Preparation method and application of acid-responsive aggregated carrot-source nano antioxidant

Abstract

The invention discloses a preparation method and application of an acid-responsive aggregated carrot-source nano antioxidant, and belongs to the technical field of medical treatment. The carrot source nano antioxidant is prepared by five steps of cleaning, crushing, filtering, gradient centrifuging and extruding, can be applied to improvement of oxidative stress, promotion of good bone remodeling and effective treatment of osteoporosis, and has good practical application value.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an acid-responsive aggregated carrot-derived nano antioxidant, belonging to the technical field of medicine. Background Art

[0002] Osteoporosis is a systemic bone disease characterized by low bone density and deterioration of bone microarchitecture. The imbalance of homeostasis between bone formation and bone resorption is the main cause of osteoporosis (Luhmann T, Germershaus O, Groll J, Meinel L: Bone targeting for the treatment of osteoporosis. J Control Release. 2012; 161:198-213.). With the aging of the population worldwide, the incidence of osteoporosis has increased rapidly (Yu B, Wang C: Osteoporosis: the result of an 'Aged' bonemicroenvironment. Trends Mol Med. 2016; 22:641-644.). Excessive production of free radicals and imbalance of the body's antioxidant defense system are considered to be the pathogenic factors of aging and various chronic degenerative diseases. More and more studies have shown that oxidative stress is one of the important pathogenesis of osteoporosis. Reactive oxygen species (ROS) produced during human metabolism include hydroxyl radicals (•OH), hydrogen peroxide (H2O2) and superoxide anions ( ), oxidative stress caused by excessive accumulation of free radicals accelerates osteoblast senescence and apoptosis, thereby significantly hindering osteogenesis (Zhao M, Kang M, Wang J, Yang R, Zhong X, Xie Q, Zhou S, Zhang Z, Zheng J, Zhang Y, et al: Stem cell-derived nanoblasts embedded in dual-layered hydrogel for programmed ROS regulation and comprehensive tissue regeneration in burn wound healing. Adv Mater. 2024; 36: e2401369.; Zhivodernikov IV, Kirichenko TV, Markina YV, Postnov AY, Markin AM: Molecular and cellular mechanisms of osteoporosis. Int J Mol Sci. 2023; 24: 15772.). Therefore, developing a new antioxidant that can alleviate oxidative stress to promote osteoblast differentiation is a key strategy for the treatment of osteoporosis.

[0003] Fruits and vegetables are rich sources of antioxidants, which play a key role in scavenging free radicals and maintaining homeostasis. The clinical application of natural antioxidants is limited due to their small molecular weight, low yield, complex purification process, instability, and poor bioavailability (Cong M, Tan S, Li S, Gao L, Huang L, Zhang HG, Qiao H: Technology insight: Plant-derived vesicles-How far from the clinical biotherapeutics and therapeutic drug carriers? Adv Drug Deliv Rev. 2022; 182:114108.). Exosome-like nanovesicles extracted from plants have attracted widespread attention as potential therapeutic candidates due to their abundant resources, low immune risk, and good biosafety. So far, nanovesicles containing various bioactive substances (including proteins, lipids, mRNA and miRNA) have been extracted from fruits and vegetables for the treatment of cancer and various inflammatory diseases (Xu X, Yuan T, Dad HA, Shi M, Huang Y, Jiang Z, Peng L: Plant exosomes as novel nanoplatforms for microRNA transfer stimulate neural differentiation of stem cells in vitro and in vivo. Nano Lett. 2021; 21:8151-8159.;Zhang L, LiS, Cong M, Liu Z, Dong Z, Zhao M, Gao K, Hu L, Qiao H: Lemon-derivedextracellular vesicle-like nanoparticles block the progression of kidneystones by antagonizing endoplasmic reticulum stress in renal tubular cells. Nano Lett. 2023; 23:1555-1563.).At the same time, exosome-like nanovesicles are rich in antioxidants such as flavonoids and polyphenols, which can be protected by the lipid bilayer membrane to prevent rapid oxidation and degradation (Zeng L, Wang H, Shi W, Chen L, Chen T, Chen G, Wang W, Lan J, Huang Z, Zhang J, Chen J: Aloe derived nanovesicle as a functional carrier for indocyanine green encapsulation and phototherapy. J Nanobiotechnol. 2021; 19:439.). Therefore, exosome-like vesicles extracted from plants are expected to become a new generation of nano antioxidants.

