Oleanolic acid modified HG type pectin nano-micelle as well as preparation method and application thereof

Abstract

The invention provides an oleanolic acid modified HG type pectin nano-micelle as well as a preparation method and application thereof, and the preparation method of the HG type pectin nano-micelle comprises the following steps: carrying out esterification reaction on 3, 3 '-dithiodipropionic acid and oleanolic acid to obtain SS-OA; sS-OA and HG type pectin are subjected to an esterification reaction, and oleanolic acid modified HG type pectin is obtained; and dissolving the oleanolic acid modified HG type pectin, and carrying out ultrasonic dispersion to obtain the oleanolic acid modified HG type pectin nano-micelle. The oleanolic acid modified HG type pectin nano-micelle combines the functional activity of HG type pectin and oleanolic acid and the redox responsiveness of 3, 3 '-dithiodipropionic acid, the nano-micelle capable of responding to ROS generated in the alcohol metabolism process is constructed, and the oleanolic acid modified HG type pectin nano-micelle has amphipathicity and good biocompatibility, and can be used for preparing the ROS-containing nano-micelle. The compound can be used for preparing medicines for relieving chronic alcoholic liver injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to an oleanolic acid-modified HG-type pectin nano-micelle and a preparation method and application thereof. Background Art

[0002] Long-term excessive drinking can cause damage to liver cells. In the continuous cycle of damage and repair, the structure of the liver will change and its function will suffer continuous damage, thus causing chronic alcoholic liver disease (ALD). The pathological development of ALD is a complex and continuous process, which includes multiple stages, each of which may overlap or occur simultaneously. These stages include fatty degeneration of the liver, fatty hepatitis, liver fibrosis and cirrhosis, and may eventually develop into hepatocellular carcinoma. Globally, with the increase in the number of drinkers and the prevalence of alcoholism, the number of people with alcoholic liver disease, the incidence rate and mortality rate have shown an increasing trend year by year. Although significant research progress has been made in the molecular pathological mechanism of ALD, there is still a lack of effective means for the treatment strategy of ALD. At present, people have not been able to successfully prevent and 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 free radicals that can induce liver oxidative stress are produced. ROS can bind to proteins and change their functional and structural properties, thereby producing new antigens to trigger immune responses, leading to inflammatory responses and tissue damage. This binding reaction produces lipid peroxides such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), the presence of which further exacerbates the severity of alcoholic liver damage. Therefore, in recent years, many researchers have continued to explore ways to reduce alcoholic liver damage by inhibiting the production of liver 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 to reactive oxygen species (ROS), thereby activating the body's antioxidant defense mechanism. This process promotes autophagy, removes damaged proteins, and effectively alleviates alcohol-induced liver damage.

[0004] Polygalacturonic acid (HG pectin, PA) is a linear polysaccharide composed of α-1,4-D-galacturonic acid units. Scientific studies have shown that HG pectin exhibits a series of beneficial physiological effects in vivo, including anti-cancer effects, immunomodulatory ability, antioxidant properties, positive regulation of intestinal flora, and lowering blood sugar levels. Oleanolic acid (OA) belongs to pentacyclic triterpenoid compounds and has multiple pharmacological activities such as liver protection, hypoglycemic, lipid-lowering, anti-cancer, and immune enhancement. Although oleanolic acid has significant potential in protecting liver health, its water solubility is poor, which affects its bioavailability in the body, thereby limiting its application in health products or drugs. All the above evidence shows that HG pectin and oleanolic acid are candidate natural compounds for preventing alcoholic liver injury. However, the efficacy of the combined use of these two ingredients in preventing alcoholic liver injury has not been reported yet. Therefore, we hypothesized that the hydrophilicity of HG pectin could improve the solubility of oleanolic acid and thus enhance its bioavailability. At the same time, we expected that these two components could work together to play a positive role in preventing alcoholic liver damage. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, and to provide an oleanolic acid-modified HG pectin nano-micelle and a preparation method and application thereof. The oleanolic acid-modified HG pectin nano-micelle has a redox-responsive polymer micelle system, and the micelle system is composed of three components: HG pectin, 3,3'-dithiodipropionic acid and oleanolic acid. The purpose is to use hydrophilic HG pectin to increase the water solubility of oleanolic acid and increase its bioavailability. At the same time, 3,3'-dithiodipropionic acid is used as a redox-responsive bond to give the entire system redox sensitivity. The nano-micelle delivery system constructed by the present invention has certain guiding significance for alleviating chronic alcoholic liver damage.

[0006] In order to achieve the above object, the present invention provides an oleanolic acid-modified HG pectin nano-micelle, wherein the oleanolic acid-modified HG pectin nano-micelle has the following structural formula I:

[0007]

[0008] The above-mentioned oleanolic acid-modified HG pectin nano-micelles further have a molecular weight of 25000-50000Da.

