Polyphenol-metal nanodot as well as preparation method and application thereof

Abstract

The invention provides a polyphenol-metal nanodot as well as a preparation method and application thereof, and belongs to the technical field of biological materials. The preparation method of the polyphenol-metal nanodot provided by the invention comprises the following steps: mixing hydrophobic polyphenol, metal salt, polyvinylpyrrolidone and a solvent, and carrying out a coordination reaction to obtain the polyphenol-metal nanodot. Polyphenols have good biomedical application potential, metal salt is added to coordinate with the polyphenols to enrich the functions of the polyphenols, and polyvinylpyrrolidone is added to further optimize the dispersity of the polyphenols in water, so that the polyphenol-metal nanodots are obtained. The nanodot has good dispersity in water, and the application range of polyphenol is widened. Experimental results show that the prepared polyphenol-metal nanodot still has good dispersity when the dispersion concentration in water is 1.24 mg / mL (wherein the concentration of rutin is about 0.86 mg / mL).

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to polyphenol-metal nanodots and a preparation method and application thereof. Background Art

[0002] Polyphenols can effectively suppress the generation of free radicals and eliminate them due to their special phenolic hydroxyl structure, and most of polyphenols are low-toxic or nontoxic, without much damage to the body, and are good natural antioxidants. However, the water solubility of polyphenols is poor, such as rutin (solubility in water is about 0.0639 mg / mL), which limits its application. Therefore, how to improve the dispersion of polyphenol materials in water is a difficult problem to expand its range of application. Summary of the invention

[0003] The purpose of the present invention is to provide a polyphenol-metal nanodot and a preparation method and application thereof. The polyphenol-metal nanodot prepared by the preparation method provided by the present invention has better dispersibility in water.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing polyphenol-metal nanodots, comprising the following steps:

[0006] The hydrophobic polyphenol, metal salt, polyvinyl pyrrolidone and solvent are mixed and coordinated to obtain polyphenol-metal nanodots.

[0007] Preferably, the hydrophobic polyphenol comprises rutin.

[0008] Preferably, the average molecular weight of the polyvinyl pyrrolidone is 8,000-58,000.

[0009] Preferably, the solvent comprises one or more of methanol, ethanol and isopropyl ketone.

[0010] Preferably, the mass ratio of the hydrophobic polyphenol to the volume ratio of the solvent is 1 mg: (0.2-1) mL.

[0011] Preferably, the metal in the metal salt includes one or more of manganese, copper, cerium and gadolinium.

[0012] Preferably, the molar ratio of the hydrophobic polyphenol to the metal ion in the metal salt is 1:(0.5-2).

[0013] Preferably, the temperature of the coordination reaction is 20-30° C., and the time of the coordination reaction is 0.5-6 h.

[0014] The present invention also provides polyphenol-metal nanodots prepared by the preparation method described in the above technical solution, wherein the particle size of the polyphenol-metal nanodots is ≤3nm.

[0015] The present invention also provides the use of the polyphenol-metal nanodots described in the above technical solution as an antioxidant.

[0016] The present invention provides a method for preparing phenol-metal nanodots, comprising the following steps: mixing hydrophobic polyphenols, metal salts, polyvinyl pyrrolidone and solvents, and performing coordination reaction to obtain polyphenol-metal nanodots. The polyphenols of the present invention themselves have good biomedical application potential, and metal salts are added to coordinate with them to enrich their functions. After adding polyvinyl pyrrolidone, their dispersibility in water is further optimized, thereby obtaining polyphenol-metal nanodots. The nanodots have good dispersibility in water, which broadens the application range of polyphenols. Experimental results show that the polyphenol-metal nanodots prepared by the present invention still have good dispersibility when the dispersion concentration in water is 1.24 mg / mL (wherein the rutin concentration is about 0.86 mg / mL, which is about 10 times the solubility of rutin monomer in water). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 TEM image of polyphenol-metal nanodots prepared in Example 1 of the present invention;

[0018] Figure 2 This is a particle size distribution diagram of polyphenol-metal nanodots prepared in Example 1 of the present invention;