[0004] Carrots are rich in carotenoids, vitamins and other nutrients that can relieve oxidative stress, but the high dose requirements for in vivo treatment and the lack of specific targeting limit their clinical application. In addition, the traditional ultracentrifugation method is prone to deformation, destruction and aggregation of heterogeneous particles in the process of repeated high-pressure purification, which affects its further application. Summary of the invention

[0005] The present invention aims to provide a preparation method and application of an acid-responsive aggregated carrot-derived nano-antioxidant. The carrot-derived nano-antioxidant is prepared by an extrusion method and can be used to improve oxidative stress, promote good bone reconstruction, and effectively treat osteoporosis.

[0006] The present invention uses an extrusion method to obtain exosome-like nanovesicles (i.e., "carrot-derived nanoantioxidants") from carrots containing various bioactive substances (such as proteins, lipids, organic oxygen compounds, and organic acids). The obtained product has the same purity as the ultracentrifugation method, complete morphology, good dispersibility, low cost-effectiveness, suitable for large-scale application, and is expected to become a promising alternative method. Carrot-derived nanoantioxidants (CNs) show excellent stability, good biocompatibility, and excellent antioxidant properties. Due to the large number of gaps between the endothelial cells and tubular channels of the bone marrow capillaries in the bone structure, CNs can passively target and accumulate in the bone tissue under the acidic microenvironment, which can improve oxidative stress, promote good bone reconstruction, and effectively treat osteoporosis.

[0007] The present invention provides a method for preparing an acid-responsive aggregated carrot-derived nano antioxidant, comprising the following steps: (1) Cleaning: Wash the carrots with distilled water, peel them, and cut them into pieces for later use; (2) Crushing: Mix 50-500 mg of carrots with 150-1000 mL of pre-cooled phosphate buffer solution (PBS) and crush using a wall crusher; the pH of the buffer solution is 7.2-7.4 and the concentration is 0.01 M; (3) Filtration: Filter the carrot juice through 100-150 mesh gauze to remove larger (100-150 μm) residues to obtain an orange-yellow transparent solution; (4) Gradient centrifugation: centrifuge the solution obtained in step (3) at 800-1200 rpm for 15-30 min and at 2000-4000 rpm for 15-30 min to remove larger fibers (1-100 μm). Centrifuge the obtained supernatant at 10000-12000 rpm for 30-60 min at 4°C and collect the supernatant. (5) Extrusion: The supernatant obtained in step (4) is extruded through 0.4 μm-0.45 μm and 0.2 μm-0.22 μm microporous filter membranes in turn. The carrot-derived nanovesicles, i.e., nano antioxidants, are successfully prepared and stored at -80°C to -20°C for future use.

[0008] Furthermore, the process parameters of the wall breaking machine are: speed of 35000-43000 rpm, power of 2200W, on for 1 min, off for 1 min, and so on for 4 to 5 cycles.

[0009] Furthermore, the microporous filter membrane in step (5) can also be replaced by a liposome extruder.

[0010] The invention provides a carrot-derived nano antioxidant, which is prepared by the method.

[0011] Furthermore, the carrot-derived nano antioxidant prepared by the above method has a uniform round structure and a characteristic lipid bilayer, a particle size of 50-200 nm, and a Zeta potential of -30 mV to -5 mV; Furthermore, the acid-responsive aggregation-antioxidant carrot-derived nano-antioxidant has a stable nanostructure under normal physiological conditions (pH 7.4) and is converted into larger particles under acidic conditions (pH 4.0).

