Oleanolic acid-modified HG-type pectin nanomicelles, their preparation methods and applications

Abstract

This invention provides oleanolic acid-modified HG-type pectin nanomicelles, their preparation method, and applications. The preparation method of the HG-type pectin nanomicelles includes: esterifying 3,3'-dithiodipropionic acid and oleanolic acid to obtain SS-OA; esterifying SS-OA and HG-type pectin to obtain oleanolic acid-modified HG-type pectin; dissolving the oleanolic acid-modified HG-type pectin and dispersing it ultrasonically to obtain oleanolic acid-modified HG-type pectin nanomicelles. The oleanolic acid-modified HG-type pectin nanomicelles of this invention combine the functional activities of HG-type pectin and oleanolic acid, as well as the redox responsiveness of 3,3'-dithiodipropionic acid, constructing nanomicelles that respond to ROS generated during alcohol metabolism. They exhibit amphiphilicity and good biocompatibility, and can be used to prepare drugs to alleviate chronic alcoholic liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry, and particularly relates to an oleanolic acid-modified HG-type pectin nanomicelles, its preparation method, and its application. Background Technology

[0002] Long-term excessive alcohol consumption damages liver cells. In the continuous cycle of damage and repair, the liver's structure changes, and its function suffers persistent impairment, leading to chronic alcoholic liver disease (ALD). The pathological development of ALD is a complex and continuous process involving multiple stages, each of which may overlap or occur simultaneously. These stages include hepatic steatosis, steatohepatitis, liver fibrosis, and cirrhosis, ultimately potentially progressing to hepatocellular carcinoma. Globally, with the increasing number of drinkers and the prevalence of alcoholism, the number of people suffering from alcoholic liver disease, its incidence, and mortality rates are showing a year-on-year upward trend. Despite significant research progress in the molecular pathological mechanisms of ALD, effective treatment strategies for ALD remain lacking, and currently, there is no successful way to prevent or treat ALD.

[0003] During the metabolism of alcohol in the liver, a large amount of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide ions, and hydroxyl radicals are produced, which can trigger oxidative stress in the liver. ROS can bind to proteins, altering their function and structural properties, thereby generating neoantigens to trigger immune responses, leading to inflammation and tissue damage. This binding effect produces lipid peroxides such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), the presence of which further exacerbates the severity of alcoholic liver injury. Therefore, in recent years, many researchers have been exploring ways to alleviate alcoholic liver injury by inhibiting the production of hepatic ROS and reducing oxidative stress. For example, Deming Li et al. (J Agr. Food Chem. , 2021, 69 (49):14856-14867) found that lactoferrin can enhance the redox regulation of cells in response to reactive oxygen species (ROS), thereby activating the body's antioxidant defense mechanisms. This process promotes autophagy, clears damaged proteins, and thus effectively alleviates alcohol-induced liver damage.

[0004] Polygalacturonic acid (HG-type pectin, PA) is a linear polysaccharide composed of α-1,4-D-galacturonic acid units. Scientific research has shown that HG pectin exhibits a series of beneficial physiological effects in vivo, including anti-cancer effects, immunomodulatory capacity, antioxidant properties, positive regulation of gut microbiota, and lowering of blood glucose levels. Oleanolic acid (OA) belongs to the pentacyclic triterpenoid class of compounds and possesses various pharmacological activities such as hepatoprotective activity, hypoglycemic activity, lipid-lowering activity, anti-cancer activity, and immune enhancement. Although oleanolic acid has significant potential in protecting liver health, its poor water solubility affects its bioavailability in vivo, thus limiting its application in health supplements or pharmaceuticals. All of the above evidence suggests that HG-type pectin and oleanolic acid are candidate natural compounds for preventing alcoholic liver injury. However, the efficacy of the combined use of these two components in preventing alcoholic liver injury has not yet been reported. Therefore, we hypothesize that the hydrophilic properties of HG-type pectin can enhance the solubility of oleanolic acid, thereby increasing its bioavailability. Simultaneously, we expect that these two components can work synergistically to play a positive role in preventing alcoholic liver injury. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide an oleanolic acid-modified HG-type pectin nanomicelle, its preparation method, and its applications. The oleanolic acid-modified HG-type pectin nanomicelle is a redox-responsive polymer micelle system composed of three components: HG-type pectin, 3,3'-dithiodipropionic acid, and oleanolic acid. The purpose is to utilize the hydrophilic HG-type pectin to improve the water solubility of oleanolic acid and increase its bioavailability. Simultaneously, 3,3'-dithiodipropionic acid is used as a redox-responsive bond, endowing the entire system with redox sensitivity. The nanomicelle delivery system constructed in this invention has certain guiding significance for alleviating chronic alcoholic liver injury.

[0006] To achieve the above objectives, the present invention provides an oleanolic acid-modified HG-type pectin nanomicelles, wherein the oleanolic acid-modified HG-type pectin nanomicelles have the following structural formula (Formula I):

[0007]

[0008] (I).

[0009] The oleanolic acid-modified HG-type pectin nanomicelles described above have a molecular weight of 25,000-50,000 Da.

[0010] The oleanolic acid-modified HG-type pectin nanomicelles mentioned above further have an oleanolic acid content of 7.0% to 8.5%.

[0011] The oleanolic acid-modified HG-type pectin nanomicelles described above further have a particle size of 400–500 nm.

