation, simultaneous mixing reaction, centrifugation and washing, and freeze-drying, specifically: Preparation of Solution A: Using deionized water as the solvent, prepare an enzyme and coenzyme mixture containing ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase, and coenzyme NAD, with a total enzyme concentration of 0.5~2.0 mg / mL. -1 The weight ratio of enzyme to NAD is 1:(4~10); Preparation of Solution B: Using deionized water as the solvent, prepare an aqueous solution containing a dispersant with a concentration of 5-10 mg / mL. -1 ; Preparation of solution C: Using deionized water as solvent, prepare an aqueous solution containing a phosphorus source with a concentration of 100~200mM; Preparation of solution D: Using deionized water as solvent, prepare an aqueous solution containing a calcium source with a concentration of 50-100 mM; At room temperature, the four solutions were pumped at a rate of 0.1–1 mL / s. -1 The rate is synchronously input to the reaction chamber of the multi-inlet vortex mixer, and the mixing reaction is carried out for 10-20 seconds under the ultrasonic assistance of 50-200W. The reaction solution was reacted at 10000 r·min -1 Centrifuge, collect the lower precipitate, wash with deionized water at least three times, add a freeze-drying protectant, freeze-dry, and store at -20℃ to obtain the final product.

[0019] The weight ratio of solutions A, B, C and D is 1:4:4:(6~7), and the preferred weight ratio is 1:4:4:6.

[0020] The preferred ultrasonic-assisted power is 100~150W.

[0021] The dispersant is at least one of acetylated allenphosphoyl hyaluronic acid (A-HA-ALN), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyoxyethylene-polyoxypropylene block copolymer (Pluronic), carboxymethyl cellulose (CMC), and sodium dodecyl sulfate (SDS), preferably acetylated allenphosphoyl hyaluronic acid.

[0022] The phosphorus source is at least one of phytic acid, sodium phytate, tripolyphosphate, sodium tripolyphosphate, pyrophosphate, sodium pyrophosphate, inositol phosphate, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, potassium tripolyphosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate, with sodium phytate being preferred.

[0023] The calcium source is at least one of calcium chloride, calcium nitrate, calcium lactate, calcium gluconate, calcium citrate, calcium acetate, calcium formate, calcium gluconate, calcium ascorbate, calcium glycerophosphate, and calcium glutamate, with calcium chloride being preferred.

[0024] The freeze-drying protectants include bovine serum albumin, sucrose, trehalose, mannitol, sorbitol, glycerol, PVP, PEG, glycine, and arginine, with bovine serum albumin being the preferred ingredient.

[0025] This application also provides an application of CaPhy-NAD nanozyme in the preparation of alcohol detoxification / hangover relief drugs, which is particularly suitable for the preparation of oral hangover relief preparations. It can effectively reduce the concentration of ethanol and acetaldehyde in the body's blood, alleviate acute liver damage caused by alcohol, and has good biocompatibility, low toxicity and side effects, and no obvious tissue damage to the body.

[0026] This application also provides a hangover remedy, comprising a pharmaceutically effective amount of CaPhy-NAD nanozyme and pharmaceutically acceptable excipients. The dosage form is an oral dosage form such as capsules, tablets, or granules. The nanozyme accounts for 10% to 50% of the mass of the preparation, and the excipients can be mannitol, sucrose, sodium carboxymethyl cellulose, etc.

[0027] The following detailed embodiments illustrate the CaPhy-NAD nanozyme, its preparation method, applications, and hangover remedies provided in this application. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; and the materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0028] Example 1: Synthesis of CaPhy-NAD Nanozymes 1) Solution preparation Phosphorus source solution C: 231 mg of sodium phytate (Na) 12 [C6H6O 24 P6) Add 45 mL of deionized water to the solid, stir and sonicate and adjust the pH to 7.0. After it is completely dissolved, make up to 50 mL to obtain a 50 mM sodium phytate solution. Seal and store at room temperature. Calcium source solution D: Add 555 mg of calcium chloride (CaCl2) solid to 45 mL of deionized water, stir and sonicate until completely dissolved, and make up to 50 mL to obtain a 100 mM calcium chloride solution. Seal and store at room temperature. Enzyme and coenzyme mixture A: Prepare a 2 mg / mL solution by using deionized water to dissolve AOx, HRP, ALDH, and NAD. -1 The mixture, with an enzyme to NAD weight ratio of 1:7, was aliquoted and stored at 4°C, and then frozen at -20°C after use. Dispersant solution B: acetylated allenphosphoated hyaluronic acid (A-HA-ALN) was prepared to a concentration of 5 mg / mL. -1 The aqueous solution should be stored at 4°C and frozen at -20°C after use.

