CN114951681A - Method for preparing platinum nanoparticles by adopting cordyceps flower extract - Google Patents
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Abstract
The invention discloses a method for preparing platinum nanoparticles by adopting cordyceps flower extract, wherein cordyceps flower is used as a raw material for biosynthesis, and the prepared platinum nanoparticles have the advantages of small particle size, uniformity, stability, high free radical scavenging capacity, high antibacterial activity and the like, and are high in synthesis efficiency and simple, convenient and rapid.
Description
Technical Field
The invention relates to a method for preparing Platinum nanoparticles (PtNPs) by utilizing Cordyceps flowers (Cordyceps flowers).
Background
In recent years, metal nanoparticles have been a hot research of great interest due to their unique bacteriostatic, anticancer, catalytic and antioxidant activities. Conventional methods for preparing metal nanoparticles, such as physical and chemical methods, generally have disadvantages of energy consumption and use of toxic chemical reagents, which may cause harm to human and environment. The biosynthesis process mainly involves plants, bacteria, fungi and the like, and the synthesis speed of metal nanoparticles by using plant extracts is reported to be faster than that of the metal nanoparticles from the fungi and the bacteria in the literature, and the plant extract-mediated biosynthesis scheme becomes an important field of nanoparticle preparation research due to simplicity and ecological friendliness.
The cordyceps flower is a cordyceps fruiting body formed by inoculating a preferred natural strain on a nutrient-rich culture medium and growing cordyceps under a proper growth environment. The application research reports of cordyceps flower mainly focus on the optimization of the extraction process of chemical components (such as polysaccharide, ergosterol and total flavonoids) and the biomedical application (such as hyperlipidemia prevention and cancer resistance). It is worth noting that some documents report the biosynthesis of metal nanoparticles (such as nano silver, nano gold and nano zinc oxide) by cordyceps militaris mycelium, but there is no research report of the synthesis of platinum nanoparticles by cordyceps flower. The applicant utilizes cordyceps flower biosynthesis PtNPs based on the difference of chemical substances contained in cordyceps flower and cordyceps militaris, and the obtained product has more excellent performance, and the cordyceps flower is easier to obtain and lower in price compared with cordyceps militaris raw materials.
Disclosure of Invention
The invention aims to provide a platinum nanoparticle and a preparation method and application thereof. The method adopts cordyceps flower as raw material for biosynthesis, and the prepared platinum nanoparticles have the advantages of small, uniform and stable particle size, high free radical scavenging capacity and antibacterial activity and the like.
The invention is realized by the following technical scheme:
a method for preparing platinum nanoparticles by adopting cordyceps flower comprises the following steps:
1) drying and grinding fresh cordyceps flower into fine powder, then dissolving the cordyceps flower fine powder in water according to a material-to-liquid ratio of 1:10-50, and carrying out water bath at the temperature of 25-90 ℃ for 0.5-4 h; centrifuging, collecting supernatant, and filtering with filter membrane with pore diameter of 0.22 μm to obtain sterile Cordyceps flower extractive solution;
2) adjusting pH of the Cordyceps flower extractive solution to 2-12 with NaOH or HCl solution;
3) h is to be 2 PtCl 6 ·6H 2 Dissolving O in distilled water to obtain H with concentration of 1mmol/L 2 PtCl 6 Adding H into the cordyceps flower extract according to the proportion of 10-90 percent of the total reaction volume 2 PtCl 6 In the solution, after being uniformly mixed, the mixture is heated at the temperature of 30-100 ℃ and stirred at high speed until the color of the solution is changed from light yellow to dark brown;
4) dialyzing the solution after the reaction by using a dialysis bag with the molecular weight cutoff of 500-5000 to ensure that the pH value is neutral, and performing vacuum freeze drying to obtain the platinum nanoparticles.
Preferably, in the step 1), the ratio of the cordyceps flower fine powder to water is 1: 10.
Preferably, in the step 1), the water bath temperature is 25 ℃ and the water bath time is 30 min.
Preferably, in the step 2), the pH of the cordyceps flower extract is adjusted to 10 by using NaOH solution.
Preferably, in the step 3), the cordyceps flower extracting solution accounts for 50% of the total reaction volume.
Preferably, in step 3), the heating temperature is 50 ℃.