[0012] The above-mentioned oleanolic acid-modified HG-type pectin nanomicelles further have a critical micelle concentration of 15.0–20.0 μg / mL.

[0013] Based on a general technical concept, the present invention also provides a method for preparing the oleanolic acid-modified HG-type pectin nanomicelles, comprising the following steps:

[0014] S1. 3,3'-dithiodipropionic acid and oleanolic acid are esterified to obtain SS-OA;

[0015] S2. The SS-OA and HG type pectin are subjected to esterification reaction to obtain oleanolic acid modified HG type pectin;

[0016] S3. Dissolve the oleanolic acid-modified HG type pectin and disperse it by ultrasonication to obtain oleanolic acid-modified HG type pectin nanomicelles.

[0017] The SS-OA has the following structural formula (II):

[0018]

[0019] (II).

[0020] The above preparation method, further, wherein S1 specifically includes:

[0021] S1-1. Dissolve 3,3'-dithiodipropionic acid in an organic reagent, add an acid-binding agent, and mix to obtain a mixed solution;

[0022] S1-2. Oleanolic acid is added to the mixed solution, and a carboxyl activating reagent is added dropwise. The reaction is carried out at room temperature to obtain SS-OA.

[0023] In the above preparation method, the molar ratio of 3,3'-dithiodipropionic acid, oleanolic acid, acid-binding agent, and carboxyl activating reagent is (1.0–1.5):1:(0.2–0.6):(1.2–1.8). Furthermore, the molar ratio of 3'-dithiodipropionic acid, oleanolic acid, 4-pyrrolylpyridine, and carboxyl activating reagent is 1.2:1:0.5:1.5.

[0024] In the above preparation method, the acid-binding agent is one or more of 4-dimethylaminopyridine, pyridine, and 4-pyrrolidinylpyridine.

[0025] In the above preparation method, the carboxyl activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0026] Furthermore, in the above preparation method, the reaction time at room temperature is 12 h to 48 h.

[0027] The above preparation method, further, wherein step S2 specifically includes:

[0028] S2-1. Dissolve SS-OA in an organic reagent, add an acid-binding agent, and obtain a mixture;

[0029] S2-2. Add HG type pectin to the mixture, add carboxyl activating reagent dropwise, and react at room temperature to obtain PA-SS-OA polymer.

[0030] In the above preparation method, the molar ratio of SS-OA, the acid-binding agent, and the carboxyl activating reagent is (1.0–1.5):(0.2–0.6):(1.2–1.8). Furthermore, the molar ratio of SS-OA, 4-pyrrolidinylpyridine, and the carboxyl activating reagent is 0.20 mmol:0.08 mmol:0.24 mmol.

[0031] In the above preparation method, the amount of HG type pectin used is 300 mg.

[0032] In the above preparation method, the acid-binding agent is one or more of 4-dimethylaminopyridine, pyridine, and 4-pyrrolidinylpyridine; the carboxyl activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0033] Furthermore, in the above preparation method, the reaction time at room temperature is 12 h to 48 h.

[0034] In the above preparation method, the ultrasonic power of the ultrasonic dispersion is 200 W to 450 W, and the ultrasonic time is 2 min to 10 min.

[0035] Based on a general technical concept, the present invention also provides the application of the oleanolic acid-modified HG-type pectin nanomicelles in the preparation of drugs or health products for alleviating chronic alcoholic liver injury.

[0036] The innovation of this invention lies in the fact that, since OA contains both carboxyl and hydroxyl groups, it preferentially undergoes self-polymerization during its reaction with PA due to the steric hindrance of PA, generating -[OA-OA]n-, thus preventing successful grafting of OA onto PA. Therefore, an innovative aspect of this invention is that, during the synthesis process, equimolar amounts of 3,3'-dithiodipropionic acid (SS) and OA are first condensed to obtain the product SS-OA. Then, SS-OA is further grafted onto PA through condensation to obtain the final product PA-SS-OA. This grafting strategy avoids the cumbersome process of adding and removing protecting groups.

[0037] Compared with the prior art, the advantages of the present invention are as follows:

[0038] (1) This invention provides an oleanolic acid-modified HG-type pectin nanomicelle, which combines the functional activity of HG-type pectin and oleanolic acid with the redox responsiveness of 3,3'-dithiodipropionic acid, and constructs nanomicelles that can respond to ROS generated during alcohol metabolism, and has amphiphilicity and good biocompatibility.

[0039] (2) This invention provides the application of oleanolic acid-modified HG-type pectin nanomicelles in the preparation of drugs to improve alcoholic liver injury. Compared with mice with alcoholic liver injury, mice gavaged with the nanomicelles of this invention showed a 11.42% decrease in liver index, a 63.0% decrease in serum ALT, 59.0% decrease in serum ALT, 59.0% decrease in serum ALT, 35.9% decrease in serum ALT, 59.0% decrease in serum TG, and 35.9% decrease in serum ALT, 59.0% decrease in serum TG, and 35.9% decrease in serum ALT, 59.0% decrease in serum ALT, 59.0% decrease in serum TG, and 35.9% decrease in serum ALT, 59.0% decrease in serum ALT, 59.0% decrease in serum ALT, 59.9% decrease in serum ALT, 59.9% decrease in serum ALT, 59.9% decrease in serum ALT, 59.9% decrease in serum ALT, 59.9% decrease in serum ALT, 59.9% decrease in serum ALT, 59.9% decrease in serum ALT, and 3 ... Attached Figure Description

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0041] Figure 1 This is a synthetic route diagram of oleanolic acid-modified HG-type pectin (PA-SS-OA) in Example 1 of the present invention.