[0009] The above-mentioned oleanolic acid-modified HG pectin nano-micelles further have an oleanolic acid content of 7.0% to 8.5%.

[0010] The above-mentioned oleanolic acid-modified HG pectin nano-micelles further have a particle size of 400-500 nm.

[0011] The above-mentioned oleanolic acid-modified HG pectin nano-micelles further have a critical micelle concentration of 15.0 to 20.0 μg / mL.

[0012] Based on a general technical concept, the present invention also provides a method for preparing the oleanolic acid-modified HG-type pectin nano-micelles, comprising the following steps:

[0013] S1, esterifying 3,3'-dithiodipropionic acid and oleanolic acid to obtain SS-OA;

[0014] S2, subjecting the SS-OA and HG pectin to an esterification reaction to obtain oleanolic acid-modified HG pectin;

[0015] S3, dissolving the oleanolic acid-modified HG pectin, and obtaining oleanolic acid-modified HG pectin nano-micelles after ultrasonic dispersion;

[0016] The SS-OA has the following structural formula II:

[0017]

[0018] In the above preparation method, further, S1 specifically comprises:

[0019] S1-1, dissolving 3,3'-dithiodipropionic acid in an organic reagent, adding an acid binding agent, and mixing to obtain a mixed solution;

[0020] S1-2, adding oleanolic acid to the mixed solution, and adding a carboxyl activation reagent dropwise, and reacting at room temperature to obtain SS-OA.

[0021] In the above preparation method, further, the molar ratio of the 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). Further, the molar ratio of the 3'-dithiodipropionic acid, oleanolic acid, 4-pyrrolidinopyridine and carboxyl activating agent is 1.2:1:0.5:1.5.

[0022] In the above preparation method, further, the acid binding agent is one or more of 4-dimethylaminopyridine, pyridine and 4-pyrrolidinopyridine.

[0023] In the above preparation method, further, the carboxyl activation reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0024] In the above preparation method, further, the reaction time at room temperature is 12h to 48h.

[0025] In the above preparation method, further, S2 specifically comprises:

[0026] S2-1, dissolving SS-OA in an organic reagent, adding an acid binding agent, and obtaining a mixed solution;

[0027] S2-2, adding HG pectin to the mixed solution, adding a carboxyl activation reagent dropwise, and reacting at room temperature to obtain a PA-SS-OA polymer.

[0028] In the above preparation method, further, the molar ratio of the SS-OA, the acid binding agent and the carboxyl activating agent is (1.0-1.5):(0.2-0.6):(1.2-1.8). Further, the molar ratio of SS-OA, 4-pyrrolidinopyridine and the carboxyl activating agent is 0.20mmol:0.08mmol:0.24mmol.

[0029] In the above preparation method, further, the amount of HG pectin used is 300 mg.

[0030] In the above preparation method, further, the acid binding agent is one or more of 4-dimethylaminopyridine, pyridine and 4-pyrrolidinylpyridine; and the carboxyl activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0031] In the above preparation method, further, the reaction time at room temperature is 12h to 48h.

[0032] In the above preparation method, further, the ultrasonic power of the ultrasonic dispersion is 200W to 450W, and the ultrasonic time is 2min to 10min.

[0033] Based on a general technical concept, the present invention also provides an application of the oleanolic acid-modified HG pectin nano-micelles in the preparation of medicines or health care products for alleviating chronic alcoholic liver damage.

[0034] The innovation of the present invention is that since OA contains both carboxyl and hydroxyl groups, when reacting with PA, due to the large steric hindrance of PA, OA will preferentially undergo self-polymerization reaction to generate -[OA-OA]n-, resulting in OA being unable to be successfully grafted onto PA. Therefore, one of the innovations of the present invention is that during the synthesis process, the product SS-OA is first obtained by condensing equimolar amounts of 3,3'-dithiodipropionic acid (SS) and OA, and then SS-OA is grafted onto PA through condensation to obtain the final product PA-SS-OA. Such a grafting strategy avoids the tediousness of adding and removing protecting groups.

[0035] Compared with the prior art, the advantages of the present invention are:

[0036] (1) The present invention provides an oleanolic acid-modified HG pectin nano-micelle, which combines the functional activities of HG pectin and oleanolic acid, and the redox responsiveness of 3,3'-dithiodipropionic acid, to construct a nano-micelle that can respond to ROS generated during alcohol metabolism, and has amphiphilicity and good biocompatibility.