[0029] 2) Nanozyme preparation The four solutions A, B, C, and D were brought back to room temperature and then pumped at a rate of 0.5 mL / s. -1 The solution was synchronously injected into the reaction chamber of the multi-inlet vortex mixer (MIVM) at a rate of 150W ultrasound-assisted mixing for 15 seconds; after the reaction was completed, the reaction solution was injected at 10000 r / min. -1 Centrifuge, calculate the enzyme and coenzyme loading rates of the supernatant using a BCA kit, wash the lower precipitate three times with deionized water, add bovine serum albumin (BSA) as a lyophilization protectant, and freeze-dry to obtain CaPhy-NAD nanozyme (AOx / HRP / ALDH@CaPhy-NAD), and store in a sealed container at -20℃.

[0030] The structure of the CaPhy-NAD nanozyme prepared in Example 1 above was characterized, and the characterization results are as follows: FIG. 1 As shown, the hydrated particle size of this nanozyme is 382 nm, PDI=0.186, pore size distribution is 10~50 nm, and the total loading of enzyme and coenzyme is 10~60 wt%.

[0031] Example 2: Detection of the catalytic activity of CaPhy-NAD nanozyme on the substrate ethanol 1) Experimental Grouping Set up a blank control group (without enzyme) and an experimental group: AOx (0.05 mL, 2 mg / mL) -1 ), AOx@CaPhy-NAD (0.05mL, 20mg・mL -1 ), AOx / HRP@CaPhy-NAD (0.05mL, 20mg・mL -1 ), AOx / HRP / ALDH@CaPhy-NAD (CaPhy-NAD nanozyme prepared in Example 1, 0.05 mL, 20 mg / mL) -1 ).

[0032] 2) Experimental Procedure Each experimental group was treated with 1 mL of 10 mmol·mL solution. -1 Mix 1 mM ethanol solution pre-diluted with PBS (pH 7.0) and stir magnetically for 10 min until the reaction is complete. Use an ethanol detection kit, an acetaldehyde detection kit, and a hydrogen peroxide detection kit to determine the ethanol concentration, acetaldehyde production, and hydrogen peroxide production in each reaction system, respectively.

[0033] 3) Experimental results (see...) FIG. 2 ) In the ethanol degradation results, the AOx / HRP / ALDH@CaPhy-NAD group showed the fastest ethanol degradation rate, significantly higher than the single enzyme group and the dual enzyme group, demonstrating the synergistic catalytic effect of the enzyme cascade system. In the acetaldehyde generation results, the AOx / HRP / ALDH@CaPhy-NAD group produced the lowest amount of acetaldehyde, indicating that ALDH can effectively catalyze the decomposition of acetaldehyde and prevent its accumulation. In the hydrogen peroxide generation results, the AOx / HRP / ALDH@CaPhy-NAD group produced the lowest amount of hydrogen peroxide, indicating that HRP can effectively catalyze the decomposition of hydrogen peroxide and achieve the self-clearance of toxic intermediates.

[0034] Example 3: Stability detection of CaPhy-NAD nanozymes Using an equal amount of natural free enzyme (AOx) as the control group, and the CaPhy-NAD nanozyme prepared according to Example 1 as the experimental group, its acid and alkali resistance, high temperature resistance, and trypsin resistance were tested. The experimental results are shown in Figure 3, and the specific data are as follows: Acid and alkali resistance: After treatment in a strongly acidic environment at pH=1 for 10 min, the catalytic activity of the CaPhy-NAD nanozyme was 93.96%, which is 1.46 times that of the free enzyme; after treatment in an alkaline environment at pH=11 for 10 min, the catalytic activity of the nanozyme was 80.59%, which is 3.33 times that of the free enzyme. High temperature resistance: After heat treatment at 67℃ for 5 min, the catalytic activity of the CaPhy-NAD nanozyme was 71.82%, while that of the free enzyme was only 47.40%. Trypsin resistance: After 60 min of trypsin treatment, the CaPhy-NAD nanozyme retained 92.17% of the relative enzyme activity, which is 1.67 times that of the free enzyme.

[0035] The above results demonstrate that the stability of CaPhy-NAD nanozymes is far superior to that of natural free enzymes, and they can resist the action of acids and digestive enzymes in the digestive tract, making them suitable for oral administration.