[0042] Figure 2 These are the UV-Vis spectra of PA, OA, DTDPA, and PA-SS-OA in Experiment 1 of this invention.

[0043] Figure 3 These are the Fourier transform infrared spectra of PA, OA, DTDPA, and PA-SS-OA in Experiment 1 of this invention.

[0044] Figure 4 These are the hydrogen nuclear magnetic resonance spectra of PA, OA, DTDPA, and PA-SS-OA from Experiment 1 of this invention.

[0045] Figure 5 These are the XRD patterns of PA, OA, DTDPA, and PA-SS-OA from Experiment 3 of this invention.

[0046] Figure 6 These are the DSC diagrams of PA, OA, DTDPA, and PA-SS-OA in Experiment 3 of this invention.

[0047] Figure 7 These are the particle size distribution diagrams (A in the figure), average particle size diagram (B in the figure), average PDI diagram (C in the figure), and average Zata potential diagram (D in the figure) of PA and PA-SS-OA in Experiment 4 of this invention.

[0048] Figure 8 These are transmission electron microscope images of PA and PA-SS-OA from Experiment 4 of this invention.

[0049] Figure 9 The figures show the fluorescence emission spectra of PA-SS-OA at different concentrations (from 0.0001 to 1 mg / mL) in Experiment 5 of this invention (Figure A) and the simulation calculation results of the critical micelle concentration (Figure B).

[0050] Figure 10 This figure shows the degradation of PA-SS-OA and OA release under redox conditions in Experiment 6 of this invention. Figure A represents the average particle size of PA-SS-OA after treatment with different concentrations of H2O2 or GSH for 24 hours; Figure B represents the PDI of PA-SS-OA after treatment with different concentrations of H2O2 or GSH for 24 hours; Figure C represents the Zeta potential of PA-SS-OA after treatment with different concentrations of H2O2 or GSH for 24 hours; and Figure D represents the amount of OA released from the PA-SS-OA micelle solution after treatment with different concentrations of H2O2 or GSH for 24 hours.

[0051] Figure 11 This is a diagram showing the results of the red blood cell hemolysis experiment in Experiment Seven of this invention.

[0052] Figure 12 The results of the cytotoxicity test in Experiment 7 of this invention are as follows.

[0053] Figure 13 The results show the changes in mouse body weight in different treatment groups in Experiment 8 of this invention.

[0054] Figure 14 The liver index of mice in different treatment groups in Experiment 8 of this invention.

[0055] Figure 15 The images show the pathological morphology and HE staining of mouse liver tissue from different treatment groups in Experiment 8 of this invention.

[0056] Figure 16 The figures show the serum levels of alanine aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B), triglycerides (TG) (C), and liver levels of alcohol dehydrogenase (ADH) (D), acetaldehyde dehydrogenase (ALDH) (E), and superoxide dismutase (SOD) (F) in mice from different treatment groups in Experiment 8 of this invention.

[0057] Figure 17 The results of changes in glutathione (GSH) (A in the figure), catalase (CAT) (B in the figure), malondialdehyde (MDA) (C in the figure), tumor necrosis factor (TNF-α) (D in the figure), interleukin-6 (IL-6) (E in the figure), and interferon-γ (IFN-γ) (F in the figure) in the livers of mice in each experimental group in Experiment 8 of this invention.

[0058] Figure 18 The relative mRNA expression results of mice in each experimental group in Experiment 8 of this invention are as follows: Nrf2 (A in the figure), GCLC (B in the figure), HO-1 (C in the figure) and NQO1 (D in the figure).

[0059] Figure 19 The relative protein expression results of Nrf2 (A in the figure), GCLC (B in the figure), HO-1 (C in the figure) and NQO1 (D in the figure) in each experimental group of mice in Experiment 8 of this invention.

[0060] Figure 20 The results are Western blot analysis of Nrf2, GCLC, HO-1 and NQO1 mice in each experimental group of Experiment 8 of this invention. Detailed Implementation

[0061] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0062] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0063] Example 1

[0064] An oleanolic acid-modified HG-type pectin nanomicelle of the present invention has the following structural formula I:

[0065]

[0066] (I)

[0067] Synthetic route according to Figure 1 The process includes the following steps:

[0068] (1) Synthesis of Product 1 (SS-OA): 3 g (14.27 mmol) of 3,3'-dithiodipropionic acid was dissolved in 50 mL of pyridine, and 4-pyrrolidinylpyridine (4-PPY, 0.5 eq) was added. The mixture was stirred for 0.5 h, and then oleanolic acid (OA, 1.0 eq) was added. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.5 eq) was added dropwise. The reaction was carried out at room temperature for 24 h. The reaction solution was concentrated by rotary evaporation under reduced pressure. The residue was dispersed in 50 mL of HCl (2 mol / L), and then extracted with ethyl acetate (3 × 40 mL). The organic layers were combined and washed successively with water and saturated brine. The organic layers were dried over anhydrous Na2SO4. After filtration and vacuum drying, a white solid SS-OA was obtained for later use.

