Natural product-based nano antioxidant for treating acute pancreatitis and preparation method of natural product-based nano antioxidant

Abstract

The invention discloses a natural product-based nano antioxidant for treating acute pancreatitis and a preparation method of the natural product-based nano antioxidant, and relates to the technical field of biological medicines. 25 mg of ferulic acid (FA); and 15 mL of 80% (v / v) ethanol-deionized water solution. According to the natural product-based nano antioxidant for treating acute pancreatitis and the preparation method thereof, the nano antioxidant effectively targets inflammatory pancreatic tissues through the specific interaction of chondroitin sulfate (CS) and CD44, and is split and released under the stimulation of pH / GSH and H2O2 in cells to generate FA and zein degradation products, the FA and zein degradation products efficiently play a synergistic role in removing ROS, and the natural product-based nano antioxidant can be used for treating acute pancreatitis. Therefore, inflammation is relieved; the nano antioxidant shows an excellent in-vivo treatment effect on acute pancreatitis and has a clinical application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a natural product-based nano antioxidant for treating acute pancreatitis and a preparation method thereof. Background Art

[0002] Acute pancreatitis (AP) is a common and fatal inflammatory disease of the exocrine pancreas, often accompanied by inflammation, edema, hemorrhage and even necrosis of the pancreas itself or distal organs. AP is associated with gallstones, smoking, drinking, diabetes, obesity and hyperlipidemia, and is one of the most common causes of hospitalization for gastrointestinal diseases worldwide. The overall incidence of AP continues to rise, severely increasing the economic burden on the public health system. Although most cases are mild and self-limiting, approximately 25% of patients develop severe AP, and the mortality rate of the patient population reaches 40%. Although AP has been widely studied in recent years, there is still a lack of effective and specific therapeutic drugs. Therefore, the development of therapeutic drugs for AP is urgent.

[0003] Although the pathogenesis of AP has not been fully elucidated, high levels of reactive oxygen species (ROS) are believed to be closely related to the occurrence and development of AP. In short, excessive production of ROS can lead to cell damage and even cell necrosis, which in turn triggers the release of damage-associated molecular patterns (DAMPs). DAMPs activate macrophages to secrete proinflammatory factors such as tumor necrosis factor-α, interleukin-1β, interleukin-6, and monocyte chemoattractant protein-1, further recruiting neutrophils and monocyte-derived macrophages to the site of pancreatic inflammation, thereby aggravating the inflammatory response and forming a vicious cycle. Based on the above theory, antioxidant therapy is considered to be a potential method for the treatment of AP. In recent years, various antioxidants including natural product antioxidants (protocatechuic acid, ferulic acid (FA), zein, α-lipoic acid, and astaxanthin) and metal nanozymes (Ce / Gd-CDs, Prussian blue, MoSe2@PVP, and Cu-MOF) have been widely studied for the treatment of AP. However, there is currently no antioxidant that can meet the clinical treatment needs of AP, mainly due to its lack of targeting to inflammatory sites, low ROS scavenging ability and insufficient intracellular drug release. Summary of the invention

[0004] The present invention provides a natural product-based nano antioxidant for treating acute pancreatitis and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] Natural product-based nano antioxidant for the treatment of acute pancreatitis and its preparation method, the required materials are:

[0007] 50 mg zein;

[0008] 25mg ferulic acid (FA);

[0009] 15 mL 80% (v / v) ethanol-deionized water solution;

[0010] 40mg chondroitin sulfate (CS);

[0011] 15 mL deionized water;

[0012] zein / FA mixed solution;

[0013] CS solution.

[0014] A method for preparing a natural product-based nano antioxidant for treating acute pancreatitis, a method for preparing materials for a natural product-based nano antioxidant for treating acute pancreatitis, 50 mg of zein and 25 mg of ferulic acid (FA) are dissolved in 15 mL of 80% (v / v) ethanol-deionized water solution, and magnetically stirred at 500 rpm for 1 hour.

[0015] Preferably, the zein / FA mixed solution is added dropwise to the CS solution under magnetic stirring at 600 rpm.

[0016] Preferably, the obtained solution is dialyzed with deionized water for 48 hours to obtain the natural product-based nano antioxidant FA@Zein-CS; FA@Zein is prepared by the same method, but without adding FA.