[0012] The present invention provides application of the acid-responsive aggregated carrot-derived nano antioxidant in preparing a medicine for treating osteoporosis.

[0013] Beneficial effects of the present invention: The present invention adopts an extrusion method to prepare a carrot-derived nano antioxidant, and the raw materials are cheap and readily available, and the extraction cost is low; the separation technology is simple, and large-scale production is easy; the purity is high, the size is uniform, and the stability is good; the biocompatibility is high, and it is rich in various bioactive components such as proteins, lipids, and organic oxides, and has the effects of improving oxidative stress, promoting osteogenesis, and the like; it responds to the acidic microenvironment of osteoporosis, can be used to prepare drugs for treating osteoporosis, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a transmission electron microscope image of the carrot-derived nano antioxidant prepared in Example 1 of the present invention.

[0015] Figure 2 This is a particle size distribution diagram of the carrot-derived nano antioxidant prepared in Example 1 of the present invention.

[0016] Figure 3 3 is the potential diagram of the carrot-derived nano antioxidant prepared in Example 1 of the present invention.

[0017] Figure 4 The particle size stability of the carrot-derived nano antioxidant prepared in Example 1 of the present invention under different conditions (pH 7.4, pH 4.0 and culture medium containing 10% FBS).

[0018] Figure 5 The potential stability of the carrot-derived nano antioxidant prepared in Example 1 of the present invention under different conditions (pH 7.4, pH 4.0 and culture medium containing 10% FBS).

[0019] Figure 6 The particle size stability of the carrot-derived nano antioxidant prepared in Example 1 of the present invention at different temperatures (4, -20, -80°C).

[0020] Figure 7 The potential stability of the carrot-derived nano antioxidant prepared in Example 1 of the present invention at different temperatures (4, -20, -80°C).

[0021] Figure 8 The stability of protein content in the carrot-derived nano antioxidant prepared from different batches of carrots in Example 1 of the present invention.

[0022] Fig. 9 It is a ratio chart of the number of super-class metabolites in each chemical category of the carrot-derived nano antioxidant described in Example 5 of the present invention.

[0023] Fig.10 This is the result of measuring the •OH scavenging ability of the carrot-derived nano antioxidant described in Example 6 of the present invention.

[0024] Fig.11 This is the result of measuring the SOD-like catalytic activity of the carrot-derived nano antioxidant described in Example 6 of the present invention.

[0025] Fig.12 This is the result of measuring the pseudo-CAT enzyme activity of the carrot-derived nano antioxidant described in Example 6 of the present invention.

[0026] Fig.13 This is an evaluation of the total antioxidant capacity of the carrot-derived nano antioxidant described in Example 6 of the present invention.

[0027] Fig.14 This is the effect of the carrot-derived nano antioxidant described in Example 7 of the present invention on cell survival rate.

[0028] Fig.15 It is the cell survival rate after incubation of the carrot-derived nano antioxidant described in Example 8 of the present invention with MC3T3-E1 cells under oxidative stress conditions.

[0029] Fig.16 It is a flow cytometry result diagram and confocal microscopy image of the intracellular reactive oxygen species (ROS) level after the carrot-derived nano antioxidant described in Example 9 of the present invention was incubated with MC3T3-E1 cells under oxidative stress conditions.

[0030] Fig.17 It is the osteogenic activity of MC3T3-E1 cells incubated with the carrot-derived nano antioxidant described in Example 10 of the present invention under oxidative stress conditions.

[0031] Fig.18 This is a DHE fluorescence image of a femoral slice of osteoporotic mice after treatment with the carrot-derived nano-antioxidant described in Example 11 of the present invention.

[0032] Fig.19 This is a Micro-CT image of osteoporotic mice treated with the carrot-derived nano-antioxidant described in Example 11 of the present invention.