[0069] SS-OA has the following structural formula (Formula II):

[0070]

[0071] (II)

[0072] (2) Synthesis of Product 2 (PA-SS-OA): 129.8 mg (0.2 mmol) of SS-OA was dissolved in 20 mL of DMSO, DMAP (0.5 eq) was added, and the mixture was stirred for 0.5 h. Then, 300 mg of pre-dissolved HG-type pectin (PA) was added, followed by dropwise addition of pre-dissolved EDCI (1.5 eq). The reaction was carried out at room temperature for 48 h. After the reaction was completed, an appropriate amount of anhydrous ethanol was added and stirred vigorously until a white precipitate appeared. The supernatant was removed by centrifugation. The precipitate was redissolved in 20 mL of DMSO and dialyzed in ultrapure water for 5 days using a dialysis bag (MWCO, 3500 Da). The dialyzed solution was then freeze-dried to obtain the PA-SS-OA polymer.

[0073] Experiment 1: Verify whether oleanolic acid in Example 1 was successfully grafted onto HG type pectin.

[0074] 1.1 Ultraviolet-Visible Spectrophotometric Analysis:

[0075] Experimental procedure: PA, OA, and PA-SS-OA samples with a concentration of 0.1 mg / mL were prepared, and the spectra of each sample in the range of 200–800 nm were measured using a UV-Vis spectrophotometer (UV-1800, Shimadzu Instruments Co. Ltd., Suzhou, China).

[0076] Figure 2 This is a UV-Vis spectrum. As can be seen from the graph, a new absorption peak appears at 282 nm in the -SS-OA spectrum. This peak is a characteristic absorption peak of OA.

[0077] 1.2 Infrared Spectroscopy Analysis:

[0078] Experimental procedure: Sample thin sections were prepared using the KBr pellet technique, and then the infrared spectra of the samples were scanned using a Fourier transform infrared spectrometer (Nicolet-iS5, Thermo Fisher, US) (wavenumber range 4000 cm⁻¹). -1 Up to 500cm -1 The resolution is set to 4 cm. -1 ).

[0079] Figure 3 This is an infrared spectrum analysis diagram. According to the Fourier transform infrared spectrum results, a new absorption peak appeared at 1690 cm⁻¹ in the PA-SS-OA spectrum. This peak is attributed to the carbonyl stretching vibration of OA and DTDPA.

[0080] 1.3 Nuclear Magnetic Resonance Spectroscopy Analysis:

[0081] Experimental procedure: The sample was diluted in 0.5 mL D2O and 0.1 mL DMSO, and the 1H NMR spectrum of the sample was measured at room temperature using a nuclear magnetic resonance spectrometer (Avance III 400M, Bruker, Germany). The chemical shift is expressed as δ (ppm).

[0082] Figure 4 Yes, the 1H NMR spectroscopy results show new chemical shift peaks at δ 0.87, 0.96, 1.05, and 1.22 in the PA-SS-OA spectrum. These peaks are attributed to hydrogen atoms on the multiple tertiary methyl groups of OA. Simultaneously, new chemical shift peaks also appear at δ 2.8 and 2.9, which are attributed to hydrogen atoms on the methylene groups in DTDPA. These results indicate the successful synthesis of PA-SS-OA.

[0083] Experiment 2: Determine the grafting rate of PA-SS-OA prepared in Example 1.

[0084] Experimental procedure: The absorbance of different concentrations of oleanolic acid at a wavelength of 282 nm was measured using a UV-Vis spectrophotometer, and a standard curve was established (y = 0.01434x + 0.00161, R0). 2 =0.9993). The absorbance of PA-SS-OA at 282 nm was measured, and the OA content was calculated using a regression equation. The OA grafting rate was calculated as follows: Grafting rate = (mass of OA groups / mass of modified polymer) × 100%.

[0085] The measured grafting rate of PA-SS-OA was (7.92±0.65)%.

[0086] Experiment 3: XRD and DSC analysis were performed on the prepared PA-SS-OA.

[0087] Experimental Procedure: An X-ray diffractometer (D2 PHASER, Bruker, Germany) was used. Conditions: Cu target, Kα rays, λ = 0.15 nm, tube voltage 40 kV, current 40 mA, scan range 5°–90°, step speed 5° / min. A differential scanning calorimeter (DSC 214 Polyma, NETZSCH, Germany) was used. Approximately 5 mg of sample was placed in an aluminum dish, with a blank dish as a control. Nitrogen was used as the carrier gas at a flow rate of 50 mL / min, and the temperature was increased from 20°C to 350°C at a scan rate of 10°C / min.

[0088] The variations in the intensity and full width at half maximum (FWHM) of the line diffraction peaks reflect the degree of crystallinity and amorphization of the particles. Figure 5 The XRD results show that PA-SS-OA exhibits a broad diffraction peak at 14.5°, a typical characteristic of amorphous diffraction, indicating that the PA crystal structure was disrupted after OA grafting. However, no characteristic peaks for OA, DTDPA, and TDPA were found in the XRD pattern of PA-SS-OA, suggesting that OA and DTDPA were successfully encapsulated within the structure in an amorphous form.

[0089] DSC analysis is used to assess the thermal stability and crystallization transformation of samples, reflecting the chemical and physical structural stability of substances at high temperatures. Figure 6 The results of DSC analysis of PA-SS-OA show that, compared with PA, the exothermic peak of PA-SS-OA shifts to a lower temperature, indicating that the original structure of PA changes after grafting and the crystallinity decreases.

[0090] Experiment 4: The particle size, zeta potential, and polydispersity index (PDI) of PA-SS-OA were determined and imaged using transmission electron microscopy.