[0037] (2) The present invention provides an application of oleanolic acid-modified HG-type pectin nano-micelles in the preparation of a drug for improving alcoholic liver injury. Compared with mice with alcoholic liver injury, the liver index of mice gavaged with the nano-micelles of the present invention decreased by 11.42%, the ALT, AST and TG contents in serum decreased by 63.0%, 59.0% and 35.9%, respectively, the ALDH, SOD and CAT activities in the liver of mice increased by 48.1%, 31.6% and 34.9%, respectively, the MDA, TNF-α, IL-6 and IFN-γ contents in the liver of mice decreased by 24.9%, 36.9%, 32.6% and 41.8%, respectively, and the nano-micelles can activate the Nrf2 antioxidant signaling pathway, effectively reduce or reverse the oxidative stress and inflammation caused by alcohol metabolism, and promote the body's state to return to a near normal level, thereby reducing alcohol damage to the liver. This protective effect is better than the use of oleanolic acid alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0039] Figure 1 The synthetic route of oleanolic acid-modified HG pectin (PA-SS-OA) in Example 1 of the present invention.

[0040] Figure 2 It is the UV-visible spectra of PA, OA, DTDPA and PA-SS-OA in Experiment 1 of the present invention.

[0041] Figure 3 It is the Fourier transform infrared spectra of PA, OA, DTDPA and PA-SS-OA in Experiment 1 of the present invention.

[0042] Figure 4 It is the hydrogen nuclear magnetic resonance spectrum of PA, OA, DTDPA and PA-SS-OA in Experiment 1 of the present invention.

[0043] Figure 5 It is the XRD pattern of PA, OA, DTDPA and PA-SS-OA in Experiment 3 of the present invention.

[0044] Figure 6 It is the DSC graph of PA, OA, DTDPA and PA-SS-OA in Experiment 3 of the present invention.

[0045] Figure 7 It is the particle size distribution diagram (A in the figure), the average particle size diagram (B in the figure), the average PDI diagram (C in the figure), and the average Zata potential diagram (D in the figure) of PA and PA-SS-OA in Experiment 4 of the present invention.

[0046] Figure 8 It is the transmission electron microscope image of PA and PA-SS-OA in Experiment 4 of the present invention.

[0047] Fig. 9 It is a fluorescence emission spectrum diagram (A in the figure) of PA-SS-OA at different concentrations (from 0.0001 to 1 mg / mL) in Experiment 5 of the present invention and a diagram of the critical micelle concentration simulation calculation results (B in the figure).

[0048] Fig.10 This is the degradation of PA-SS-OA and the release of OA under redox conditions in Experiment 6 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 release of OA from the PA-SS-OA micellar solution after treatment with different concentrations of H2O2 or GSH for 24 hours.

[0049] Fig.11 This is a graph showing the results of the red blood cell hemolysis experiment in Experiment 7 of the present invention.

[0050] Fig.12 This is the result of the cell toxicity test in Experiment 7 of the present invention.

[0051] Fig.13 The figures are the weight change results of mice in different treatment groups in Experiment 8 of the present invention.

[0052] Fig.14 These are the liver indexes of mice in different treatment groups in Experiment 8 of the present invention.

[0053] Fig.15 These are the pathological morphology and HE staining images of the liver tissues of mice in different treatment groups in Experiment 8 of the present invention.

[0054] Fig.16 These are the results of alanine aminotransferase (ALT) (A in the figure), aspartate aminotransferase (AST) (B in the figure), triglyceride (TG) (C in the figure) in the serum of mice in each experimental group of mice in different treatment groups in Experiment 8 of the present invention, and alcohol dehydrogenase (ADH) (D in the figure), acetaldehyde dehydrogenase (ALDH) (E in the figure), and superoxide dismutase (SOD) (F in the figure) in the liver.

[0055] Fig.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 gamma (IFN-γ) (F in the figure) in the liver of mice in each experimental group in Experiment 8 of the present invention.

[0056] Fig.18 The relative expression results of mRNA 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 the present invention.

[0057] Fig.19 The relative 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) proteins in each experimental group of mice in Experiment 8 of the present invention.

[0058] Fig. 20 These are the results of Western blot analysis of Nrf2, GCLC, HO-1 and NQO1 in mice of each experimental group in Experiment 8 of the present invention. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0060] The materials, reagents and instruments used in the following examples can all be purchased from commercial channels. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.

[0061] Example 1

[0062] The oleanolic acid-modified HG-type pectin nano-micelles of the present invention have the following structural formula I:

[0063]

[0064] The synthetic route is as follows Figure 1 The specific steps include:

[0065] (1) Synthesis of product 1 (SS-OA): 3 g (14.27 mmol) of 3,3'-dithiodipropionic acid was dissolved in 50 mL of pyridine, 4-pyrrolidinylpyridine (4-PPY, 0.5 eq) was added, and the mixture was stirred for 0.5 h. Oleanolic acid (OA, 1.0 eq) was then added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.5 eq) dissolved in advance was added dropwise. The mixture was reacted at room temperature for 24 h. The reaction solution was concentrated by rotary evaporation under reduced pressure, and the residue was dispersed in 50 mL of HCl (2 mol / L). The mixture was then extracted with ethyl acetate (3×40 mL). The organic layers were combined, washed with water and saturated brine in turn, and dried over anhydrous Na2SO4. The mixture was filtered and dried under vacuum to obtain a white solid SS-OA for later use.