[0033] Fig. 20 This is the evaluation of bone-related parameters after treating osteoporotic mice with the carrot-derived nano antioxidant described in Example 11 of the present invention. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the examples. It should be understood that these examples are only for illustrative purposes and are in no way intended to limit the scope of the present invention. The raw materials described in the following examples are all commercially available. Example 1

[0035] A method for preparing a carrot-derived nano antioxidant comprises the following steps: (1) Cleaning: Wash the carrots with distilled water, peel them, and cut them into pieces for later use; (2) Crushing: Mix 100 mg of carrots with 150 mL of pre-cooled phosphate buffer solution (PBS) and crush them using a wall-breaking machine. The wall-breaking machine was set to gear 7 (the process parameters of the wall-breaking machine were: speed 42,000 rpm, power 2,200 W), on for 1 min, off for 1 min, and this was repeated for 4 cycles. (3) Filtration: Filter the carrot juice through 100-mesh gauze to remove the residue to obtain an orange-yellow transparent solution; (4) Gradient centrifugation: Use a centrifuge to centrifuge the solution obtained in step (3) at 1000 × g for 20 min and 3000 × g for 20 min, and then centrifuge the obtained supernatant at 10000 × g for 40 min at 4°C. (5) Extrusion: The supernatant obtained in step (4) was extruded through 0.45 and 0.2 μm microporous filter membranes in turn, and the carrot-derived nano-antioxidant was successfully prepared and stored at -80°C for subsequent experiments. Example 2

[0036] A method for preparing a carrot-derived nano antioxidant comprises the following steps: (1) Cleaning: Wash the carrots with distilled water, peel them, and cut them into pieces for later use; (2) Crushing: Mix 50 mg of carrots with 100 mL of pre-cooled phosphate buffer solution (PBS) and crush them using a wall-breaking machine. The wall-breaking machine was set to gear 6 (the process parameters of the wall-breaking machine were: speed 41,000 rpm, power 2,200 W), and the mixture was turned on for 1 min and off for 1 min for 5 cycles. (3) Filtration: Filter the carrot juice through 100-mesh gauze to remove the residue to obtain an orange-yellow transparent solution; (4) Gradient centrifugation: Use a centrifuge to centrifuge the solution obtained in step (3) at 800 × g for 20 min and 3000 × g for 20 min, and then centrifuge the obtained supernatant at 12000 × g for 30 min at 4°C. (5) Extrusion: The supernatant obtained in step (4) was extruded through 0.45 and 0.2 μm microporous filter membranes in turn, and the carrot-derived nano-antioxidant was successfully prepared and stored at -80°C for subsequent experiments. Example 3

[0037] A method for preparing a carrot-derived nano antioxidant comprises the following steps: (1) Cleaning: Wash the carrots with distilled water, peel them, and cut them into pieces for later use; (2) Crushing: Mix 200 mg of carrots with 500 mL of pre-cooled phosphate buffer solution (PBS) and crush them using a wall-breaking machine. The wall-breaking machine was set to gear 8 (the process parameters of the wall-breaking machine were: speed 43,000 rpm, power 2200 W), and the mixture was turned on for 1 min and off for 1 min for 4 cycles. (3) Filtration: Filter the carrot juice through 100-mesh gauze to remove the residue to obtain an orange-yellow transparent solution; (4) Gradient centrifugation: Use a centrifuge to centrifuge the solution obtained in step (3) at 1000 × g for 20 min and 3000 × g for 20 min, and then centrifuge the obtained supernatant at 10000 × g for 40 min at 4°C. (5) Extrusion: The supernatant obtained in step (4) was extruded through 0.4 and 0.2 μm microporous filter membranes in turn, and the carrot-derived nano antioxidant was successfully prepared and stored at -80°C for subsequent experiments. Example 4