[0017] Characterization of the preparation of natural product-based nano-antioxidants for the treatment of acute pancreatitis, the preparation method of natural product-based nano-antioxidants for the treatment of acute pancreatitis, the hydrated particle size and Zeta potential of FA@Zein-CS were determined by Malvern laser particle size analyzer, and its morphology was characterized by HITACHI transmission electron microscopy.

[0018] Preferably, the hydrodynamic diameter of FA@Zein-CS is 134.0 nm, which is smaller than the hydrodynamic diameter of FA@Zein (200.1 nm). CS can hybridize with FA@Zein through electrostatic interaction, thereby stabilizing the colloidal system and resulting in a smaller particle size.

[0019] Preferably, the zeta potential of FA@Zein-CS is -32.2 mV, and compared with FA@Zein, FA@Zein-CS exhibits a significant surface charge reversal (42.6 mV).

[0020] Preferably, the negatively charged surface may exhibit improved colloidal stability and increased blood circulation time compared to the positively charged nanosystems. Transmission electron microscopy images showed that the morphology of FA@Zein-CS was spherical.

[0021] Preferably, FA@Zein-CS is diluted with 80% ethanol solution, and then FA is extracted by ultrasound-assisted method. The content of FA in the dispersion is determined by using a Shimadzu UV-2600i spectrophotometer.

[0022] Preferably, the absorbance at 321 nm is recorded, and the drug loading and encapsulation efficiency are calculated by the following formula:

[0023] Drug loading (%) = (mass of FA in FA@Zein-CS / mass of FA@Zein-CS) × 100%

[0024] Encapsulation rate (%) = (FA mass in FA@Zein-CS / FA input amount) × 100%

[0025] The measurement results showed that the drug loading and encapsulation efficiency of FA in FA@Zein-CS were approximately 70% and 52%, respectively.

[0026] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0027] 1. The present invention provides a natural product-based nano antioxidant for the treatment of acute pancreatitis and a preparation method thereof. The nano antioxidant effectively targets inflammatory pancreatic tissue through the specific interaction between chondroitin sulfate (CS) and CD44, and cleaves and releases FA and zein degradation products under the stimulation of intracellular pH / GSH and H2O2. The two effectively and synergistically clear ROS, thereby reducing inflammation. The nano antioxidant shows excellent in vivo therapeutic effect on acute pancreatitis and has clinical application prospects.

[0028] 2. The present invention provides a natural product-based nano antioxidant for the treatment of acute pancreatitis and a preparation method thereof, which overcomes the shortcomings of antioxidants such as poor targeting of inflammatory sites, low ROS scavenging ability and insufficient intracellular drug release; the nano antioxidant uses CS as a targeting ligand for CD44, zein as a response controlled release carrier, and FA and zein degradation products as synergistic antioxidant parts, and shows a stronger antioxidant effect than single FA, zein and FA@Zein, meeting the clinical treatment needs of acute pancreatitis.

[0029] 3. The present invention provides a natural product-based nano antioxidant for the treatment of acute pancreatitis and a preparation method thereof. The nano antioxidant can not only efficiently target the inflamed pancreatic tissue through the specific binding of CS and CD44, but also effectively initiate the release of FA and zein degradation products in response to intracellular pH / GSH / ROS, thereby achieving a synergistic antioxidant effect; in addition, since all components are derived from natural products, the nano antioxidant has good biocompatibility; in vivo experimental results show that the nano antioxidant significantly reduces pancreatic structural damage, restores pancreatic function, and reduces serum amylase and lipase by 61.8% and 82.8%, respectively; this natural product-based nano antioxidant has great clinical application potential for AP. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A schematic diagram of the natural product-based nano antioxidant for treating acute pancreatitis of the present invention;

[0031] Figure 2 The results are the characterization results of the particle size, potential and morphology of the natural product-based nano antioxidant of the present invention;

[0032] Figure 3 The UV and IR characterization results of the natural product-based nano antioxidants in the present invention;

[0033] Figure 4 The results are the characterization results of the in vitro responsive release of the natural product-based nano antioxidants in the present invention;