[0091] 4.1 Experimental procedure for particle size, zeta potential, and polydispersity index (PDI) detection: Prepare 0.1 mg / mL solutions of each sample, and then use a dynamic light scattering particle size analyzer (Zetasizer Nano ZS 90, Malvern, UK) to measure the particle size distribution (PSD), zeta potential, and polydispersity index (PDI) of the samples.

[0092] Figure 7 Figure 1 shows the particle size (A), average particle size (B), PDI (C), and zeta potential (D) of PA-SS-OA. As can be seen from the figure, the average particle size of PA-SS-OA is 446.20 ± 43.19 nm, the PDI is 0.3357 ± 0.0213, and the zeta potential is -29.98 ± 3.04. The particle size of PA-SS-OA is larger than that of PA. This is likely because after OA is grafted onto PA, the strong hydrophobic properties of OA result in a more porous micelle structure, thus increasing the particle size. The relatively small PDI value of PA-SS-OA indicates a more uniform micelle size distribution.

[0093] 4.2 Experimental steps for observing the morphology and size of samples using a transmission electron microscope (H-7650, Hitachi, Japan): Take a certain volume of sample aqueous solution (0.5 mg / mL) and gently drop it onto a copper grid covered with a carbon support film. Use filter paper to absorb the excess solution. After air drying, counterstain with phosphotungstic acid solution for 2 minutes. Then, allow it to air dry at room temperature before observing it under a transmission electron microscope.

[0094] Figure 8 The images show the results of transmission electron microscopy (TEM). In the figure, A represents PA, and B represents PA-SS-OA. As can be seen from the images, both PA and PA-SS-OA exhibit an approximately spherical morphology under TEM. The particle size of PA-SS-OA is larger than that of PA, indicating that the morphology of PA becomes more porous after grafting OA onto it.

[0095] Experiment 5: Determination of the critical micelle concentration of PA-SS-OA.

[0096] Experimental steps: First, accurately prepare a solution with a concentration of 6.0 × 10⁻⁶. -5A 1 mol / L pyrene-acetone solution was prepared. Next, 50 μL of the prepared pyrene-acetone solution was added to nine 10 mL volumetric flasks and placed in a fume hood to allow the acetone to evaporate naturally. Then, a 1 mg / mL PA-SS-OA solution was prepared. Subsequently, different volumes of sample solution were transferred to each volumetric flask containing pyrene, and the volume was adjusted to 10 mL with deionized water. The samples were then allowed to stand at room temperature for 2 hours. To ensure sufficient pyrene inclusion in the hydrophobic core of the micelles, the samples were placed in a 37°C constant-temperature shaker for 1 hour, followed by sonication at room temperature for 1 hour. Finally, the fluorescence intensity of the samples was measured using a fluorescence spectrophotometer (e.g., RF-5301PC, Shimadzu, Japan) (excitation wavelength 334 nm, emission wavelength range 350-450 nm, excitation slit and emission slit 3 nm and 5 nm, respectively). The fluorescence intensity was recorded at 373 nm (I...). 373 ) and 384 nm (I 384 The fluorescence intensity at point (I) is plotted on the x-axis as the logarithm of the solution concentration (Log C). 373 with I 384 The ratio (I) 373 / I 384 Use y as the vertical axis to plot the image.

[0097] Figure 9 The results of the critical micelle concentration determination are shown in Figure A, which is the fluorescence emission spectrum of PA-SS-OA at different concentrations (from 0.0001 to 1 mg / mL), and Figure B is the simulation calculation result of the critical micelle concentration. It can be seen from the figure that the lower the concentration, the more stable the micelles are. The measured CMC value of PA-SS-OA is 0.0171 mg / L.

[0098] Experiment 6: Investigate the degradation of PA-SS-OA and the release behavior of oleanolic acid under oxidative / reduction conditions.

[0099] 6.1. Simulating oxidation and reduction environments using H2O2 and GSH. Experimental procedure: First, certain amounts of hydrogen peroxide (H2O2, final concentrations of 20 µM and 1 mM, respectively) and glutathione (GSH, final concentrations of 1 mM and 5 mM, respectively) were added to a micelle solution prepared with distilled water. Then, we used a dynamic light scattering particle size analyzer (Zetasizer Nano ZS90, Malvern, UK) to monitor the changes in micelle size, Zeta potential, and PDI at 0 h, 4 h, 12 h, and 24 h.

[0100] 6.2. A systematic evaluation of the in vitro OA release behavior of PA-SS-OA micelles under oxidative and reducing conditions was conducted using dialysis. Experimental procedure: First, 5 mg of micelles were dissolved in 5 mL of the corresponding release medium (distilled water containing 20 µM, 1 mM, 1 mM, and 5 mM GSH, respectively). The solution was then rapidly transferred into a dialysis bag with a molecular weight of 3.5 kDa. The dialysis bag was immersed in 50 mL of the corresponding release medium and shaken at 37 °C and 100 rpm in a constant-temperature shaker. At preset time points (0.5, 4, 12, 24, and 36 hours), 5.0 mL of release medium was removed, and an equal volume of fresh medium was added to maintain a constant volume. Finally, the OA content in the release medium was quantitatively analyzed using a UV-Vis spectrophotometer (UV-1800, Shimadzu Instruments Co., Ltd., Suzhou, China).