[0038] In this example, the CNs prepared in Example 1 were tested and characterized in the following aspects: The morphology of CNs was determined by transmission electron microscopy (TEM). The results showed that CNs had a uniform round structure and a characteristic lipid bilayer ( Figure 1 ); The size distribution and zeta potential of CNs were measured by dynamic light scattering (DLS) analysis, and the average size of CNs was found to be 137.8 nm ( Figure 2 ), which is consistent with the transmission electron microscopy results, the Zeta potential is -16.5 mV ( Figure 3 ); CNs were dispersed in PBS buffer (pH 4.0 and 7.4) or basal medium containing 10% fetal bovine serum (FBS) and incubated at 37°C for different time periods. DLS was used to detect the size distribution and zeta potential at different time points to evaluate the physiological stability of CNs under different conditions. The test results showed that CNs had a stable nanostructure under normal physiological conditions (pH 7.4) and in the presence of FBS, and would be converted into larger particles under pH 4.0, indicating that CNs had a strong retention capacity in the acidic microenvironment of osteoporosis ( Figure 4 , 5); CNs were dispersed in PBS (pH 7.4) and stored at 4, -20, and -80°C, and the particle size distribution was measured weekly ( Figure 6 ) and zeta potential ( Figure 7 ) to test the storage stability of CNs at different temperatures. The test results showed that CNs maintained a stable structure after two weeks; Carrots purchased from different batches were used for the experiment, and the experimental process was the same as in Example 1. CNs were extracted, and the protein content was quantified using a BCA test kit to detect the batch stability of CNs. The CNs measurement of each group was repeated 3 times, and the average value was taken. The measurement results showed that the CNs preparation had good batch stability ( Figure 8 ); It shows that the extrusion method provided by the present invention is highly practical. Example 5

[0039] In this example, high-resolution non-targeted metabolomics analysis was performed on the CNs prepared in Example 1, and metabolites in the CNs were detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), including the following steps: (1) Sample extraction: After slowly thawing at 4°C, take an appropriate amount of sample and add it to pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortex mix, sonicate at low temperature for 30 min, let stand at -20°C for 10 min, centrifuge at 14000 g at 4°C for 20 min, take the supernatant and vacuum dry, add 100 μL acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v) for re-dissolution during mass spectrometry analysis, vortex, centrifuge at 14000 g at 4°C for 15 min, and take the supernatant for sampling and analysis; (2) Chromatography-mass spectrometry analysis: The samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) C-18 column, and the primary and secondary spectra of the samples were collected using an AB Triple TOF 6600 mass spectrometer; (3) Data analysis: The data extracted by MSDAIL software are first subjected to metabolite structure identification and data preprocessing, then the experimental data quality is evaluated, and finally data analysis is performed.

[0040] The metabolite identification results obtained by statistical analysis of Example 5 are as follows: After the positive and negative ion modes were combined, total-metabolites were identified, and a total of 286 metabolites were detected. According to their chemical classification information, they were classified and counted into 11 superclasses. The specific proportion of each metabolite is as follows: Fig. 9 shown. Example 6

[0041] In this example, the antioxidant activity of CNs prepared in Example 1 was determined, including the following aspects: The hydroxyl radical (•OH) scavenging ability assay kit was used to determine the •OH scavenging ability of CNs. H2O2 / Fe3+ produced hydroxyl radicals through the Fenton reaction, which converted o-phenanthroline-Fe 2+ Aqueous solution Fe 2+ Oxidized to Fe3+ , resulting in a decrease in the absorbance at 536 nm. The degree of inhibition of the rate of decrease in the absorbance at 536 nm of the sample reflects the ability of the sample to scavenge hydroxyl free radicals. The absorbance (OD value) at 536 nm was measured by an enzyme-labeled instrument, and the calculation formula was: • OH scavenging rate (%) = (A 样品 -A 对照组 ) / (A 空白 -A 对照组 )×100%. The test results showed that the •OH scavenging rate of CNs increased significantly with the increase of concentration. When the concentration of CNs was 1600μg / mL, its scavenging rate reached 57.2%, indicating that CNs had a large •OH scavenging capacity ( Fig.10 ).