[0034] Figure 5 This is the result of the natural product-based nano antioxidants in the present invention protecting cells from oxidative stress damage;

[0035] Figure 6 It is the antioxidant result of the natural product-based nano antioxidant in the present invention;

[0036] Figure 7 The results of the natural product-based nano antioxidant in the present invention in reducing lipid peroxides;

[0037] Figure 8 The anti-inflammatory effect of the natural product-based nano antioxidant in the present invention;

[0038] Fig. 9 The expression results of CD44 in pancreatic tissues of normal mice and mice with acute pancreatitis in the present invention;

[0039] Fig.10 The effect of cells taking up natural product-based nano antioxidants in the present invention;

[0040] Fig.11 This is the result of the in vivo targeting of the natural product-based nano antioxidant in the present invention;

[0041] Fig.12 This is the effect of the natural product-based nano antioxidant in the present invention on treating acute pancreatitis in vivo. DETAILED DESCRIPTION

[0042] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0043] like Figure 1-Figure 12 As shown, natural product-based nano antioxidants and preparation methods thereof for the treatment of acute pancreatitis.

[0044] Example 1

[0045] Preparation and characterization of natural product-based nano antioxidants

[0046] 50 mg of corn alcohol-soluble protein (zein) and 25 mg of ferulic acid (FA) were dissolved in 15 mL of 80% (v / v) ethanol-deionized water solution and magnetically stirred at 500 rpm for 1 h. 40 mg of chondroitin sulfate (CS) was slowly added to 15 mL of deionized water and stirred at 500 rpm for 30 min. Then, the zein / FA mixed solution was added dropwise to the CS solution under magnetic stirring at 600 rpm. Finally, the resulting solution was dialyzed with deionized water for 48 h to obtain the natural product-based nano antioxidant FA@Zein-CS. FA@Zein was prepared using the same method, but without the addition of FA.

[0047] The hydrated particle size and Zeta potential of FA@Zein-CS were measured by Malvern laser particle size analyzer, and its morphology was characterized by HITACHI transmission electron microscope. Figure 2 As shown in Figure a, the hydrodynamic diameter of FA@Zein-CS is 134.0nm, which is smaller than the hydrodynamic diameter of FA@Zein (200.1nm). We speculate that CS can hybridize with FA@Zein through electrostatic interaction, thereby stabilizing the colloidal system and causing the particle size to decrease. The zeta potential measurement results are shown in Figure 2. Figure 2 As shown in Fig. b, the zeta potential of FA@Zein-CS is -32.2 mV, and FA@Zein-CS exhibits a significant surface charge reversal (42.6 mV) compared to FA@Zein. In general, negatively charged surfaces may exhibit improved colloidal stability and increased blood circulation time compared to positively charged nanosystems. In addition, the TEM image shows that the morphology of FA@Zein-CS is spherical ( Figure 2 c).

[0048] The drug loading and encapsulation efficiency of FA were determined by UV spectrophotometer. Specifically, FA@Zein-CS was diluted with 80% ethanol solution, and then FA was extracted by ultrasound-assisted method. The content of FA in the dispersion was determined by Shimadzu UV-2600i spectrophotometer. The absorbance at 321nm was recorded, and the drug loading and encapsulation efficiency were calculated by the following formula:

[0049] Drug loading (%) = (mass of FA in FA@Zein-CS / mass of FA@Zein-CS) × 100%

[0050] Encapsulation rate (%) = (FA mass in FA@Zein-CS / FA input amount) × 100%

[0051] The measurement results showed that the drug loading and encapsulation efficiency of FA in FA@Zein-CS were approximately 70% and 52%, respectively.