[0101] Depend on Figure 10 This figure shows the degradation of PA-SS-OA and the release of OA under redox conditions in Example 1 of the present invention (A in the figure is the average particle size of PA-SS-OA after treatment with different concentrations of H2O2 or GSH for 24 hours; B in the figure is the PDI of PA-SS-OA after treatment with different concentrations of H2O2 or GSH for 24 hours; C in the figure is the Zeta potential of PA-SS-OA after treatment with different concentrations of H2O2 or GSH for 24 hours; D in the figure is the amount of OA released from the PA-SS-OA micelle solution after treatment with different concentrations of H2O2 or GSH for 24 hours).

[0102] As shown in the figure, after 24 hours of treatment with 20 μM H₂O₂ and 1 mM GSH, the particle size, Zeta potential, and PDI of the PA-SS-OA micelle solution did not show significant changes. After treatment with 1 mM H₂O₂ and 5 mM GSH, the particle size of the PA-SS-OA micelle solution increased from 461.6 nm at 0 h to 1154.61 nm and 969.78 nm at 24 h, respectively; the PDI increased from 0.3363 to 0.6128 and 0.6021, respectively; and the Zeta potential decreased from -31.28 mV to -11.32 mV and -14.29 mV, respectively. These results indicate that under the above conditions, the sulfur bonds in the PA-SS-OA micelle solution may break, leading to the detachment of the PA shell from the micelles, which in turn changes the hydrophilic-hydrophobic ratio of the micelles, ultimately resulting in micelle dissociation. This suggests that PA-SS-OA exhibits dual redox response. Under conditions of 20 μM H₂O₂ and 1 mM GSH, the OA release rates of the PA-SS-OA micelle solution after 48 hours were 9.65% and 7.68%, respectively. These data indicate that these micelles exhibit good stability under normal physiological conditions, meeting the design requirements. However, under conditions of 1 mM H₂O₂ and 5 mM GSH, the OA release rates of the PA-SS-OA micelle solution after 48 hours were even higher, reaching 80.61% and 75.17%, respectively. These results indicate that the PA-SS-OA micelle solution can release OA more quickly and effectively in response to both oxidative and reducing stimuli.

[0103] Experiment 7: Evaluation of the toxicological properties of PA-SS-OA.

[0104] 7.1 Hemolysis Assay: Different concentrations (0.1, 0.5, 1 mg / mL) of PA-SS-OA solution were mixed with an equal volume of 2% erythrocyte suspension and incubated at 37℃ and 200 rpm for 0.5, 1, 2, and 3 h respectively. The samples were removed and centrifuged at 1500 rpm for 8 min to remove intact erythrocytes. 100 µL of the supernatant was transferred to a 96-well plate, and the absorbance at 540 nm was measured using a microplate reader (Synergy H1, BIOTEK, USA). The erythrocyte suspension group incubated with PBS buffer (pH 7.4) served as the negative control, and the deionized water group served as the positive control. The hemolysis rate (%) was calculated using the formula: Hemolysis rate (%) = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100%.

[0105] Figure 11The hemolysis assay is used to assess the degree of damage the material causes to red blood cells. As shown in the figure, when the PA-SS-OA micelle concentration is between 0.5 mg / mL and 2.0 mg / mL, and the hemolysis time is between 0.5 hours and 3 hours, the hemolysis rate is less than 5%. This indicates that PA-SS-OA does not cause hemolysis during in vivo circulation and possesses good biocompatibility.

[0106] 7.2. Cell Toxicology Experiments: Normal mouse hepatocytes (AML12) (5000 cells / well) were seeded into 96-well plates and incubated overnight at 37°C in a 5% CO2 cell culture incubator (3111, Thermo, USA). Cells were then incubated for 24 h and 48 h with different concentrations of the sample (2.5, 5, 10, 20 μg / mL). Cell viability was then determined using a CCK-8 assay kit (Dojindo, Kumamoto, Japan). 10 μL of CCK-8 solution was added to each well of the 96-well plate, and after incubation for 2 h, the absorbance at 450 nm was measured using a microplate reader (synergy H1, BIOTEK, USA). Untreated cells served as a positive control, and each sample was tested in 6 replicates. Cell viability was calculated as follows: Cell viability (%) = (OD sample - OD blank) / (OD positive control - OD blank) × 100%.

[0107] Figure 12 The results of the cytotoxicity test are shown in the figure. As can be seen from the figure, within the concentration range of 0.5 to 20 µg / mL, after 24 hours and 48 hours of co-incubation with PA-SS-OA and AML-12 cells, the cell viability remained above 95%, which indicates that PA-SS-OA has no significant toxic effect on AML-12 cells.

[0108] Experiment 8: Animal experiments to evaluate the effect of PA-SS-OA in alleviating chronic alcoholic liver injury.

[0109] Experimental steps:

[0110] (1) Grouping and administration: Seventy C57BL / 6J mice were randomly divided into seven groups of ten each. Group 1 (normal group), Group 2 (alcohol model group), Group 3 (oleanolic acid, OA, 0.04 mg / 20 g), Group 4 (polygalacturonic acid, PA, 1 mg / 20 g), Group 5 (polygalacturonic acid and oleanolic acid mixture, PA: 1 mg / 20 g; OA: 0.04 mg / 20 g), Group 6 (low-dose administration group, PA-SS-OA, 0.5 mg / 20 g), and Group 7 (high-dose administration group, PA-SS-OA, 1 mg / 20 g).