[0042] The SOD-like catalytic activity of CNs was detected using a SOD kit. CNs of different concentrations were thoroughly mixed with the working solution, incubated in a 37°C water bath for 40 min, and then allowed to stand at room temperature for 10 min after adding the color developer. The absorption curve was measured using an ultraviolet spectrophotometer (UV-Vis). The decrease in the absorbance value of the sample group relative to the control group at 550 nm indicated the activity of CNs to superoxide anion free radicals ( ) and took photos to record the color changes after the reaction of different concentrations of CNs. The results showed that with the increase of CNs concentration, the absorbance value at 550 nm decreased from 0.61 to 0.06, indicating that CNs has a strong pseudo-SOD enzyme activity ( Fig.11 ).

[0043] The hydrogen peroxide (H2O2) detection kit was used to detect the scavenging effect of CNs on H2O2. CNs of different concentrations were incubated with H2O2 (50 mM) at room temperature, and the scavenging rate of H2O2 was measured at 560 nm at different time intervals (10, 60 min) using a microplate reader. The results showed that CNs had a significant scavenging efficiency for H2O2. At a concentration of 1600 μg / mL, about 96.9% of hydrogen peroxide was eliminated after 60 min, indicating that CNs had strong CAT-like enzyme activity ( Fig.12 ).

[0044] The total antioxidant activity of CNs was detected using the ABTS method. CNs of different concentrations were mixed with the pre-prepared ABTS working solution at room temperature for 6 min, and the resulting mixture was detected by UV-Vis absorption spectroscopy. The formula is free radical scavenging rate (%) = (A 空白 -A 样品 ) / A 空白 × 100. After treatment with different concentrations of CNs, ABTS •+The characteristic absorption peak at 734 nm decreased significantly, and its free radical scavenging rate increased significantly with the increase of concentration, indicating that CNs have excellent total antioxidant capacity ( Fig.13 ). Example 7

[0045] In this example, the effects of the CNs prepared in Example 1 on cell survival rate were specifically implemented as follows: Using alarmBlue TM The HS cell viability detection kit was used to measure the survival rate of osteoblast MC3T3-E1 cells at 1, 3, and 5 days after administration of CNs at different concentrations (50, 100, 200, and 400 μg / mL). The absorbance value at 600 nm was measured using an ELISA instrument as a reference wavelength, and the absorbance value (OD) at a wavelength of 570 nm was measured. The cell survival rate (%) = (OD of the experimental group-OD of the blank group) / (OD of the control group-OD of the blank group) × 100%. The experimental results showed that CNs at different concentrations of 0~400 μg / mL can significantly promote the proliferation of MC3T3-E1 cells. In particular, after treatment with 400 μg / mL CNs, cell viability increased by 3.3 times from day 1 to day 5, indicating that CNs has good biocompatibility and the ability to promote osteoblast proliferation ( Fig.14 ). Example 8

[0046] In this example, the protective effect of CNs prepared in Example 1 on H2O2-induced ME3T3-E1 cell damage was investigated by the following specific implementation steps: MC3T3-E1 cells were seeded on 96-well plates. After the cells adhered to the wall, the cell culture supernatant was aspirated and different concentrations of CNs were added to the drug groups. After 24 hours, DMEM medium containing H2O2 (1mM) was added to both the drug group and the H2O2 group for 6 hours. The old medium was aspirated, the cells were washed twice with sterile PBS, and serum-free medium containing 10% CCK-8 was added. The cells were placed in a CO2 incubator and incubated for 2 hours. The absorbance value was measured at 450 nm using an ELISA reader, and the cell survival rate of different treatment groups was calculated. The results showed that MC3T3-E1 cells were treated with H2O2 to induce oxidative stress to simulate the harmful microenvironment of osteoporosis. 1 mM H2O2 reduced the cell survival rate to 53.1%. After being treated with 200 and 400μg / mL CNs for 24 hours, the cell viability increased to 78.5% and 82.8%, respectively, indicating that CNs can effectively protect cells from damage by ROS ( Fig.15 ). Example 9