[0052] Example 2

[0053] UV and IR spectroscopy characterization of natural product-based nano antioxidants

[0054] The loading of FA and the hybridization of CS were confirmed by the differences in UV absorption and FTIR spectra between FA, CS, zein, Zein@FA, and FA@Zein-CS. Figure 3 As shown in Figure a, when FA is loaded into zein nanoparticles, the characteristic peak of zein red-shifts from 278.0nm to 287.5nm, and the characteristic absorption peak of FA appears, indicating that FA is successfully loaded. In contrast, the absorption peaks of zein and FA in FA@Zein-CS are slightly shifted, and the characteristic peak of CS at 259.1nm is completely covered, confirming the successful hybridization of CS. The infrared spectra of zein, FA, FA@Zein, CS and FA@Zein-CS are shown in Figure 3. Figure 3 As shown in b, zein is 1642.62cm -1 There is a typical peak at 1646.53 cm, which is related to the stretching vibration of its carbonyl group. After the formation of FA@Zein nanoparticles, the peak red-shifted to 1646.53 cm -1 , proving that hydrophobic interaction occurred between FA and Zein. For FA@Zein-CS nanoantioxidant, the carbonyl peak shifted to 1648.54 cm due to the hydrophobic and electrostatic interactions between CS and zein. -1 Due to the hydrogen bonding between CS and zein, the hydroxyl peak increases from 3288.35 cm -1 (zein) and 3304.30cm -1 (CS) redshifted to 3299.27 cm -1In addition, no FA at 1619.42 cm was observed in FA@Zein and FA@Zein-CS. -1 、1599.81cm -1 and 1267.17cm -1 The characteristic peak at , confirming the successful loading of FA.

[0055] Example 3

[0056] In vitro responsive release characteristics of natural product-based nano antioxidants

[0057] The cumulative release curve of FA was determined by dispersing FA@Zein-CS in different pH or redox environments. To investigate the effect of pH on the release of FA from FA@Zein-CS, 5 mL of FA@Zein-CS (FA concentration of 1.5 mg / mL) was dialyzed in 100 mL of PBS at pH 5.5 or pH 7.4, respectively. To study the effect of redox stimulation on FA release, 5 mL of FA@Zein-CS (FA concentration of 1.5 mg / mL) was dialyzed in 100 mL of PBS containing different concentrations of glutathione (GSH) and hydrogen peroxide (H2O2), respectively. At the designated time points, 2 mL of culture medium was removed and replenished with fresh PBS solution. FA was measured using a UV-visible spectrophotometer. The in vitro release rate of FA was calculated according to the following formula:

[0058] Drug release (%) = (weight of released FA / weight of total loaded FA) × 100%

[0059] The pH-responsive release results of FA are shown in Figure 4 As shown in Figure a, after FA@Zein-CS was incubated in PBS at pH = 7.4 for 48 h, approximately 45.4% of FA was released from FA@Zein-CS. As the pH value decreased to 5.5, the cumulative release of FA increased to 55.3%. In addition, the reduction-responsive release characteristics of FA in FA@Zein-CS were determined using GSH as a reducing agent. Figure 4 As shown in Figure b, after incubation in a PBS solution containing GSH (10 mM) for 48 h, the cumulative release of FA reached 83.3%, which was much higher than the cumulative release in a PBS solution containing 1 mM GSH. Given the increased level of ROS in activated macrophages, we also investigated the responsiveness of FA@Zein-CS in PBS solutions containing different concentrations of H2O2 (100 μM and 1 mM), and further determined the release curve of FA. The results are shown in Figure 4As shown in Figure c, after FA@Zein-CS was exposed to PBS containing H2O2 (100uM) for 48h, about 71.3% of FA was released from FA@Zein-CS. In comparison, as the H2O2 concentration increased to 1mM, the cumulative release of FA increased significantly to 92.2%, indicating that FA@Zein-CS has a strong H2O2 response characteristic.

[0060] Example 4

[0061] Natural product-based nanoantioxidants protect cells from oxidative stress

[0062] RAW264.7 cells were grown at 4 × 10 4 After incubation for 12 h, the cells were inoculated in a 24-well plate at a density of 10 cells / mL. The cells were then pre-incubated with FA (14 μg / mL), zein (6 μg / mL), FA@Zein ([FA] = 14 μg / mL, [zein] = 6 μg / mL), and FA@Zein-CS ([FA] = 14 μg / mL, [zein] = 6 μg / mL) for 3 h, and then co-incubated with 300 μM H2O2 for 24 h. After washing with PBS, the cells were stained with calcein-AM / PI, and the fluorescence images were observed under a fluorescence inverted microscope. The results are shown in Figure 2. Figure 5 As shown, RAW264.7 cells stimulated by H2O2 showed obvious red fluorescence, indicating that ROS triggered a large amount of cell necrosis. The red fluorescence intensity of cells treated with zein or FA alone did not decrease significantly, which was attributed to their limited antioxidant capacity and nonspecific distribution. The red fluorescence intensity of the FA@Zein group was significantly reduced, which may be due to the synergistic scavenging effect of zein and FA on ROS. In comparison, FA@Zein-CS significantly reduced the red fluorescence intensity to a level comparable to that of the normal group, which is closely related to its efficient uptake by oxidatively damaged cells. The above results show that FA@Zein-CS has a significant effect in protecting cells from oxidative stress damage.