[0019] Figure 3 The XRD pattern of polyphenol-metal nanodots prepared in Example 1 of the present invention;

[0020] Figure 4 The infrared spectra of polyphenol-metal nanodots, rutin and polyvinyl pyrrolidone prepared in Example 1 of the present invention;

[0021] Figure 5 A polyphenol-metal nanodot aqueous dispersion system diagram and a red laser irradiation diagram prepared with physiological saline and polyphenol-metal nanodots prepared in Example 1;

[0022] Figure 6 This is a cell MTT experiment diagram of polyphenol-metal nanodots prepared in Example 1 of the present invention;

[0023] Figure 7 This is a diagram of the hemolysis experiment of the polyphenol-metal nanodots prepared in Example 1 of the present invention;

[0024] Figure 8 This is a graph showing the DPPH removal efficiency of polyphenol-metal nanodots prepared in Example 1 of the present invention;

[0025] Fig. 9 This is a spectrum corresponding to the DPPH removal efficiency of the polyphenol-metal nanodots prepared in Example 1 of the present invention;

[0026] Fig.10 This is a graph showing the intracellular antioxidant performance of the polyphenol-metal nanodots prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a method for preparing polyphenol-metal nanodots, comprising the following steps:

[0028] The hydrophobic polyphenol, metal salt, polyvinyl pyrrolidone and solvent are mixed and coordinated to obtain polyphenol-metal nanodots.

[0029] Unless otherwise specified, the present invention has no particular limitation on the sources of the raw materials, and commercially available products known to those skilled in the art may be used.

[0030] In the present invention, the hydrophobic polyphenol is preferably rutin.

[0031] In the present invention, the metal in the metal salt preferably includes one or more of manganese, copper, cerium and gadolinium, and more preferably manganese.

[0032] In the present invention, the metal salt preferably includes metal hydrochloride, metal nitrate or metal sulfate, and more preferably includes manganese chloride tetrahydrate, manganese nitrate, manganese sulfate, copper chloride, copper nitrate, copper sulfate, cerium chloride heptahydrate, cerium nitrate, cerium sulfate, gadolinium chloride hexahydrate, gadolinium nitrate or gadolinium sulfate.

[0033] In the present invention, the molar ratio of the hydrophobic polyphenol to the metal ion in the metal salt is preferably 1:(0.5-2), more preferably 1:(0.5-1). The present invention limits the molar ratio of the hydrophobic polyphenol to the metal ion in the metal salt to the above range, which can further ensure the coordination effect between the metal ion and the polyphenol in the polyphenol-metal nanodots.

[0034] In the present invention, the average molecular weight of the polyvinyl pyrrolidone is preferably 8000 to 58000 or the K value is preferably 13 to 32, more preferably the average molecular weight is 10000 or the K value is 13 to 18. The present invention limits the average molecular weight or K value of polyvinyl pyrrolidone to the above range, which can effectively ensure the dispersibility of polyphenol-metal nanodots in water.

[0035] In the present invention, the mass ratio of the hydrophobic polyphenol to polyvinyl pyrrolidone is preferably 1:(0.1-0.2), more preferably 1:0.11. The present invention limits the mass ratio of the hydrophobic polyphenol to polyvinyl pyrrolidone within the above range, which can better improve the dispersibility of the polyphenol-metal nanodots in water, while ensuring that the proportion of the active pharmaceutical ingredient in the dosage form is high. If the dispersibility of the polyphenol-metal nanodots in water needs to be further improved in a specific application, the amount of polyvinyl pyrrolidone added can be appropriately increased.

[0036] In the present invention, the solvent preferably includes one or more of methanol, ethanol and isopropyl ketone, and more preferably ethanol.

[0037] In the present invention, the mass ratio of the hydrophobic polyphenol to the volume ratio of the solvent is preferably 1 mg: (0.2-1) mL, more preferably 1 mg: 0.6 mL. The present invention limits the mass ratio of the hydrophobic polyphenol to the volume ratio of the solvent to the above range, so that the raw materials can be fully dissolved.