[0036] Example 4: Biocompatibility Detection of CaPhy-NAD Nanozymes Using human umbilical vein endothelial cells (HUVECs) as the experimental subject, the cytotoxicity, biosafety after catalytic reaction, and hemolytic activity of the CaPhy-NAD nanozyme prepared in Example 1 were tested. The experimental results are shown in Figures 4 and 11. Cytotoxicity assay: The CaPhy-NAD nanozyme prepared in Example 1 was serially diluted with PBS to 0, 50, 100, 200, 300, 400, 500, 600, and 700 μg / mL. -1After co-incubating with HUVEC cells for 24 hours, cell viability was detected by CCK-8 assay. The results showed that cell viability remained above 90% in all concentration groups, with no obvious cytotoxicity. Biosafety after catalytic reaction: 400 μg / mL -1 The CaPhy-NAD nanozyme prepared in Example 1 was co-incubated with HUVEC cells in a culture medium containing 500 μM ethanol for 6 h. The results of live and dead staining of human umbilical vein endothelial cells (HUVEC) in different experimental groups were obtained. FIG. 4 The results showed that the number of dead cells in the CaPhy-NAD nanozyme group was much smaller than that in the free enzyme group, and the intensity of intracellular oxidative stress was significantly reduced. Hemolytic activity assay ( FIG. 11 CaPhy-NAD nanozymes at concentrations of 100–500 μg / mL -1 It showed no significant hemolytic activity within the concentration range, indicating good blood compatibility.

[0037] Example 5: Detection of Lyophilized Activity Retention of CaPhy-NAD Nanozymes Using bovine serum albumin (BSA) as a lyophilization protectant, two groups (+BSA and -BSA) were set up to freeze-dry the CaPhy-NAD nanozyme prepared in Example 1. The particle size change and enzyme activity before and after lyophilization were detected. The experimental results are shown in [Figure number missing]. FIG. 5 : The particle size of the nanozyme did not change significantly after freeze-drying, and its structure remained stable. After lyophilization, the +BSA group retained 99.32% of the catalytic activity of AOx and 94.37% of the catalytic activity of ALDH; while the -BSA group retained only about 50% of the enzyme activity after lyophilization, proving that the lyophilization protectant can effectively preserve the catalytic activity of nanozymes.

[0038] Example 6: Detection of the in vivo alcohol detoxification and liver protection effects of CaPhy-NAD nanozyme An acute alcohol poisoning model was established using 8-week-old BABL / C mice. FIG. 6 The complete experimental procedure for administering alcohol via gavage to mice, including sample collection and indicator detection, is demonstrated below: Model establishment: Anhydrous ethanol was diluted to 40% (v / v) with PBS and administered to each mouse by gavage at a concentration of 5.5 g / kg. -1 When alcohol is applied, mice lose their righting reflex, indicating intoxication.

[0039] Experimental groups: Blank group, PBS group, AOx@CaPhy-NAD group, AOx / HRP@CaPhy-NAD group, AOx / HRP / ALDH@CaPhy-NAD group.

[0040] Sample collection: Blood samples were collected from the orbital cavity of mice at 30, 60, 90 and 180 min after gavage administration of alcohol to detect the concentrations of ethanol and acetaldehyde in the blood; blood samples were collected from the eyeballs 24 h after gavage administration to detect blood biochemical indicators (ALT, AST, BUN, CRE), and the heart, liver, spleen, lung and kidney of mice were collected for tissue sectioning and H&E staining, and the liver was collected to detect the MDA content.

[0041] Experimental results: Blood ethanol / acetaldehyde concentration ( FIG. 7 Mice in the AOx / HRP / ALDH@CaPhy-NAD group had significantly lower blood ethanol and acetaldehyde concentrations than other groups within 30-180 minutes after drinking alcohol, and the rate of decrease was the fastest. liver oxidative stress ( FIG. 8 The MDA content in the liver of mice in the AOx / HRP / ALDH@CaPhy-NAD group was significantly reduced, and the ALT level returned to the normal range, demonstrating that the nanozyme can alleviate alcohol-induced liver oxidative damage. Blood biochemical indicators ( FIG. 9 The ALT and AST levels of mice in the AOx / HRP / ALDH@CaPhy-NAD group were significantly lower than those in the PBS group, while BUN and CRE showed no significant abnormalities, proving that the nanozyme did not cause damage to the liver and kidneys. Histopathology FIG. 10 H&E staining of heart, liver, spleen, lung, and kidney tissue sections from mice in each nanozyme group showed no obvious pathological damage, and there was no significant difference compared with the Blank group, demonstrating that the nanozyme has high in vivo biosafety.