[0063] Example 5

[0064] Evaluation of the antioxidant effect of natural product-based nanoantioxidants

[0065] RAW264.7 cells were plated at 1.2 × 10 6The cells were inoculated at a density of 100 μg / mL in a 6-well plate and cultured for 12 h, and then pretreated with [lipopolysaccharide + caerulein] ([lipopolysaccharide] = 500 ng / mL, [caerulein] = 0.2 μg / mL) for 18 h. After the stimulation, the cells were incubated with FA (14 μg / mL), zein (6 μg / mL), FA@Zein ([FA] = 14 μg / mL, [zein] = 6 μg / mL) and FA@Zein-CS ([FA] = 14 μg / mL, [zein] = 6 μg / mL) for 3 h. After washing with PBS three times, the intracellular ROS fluorescence image was recorded using a fluorescent inverted microscope, and the intracellular fluorescence intensity was quantified using a flow cytometer. RAW cells without any treatment were set as the control group, and cells induced only with [lipopolysaccharide + caerulein] were set as the model group. The results are shown in Figure 6 As shown in a-6b, compared with the control group, RAW264.7 cells stimulated by [lipopolysaccharide + caerulein] showed obvious green fluorescence, indicating that the oxidative stress cell model was successfully constructed. FA@Zein-CS reduced the fluorescence intensity to a level comparable to that of the control group and increased the intracellular ROS elimination rate to 94.1%, which were 4.6, 3.6, and 1.6 times that of zein, FA, and FA@Zein, respectively.

[0066] Example 6

[0067] Inhibitory effect of natural product-based nano antioxidants on lipid peroxide malondialdehyde

[0068] RAW264.7 cells were cultured at 1.2 × 10 6 The cells were inoculated at a density of 100 cells / well in a 6-well plate and incubated for 12 hours. After incubation with [lipopolysaccharide + caerulein] for 18 hours, the cells were treated with different preparations for 3 hours. Subsequently, the cells were collected and lysed. After centrifugation at 15,000×g and 4°C for 20 minutes, malondialdehyde (MDA) detection was performed. RAW cells without any treatment were set as the control group, and cells induced only with [lipopolysaccharide + caerulein] were set as the model group. The results are shown in Figure 7 As shown, [lipopolysaccharide + caerulein] induced macrophages to produce high levels of MDA. Unexpectedly, FA@Zein-CS reduced intracellular MDA by 88.3%, which was 2.1, 2.0, and 1.4 times that of zein, FA, and FA@Zein, respectively.

[0069] Example 7

[0070] Evaluation of anti-inflammatory activity of natural product-based nanoantioxidants

[0071] RAW264.7 cells (1.2×10 6 / well) were inoculated in a 6-well plate and cultured for 12 hours. After stimulating the cells with [lipopolysaccharide + caerulein] for 18 hours, different preparations were added and incubated for 3 hours. Then, typical pro-inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and mononuclear cell protein (MCP-1), were determined using an ELISA kit. RAW cells without any treatment were set as the control group, and cells induced only with [lipopolysaccharide + caerulein] were set as the model group. The results are shown in Figure 8 As shown in a-8d, [lipopolysaccharide + caerulein]-induced RAW264.7 cells highly expressed pro-inflammatory factors such as TNF-α, IL-1β, IL-6, and MCP-1. In particular, FA@Zein-CS was able to reduce TNF-α, IL-1β, IL-6, and MCP-1 to levels comparable to those of the normal control group.