Preferably, the dialysis bag has a molecular weight cut-off of 1000.
The invention has the beneficial effects that:
(1) the method is simple and rapid. The invention uses a biosynthesis method to prepare the platinum nanoparticles, the used raw materials are natural and environment-friendly, the reaction time is only about 1 hour, and other strict condition control except the temperature is not needed in the reaction process, so the method has the advantages of high synthesis efficiency, simple and convenient method and the like.
(2) According to the invention, through comparing various biological resources, cordyceps flower is finally screened out to be used as a synthetic raw material, the prepared nano particles have more excellent particle size distribution and oxidation resistance and antibacterial performance, and the raw material is easy to obtain and low in price.
Drawings
FIG. 1: influence of extraction temperature and time on antioxidant activity of Cordyceps militaris extract.
FIG. 2: the effect of the extract liquid ratio on the antioxidant activity of the cordyceps flower extract.
FIG. 3: influence of pH of cordyceps flower extract on particle size of PtNPs.
FIG. 4: cordyceps flower extract and H 2 PtCl 6 Influence of volume ratio of solution on particle size of PtNPs.
FIG. 5: influence of reaction temperature on the particle size of PtNPs.
FIG. 6: the particle size distribution curves of the PtNPs synthesized by different biological resources are shown in the specification, wherein a is the PtNPs synthesized by utilizing cordyceps flower dry powder aqueous extract, b is the PtNPs synthesized by utilizing fresh cordyceps flower aqueous extract, c is the PtNPs synthesized by utilizing tomato aqueous extract, and d is the PtNPs synthesized by utilizing cordyceps militaris mycelium aqueous extract.
FIG. 7: transmission electron microscopy images of PtNPs synthesized from cordyceps flower dry powder aqueous extract.
FIG. 8: transmission electron microscope images of PtNPs synthesized by using fresh cordyceps flower aqueous extracts.
FIG. 9: transmission electron microscopy images of PtNPs synthesized using tomato aqueous extracts.
FIG. 10: transmission electron microscopy images of PtNPs synthesized from aqueous extracts of mycelia of Cordyceps militaris.
FIG. 11: the obtained information of the particle size is shown in fig. 7.
FIG. 12: the antioxidant activity of PtNPs synthesized by different biological resources is utilized, wherein a is PtNPs synthesized by utilizing cordyceps flower dry powder aqueous extract, b is PtNPs synthesized by utilizing fresh cordyceps flower aqueous extract, c is PtNPs synthesized by utilizing tomato aqueous extract, and d is PtNPs synthesized by utilizing cordyceps militaris mycelium aqueous extract.
FIG. 13: the antibacterial activity of PtNPs, wherein a-d is escherichia coli, e-h is salmonella typhimurium, i-l is bacillus subtilis, and m-p is staphylococcus aureus. a and b, e and f, i and j, m and n respectively represent strains in a test group under different magnification; c and d, g and h, k and l, o and p represent control strains at different magnifications, respectively.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1 extraction of Cordyceps flowers
Dissolving the cordyceps flower fine powder in distilled water according to different material-liquid ratios (1:10, 1:20, 1:30, 1:40 and 1:50), and stirring the mixture for different times (0.5h, 1.0h, 2.0h, 3.0h and 4.0h) in water bath at different temperatures (25 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃); centrifuging (11000r/min, 30min), retaining supernatant, filtering the supernatant with filter membrane with pore diameter of 0.22 μm to obtain sterile Cordyceps flower extractive solution, and storing at 4 deg.C. And (3) measuring the DPPH free radical clearance rate of the cordyceps flower extracting solution prepared under various conditions, and further preliminarily evaluating the metal ion reducing capacity of the cordyceps flower extracting solution.
As can be seen from FIGS. 1 and 2, the Cordyceps flower extract has strong antioxidant activity when the Cordyceps flower dry powder and distilled water are mixed according to a ratio of 1:10, and the mixture is bathed in water at 25 deg.C for 30 min.