[0047] In this example, the CNs prepared in Example 1 were subjected to intracellular reactive oxygen species (ROS) determination, and the specific implementation steps were as follows: The fluorescent probe DCFH-DA in the ROS detection kit was used to detect the intracellular ROS level. MC3T3-E1 cells were cultured at 3×10 5 / well cultured in a 6-well plate, incubated with 200 μg / mL (protein concentration) CNs for 24 hours, then treated with 1 mM hydrogen peroxide for 6 hours, washed twice with PBS, incubated with serum-free medium and 2 μM DCFH-DA at 37 ° C for 30 min, and then washed twice with PBS again to remove unlabeled DCFH-DA. The fluorescence intensity was characterized by flow cytometry and CLSM. The test results showed that the ROS level of MC3T3-E1 cells in the oxidative stress model group was severely increased. However, after the cells were treated with CNs, the green fluorescence signal was significantly reduced and the number of positively stained cells decreased (13.9%), indicating that it has a strong ROS elimination ability in vitro ( Fig.16 ).

[0048] Embodiment 10: In this example, the osteogenic differentiation ability of CNs prepared in Example 1 under oxidative stress conditions was evaluated, and the specific implementation steps were as follows: To examine the expression of alkaline phosphatase (ALP), an early osteogenic marker of MC3T3-E1 cells, cells were seeded at a density of 5000 cells / well in 6-well plates and treated with different concentrations of CNs for 24 h, followed by stimulation with H2O2-containing medium for 6 h. After 3 days, cells were fixed with paraformaldehyde (4%), stained with BCIP / NBT alkaline phosphatase colorimetric kit and observed under a microscope. In order to detect the production of mineralized nodules, a marker of osteoblast differentiation and maturation, MC3T3-E1 cells were stained with Alizarin Red (ARS). After 5 days of culturing MC3T3-E1 cells with different concentrations of CNs (50, 100, 200, and 400 μg / mL) under oxidative stress conditions, cells were fixed with 4% paraformaldehyde solution, washed twice with PBS, and stained with ARS solution. Images were recorded with a DM500 microscope and semi-quantified using ImageJ software. ALP staining images showed that CNs effectively alleviated H2O2-induced oxidative stress and reprogrammed osteoblasts, with the highest ALP expression at concentrations above 200 μg / mL. In addition, the amount of calcium mineral deposition by ARS staining in the hydrogen peroxide-stimulated group was lower than that in the control group. However, after incubation with 200 μg / mL CNs, a significant increase in calcium mineral deposition was observed ( Fig.17 ).

[0049] Embodiment 11: In this example, the CNs prepared in Example 1 were evaluated for their anti-oxidative stress and osteogenic activities in vivo, and the specific implementation steps were as follows: Eight-week-old female C57BL / 6J mice underwent bilateral ovariectomy to induce an osteoporotic (OVX) mouse model. One week after surgery, once the wounds healed, the mice were divided into four groups, with 5 mice in each group. The groups were as follows: sham group (Sham), model group (OVX), OVX mouse tail vein injection of CNs (2.5 mg / kg) treatment group (OVX+CNs), and positive control group: OVX mouse tail vein injection of estradiol (E2, 0.5 mg / kg) treatment group (OVX + E2). The entire treatment period lasted for 8 weeks, and the femoral tissues of the mice were collected after the end. DHE fluorescent probe staining was used to detect the level of ROS on femoral sections; Micro-CT scanning was used to observe the changes in bone microstructure and analyze and calculate bone-related parameters.