[0111] (2) Modeling: Mice were administered the drug by gavage at a fixed time each afternoon according to the set dosage (the normal group and the model group were given the same volume of physiological saline). After each group was administered the drug, except for the normal group which was given the same volume of physiological saline, the mice in the other groups were administered 52% Niulanshan Erguotou by gavage. The dosage was 0.16 mL / 20 g in the first week, 0.18 mL / 20 g in the second week, 0.20 mL / 20 g in the third week, and 0.22 mL / 20 g from the fourth to the sixth week. During the modeling period, the weight of the mice was weighed and recorded every 7 days.

[0112] (3) Specimen collection: At the end of week 6, all mice were fasted overnight but allowed free access to water for 12 hours. Blood was collected from the heart after abdominal anesthesia and placed in 1.5 mL EP tubes. After standing at room temperature, the blood was centrifuged at 12,000 rpm for 10 min. The supernatant was collected, aliquoted, and stored at -80℃. The liver was dissected and the entire liver tissue was cut and weighed. The right lobe of the liver was cut off with a blade and fixed with 4% paraformaldehyde. The remaining liver tissue was stored at -80℃.

[0113] (4) Calculation of liver index and observation of sections: Liver index (%) = liver weight (g) / body weight (g) × 100%. After dehydration, embedding, sectioning and HE staining, the morphology of the liver of each group of mice was observed using an optical microscope (Primostar 3, ZEISS, Germany).

[0114] (5) Serum and liver biochemical index determination: The ALT, AST, and TG levels in the serum of each group were detected according to the methods of the purchased kits. Approximately 0.1g of liver tissue was cut, 1mL of physiological saline was added, and the tissue was thoroughly ground using a homogenizer (JXFSTPRP-64, Shanghai Jingxin, China). The homogenate was then centrifuged at 8000 r / min for 15 min at 4°C to obtain the supernatant. The levels of ADH, ALDH, SOD, CAT, GSH, MDA, ROS, TNF-α, IFN-γ, and IL-6 in the liver tissue were determined according to the methods of the purchased kits.

[0115] (6) RNA extraction and mRNA quantification: Total RNA was extracted from the liver tissue of experimental mice, and the mRNA was reverse transcribed into cDNA. Subsequently, the mRNA expression levels of Nrf2, GCLC, HO-1, and NQO1 were detected using a real-time PCR instrument (ABI 7500, Applied Biosystems, USA), with β-actin used as an internal reference gene. —ΔΔCt The method calculates the relative mRNA expression levels of each gene group.

[0116] (7) Expression of Nrf2, GCLC, HO-1 and NQO1 proteins in liver tissue: Total protein was extracted from liver tissue using the RIPA lysis method, and the protein concentration of the samples was determined using the BCA protein concentration kit. 30 μg of total protein was loaded onto a membrane and separated by 10% SDS-PAGE electrophoresis. The membrane was then electroporated to a 0.45 μm PVDF membrane and blocked with protein-free rapid blocking powder for 30 min. Primary antibodies Nrf2 (1:1000), GCLC (1:1000), HO-1 (1:2000), NQO1 (1:1000), and β-actin (1:2000) were added, and the membrane was incubated overnight at 4 °C. After washing with TBST, secondary antibody Ig (1:5000) was added, and the membrane was incubated at room temperature for 1.5 h. After washing with TBST, the membrane was placed in a V3 Western blot detection and analysis system, and ECL chemiluminescent substrate was added for exposure and imaging. Protein expression was analyzed using Bio-Rad Image Lab Software 5.2 with total protein quantification (relative expression level calculated using β-actin as an internal reference).

[0117] Figure 13 The figure shows the changes in body weight of mice in different treatment groups. As can be seen from the figure, after gavage administration of alcohol and other drugs, except for group 1 (normal group), group 4 (PA group), and group 5 (PA / OA), the average body weight of mice in all other groups decreased after the first week. This indicates that gavage administration of alcohol affects the appetite and food intake of mice, thus affecting body weight changes. From the second week onwards, the average body weight of mice in all groups increased weekly.

[0118] Figure 14 The figure shows the liver index of mice in different treatment groups. As can be seen from the figure, compared with group 2 (alcohol model group), the liver index of group 6 (low-dose PA-SS-OA group) and group 7 (high-dose PA-SS-OA group) decreased significantly by 9.96% and 11.42%, respectively, indicating that PA-SS-OA can reverse this abnormality.

[0119] Figure 15The images show the pathological morphology and HE staining of mouse liver tissue from different treatment groups. As can be seen from the images: In the pathological morphology images, group 2 showed fat droplets and vacuoles of varying sizes on the surface of the liver tissue. The surface condition of the liver tissue in the other groups was not significantly different from group 1. In the HE staining images, in group 2, hepatocytes were blurred, nuclei were shrunken or even disappeared, cells were swollen, and intracellular vacuoles were present, indicating significant pathological changes in the liver tissue of the alcohol model group. In groups 6 and 7, the liver tissue structure and hepatocyte arrangement were more orderly, and the number of inflammatory cells and fat droplets and vacuoles were significantly reduced compared to group 2. In conclusion, this suggests that PA-SS-OA can alleviate alcohol-induced liver damage to a certain extent.