[0066] SS-OA has the following structural formula II:

[0067]

[0068] (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 HG pectin (PA) dissolved in advance was added, and EDCI (1.5 eq) dissolved in advance was added dropwise. The mixture was reacted for 48 h at room temperature. After the reaction was completed, an appropriate amount of anhydrous ethanol was added and stirred vigorously. A white precipitate appeared, and the supernatant was removed by centrifugation. The precipitate was re-dissolved in 20 mL of DMSO and dialyzed in ultrapure water for 5 days using a dialysis bag (MWCO, 3500 Da). Then, the dialyzed solution was freeze-dried to obtain a PA-SS-OA polymer.

[0069] Experiment 1: Verify whether oleanolic acid in Example 1 is successfully grafted onto HG pectin.

[0070] 1.1. UV-Vis spectrophotometric analysis:

[0071] Experimental procedures: 0.1 mg / mL PA, OA, and PA-SS-OA samples were prepared, and the spectra of each sample in the range of 200 to 800 nm were measured using an ultraviolet-visible spectrophotometer (UV-1800, Shimadzu Instruments Co. Ltd., Suzhou, China).

[0072] Figure 2 It is a UV-visible light spectrum. From the figure, we can see that in the spectrum of -SS-OA, a new absorption peak appears at 282nm, which is the characteristic absorption peak of OA.

[0073] 1.2. Infrared spectrum analysis:

[0074] Experimental steps: The sample slices were prepared by KBr pressing technology, and then the samples were scanned by infrared spectrum (wave number range 4000cm) using Fourier transform infrared spectrometer (Nicolet-iS5, Thermo Fisher, US). -1 Up to 500cm -1 , the resolution is set to 4cm -1 ).

[0075] Figure 3 It is an infrared spectrum analysis chart. According to the results of Fourier infrared spectrum, in the spectrum of PA-SS-OA, a new absorption peak appeared at 1690cm-1, which was attributed to the carbonyl stretching vibration of OA and DTDPA.

[0076] 1.3. Nuclear magnetic resonance spectroscopy analysis:

[0077] Experimental procedures: 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), and the chemical shift was expressed in δ (ppm).

[0078] Figure 4 The results of the nuclear magnetic resonance hydrogen spectrum show that in the spectrum of PA-SS-OA, new chemical shift peaks appeared at δ0.87, 0.96, 1.05 and 1.22, which are attributed to the hydrogen on the multiple tertiary methyl groups of OA. At the same time, new chemical shift peaks also appeared at δ2.8 and 2.9, which are attributed to the hydrogen on the methylene group in DTDPA. These results show that PA-SS-OA was successfully synthesized.

[0079] Experiment 2: Determine the grafting rate of PA-SS-OA prepared in Example 1.

[0080] Experimental steps: Use UV-visible spectrophotometer to measure the absorbance of different concentrations of oleanolic acid at 282nm wavelength, and establish a standard curve (y=0.01434x+0.00161, R 2 =0.9993). The absorbance of PA-SS-OA at 282 nm was measured, and the OA content was calculated by regression equation. The grafting rate of OA was calculated as follows: grafting rate = (OA group mass / modified polymer mass) × 100%.

[0081] The PA-SS-OA grafting rate was measured to be (7.92±0.65)%.

[0082] Experiment 3: XRD and DSC analysis of the prepared PA-SS-OA.

[0083] Experimental steps: X-ray diffractometer (D2PHASER, Bruker, Germany) was used under the following conditions: Cu target, Kα ray, λ=0.15 nm, tube voltage 40 kV, current 40 mA, scanning range 5°-90°, step speed 5° / min. Differential scanning calorimeter (DSC 214Polyma, NETZSCH, Germany) was used for determination: about 5 mg of sample was placed in an aluminum pan, a blank pan was used for comparison, nitrogen was used as carrier gas, the flow rate was 50 mL / min, and the temperature was raised from 20°C to 350°C at a scanning rate of 10°C / min.

[0084] The changes in the intensity and half-peak width of the line diffraction peak reflect the crystallinity and amorphism degree of the particles. Figure 5 The XRD results show that PA-SS-OA has a broad diffraction peak at 14.5°, which is a typical amorphous diffraction feature, indicating that the PA crystal structure is destroyed after grafting OA. However, no characteristic peaks of OA, DTDPA and TDPA were found in the XRD spectrum of PA-SS-OA, indicating that OA and DTDPA have been successfully encapsulated in the structure in an amorphous form.