EXAMPLE 2 adjustment of pH value
Mixing Cordyceps flower dry powder with distilled water at a ratio of 1:10, stirring in water bath at 25 deg.C for 30min, centrifuging, filtering, sterilizing to obtain Cordyceps flower extractive solution with pH of about 6.0, and adjusting pH to 2.0-12.0 with NaOH solution or HCl solution respectively; adding the extractive solution into 1mmol/L H according to 50% of total reaction volume 2 PtCl 6 After mixing well in the solution, the mixture was heated at 50 ℃ and stirred at high speed for 1h, and the color of the mixture changed from light yellow to dark brown. The obtained product is dialyzed by a dialysis bag with the molecular weight cut-off of 1000 until the pH value is neutral, and the average particle size of the nano particles in the solution is measured by adopting a dynamic light scattering method.
As can be seen from fig. 3, when the pH of the extract solution was adjusted to 10.0, the average particle size of the synthesized PtNPs was smaller, and the quality was more stable and uniform.
Example 3 optimization of conditions for PtNPs Synthesis
Mixing Cordyceps flower dry powder with distilled water at a ratio of 1:10, stirring in water bath at 25 deg.C for 30min, centrifuging, filtering to remove bacteria, and adding NaOH solutionThe pH was adjusted from the original 6.0 to 10.0. Adding 1mmol/L H into the extractive solution at different total reaction volume ratio (10%, 30%, 50%, 70%, 90%) 2 PtCl 6 After mixing the solution evenly, heating and stirring at high speed for 1h at different temperatures (30 ℃, 50 ℃, 70 ℃, 90 ℃, 100 ℃) and the color of the mixture changes from light yellow to dark brown. The obtained product is dialyzed by a dialysis bag with the molecular weight cutoff of 1000 until the pH is neutral, and the average particle size of the nano particles in the solution is measured by a dynamic light scattering method.
As can be seen from FIGS. 4 and 5, the extract was mixed with H 2 PtCl 6 After the solutions are uniformly mixed according to the volume ratio of 1:1, the solutions are heated at 50 ℃ and stirred at high speed for 1h, so that the prepared PtNPs have smaller average particle size and more stable quality.
Example 4 characterization of PtNPs Synthesis Using different biological resources
The particle size distribution curve of the 4 kinds of nano particles is measured by adopting a dynamic light scattering method, the particle dispersibility and the particle size are preliminarily evaluated, and the morphology is further observed by adopting a transmission electron microscope. Wherein, the extraction method of the fresh cordyceps flower aqueous extract, the tomato aqueous extract, the cordyceps militaris mycelium aqueous extract and the process for synthesizing the PtNPs all use the optimal processes optimized in the embodiments 1-3.
As can be seen from FIG. 6, the PtNPs synthesized by the cordyceps flower dry powder aqueous extract have better monodispersity and smaller average particle size, while other biological resources such as fresh cordyceps flower, tomato and cordyceps militaris mycelium have low concentration of compounds capable of undergoing reduction reaction with platinum and low content of protein and polysaccharide substances capable of stabilizing nanoparticles, so that the synthesized PtNPs have polydispersity and nonuniform particle size and are easy to aggregate into large particles. From comparison of fig. 7 to 10, it can be seen that PtNPs synthesized from cordyceps flower dry powder are most dispersed in the visual field, and other nanoparticles show different degrees of agglomeration or large size, and the results are consistent with the data measured by the dynamic light scattering method, and further, the particle size in fig. 7 is measured to obtain fig. 11, and it can be seen that the average particle size of PtNPs synthesized from cordyceps flower dry powder is 13.34 ± 4.06 nm.
Example 5 determination of antioxidant Activity of PtNPs synthesized Using different biological resources
The antioxidant activity of PtNPs synthesized by different biological resources at different concentrations (0.50-125.00. mu.g/mL) was evaluated by DPPH free radical scavenging ability test.
As can be seen from FIG. 12, as the sample concentration increased from 0.50. mu.g/mL to 125.00. mu.g/mL, the DPPH radical clearance of PtNPs synthesized with the cordyceps flower dry powder aqueous extract increased from 27.77% to 44.00%, the radical clearance of PtNPs synthesized with the fresh cordyceps flower aqueous extract increased from 4.50% to 12.66%, the radical clearance of PtNPs synthesized with the tomato aqueous extract increased from 3.12% to 11.40%, and the radical clearance of PtNPs synthesized with the cordyceps militaris mycelia aqueous extract increased from 5.31% to 17.08%. The antioxidant activity of the 4 PtNPs is concentration-dependent, but under the same concentration, the DPPH free radical clearance of the PtNPs synthesized by the cordyceps flower dry powder aqueous extract is far higher than that of the other 3PtNPs, and the PtNPs have obvious antioxidant activity under low concentration.