[0120] Figure 16 The figures show the serum levels of alanine aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B), triglycerides (TG) (C), and liver levels of alcohol dehydrogenase (ADH) (D), aldehyde dehydrogenase (ALDH) (E), and superoxide dismutase (SOD) (F) in mice from different treatment groups. Different letters in the figures indicate that the differences between the groups are statistically significant at the p < 0.05 level.

[0121] Figure 17 The figures show the changes in glutathione (GSH) (A) in the liver of mice in each experimental group, as well as the changes in catalase (CAT) (B), malondialdehyde (MDA) (C), tumor necrosis factor (TNF-α) (D), interleukin-6 (IL-6) (E), and interferon-γ (IFN-γ) (F). Different letters in the figures indicate that the differences between the groups are statistically significant at the p < 0.05 level.

[0122] Figure 18 The figures show the relative mRNA expression of Nrf2 (A in the figure), GCLC (B in the figure), HO-1 (C in the figure), and NQO1 (D in the figure) in mice from each experimental group. Different letters in the figure indicate that the differences between the groups are statistically significant at the p<0.05 level.

[0123] Figure 19 The figures show the relative protein expression of Nrf2 (A in the figure), GCLC (B in the figure), HO-1 (C in the figure), and NQO1 (D in the figure) in mice from each experimental group. Different letters in the figure indicate that the differences between the groups are statistically significant at the p<0.05 level.

[0124] Figure 20 The results are Western blot analysis of Nrf2, GCLC, HO-1 and NQO1 mice from each experimental group.

[0125] As shown in the figure, compared with group 2, the liver index of mice in group 7 decreased by 11.42%, and the serum ALT, AST, and TG levels decreased by 63.0%, 59.0%, and 35.9%, respectively. The activities of ALDH, SOD, and CAT in the liver increased by 48.1%, 31.6%, and 34.9%, respectively, while the levels of MDA, TNF-α, IL-6, and IFN-γ in the liver decreased by 24.9%, 36.9%, 32.6%, and 41.8%, respectively. Compared with group 2, the protein expression levels of Nrf2 and its downstream targets GCLC, HO-1, and NQO1 in groups 6 and 7 showed a significant restorative upregulation. This indicates that PA-SS-OA can activate the Nrf2 antioxidant signaling pathway, effectively reducing or reversing oxidative stress and inflammation induced by alcohol metabolism, promoting the body's condition to near-normal levels, thereby alleviating alcohol damage to the liver. This protective effect is superior to oleanolic acid alone.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. An oleanolic acid-modified HG-type pectin nanomicelle, characterized in that, The oleanolic acid-modified HG-type pectin nanomicelles have the following structural formula (Formula I): （Ⅰ）； The molecular weight of the HG-type pectin nanomicelles is 25,000 Da to 50,000 Da; And / or, the percentage content of oleanolic acid in the HG-type pectin nanomicelles is 7.0% to 8.5%; And / or, the particle size of the HG-type pectin nanomicelles is 400 nm to 500 nm; And / or, the critical micelle concentration of the HG-type pectin nanomicelles is 15.0 μg / mL to 20.0 μg / mL.

2. A method for preparing oleanolic acid-modified HG-type pectin nanomicelles as described in claim 1, characterized in that, The preparation method includes the following steps: S1. 3,3'-dithiodipropionic acid and oleanolic acid are esterified to obtain SS-OA; S2. The SS-OA and HG type pectin are subjected to esterification reaction to obtain oleanolic acid modified HG type pectin; S3. Dissolve the oleanolic acid-modified HG type pectin and disperse it by ultrasonication to obtain oleanolic acid-modified HG type pectin nanomicelles. The SS-OA has the following structural formula (II): （Ⅱ）。 3. The preparation method according to claim 2, characterized in that, S1 includes: S1-1. Dissolve 3,3'-dithiodipropionic acid in an organic solution, add an acid-binding agent, and mix to obtain a mixed solution; S1-2. Oleanolic acid is added to the mixed solution, and a carboxyl activating reagent is added dropwise. The reaction is carried out at room temperature to obtain SS-OA.

4. The preparation method according to claim 3, characterized in that, The molar ratio of 3,3'-dithiodipropionic acid, oleanolic acid, acid-binding agent, and carboxyl activating agent is (1.0–1.5):1:(0.2–0.6):(1.2–1.8). And / or, the acid-binding agent is one or more of 4-dimethylaminopyridine, pyridine, and 4-pyrrolidinylpyridine; the carboxyl activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; And / or, the reaction time at room temperature is 12h to 24h.

5. The preparation method according to claim 2, characterized in that, S2 specifically includes: S2-1. Dissolve SS-OA in an organic reagent, add an acid-binding agent, and obtain a mixture; S2-2. Add HG type pectin to the mixture, add carboxyl activating reagent dropwise, and react at room temperature to obtain PA-SS-OA polymer.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the SS-OA, acid-binding agent, and carboxyl activating agent is (1.0–1.5):(0.2–0.6):(1.2–1.8). And / or, the acid-binding agent is one or more of 4-dimethylaminopyridine, pyridine, and 4-pyrrolidinylpyridine; the carboxyl activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; And / or, the reaction time at room temperature is 12h to 24h.

7. The preparation method according to any one of claims 2 to 6, characterized in that, The ultrasonic power for ultrasonic dispersion is 200W to 450W, and the ultrasonic time is 2 min to 10 min.

8. The use of the oleanolic acid-modified HG-type pectin nanomicelles as described in claim 1 in the preparation of a drug to alleviate chronic alcoholic liver injury.

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