[0085] DSC analysis is used to evaluate the thermal stability and crystallization transformation of samples, which reflects the chemical and physical structure stability of substances at high temperatures. Figure 6 This is the DSC analysis result of PA-SS-OA. It can be seen from the figure that compared with PA, the exothermic peak of PA-SS-OA shifts to a lower temperature, that is, the original structure of PA after grafting changes and the crystallinity decreases.

[0086] Experiment 4: Determine the particle size, Zeta potential, and polydispersity index (PDI) of PA-SS-OA and take transmission electron microscopy photos.

[0087] 4.1 Experimental steps for particle size, zeta potential, and polydispersity index (PDI) detection: 0.1 mg / mL sample solutions were prepared, and then the particle size distribution (PSD), zeta potential, and polydispersity index (PDI) of the samples were measured using a dynamic light scattering particle size analyzer (Zetasizer Nano ZS 90, Malvern, UK).

[0088] Figure 7 The particle size (A), average particle size (B), PDI (C), and Zeta potential (D) of PA-SS-OA. It can be seen from the figure that the average particle size of PA-SS-OA is 446.20±43.19nm, the PDI is 0.3357±0.0213, and the zeta is -29.98±3.04. The particle size of PA-SS-OA is larger than that of PA. This may be because after PA is grafted with OA, the strong hydrophobic property of OA causes the formed micelle structure to be looser, thereby increasing the particle size. The PDI value of PA-SS-OA is relatively small, indicating that the size distribution of the formed micelles is relatively uniform.

[0089] 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 on the copper mesh covered with a carbon support film. Use filter paper to absorb the excess solution. After natural drying, use phosphotungstic acid solution to re-stain for 2 minutes, then dry naturally at room temperature and observe under a transmission electron microscope.

[0090] Figure 8 The results of transmission electron microscopy observation are shown in Figure A, and Figure B is PA-SS-OA. It can be seen from the figure that both PA and PA-SS-OA are approximately spherical in shape under transmission electron microscopy, and the particle size of PA-SS-OA is larger than that of PA, which indicates that after PA is grafted with OA, the morphology becomes more fluffy.

[0091] Experiment 5: Determination of the critical micelle concentration of PA-SS-OA.

[0092] Experimental steps: First, accurately prepare a concentration of 6.0×10 -5mol / L of pyrene acetone solution. Next, take 50 μL of the prepared pyrene acetone solution, add it to 9 10 mL volumetric flasks, and place it in a fume hood to allow the acetone to evaporate naturally. Then, prepare a 1 mg / mL PA-SS-OA solution. Subsequently, transfer different volumes of sample solution to each volumetric flask containing pyrene, dilute to 10 mL with deionized water, and let stand at room temperature for 2 hours. In order to allow pyrene to fully enter the hydrophobic core of the micelle, the sample was placed in a constant temperature shaker at 37°C for 1 hour, and then ultrasonically treated at room temperature for 1 hour. Finally, use a fluorescence spectrophotometer (such as RF-5301PC, Shimadzu, Japan) to measure the fluorescence intensity of the sample (excitation wavelength 334 nm, emission wavelength range 350-450 nm, excitation slit and emission slit 3 nm and 5 nm respectively). Record 373 nm (I 373 ) and 384nm(I 384 ) at the point of fluorescence intensity. The logarithm of the solution concentration (Log C) is used as the horizontal axis, I 373 with I 384 The ratio (I 373 / I 384 ) as the vertical axis and draw the image.

[0093] Fig. 9 Figure 5 is the critical micelle concentration measurement result. Figure A is the fluorescence emission spectrum of PA-SS-OA at different concentrations (from 0.0001 to 1 mg / mL), and Figure B is the critical micelle concentration simulation calculation result. It can be seen from the figure that the lower the concentration, the more stable the micelle. The measured CMC value of PA-SS-OA is 0.0171 mg / L.

[0094] Experiment 6: Investigate the degradation of PA-SS-OA and the release behavior of oleanolic acid under oxidation / reduction environment.

[0095] 6.1. Use H2O2 and GSH to simulate oxidative and reductive environments. Experimental steps: First, a certain amount of hydrogen peroxide (H2O2, final concentrations of 20μM and 1mM, respectively) and glutathione (GSH, final concentrations of 1mM and 5mM, respectively) were added to the micelle solution prepared with distilled water. Then, we used a dynamic light scattering particle size analyzer (Zetasizer Nano ZS 90, Malvern, UK) to measure the particle size, Zeta potential and PDI changes of the micelles at 0h, 4h, 12h and 24h.