[0050] The experimental results of Example 11 are analyzed as follows: (1) The ROS fluorescence intensity in the OVX model group was higher than that in the sham operation group, indicating that the oxidative stress in the microenvironment associated with osteoporosis was enhanced. After treatment with 2.5 mg / kg CNs, the ROS level decreased to 86.2% compared with the OVX group, effectively alleviating the oxidative stress in the body ( Fig.18 ); (2) Micro-CT and 3D reconstruction techniques were used to observe bone regeneration in mice. Cross-sectional 2D images and 3D reconstruction images showed that OVX mice had severe bone loss, decreased bone density, and destroyed bone microstructure. However, after CNs treatment, trabecular bone was observed to have better connectivity and organizational structure, similar to the results of the E2-positive group ( Fig.19 ); (3) In the quantitative results of bone-related parameters, the BV / TV (3.6%) and BMD (0.11 g cm -3 )、Tb.N(0.87 mm -1 ) and Tb.Th (0.04 mm) values ​​were lower, and Tb.Sp (0.36 mm) value was higher. However, BV / TV (%), BMD (g cm -3 )、Tb.N (mm -1 ), Tb.Th (mm) and Tb.Sp (mm) values ​​are 7.0% and 0.16 g cm -3 , 1.37mm -1 , 0.05 mm and 0.29 mm. There was no significant difference between the CNs group and the sham operation group. CNs showed great potential as nano antioxidants in bone tissue preservation and reversal of osteoporosis ( Fig. 20 ).

[0051] The above is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an acid-responsive aggregated carrot-derived nano antioxidant, characterized in that The following steps are involved: (1) Cleaning: Wash the carrots with distilled water, peel them, and cut them into pieces for later use; (2) Crushing: Mix 50-500 mg of carrots with 150-1000 mL of pre-cooled phosphate buffer solution and crush using a wall crusher; (3) Filtration: Filter the carrot juice through gauze to remove the residue and obtain an orange-yellow transparent solution; (4) Gradient centrifugation: centrifuge the solution obtained in step (3) at 800-1200 rpm for 15-30 min and at 2000-4000 rpm for 15-30 min to remove larger fibers, and centrifuge the obtained supernatant at 10000-12000 rpm for 30-60 min at 4°C to collect the supernatant; (5) Extrusion: The supernatant obtained in step (4) is sequentially passed through 0.4 μm-0.45 μm and 0.2 μm-0.22 μm microporous filter membranes to extrude carrot-derived nanovesicles, i.e., the nano antioxidant is successfully prepared and stored at -80°C to -20°C for future use.

2. The method for preparing the acid-responsive aggregated carrot-derived nano antioxidant according to claim 1, characterized in that: The pH range of phosphate buffer solution is 7.2-7.4 and the concentration is 0.01M.

3. The method for preparing the acid-responsive aggregated carrot-derived nano antioxidant according to claim 1, characterized in that: The process parameters of the wall breaking machine are: speed of 35000-43000 rpm, power of 2200W, on for 1 min, off for 1 min, and so on for 4 to 5 cycles.

4. The method for preparing the acid-responsive aggregated carrot-derived nano antioxidant according to claim 1, characterized in that: In step (3), the gauze has a mesh size of 100-150.

5. The method for preparing the acid-responsive aggregated carrot-derived nano antioxidant according to claim 1, characterized in that: In step (5), a liposome extruder is used instead of a microporous filter to extrude carrot-derived nanovesicles.

6. The method for preparing the acid-responsive aggregated carrot-derived nano antioxidant according to claim 1, characterized in that: The prepared carrot-derived nano antioxidant has a uniform round structure and a characteristic lipid bilayer, a particle size of 50-200nm, and a Zeta potential of -30mV~-5mV; the carrot-derived nano antioxidant has a stable nano structure under normal physiological conditions and will be converted into larger particles under acidic conditions.

7. Use of the acid-responsive aggregated carrot-derived nano antioxidant prepared by the preparation method according to any one of claims 1 to 6 in the preparation of a drug for treating osteoporosis.

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