[0038] In the present invention, the mixing of the hydrophobic polyphenol, the metal salt, polyvinyl pyrrolidone and the solvent is preferably carried out by mixing polyvinyl pyrrolidone and the solvent, and then adding the hydrophobic polyphenol and the metal salt.

[0039] In the present invention, the mixing is preferably carried out under stirring conditions. The present invention has no particular limitation on the stirring method and rate, and the raw materials can be uniformly mixed using a stirring method and rate well known to those skilled in the art.

[0040] In the present invention, the temperature of the coordination reaction is preferably 20-30°C, more preferably 25°C; the time of the coordination reaction is preferably 0.5-6h, more preferably 2-3h; the coordination reaction is preferably carried out under stirring. The present invention has no special restrictions on the stirring mode and rate, and the stirring mode and rate well known to those skilled in the art can be used. The present invention limits the temperature and time of the coordination reaction within the above range, so that the reaction can be fully carried out.

[0041] After the coordination reaction is completed, the product of the coordination reaction is preferably dialyzed and dried in sequence to obtain polyphenol-metal nanodots.

[0042] In the present invention, the interception amount during dialysis is preferably 800-10000; the dialysis time is preferably 3-8 hours, more preferably 4 hours. The present invention limits the interception amount and time during dialysis to the above ranges, which can fully remove unbound particles and raw materials.

[0043] In the present invention, the drying is preferably freeze-drying. The present invention has no particular limitation on the temperature and time of freeze-drying, and freeze-drying temperature and time well known to those skilled in the art can be used.

[0044] The present invention also provides polyphenol-metal nanodots prepared by the preparation method described in the above technical solution.

[0045] In the present invention, the particle size of the polyphenol-metal nanodots is preferably ≤3 nm. The polyphenol-metal nanodots prepared in the present invention have excellent dispersibility in water.

[0046] The present invention also provides the use of the polyphenol-metal nanodots described in the above technical solution as an antioxidant.

[0047] The present invention has no special limitation on the operation of the application, and the technical solutions of the application well known to those skilled in the art may be adopted.

[0048] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1

[0050] At room temperature, polyvinyl pyrrolidone PVP K13-18 (average molecular weight 10000) and ethanol were mixed, and then rutin (the mass ratio of rutin to the volume of ethanol was 1 mg:0.6 ml, and the mass ratio of rutin to polyvinyl pyrrolidone PVP K13-18 was 1:0.11) and MnCl2·4H2O (the molar ratio of manganese ions in rutin and MnCl2·4H2O was 1:1) were added, and the coordination reaction was carried out under stirring conditions for 3 hours. After the reaction was completed, it was dialyzed with a dialysis bag with a cutoff volume of 10000 for 4 hours, and freeze-dried at -60°C for 2 days to obtain polyphenol-metal nanodots, which were recorded as Rutin-Mn-PVPNDs.

[0051] Example 2

[0052] The MnCl2·4H2O in Example 1 was replaced by cupric chloride, and the other parameters were the same as those in Example 1.

[0053] Example 3

[0054] The MnCl2·4H2O in Example 1 was replaced by cerium chloride heptahydrate, and the other parameters were the same as those in Example 1.

[0055] Example 4

[0056] The MnCl2·4H2O in Example 1 was replaced by gadolinium chloride hexahydrate, and the other parameters were the same as those in Example 1.

[0057] The polyphenol-metal nanodots prepared in Example 1 were observed using a transmission electron microscope, and the obtained TEM image is as follows: Figure 1 As shown. Figure 1 It can be seen that the microscopic morphology of nanoparticles is in the form of nanodots with a particle size of ≤3nm.

[0058] The particle size distribution of polyphenol-metal nanodots prepared in Example 1 is shown in FIG. Figure 2 As shown. Figure 2 It can be seen that the particle size of the nanoparticles ranges from 0.5 to 3 nm, and the particle size conforms to the normal distribution.