[0096] 6.2. The in vitro OA release behavior of PA-SS-OA micelles under oxidative and reductive conditions was systematically evaluated by dialysis. Experimental steps: First, 5 mg of micelles were dissolved in 5 mL of the corresponding release medium (distilled water containing 20 μM, 1 mM and 1 mM, 5 mM GSH, respectively). Subsequently, the solution was quickly loaded 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 oscillated in a constant temperature oscillator at 37 ° C and 100 rpm. At the preset time points (0.5, 4, 12, 24 and 36 hours), 5.0 mL of the release medium was taken out and supplemented with an equal volume of fresh medium to maintain a constant volume. Finally, the OA content in the release medium was quantitatively analyzed using a UV-visible spectrophotometer (UV-1800, Shimadzu Instruments Co., Ltd., Suzhou, China).

[0097] Depend on Fig.10 It is 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 release of OA from PA-SS-OA micellar solution after treatment with different concentrations of H2O2 or GSH for 24 hours).

[0098] It can be seen from the figure that after being treated with 20μM H2O2 and 1mM GSH for 24 hours, the particle size, Zeta potential and PDI of the PA-SS-OA micelle solution did not show significant changes. After being treated with 1mM H2O2 and 5mM GSH, the particle size of the PA-SS-OA micelle solution increased from 461.6nm at 0h to 1154.61nm and 969.78nm at 24h, respectively, the PDI increased from 0.3363 to 0.6128 and 0.6021, respectively, and the Zeta potential decreased from -31.28mV to -11.32mV and -14.29mV, respectively. These results indicate that the sulfide bonds of the PA-SS-OA micelle solution may break under the above conditions, resulting in the shedding of the PA shell of the micelle, which in turn changes the hydrophilic and hydrophobic ratio of the micelle, and ultimately leads to the dissociation of the micelle, indicating that PA-SS-OA has dual oxidation / reduction responsiveness. Under the conditions of 20μM H2O2 and 1mM GSH, the OA release of PA-SS-OA micelle solution in 48 hours was 9.65% and 7.68%, respectively. These data show that under normal physiological conditions, these micelles exhibit good stability and meet the design requirements. However, under the conditions of 1mM H2O2 and 5mM GSH, the OA release of PA-SS-OA micelle solution in 48 hours was as high as 80.61% and 75.17%. The results show that PA-SS-OA micelle solution can release OA faster and more effectively in response to oxidative and reductive stimuli.

[0099] Experiment 7: Evaluation of the toxicity of PA-SS-OA.

[0100] 7.1. Red blood cell hemolysis experiment. Experimental steps: PA-SS-OA solution of different concentrations (0.1, 0.5, 1 mg / mL) was mixed with an equal volume of 2% red blood cell suspension and incubated in a shaker at 37°C and 200 rpm for 0.5, 1, 2, and 3 h. The sample was taken out and centrifuged at 1500 rpm for 8 min to remove intact red blood cells. 100 μL of supernatant was placed in a 96-well plate and the absorbance at 540 nm was detected using an ELISA reader (synergy H1, BIOTEK, USA). The red blood cell suspension group incubated with PBS buffer (pH 7.4) was used as a negative control, and the deionized water group was used as a positive control. The hemolysis rate was calculated using the formula: hemolysis rate (%) = (OD sample-OD negative control) / (OD positive pair-OD negative control) × 100%.

[0101] Fig.11The hemolysis test is used to evaluate the damage of the material to red blood cells. It can be seen from the figure that when the concentration of PA-SS-OA micelles is between 0.5mg / mL and 2.0mg / mL, and the hemolysis time is between 0.5 hours and 3 hours, the hemolysis rate is less than 5%. This shows that PA-SS-OA does not cause hemolysis during the circulation process in the body and has good biosafety.

[0102] 7.2. Cytotoxicity experiment, experimental steps: Mouse normal liver cells (AML12) (5000 cells / well) were inoculated into 96-well plates and incubated overnight in a 37°C, 5% CO2 cell culture incubator (3111, Thermo, USA). Then the cells were incubated with different concentrations of samples (2.5, 5, 10, 20 μg / mL) for 24 h and 48 h, respectively. Next, the cell viability was determined according to the CCK-8 assay kit (Dojindo, Kumamoto, Japan). 10 μL of CCK-8 solution was added to each well of the 96-well plate. After incubation for 2 h, the absorbance value of the sample at 450 nm was detected by an enzyme reader (synergy H1, BIOTEK, USA). Untreated cells were set as positive controls, and 6 replicates were set for each sample. The formula for calculating cell viability is: Cell viability (%) = (OD sample-OD blank) / (OD positive control-OD blank) × 100%.

[0103] Fig.12 This is the result of the cell toxicity test. It can be seen from the figure that within the concentration range of 0.5 to 20 μg / mL, after PA-SS-OA was co-incubated with AML-12 cells for 24 hours and 48 hours, the cell survival rate remained above 95%, indicating that PA-SS-OA had no significant toxic effect on AML-12 cells.

[0104] Experiment 8: Animal experiment to evaluate the effect of PA-SS-OA in alleviating chronic alcoholic liver damage.