[0072] Example 8

[0073] Expression of CD44 in pancreatic tissues of normal mice and mice with acute pancreatitis

[0074] Quantitative proteomics was used to analyze the expression of CD44 in pancreatic tissue. Specifically, 12 mice were randomly divided into two groups, a normal group and a model group. C57BL / 6J mice were intraperitoneally injected with caerulein (50 μg / kg) 7 times at 1 h intervals, and lipopolysaccharide (10 mg / kg) was injected at the same time in the last injection to establish an animal model of acute pancreatitis. Both groups of mice were euthanized, and appropriate amounts of pancreatic tissue were taken into a mortar pre-cooled with liquid nitrogen and fully ground into powder with liquid nitrogen. Four times the volume of lysis buffer (1% SDC, 1% protease inhibitor) was added to each group of samples, and ultrasonic lysis was performed. Centrifuge at 4°C, 12000g for 10 min, transfer the supernatant to a new centrifuge tube, and use the BCA kit to determine the protein concentration. Take an equal amount of protein from each sample for enzymatic hydrolysis, adjust the volume to the same with lysis buffer, add dithiothreitol (DTT) to a final concentration of 5 mM, and reduce at 56°C for 30 min. Then, iodoacetamide (IAA) was added to a final concentration of 11 mM, and the cells were incubated at room temperature in the dark for 15 min. TEAB was added to dilute urea to ensure that the concentration was less than 2 M. Trypsin was added at a ratio of 1:50 (protease: protein, m / m) and the digestion was carried out overnight. Trypsin was then added at a ratio of 1:100 (protease: protein, m / m) and the digestion was continued for 4 h. The peptides were dissolved in liquid chromatography mobile phase A and separated using the NanoElute ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile-water solution containing 0.1% formic acid. Liquid phase gradient settings: 0-14 min, 6%-24% B; 14-16 min, 24%-35% B; 16-18 min, 35%-80% B; 18-20 min, 80% B, and the flow rate was maintained at 500 nl / min. After the peptides were separated by an ultra-high performance liquid phase system, they were injected into the Capillary ion source for ionization and then into the timsTOF Pro mass spectrometer for data acquisition. The ion source voltage was set to 1.75 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using TOF. The data acquisition mode used the data-independent parallel accumulation serial fragmentation (dia-PASEF) mode, and the primary mass spectrometry scan range was set to 300-1500 m / z. After one primary mass spectrum was acquired, 20 PASEF mode acquisitions were performed, and the secondary mass spectrometry scan was in the range of 400-850, with every 7 m / z as a window. The measurement results are as follows: Fig. 9 As shown, the expression level of CD44 in pancreatic tissue of mice with acute pancreatitis was 7.6 times that of pancreatic tissue of mice in the normal group, indicating that the expression of CD44 in pancreatic tissue of acute pancreatitis was abnormally increased.

[0075] Example 9

[0076] Cellular uptake of nano-antioxidants

[0077] RAW264.7 cells (1×10 5 / well) were inoculated in a 12-well plate and incubated for 12 hours. After incubation with [lipopolysaccharide + caerulein] for 18 hours, the cells were treated with coumarin 6 (Cou)-labeled FA@Zein and FA@Zein-CS for 3 hours. After incubation, the cells were washed three times with cold PBS, and then the cell nuclei were stained with hoechst33342 and observed under a fluorescence inverted microscope. The results are shown in Fig.10 As shown in a-10b, the green fluorescence signal of Cou in the FA@Zein-CS group was significantly higher than that in the FA@Zein group, indicating that the binding of CS to CD44 highly expressed in RAW264.7 cells promoted the cellular uptake of nano-antioxidants.

