CN108743609B - Preparation method and application of nanoparticles capable of inhibiting Abeta polypeptide aggregation and eliminating active oxygen - Google Patents
Preparation method and application of nanoparticles capable of inhibiting Abeta polypeptide aggregation and eliminating active oxygen Download PDFInfo
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Abstract
The invention relates to the field of nano-selenium medicine preparation, and particularly discloses a preparation method and application of nanoparticles for inhibiting A beta polypeptide aggregation and eliminating active oxygen. The nano particles are nano selenium modified by resveratrol. Resveratrol has the ability to scavenge active oxygen, but its water solubility is low and its ability to inhibit the aggregation of a β induced by metal ions is weak. Compared with the resveratrol serving as a modifier, the resveratrol nano-selenium prepared by the invention can inhibit the aggregation of metal ions induced by Abeta, has the effect of clearing active oxygen and can be applied to the preparation of the medicine for resisting Alzheimer's syndrome. The product system is simple, the method is simple, and the product can be directly stored and used. The nano-selenium selected by the invention greatly improves the problems of low water solubility and low bioavailability of the resveratrol, and increases the capability of the resveratrol in inhibiting the aggregation of metal ions induced by Abeta.
Description
Technical Field
The invention relates to the field of nano-selenium medicine preparation, and in particular relates to a resveratrol nano-selenium medicine and a preparation method and application thereof.
Background
Alzheimer's Disease (AD) is a common neurodegenerative disease, with the clinical manifestations mainly being decline of memory function and cognitive ability. In china, the incidence of AD is also increasing dramatically as the aging problem of the population becomes increasingly severe. Since the severity of the AD condition greatly affects the quality of life of patients and their families, the development of AD drugs is highly necessary and urgent. The main pathological feature of AD is the discovery of amyloid plaques and the presence of fibrillar tangles within neuronal cells in the brain. Among them, amyloid plaques are a potent neuropathological marker of AD. A β polypeptides are the major components that make up amyloid plaques. Although the pathogenesis of AD is currently unclear, the amyloid deposition hypothesis is considered as the core hypothesis of AD pathogenesis. The hypothesis suggests that the fibers formed by the aggregation of a β and its various forms of aggregates can trigger a series of pathological changes in the brain of AD patients, such as synaptic damage, inflammation, and neuronal cell death. On the other hand, aggregated a β is a promoter of generation of active oxygen and active nitrogen radicals. The nervous system is an organ with high oxygen consumption, and in the elderly, the appearance of a large number of active oxygen radicals leads to the appearance of oxidative stress in the brain of patients due to the large loss of oxygen and low antioxidant levels. Active oxygen can cause lipid oxidation, DNA damage, and protein oxidation on cell membranes. Therefore, the inhibition of the spontaneous aggregation of the A beta polypeptide and the simultaneous elimination of active oxygen have very important significance for the treatment of AD.
Plant polyphenols are secondary metabolites of higher plants. At present, polyphenol has proved to have various biological functions, such as strong antioxidation, prevention of chronic diseases such as hyperglycemia, hyperlipidemia, cardiovascular and cerebrovascular diseases and the like, anticancer and resistance of nervous diseases. Resveratrol in polyphenols has high biological activity, but has the disadvantages of low water solubility, low bioavailability and the like. The appearance of the nano material provides a new way for solving the application of polyphenol in the aspect of biological medicine. The nano material has the advantages of small particle size, large specific surface area, high surface energy and the like. At the same time, the conformation of proteins and polypeptides adsorbed to the surface of the nanoparticles is often changed, which affects their biological function and aggregation. However, it is still unknown whether resveratrol-modified nanomaterials can inhibit metal ion-induced fibrotic aggregation of a β polypeptides while scavenging active oxygen.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a nanoparticle which can inhibit the aggregation of Abeta polypeptide induced by metal ions and can remove active oxygen, so as to solve the defects of low water solubility and low bioavailability of resveratrol in polyphenol.
The invention also aims to provide a preparation method of resveratrol nano-selenium for inhibiting aggregation of Abeta polypeptide induced by metal ions and eliminating active oxygen.
The invention also aims to provide application of the resveratrol nano-selenium for inhibiting the aggregation of Abeta polypeptide induced by metal ions and eliminating active oxygen in preparing the medicine for resisting Alzheimer's disease.