[0105] Experimental steps:

[0106] (1) Grouping and administration: Seventy C57BL / 6J mice were randomly divided into 7 groups, with 10 mice in each group. 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).

[0107] (2) Modeling: The mice were first gavaged with the set dose at a fixed time every afternoon (the normal group and the model group were given an equal volume of saline). After each group was given the drug, except for the normal group which was given an equal volume of saline, the mice in the other groups were gavaged with 52% Niulanshan Erguotou, 0.16mL / 20g in the first week, 0.18mL / 20g in the second week, 0.20mL / 20g in the third week, and 0.22mL / 20g from the fourth to the sixth week. During the modeling period, the weight of the mice was weighed and recorded every 7 days.

[0108] (3) Specimen collection: At the end of the sixth week, all mice were fasted overnight but not watered for 12 h. After abdominal anesthesia, blood was collected from the heart 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 and packaged and stored in a -80°C refrigerator. The liver was dissected and the entire liver tissue was cut and weighed. The right lobe of the liver was cut with a blade and fixed with 4% paraformaldehyde. The remaining liver tissue was stored in a -80°C refrigerator.

[0109] (4) Calculation of liver index and section observation: Liver index (%) = liver mass (g) / body mass (g) × 100%. After dehydration, embedding, sectioning and HE staining, the liver morphology of mice in each group was observed using an optical microscope (Primostar 3, ZEISS, Germany).

[0110] (5) Determination of serum and liver biochemical indices: The ALT, AST and TG levels in the serum of each group were detected according to the method of the purchased kit. About 0.1 g of liver tissue was cut, 1 mL of normal saline was added, and it was fully ground using a grinder (JXFSTPRP-64, Shanghai Jingxin, China), and then centrifuged at 8000 r / min for 15 min at 4°C to obtain the supernatant. The ADH, ALDH, SOD, CAT, GSH, MDA, ROS, TNF-α, IFN-γ, and IL-6 levels in the liver tissue were determined according to the method of the purchased kit.

[0111] (6) RNA extraction and mRNA quantification: Total RNA was extracted from the liver tissue of experimental mice, and the mRNA was reverse transcribed to synthesize cDNA. Subsequently, the mRNA expression levels of Nrf2, GCLC, HO-1, and NQO1 were detected by fluorescence quantitative PCR instrument (ABI 7500, Applied Biosystems, USA), using β-actin as the internal reference gene. —ΔΔCt Methods The relative mRNA expression of each gene group was calculated.

[0112] (7) Expression of Nrf2, GCLC, HO-1, and NQO1 proteins in liver tissue: The total protein of liver tissue was extracted by RIPA lysis method, and the protein concentration of the sample was determined by BCA protein concentration kit. 30 μg of total protein was loaded and separated by 10% SDS-PAGE electrophoresis, then electrotransferred to a 0.45 μm PVDF membrane. After blocking with protein-free rapid blocking solution 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 respectively, and incubated at 4°C overnight. After washing with membrane washing buffer (TBST), secondary antibody Ig (1:5000) was added and incubated at room temperature for 1.5 h. After washing with TBST, the membrane was placed in a V3 Western blotting detection and analysis system and ECL chemiluminescent substrate was added for exposure and imaging. The protein expression was analyzed by the total protein quantification method using Bio-Rad Image Lab Software 5.2 (relative expression was calculated using β-actin as an internal reference).

[0113] Fig.13 The results of the weight changes of mice in different treatment groups. As can be seen from the figure: after the mice were gavaged with alcohol and drug administration, except for Group 1 (normal group), Group 4 (PA group), and Group 5 (PA / OA), the average weight of mice in other groups decreased after the first week. This shows that gavage with alcohol will affect the appetite of mice, affect the amount of food intake, and thus affect the change of body weight. Starting from the second week, the average weight of mice in each group increased every week.

[0114] Fig.14 The liver index of mice in different treatment groups. It can be seen from the figure that compared with the second group (alcohol model group), the liver index of the sixth group (PA-SS-OA low-dose group) and the seventh group (PA-SS-OA high-dose group) decreased significantly by 9.96% and 11.42%, respectively, indicating that PA-SS-OA can reverse this abnormality.

[0115] Fig.15 The following are the pathological morphology and HE staining images of the liver tissue of mice in different treatment groups. It can be seen from the figure that in the pathological morphology, fat droplets and vacuoles of different sizes appeared on the surface of the liver tissue in Group 2. The surface conditions of the liver tissues in the other groups observed with the naked eye were not much different from those in Group 1. In the HE staining image, in Group 2, the liver cells were blurred, the nuclei were shrunk or even disappeared, the cells were swollen, and there were vacuoles in the cells, indicating that the pathological changes of the liver tissue in the alcohol model group were obvious. The liver tissue structure and liver cells of mice in Groups 6 and 7 were arranged more neatly, and the inflammatory cells and fat droplet vacuoles were significantly reduced compared with those in Group 2. In summary, it can be shown that PA-SS-OA can alleviate the damage of alcohol to the liver to a certain extent.