[0078] Example 10

[0079] In vivo targeting effects of natural product-based nanoantioxidants

[0080] FA@Zein ([FA] = 35 mg / kg, [zein] = 15 mg / kg) and FA@Zein-CS ([FA] = 35 mg / kg, [zein] = 15 mg / kg) labeled with indocyanine green (ICG) were injected into mice with acute pancreatitis induced by [lipopolysaccharide + cerulein] and normal mice, respectively. The pancreas and major organs were isolated 3, 6, and 12 hours after injection, respectively, and near-infrared imaging and quantitative analysis were performed. The results are shown in Fig.11 As shown in the figure, 3 hours after injection, the average fluorescence signals in the pancreatic tissues of normal mice treated with FA@Zein and FA@Zein-CS were weak, about 3.9 and 5.5, respectively, and the fluorescence intensity decreased in a time-dependent manner. In comparison, 3 hours after injection of FA@Zein and FA@Zein-CS, the pancreatic tissues of mice with acute pancreatitis showed stronger ICG fluorescence signals, with average fluorescence intensities of about 133.9 and 26.7, respectively. 6 hours after injection, the fluorescence intensity of the pancreatic tissues of mice with acute pancreatitis treated with FA@Zein-CS was still significantly higher than that of the FA@Zein group, indicating that FA@Zein-CS can be highly taken up by the inflamed pancreas. 12 hours after injection, relatively significant fluorescence intensity could still be monitored in the pancreas of mice with acute pancreatitis treated with FA@Zein-CS, while the fluorescence signal in the pancreas of mice in the FA@Zein group was significantly reduced. This targeting ability of CS modification can promote the long-term retention of natural product-based nanoantioxidants in inflamed pancreatic tissues.

[0081] Embodiment 11

[0082] In vivo treatment of acute pancreatitis with natural product-based nanoantioxidants

[0083] After a one-week adaptation period, the mice were randomly divided into the following six groups: control group, model group, zein group, FA group, FA@Zein group and FA@Zein-CS group. After fasting for 12 hours, the control group mice were intraperitoneally injected with normal saline, and the other five groups of mice were intraperitoneally injected with caerulein (50ug / kg) seven times at 1-hour intervals into C57BL / 6J mice, and lipopolysaccharide (10mg / kg) was injected at the same time in the last time to establish an animal model of acute pancreatitis. The above-mentioned modeling mice were treated twice by tail vein injection of normal saline, zein (15mg / kg), FA (35mg / kg), FA@Zein ([FA]=35mg / kg, [zein]=15mg / kg) and FA@Zein-CS ([FA]=35mg / kg, [zein]=15mg / kg), respectively. The treatment time was 1h and 5h after the injection of caerulein (such as Fig.12 a). The mice in the control group and the model group were injected with the same volume of normal saline. The pancreas and blood were collected 48 hours later. The pancreas was fixed by perfusion with 4% paraformaldehyde, and paraffin sections were prepared for HE staining. The blood was centrifuged at 4°C (3000r / min, 10min) to obtain the supernatant, and then the levels of serum amylase and serum lipase were measured using the corresponding specific kits. After 2 intravenous treatments, the entire pancreas was collected and photographed. The pictures showed that the pancreas of the acute pancreatitis model mice showed obvious collapse, congestion and edema ( Fig.12 b). The FA@Zein-treated group showed reduced congestion and edema in the pancreatic tissue. Notably, the degree of pancreatic collapse, congestion, and edema in the FA@Zein-CS group was significantly reduced or even returned to normal. The reduction in pancreatic mass ratio in the FA@Zein-CS group confirmed the reduction in edema ( Fig.12 c) HE staining results of pancreatic sections are as follows Fig.12 As shown in Figure d, [lipopolysaccharide + caerulein]-induced mice showed obvious diffuse widening of intralobular spaces, destruction of normal pancreatic architecture, and extensive necrosis and inflammatory infiltration, demonstrating the successful establishment of acute pancreatitis model mice. Treatment with zein and FA alone only partially alleviated the pathological damage. In contrast, the FA@Zein-treated group showed a significant reduction in widening of intralobular spaces, pancreatic tissue destruction, and inflammatory infiltration. It is worth noting that the efficacy of FA@Zein was much lower than that of the FA@Zein-CS-treated group. Histopathological scores also showed that the scores of the FA@Zein-CS-treated group were significantly reduced compared with the zein, FA, and FA@Zein-treated groups, indicating that FA@Zein-CS significantly improved pancreatic damage ( Fig.12 e) Serum amylase and lipase levels Fig.12As shown in f-12g, [lipopolysaccharide + caerulein] stimulation led to a sharp increase of 4.0-fold and 13.9-fold in serum amylase and lipase levels, respectively, compared with the normal group of mice. Injection of zein and FA alone only reduced amylase by 22.2% and 34.4%, and lipase by 20.2% and 29.9%, respectively. Treatment with FA@Zein reduced amylase and lipase by 49.3% and 56.1%, respectively. Surprisingly, the amylase and lipase levels of mice treated with FA@Zein-CS were significantly reduced by 61.8% and 82.8%, respectively. The significant therapeutic effect of FA@Zein-CS on acute pancreatitis can be attributed to the targeting ability of CS-mediated inflamed pancreas, the intracellular response release characteristics, and the synergistic antioxidant effects of zein and FA.