The technical problem to be solved by the invention is realized by the following technical scheme:
a preparation method of resveratrol nano-selenium for inhibiting metal ion induced Abeta polypeptide aggregation and eliminating active oxygen comprises the following steps:
respectively collecting resveratrol and Na2SeO3Mixing, dropwise adding sodium borohydride, stirring, cooling, standing and centrifuging to obtain resveratrol nano-selenium.
The Na is2SeO3The concentration of the solution is 0.02-0.1 mol/L.
The concentration of the resveratrol solution is 0.02-0.1 mol/L.
The reaction temperature is adjusted to be in the range of 25-50 ℃.
The pH value is adjusted to be 2-12.
The concentration of the sodium borohydride solution is 0.02-0.1 mol/L.
Preferably, the resveratrol and Na2SeO3The molar ratio is 1: 1-8: 1.
Preferably, the sodium borohydride is mixed with Na2SeO3The molar ratio is 1: 1-4: 1.
Most preferably, the resveratrol and Na2SeO3The molar ratio is 4: 1; sodium borohydride and Na2SeO3Is 1: 1.
Preferably, the reaction temperature is 35 ℃.
Preferably, the pH is 6.
Preferably, the stirring is carried out at 25-50 ℃ for 10-60 minutes.
Preferably, the cooling and standing time is 12-24 h.
Preferably, the nanosol produced can be stored at 4 ℃.
Preferably, the nanoparticles obtained after centrifugation are washed with ultrapure water. The prepared solution is prepared by adopting ultrapure water.
The resveratrol nano-selenium can be applied to the preparation of the medicine for treating the Alzheimer disease.
The resveratrol nano-selenium is applied as an A beta aggregation inhibitor.
The resveratrol nano-selenium is used as an antioxidant.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the resveratrol nano-selenium can inhibit aggregation of Abeta polypeptide induced by metal ions, and can clear active oxygen and reduce oxidation damage;
(2) the resveratrol provided by the invention has stronger oxidation resistance, but does not have the capability of inhibiting the aggregation of Abeta polypeptide induced by metal ions. The resveratrol nano-selenium not only has the strong oxidation resistance of resveratrol, but also shows the capability of inhibiting the aggregation of Abeta polypeptide induced by metal ions, thereby simultaneously improving the biological activity of the resveratrol.
(3) The resveratrol nano-selenium prepared by the invention has simple preparation process and stable product system, and can be directly stored and used.
Drawings
Fig. 1 is a morphological diagram of resveratrol nano-selenium.
Fig. 2 is a graph showing the inhibition of metal ion-induced a β 42 polypeptide aggregation by resveratrol nano-selenium.
Fig. 3 is a graph showing the effect of resveratrol nano-selenium on metal ion-induced a β 42 aggregation and morphology.
Fig. 4 shows the binding capacity of resveratrol nano-selenium and a beta 42 polypeptide.
Fig. 5 is a graph of the effect of resveratrol nano-selenium on the induction of intracellular reactive oxygen species by Α β 42 aggregates.
Fig. 6 is a graph of the effect of resveratrol nano-selenium on a β 42 aggregate-induced apoptosis of PC12 cells.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
Example 1 preparation of resveratrol nano-selenium
1) Weighing 0.173 g of sodium selenite, and diluting to 10 mL with distilled water to prepare 0.1mol/L storage solution; simultaneously weighing 0.143g of resveratrol, and diluting to 25 mL with 60% ethanol to constant volume to prepare 0.025 mol/L storage solution; 0.0378g of sodium borohydride is weighed, and distilled water is used for fixing the volume to 10 mL to prepare 0.1mol/L storage solution.
2) Mixing 0.2 mL of sodium selenite stock solution with resveratrol in different proportions, stirring at 400 rpm/min, adjusting reaction temperature to 25-50 deg.C, and adjusting pH to 2-12. Gradually dropwise adding sodium borohydride with different proportions, and continuously stirring for 10 to 60 minutes. The molar ratio of the sodium selenite to the resveratrol or the sodium borohydride is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8 respectively. Wherein, the sample with the molar ratio of sodium selenite to resveratrol of 1:4 is most stable at 35 ℃ and pH of 6, and the molar ratio of sodium selenite to sodium borohydride of 1: 1. After stirring for 30 minutes the solution turned brick red in color. And (3) placing the solution at 4 ℃ for 12 h, performing centrifugal separation, removing supernatant, washing, and fixing the volume to 10 mL to obtain the resveratrol nano-selenium. Observed by a Transmission Electron Microscope (TEM) of TEANAI-10 type, as shown in FIG. 1. A dispersion of nanoparticles having a particle size of about 100 nm was obtained. The nanoparticles can stably exist at room temperature and are easy to store.