[0116] Fig.16The results of alanine aminotransferase (ALT) (A in the figure), aspartate aminotransferase (AST) (B in the figure), triglyceride (TG) (C in the figure) in the serum and alcohol dehydrogenase (ADH) (D in the figure), acetaldehyde dehydrogenase (ALDH) (E in the figure), superoxide dismutase (SOD) (F in the figure) in the liver of mice in each experimental group under different treatment groups; different letters in the figure indicate that the differences between the groups are statistically significant at the level of p<0.05.

[0117] Fig.17 These are the 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 gamma (IFN-γ) (F in the figure) in the livers of mice in each experimental group. Different letters in the figure indicate that the differences between the groups are statistically significant at the level of p<0.05.

[0118] Fig.18 These are the relative mRNA expressions 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 of each experimental group. Different letters in the figure indicate that the differences between the groups are statistically significant at the level of p < 0.05.

[0119] Fig.19 These are the relative protein expressions 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 of each experimental group. Different letters in the figure indicate that the differences between the groups are statistically significant at the level of p < 0.05.

[0120] Fig. 20 These are the results of Western blot analysis of Nrf2, GCLC, HO-1 and NQO1 in mice from each experimental group.

[0121] As shown in the figure, compared with group 2, the liver index of mice in group 7 decreased by 11.42%, the ALT, AST and TG contents in serum decreased by 63.0%, 59.0% and 35.9%, respectively, the ALDH, SOD and CAT activities in the liver of mice increased by 48.1%, 31.6% and 34.9%, respectively, and the MDA, TNF-α, IL-6 and IFN-γ contents in the liver of mice 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 target proteins GCLC, HO-1 and NQO1 in groups 6 and 7 showed significant restorative upregulation. This indicates that PA-SS-OA can activate the Nrf2 antioxidant signaling pathway, effectively reduce or reverse the oxidative stress and inflammation caused by alcohol metabolism, and restore the body state to near normal levels, thereby reducing alcohol damage to the liver. This protective effect is better than the use of oleanolic acid alone.

[0122] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. An oleanolic acid-modified HG-type pectin nano-micelle, characterized in that: The oleanolic acid modified HG pectin nano-micelles have the following structural formula I:

2. The HG-type pectin nano-micelles modified with oleanolic acid according to claim 1, characterized in that: The molecular weight of the HG pectin nano-micelles is 25000Da to 50000Da; And / or, the percentage of oleanolic acid in the HG pectin nano-micelles is 7.0% to 8.5%; And / or, the particle size of the HG pectin nanomicelles is 400nm to 500nm; And / or, the critical micelle concentration of the HG pectin nanomicelles is 15.0 μg / mL to 20.0 μg / mL.

3. A method for preparing the oleanolic acid-modified HG pectin nanomicelles according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: S1, esterifying 3,3'-dithiodipropionic acid and oleanolic acid to obtain SS-OA; S2, subjecting the SS-OA and HG pectin to an esterification reaction to obtain oleanolic acid-modified HG pectin; S3, dissolving the oleanolic acid-modified HG pectin, and obtaining oleanolic acid-modified HG pectin nano-micelles after ultrasonic dispersion; The SS-OA has the following structural formula II:

4. The preparation method according to claim 3, characterized in that: The S1 includes: S1-1, dissolving 3,3'-dithiodipropionic acid in an organic solution, adding an acid binding agent, and mixing to obtain a mixed solution; S1-2, adding oleanolic acid to the mixed solution, and adding a carboxyl activation reagent dropwise, and reacting at room temperature to obtain SS-OA.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the 3,3'-dithiodipropionic acid, oleanolic acid, acid binding agent and carboxyl activation 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 activation reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; And / or, the reaction time at room temperature is 12 h to 24 h.

6. The preparation method according to claim 3, characterized in that: The S2 specifically includes: S2-1, dissolving SS-OA in an organic reagent, adding an acid binding agent, and obtaining a mixed solution; S2-2, adding HG pectin to the mixed solution, adding a carboxyl activation reagent dropwise, and reacting at room temperature to obtain a PA-SS-OA polymer.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the SS-OA, the acid binding agent and the carboxyl activation 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 activation reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; And / or, the reaction time at room temperature is 12 h to 24 h.

8. The preparation method according to any one of claims 3 to 7, characterized in that: The ultrasonic power of the ultrasonic dispersion is 200W to 450W, and the ultrasonic time is 2min to 10min.

9. Use of the oleanolic acid-modified HG pectin nano-micelles according to claim 1 or 2 in the preparation of a drug for alleviating chronic alcoholic liver damage.

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