[0059] The XRD pattern of polyphenol-metal nanodots prepared in Example 1 is as follows: Figure 3 As shown. Figure 3 It can be seen that the synthesized polyphenol-metal nanodots have an amorphous structure.

[0060] The infrared spectrum of the polyphenol-metal nanodots prepared in Example 1 is as follows Figure 4 As shown. Figure 4 It can be seen that the FTIR spectra of Rutin-Mn-PVP NDs are at 1288cm -1 -1297cm -1 and 1652cm -1 -1664cm -1 Band (shared by rutin and PVP), 1062cm -1 -1075cm -1 and 1012cm -1 -1018cm -1 The band (specific to rutin) showed characteristic absorption peaks of rutin and PVP, confirming the presence of rutin and PVP in the product. Compared with the FTIR spectrum of original rutin, the spectrum of Rutin-Mn-PVPNDs was at 525cm -1 It shows an increase at the nanodots, reflecting the formation of Mn-O bonds in the nanodots, proving the successful coordination of polyphenols with Mn ions through phenolic hydroxyl groups.

[0061] The polyphenol-metal nanodots prepared in Example 1 were dispersed in water to obtain a polyphenol-metal nanodot water dispersion system with a concentration of 1.24 mg / mL. The polyphenol-metal nanodot (Rutin-Mn-PVPNDs) water dispersion system prepared by physiological saline and the polyphenol-metal nanodots prepared in Example 1 was irradiated with red laser to observe its Tyndall effect. The results are as follows: Figure 5 shown. Figure 5From left to right in the figure are the physiological saline image, the physiological saline red laser irradiation image, the Rutin-Mn-PVP NDs water dispersion system image, and the Rutin-Mn-PVP NDs water dispersion system red laser irradiation image. The dispersion concentration of Rutin-Mn-PVPNDs in the above Rutin-Mn-PVP NDs water dispersion system is 1.24 mg / mL, and the corresponding rutin concentration is about 0.86 mg / mL. Figure 5 It can be seen that when the aqueous dispersion system of the nanodots prepared by the present invention has a dispersion concentration of 1.24 mg / mL (wherein the rutin concentration is about 0.86 mg / mL, which is about 10 times the solubility of rutin monomer in water), the system has good dispersibility, light yellow color, and a more obvious Tyndall effect.

[0062] Cell MTT assay: L929 cells were seeded in 96-well plates at a density of 10 cells per well. 4 After the cells adhered, the Rutin-Mn-PVPNDs prepared in Example 1 were added to the culture medium at gradient concentrations and cultured overnight. Subsequently, 10 μL of MTT solution (5 mg / mL) was added to each well and incubated for another 4 hours. After that, the remaining culture medium in each well was aspirated and DMSO (150 μL per well) was used to dissolve the formazan. The absorbance of all the above wells at 570 nm was analyzed using an ELISA reader. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that as the concentration of the material in the culture medium increased to 480 μg / mL, the cell activity remained good and there was no significant difference from the control group.

[0063] Hemolysis experiment: Fresh blood was collected from SD rats and stabilized with sodium citrate. Plasma and red blood cells were separated by centrifugation for further use. In order to carry out the hemolysis experiment, the red blood cell sample was repeatedly washed with saline until the supernatant was clear and diluted with saline (1:2). Subsequently, the diluted red blood cell suspension (0.2 mL) was mixed with water (0.8 mL), saline (0.8 mL) and saline containing Rutin-Mn-PVPNDs prepared in Example 1 with a concentration gradient to establish a positive group (hemolysis group, i.e., red blood cell water suspension), a negative group (red blood cell saline suspension) and an experimental group (red blood cell material saline suspension). All samples were vortexed and incubated together at room temperature for 3 hours. After centrifugation, the absorbance of the supernatant at 541 nm was measured. Hemolysis rate (%) = [(As-An) / (Ap-An)] × 100, where Ap, An and As are the absorbances of the positive group, negative group and experimental group at 541 nm, respectively. The hemolysis results are shown in the figure. Figure 7 As shown. Figure 7 It can be seen that as the material concentration in the suspension increased to 60 μg / mL, no hemolysis occurred.