Example 2 inhibitory Effect of resveratrol Nano-selenium on aggregation of Abeta 42 Polypeptides induced by Metal ions
Taking 35 mu M A beta 42 polypeptide and respectively mixing with 70 mu M CuCl2And incubating resveratrol with different concentrations and the resveratrol nano-selenium prepared in example 1 for 0-5 days. From the incubation solution 50. mu.L was taken daily and mixed with 200. mu.L of 15. mu.M ThT and incubated in the dark for 15 min. The fluorescence intensity of ThT was measured on a fluorescence spectrophotometer. The excitation wavelength for ThT is 440 nm and the emission wavelength is 490 nm. The experimental result is shown in fig. 2, resveratrol can not obviously inhibit aggregation of Abeta 42 polypeptide induced by metal ions, and ThT fluorescence is still maintained at higher intensity. With the increase of the resveratrol nano-selenium concentration and the decrease of ThT fluorescence, the lag phase of the aggregation of the A beta 42 polypeptide induced by the metal ions is prolonged, and the veratryl alcohol nano-selenium is an effective inhibitor for the aggregation of the A beta 42 polypeptide induced by the metal ions and can start to inhibit the aggregation of the polypeptide at the early stage of the aggregation.
Example 3 Effect of resveratrol Nano-selenium on the Metal ion-induced aggregation morphology of Abeta 42
Taking 35 mu M A beta 42 and 70 mu M CuCl respectively2And incubating resveratrol with different concentrations and the resveratrol nano-selenium prepared in example 1 for 3 days. From the incubation solution10 mu L of the solution is dropped on a copper net, dried for 10 minutes, and then 5 mu L of 1.5% (w/v) phosphotungstic acid is dropped for dyeing. And then observing an influence graph of resveratrol nano-selenium on the aggregation form of the metal ion-induced Abeta 42 on a transmission electron microscope. As shown in fig. 3, the metal ions can induce a β 42 aggregation, and bulk a β 42 aggregates can be observed. Resveratrol can not obviously inhibit the aggregation of Abeta 42 polypeptide induced by metal ions, while resveratrol nano-selenium can obviously inhibit the aggregation of Abeta 42 polypeptide induced by metal ions, and only a small amount of aggregates are observed. Therefore, the resveratrol nano-selenium can effectively inhibit the aggregation of Abeta 42 polypeptide induced by metal ions compared with resveratrol.
Example 4 resveratrol Nanoselenide binding ability to Abeta 42
Firstly, the combination of resveratrol nano selenium and A beta 42 fiber is detected. After the Abeta 42 polypeptide is incubated for 3 days, Abeta 42 fibers are formed, then 60 mu g/mL of nanoparticles are added for incubation for 6 h, and then the binding capacity of the nanoparticles and the Abeta 42 fibers is observed on a transmission electron microscope. The experimental result is shown in fig. 4A, and resveratrol nano-selenium is combined on the A beta 42 fiber. And then detecting the combination of resveratrol nano-selenium and A beta 42 monomer by adopting a resonance light scattering method. The resonant light scattering intensity is related to the size of the scattering particles. Diluting 2 mL of 0.05 mu g/mL resveratrol nano-selenium, and adding 5 mu L A beta 42 monomer to make the concentration of the A beta 42 monomer in the final volume from 5X 10-5To 50X 10-5μ g/mL. The simultaneous fluorescence spectra from 200 nm to 800 nm, Δ λ =0 nm, were detected on a Cary Eclipse fluorescence spectrophotometer with both excitation and emission slit widths set at 5 nm. The experimental result is shown in fig. 4B, in the synchronous fluorescence spectrum, the resveratrol nano-selenium can detect a corresponding characteristic peak, and the intensity of the characteristic peak increases with the increase of the concentration of the a β 42 monomer after the a β 42 monomer is added. It is understood that veratryl alcohol nano selenium can be combined with A beta 42 fiber or monomer, and the combination can affect the metal ions to induce A beta 42 aggregation. Therefore, the resveratrol nano-selenium can inhibit the aggregation of the Abeta 42 polypeptide induced by metal ions more effectively than resveratrol.