Example 6 measurement of bacteriostatic Activity of PtNPs
From the results, the PtNPs synthesized by the cordyceps flower dry powder aqueous extract have obvious advantages, and the morphological change of the PtNPs synthesized by exposing 4 bacteria (Escherichia coli, Salmonella typhimurium, Bacillus subtilis and Staphylococcus aureus) to the 100 mu g/mL cordyceps flower dry powder aqueous extract for 15min is observed by a scanning electron microscope so as to evaluate the bacteriostatic activity of the PtNPs.
From fig. 13, it can be seen that PtNPs synthesized from the cordyceps flower dry powder aqueous extract can destroy the bacterial structure in a short time (15min), so that the bacterial structure has the phenomena of cell perforation, cell rupture, content leakage and the like, and the destruction effect of the PtNPs on gram-negative bacteria is stronger than that of gram-positive bacteria.
Claims (9)
1. A method for preparing platinum nanoparticles by adopting cordyceps flower is characterized by comprising the following steps:
1) drying and grinding fresh cordyceps flower into fine powder, then dissolving the cordyceps flower fine powder in water according to a material-to-liquid ratio of 1:10-50, and carrying out water bath at the temperature of 25-90 ℃ for 0.5-4 h; centrifuging, collecting supernatant, and filtering with filter membrane with pore diameter of 0.22 μm to obtain sterile Cordyceps flower extractive solution;
2) adjusting pH of the Cordyceps flower extractive solution to 2-12 with NaOH or HCl solution;
3) h is to be 2 PtCl 6 ·6H 2 Dissolving O in distilled water to obtain H with concentration of 1mmol/L 2 PtCl 6 Adding H into the cordyceps flower extract according to the proportion of 10-90 percent of the total reaction volume 2 PtCl 6 In the solution, after being uniformly mixed, the mixture is heated at the temperature of 30-100 ℃ and stirred at high speed until the color of the solution is changed from light yellow to dark brown;
4) dialyzing the solution after reaction by using a dialysis bag with the molecular weight cutoff of 500-5000-.
2. The method for preparing platinum nanoparticles according to claim 1, characterized in that: in the step 1), the material-to-liquid ratio of the cordyceps flower fine powder to water is 1: 10.
3. The method for preparing platinum nanoparticles according to claim 1, characterized in that: in the step 1), the water bath temperature is 25 ℃, and the water bath time is 30 min.
4. The method for preparing platinum nanoparticles according to claim 1, characterized in that: in the step 2), the pH value of the cordyceps flower extracting solution is adjusted to 10 by using NaOH solution.
5. The method for preparing platinum nanoparticles according to claim 1, characterized in that: in the step 3), the cordyceps flower extracting solution accounts for 50% of the total reaction volume.
6. The method for preparing platinum nanoparticles according to claim 1, characterized in that: in the step 3), the heating temperature is 50 ℃.
7. The method for preparing platinum nanoparticles according to claim 1, characterized in that: the molecular weight cut-off of the dialysis bag is 1000.
8. Platinum nanoparticles prepared according to the method of any one of claims 1 to 7.
9. Use of the platinum nanoparticles of claim 8 in the preparation of anti-oxidant and bacteriostatic drugs.
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| PCT/CN2023/109086 WO2023227145A1 (en) | 2022-05-27 | 2023-07-25 | Preparation method for platinum nanoparticles, platinum nanoparticles prepared therefrom, and application of platinum nanoparticles |
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| WO2023227145A1 (en) * | 2022-05-27 | 2023-11-30 | 开贝科技(苏州)有限公司 | Preparation method for platinum nanoparticles, platinum nanoparticles prepared therefrom, and application of platinum nanoparticles |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023227145A1 (en) * | 2022-05-27 | 2023-11-30 | 开贝科技(苏州)有限公司 | Preparation method for platinum nanoparticles, platinum nanoparticles prepared therefrom, and application of platinum nanoparticles |
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