[0064] In vitro antioxidant properties

[0065] DPPH removal efficiency and corresponding spectrum

[0066] Experimental method: DPPH has a characteristic absorbance at 519 nm. Different concentrations of Rutin-Mn-PVPNDs prepared in Example 1 were added to an ethanol solution containing DPPH (5 mg / mL) and reacted for 30 minutes to evaluate its DPPH scavenging ability. The scavenging percentage was calculated as follows: scavenging percentage (%) = [(A0-A) / A0] × 100, where A0 is the absorbance of the control group and A is the absorbance of the mixture after the reaction. The results are shown in Figure 2. Figures 8-9 As shown. Figures 8-9 As can be seen from the figure, as the concentration of Rutin-Mn-PVPNDs gradually increased to 60 μg / mL, the DPPH clearance rate gradually increased to about 60% within 30 minutes ( Figure 8 ), after the reaction, the color of the reaction system changes from dark to light as the concentration of the added nanoparticles increases, corresponding to the absorbance at 519nm showing a gradient decrease ( Fig. 9 ).

[0067] Intracellular antioxidant

[0068] Experimental method: L929 cells were seeded in six-well plates at a density of 10 cells per well. 6 cells. After the cells adhered, simple culture medium, culture medium containing Rutin-Mn-PVPNDs (600 μg / mL) prepared in Example 1, culture medium containing H2O2 (2.5 mM), and culture medium containing H2O2 (2.5 mM) and Rutin-Mn-PVP NDs (600 μg / mL) prepared in Example 1 were added to the ctrl group, NDs group, H2O2 group, and H2O2+NDs group well plates, respectively, and incubated with the cells for 30 minutes before removing the culture medium. Afterwards, the DCFH-DA fluorescent probe was used to detect the intracellular ROS content of different groups of cells to evaluate the intracellular ROS scavenging ability of Rutin-Mn-PVPNDs. The results are as follows: Fig.10 As shown. Fig.10 It can be seen that the fluorescence levels of cells in the ctrl group and the NDs group are consistent, indicating that the material itself does not cause ROS accumulation in the cells; the fluorescence level of cells in the H2O2 group is significantly higher than that in the ctrl group, indicating that the ROS level in cells induced by H2O2-induced oxidative stress is significantly increased; the fluorescence level of cells in the H2O2+NDs group is significantly lower than that in the H2O2 group, indicating that the material can effectively reduce the ROS level in cells induced by H2O2-induced oxidative stress.

[0069] In summary, the polyphenol-metal nanodots prepared by the present invention have good dispersibility, biosafety and antioxidant properties.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing polyphenol-metal nanodots, comprising the following steps: The hydrophobic polyphenol, metal salt, polyvinyl pyrrolidone and solvent are mixed and coordinated to obtain polyphenol-metal nanodots.

2. The preparation method according to claim 1, characterized in that: The hydrophobic polyphenols include rutin.

3. The preparation method according to claim 1, characterized in that: The average molecular weight of the polyvinyl pyrrolidone is 8000-58000.

4. The preparation method according to claim 1, characterized in that: The solvent includes one or more of methanol, ethanol and isopropyl ketone.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the hydrophobic polyphenol to the volume ratio of the solvent is 1 mg: (0.2-1) mL.

6. The preparation method according to claim 1, characterized in that: The metal in the metal salt includes one or more of manganese, copper, cerium and gadolinium.

7. The preparation method according to claim 1, characterized in that: The molar ratio of the hydrophobic polyphenol to the metal ion in the metal salt is 1:(0.5-2).

8. The preparation method according to claim 1, characterized in that: The temperature of the coordination reaction is 20-30° C., and the time of the coordination reaction is 0.5-6 hours. 9 . The polyphenol-metal nanodots prepared by the preparation method according to claim 1 , wherein the particle size of the polyphenol-metal nanodots is ≤3 nm.

10. Use of the polyphenol-metal nanodots according to claim 9 as an antioxidant.

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