[0042] In summary, this application successfully developed a CaPhy-NAD nanozyme. This nanozyme, achieved through a co-precipitation method combined with ultrasound-assisted processing, efficiently loaded and encapsulated ethanol oxidase, horseradish peroxidase, and aldehyde dehydrogenase (AOx / HRP / ALDH) into nanoparticles. The overall synthesis method is simple, easy to perform, and highly efficient. The outer shell of the CaPhy-NAD nanoparticles possesses excellent acid and alkali resistance and trypsin resistance, effectively protecting the encapsulated enzymes from degradation and inactivation in the digestive tract, providing crucial assurance for oral administration of this nanozyme. In the acidic environment of the stomach, the nanoparticles release a small amount of CaPhy-NAD nanoparticles in a sustained manner. 2+ It can act as an activator of acetaldehyde dehydrogenase, significantly enhancing the catalytic efficiency of ALDH. Simultaneously, AOx, HRP, and ALDH undergo highly efficient confined cascade catalytic reactions within the pores of the nanoparticles. The toxic intermediate H2O2 produced during alcohol metabolism can be simultaneously accelerated by HRP catalysis, promoting the oxidation and decomposition of ethanol and the oxidation of NADH, ultimately achieving self-clearance of toxic intermediates related to alcohol metabolism, as well as the reduction of NAD... +The CaPhy-NAD nanozyme exhibits excellent biocompatibility, completing its alcohol detoxification catalysis in vivo after oral administration and being naturally metabolized by the body without the risk of organ accumulation.

[0043] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A CaPhy-NAD nanozyme, characterized in that, The CaPhy-NAD nanozyme is composed of calcium phytate nanoparticles as a carrier, and ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase, and coenzyme loaded on the calcium phytate nanoparticle carrier; the calcium phytate nanoparticles release a small amount of Ca under acidic conditions. 2+ It can activate the catalytic activity of acetaldehyde dehydrogenase. Ethanol oxidase, acetaldehyde dehydrogenase, and horseradish peroxidase undergo a confined cascade reaction within the pores of calcium phytate nanoparticles, achieving the cyclic regeneration of coenzymes and simultaneously removing three toxic substances: ethanol, acetaldehyde, and hydrogen peroxide.

2. A method for preparing the CaPhy-NAD nanozyme according to claim 1, characterized in that, include: Using deionized water as a solvent, prepare the following solutions: A) a mixed solution containing ethanol oxidase, acetaldehyde dehydrogenase, horseradish peroxidase, and coenzyme; B) an aqueous solution containing a dispersant; C) an aqueous solution containing a phosphorus source; and D) an aqueous solution containing a calcium source. Solutions A, B, C, and D are simultaneously fed into a vortex mixer and mixed under ultrasonic assistance. After the reaction is completed, the mixture is centrifuged, washed, and lyophilized to obtain the CaPhy-NAD nanozyme.

3. The method for preparing CaPhy-NAD nanozymes according to claim 2, characterized in that, In solution A, the weight ratio of ethanol oxidase, acetaldehyde dehydrogenase, and horseradish peroxidase to coenzyme is 1:(4~10); the concentration of enzymes in solution A is 0.5~2.0 mg·mL. -1 .

4. The method for preparing CaPhy-NAD nanozymes according to claim 2, characterized in that, The calcium source is at least one of calcium chloride, calcium nitrate, calcium lactate, calcium gluconate, calcium citrate, calcium acetate, calcium formate, calcium gluconate, calcium ascorbate, calcium glycerophosphate, and calcium glutamate; the concentration of the calcium source in solution D is 50-100 mM.

5. The method for preparing CaPhy-NAD nanozymes according to claim 2, characterized in that, The phosphorus source is at least one of phytic acid, sodium phytate, tripolyphosphate, sodium tripolyphosphate, pyrophosphate, sodium pyrophosphate, inositol phosphate, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, potassium tripolyphosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; the concentration of the phosphorus source in solution C is 100~200mM.

6. The method for preparing CaPhy-NAD nanozymes according to claim 2, characterized in that, The dispersant is at least one selected from acetylated alendronate hyaluronic acid, polyacrylic acid, polyvinyl alcohol, polyoxyethylene-polyoxypropylene block copolymer, carboxymethyl cellulose, and sodium dodecyl sulfate; the concentration of the dispersant in solution B is 5-10 mg / mL. -1 .

7. The method for preparing CaPhy-NAD nanozymes according to claim 2, characterized in that, The weight ratio of solutions A, B, C, and D is 1:4:4:(6~7); the ultrasonic-assisted power is 50~200W; and the addition rate of each solution is 0.1mL·s. -1 ~1mL・s -1 The mixing reaction time is 10~20s.

8. The method for preparing CaPhy-NAD nanozymes according to claim 2, characterized in that, The weight ratio of solution A, solution B, solution C and solution D is 1:4:4:6; the ultrasonic auxiliary power is 100~150W.

9. The use of the CaPhy-NAD nanozyme according to claim 1 in the preparation of alcohol detoxification / hangover relief drugs.

10. A hangover remedy, characterized in that, The hangover remedy comprises a pharmaceutically effective amount of the CaPhy-NAD nanozyme as described in claim 1, and pharmaceutically acceptable excipients.

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