Example 5 inhibition of A beta 42 aggregate-induced intracellular reactive oxygen species by resveratrol Nanoselenium
Abeta 42 aggregate induced finesIntracellular ROS production was detected by DCFH-DA fluorescence changes. Abeta 42 polypeptide (35. mu.M) with 70. mu.M CuCl2And 60 mu g/mL resveratrol nano-selenium or resveratrol is incubated in a cell culture medium at 37 ℃ for 3 days. PC12 cells were then incubated with these incubation solutions for a further 72 h. PC12 cells were digested with pancreatin, 10 mM DCFH-DA was added, incubated for 15min in the absence of light, and the intracellular ROS content was subsequently detected by flow cytometry. The excitation and emission wavelengths were 488 nm and 525 nm, respectively. The results of the experiment are shown in FIG. 5, Cu2+The ions can remarkably promote the aggregation of the A beta 42 monomer, thereby promoting the active oxygen content in PC12 cells to be remarkably increased. The resveratrol nano-selenium obviously reduces the content of active oxygen in cells, and the reduction degree is better than that of resveratrol.
Example 6 Effect of resveratrol Nanoselenium on A β 42 aggregate-induced apoptosis
DNA fragments generated by a β 42 aggregate-induced apoptosis were detected using TUNEL kit. 35 μ M A β 42 monomer with 70 μ M CuCl2And 60 mu g/mL resveratrol nano-selenium or resveratrol is incubated in a cell culture medium at 37 ℃ for 3 days. PC12 cells were seeded onto sterile slides and incubated with the above incubation solution for 48 h. Cells were fixed with 4% paraformaldehyde solution for 15 min. After washing with PBS, it was permeabilized with 0.1% Triton X-100 for 15 min. Staining was performed using TUNEL detection kit, followed by DAPI staining for 10 min, and observation of apoptotic nuclei on a Nikon Eclipse TE 2000-E fluorescence microscope (200X). The experimental result is shown in fig. 6, the resveratrol nano-selenium can obviously reduce A beta 42 aggregate induced apoptosis, and the effect is also superior to that of resveratrol.
Claims (5)
1. A method for preparing nanoparticles which can inhibit Abeta polypeptide aggregation and eliminate active oxygen is characterized in that: the nano particles are nano selenium modified by resveratrol; the preparation method comprises the following steps:
mixing resveratrol with Na2SeO3 Preparing a solution A, adjusting the reaction temperature and pH, dropwise adding sodium borohydride, stirring, cooling, standing and centrifuging to obtain nanoparticles;
wherein the concentration of the resveratrol solution is 0.02-0.1 mol/L;
wherein Na is used for preparing the solution A2SeO3The concentration of (A) is 0.02-0.1 mol/L;
in the solution A, the resveratrol and Na are used2SeO3The molar ratio of the total amount is 1: 1-8: 1;
sodium borohydride and Na are used2SeO3The molar ratio of (a) to (b) is 1:1 to 4: 1;
the reaction temperature range is 25-50 ℃.
2. The method of claim 1, wherein the method comprises the steps of: adjusting the pH value to 2-12.
3. The method of claim 1, wherein the method comprises the steps of: the concentration of the sodium borohydride is 0.02-0.1 mol/L.
4. The method of claim 1, wherein the method comprises the steps of: after dropwise adding sodium borohydride, stirring for 10-60 minutes; and cooling, standing and cooling to room temperature and standing for 12-24 h.
5. The application of the nanoparticle which is prepared by the preparation method according to any one of claims 1 to 4 and has the functions of inhibiting A beta polypeptide aggregation and scavenging active oxygen in the preparation of the anti-Alzheimer's disease drug.
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| CN112451514A (en) * | 2020-11-26 | 2021-03-09 | 江西农业大学 | Dihydromyricetin nano-selenium and preparation method and application thereof |
| CN114522222B (en) * | 2022-02-22 | 2024-04-26 | 天津大学 | Preparation method of albumin/manganese dioxide composite nano particle and application of albumin/manganese dioxide composite nano particle in inhibiting beta amyloid aggregation and removing active oxygen |
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| CN103948935A (en) * | 2014-04-15 | 2014-07-30 | 暨南大学 | Nano-particle for inhibiting aggregation of alpha-beta and metal ions induced alpha-beta, as well as preparation and application |
| WO2016094189A1 (en) * | 2014-12-10 | 2016-06-16 | Albert Einstein College Of Medicine, Inc. | Nanoparticle compositions and methods thereof to restore vascular integrity |
| CN105617358A (en) * | 2015-12-29 | 2016-06-01 | 广州市戴菊科技发展有限公司 | Medical foods with antitumor function and preparation method thereof |
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