[0084] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A natural product-based nano antioxidant for the treatment of acute pancreatitis, characterized in that: The materials needed are: 50mg zein; 25mg ferulic acid; 15 mL 80% (v / v) ethanol-deionized water solution; 40mg chondroitin sulfate; 15 mL deionized water; zein / FA mixed solution; CS solution.

2. A method for preparing a natural product-based nano antioxidant for the treatment of acute pancreatitis, based on the method for preparing the material of the natural product-based nano antioxidant for the treatment of acute pancreatitis according to claim 1, characterized in that: 50 mg of zein and 25 mg of ferulic acid were dissolved in 15 mL of 80% (v / v) ethanol-deionized water solution and magnetically stirred at 500 rpm for 1 h.

3. The method for preparing the natural product-based nano antioxidant for treating acute pancreatitis according to claim 2, characterized in that: The zein / FA mixed solution was added dropwise into the CS solution under magnetic stirring at 600 rpm.

4. The method for preparing the natural product-based nano antioxidant for treating acute pancreatitis according to claim 3, characterized in that: The resulting solution was dialyzed with deionized water for 48 h to obtain the natural product-based nano antioxidant FA@Zein-CS; FA@Zein was prepared using the same method but without adding FA.

5. Characterization of the preparation of a natural product-based nano antioxidant for the treatment of acute pancreatitis, which is achieved based on the method for preparing a natural product-based nano antioxidant for the treatment of acute pancreatitis according to any one of claims 2 to 4, characterized in that: The hydrated particle size and Zeta potential of FA@Zein-CS were measured by Malvern laser particle size analyzer, and its morphology was characterized by HITACHI transmission electron microscopy.

6. Characterization of the preparation of the natural product-based nano antioxidant for the treatment of acute pancreatitis according to claim 5, characterized in that: The hydrodynamic diameter of FA@Zein-CS is 134.0 nm, which is smaller than that of FA@Zein (200.1 nm). CS can hybridize with FA@Zein through electrostatic interaction, thereby stabilizing the colloidal system and resulting in a smaller particle size.

7. Characterization of the preparation of the natural product-based nano antioxidant for the treatment of acute pancreatitis according to claim 5, characterized in that: The zeta potential of FA@Zein-CS is -32.2 mV, and compared with FA@Zein, FA@Zein-CS exhibits a significant surface charge reversal (42.6 mV).

8. Characterization of the preparation of the natural product-based nano antioxidant for the treatment of acute pancreatitis according to claim 5, characterized in that: Compared with positively charged nanosystems, the negatively charged surface may exhibit improved colloidal stability and increased blood circulation time. Transmission electron microscopy images showed that the morphology of FA@Zein-CS was spherical.

9. Characterization of the preparation of the natural product-based nano antioxidant for the treatment of acute pancreatitis according to claim 5, characterized in that: FA@Zein-CS was diluted with 80% ethanol solution, and then FA was extracted by ultrasound-assisted method; the content of FA in the dispersion was determined by Shimadzu UV-2600i spectrophotometer.

10. Characterization of the preparation of the natural product-based nano antioxidant for the treatment of acute pancreatitis according to claim 5, characterized in that: The absorbance at 321 nm was recorded, and the drug loading and encapsulation efficiency were calculated by the following formula: Drug loading (%) = (mass of FA in FA@Zein-CS / mass of FA@Zein-CS) × 100% Encapsulation rate (%) = (FA mass in FA@Zein-CS / FA input amount) × 100% The measurement results showed that the drug loading and encapsulation efficiency of FA in FA@Zein-CS were approximately 70% and 52%, respectively.

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