KR102058087B1 - A method for producing nanoparticles using a Dryopteris crassirhizoma extract and nanoparticles prepared therefrom - Google Patents

Abstract

The present invention relates to a method for producing nanoparticles using the spectral extract and nanoparticles prepared therefrom. Nanoparticles prepared using the extract according to the present invention shows a size of 5 to 60 nm and exhibits good antimicrobial activity against harmful bacteria, it can be applied as an antibacterial and disinfectant.

Description

A method for producing nanoparticles using a Dryopteris crassirhizoma extract and nanoparticles prepared therefrom}

The present invention relates to a method for producing nanoparticles using the spectral extract and nanoparticles prepared therefrom.

Metal nanoparticles and composites thereof are widely used in biomedical applications such as drug delivery, antibacterial and medical devices due to their small size, high surface area to volume ratio, inert properties, stability, high dispersion, non-cytotoxicity and biocompatibility. (Non Patent Literature 1, Non Patent Literature 2).

On the other hand, the development of a photocatalytic reaction sensitized by a high-brightness LED (light-emitting diode) has recently been progressing significantly. When using an LED lamp as a light source, local surface plasmon resonance (SPR) effects (Non Patent Literature 3, Non Patent Literature 4), morphology and optical property control (Non Patent Literature 5), improved film performance, high There are advantages such as photon efficiency, generation of low voltage electricity, power stability, accuracy and low cost (Non-Patent Document 6).

The development of reliable and environmentally friendly chemical processes for biological synthesis of nanomaterials is important for the clinical application of antibiotics (Non-Patent Document 7). One potential solution to the development of green nanomaterials with high medicinal value is to use medicinal plant extracts as reducing and capping agents.

Accordingly, the present inventors completed the present invention by developing a composition exhibiting excellent antimicrobial activity by preparing silver nanoparticles (AgNPs) using a spectator (Dryopteris crassirhizoma, SM) root stem extract as a reducing agent and a capping agent.

 A.K. Khan, R. Rashid, G. Murtaza, A. Zahra, Gold nanoparticles: synthesis and appli-cations in drug delivery, Trop. J. Pharm. Res. 13 (2014) 1169-1177.  P. Velmurugan, S.M. Lee, M. Cho, J. H. Park, S.K. Seo, H. Myung, K.S. Bang, B.T. Oh, An-tibacterial activity of silver nanoparticle-coated fabric and leather against odor and skin infection causing bacteria, Appl. Microbiol. Biotechnol. 19 (2014) 8179-8189.  X. Gu, T. Qiu, W. Zhang, P.K. Chu, Light-emitting diodes enhanced by localized sur-face plasmon resonance, Nanoscale Res. Lett. 6 (2011) 199-203.  V. Kumar, D.K. Singh, S. Mohan, S. H. Hasan, Photo-induced biosynthesis of silver nanoparticles using aqueous extract of Erigeron bonariensis and its catalytic activity against Acridine Orange, J. Photochem. Photobiol. B 155 (2016) 39-50.  K.G. Stamplecoskie, J.C. Scaiano, Light emitting diode irradiation can control the morphology and optical, properties of silver nanoparticles, J. Am. Chem. Soc. 132 (2010) 1825-1827.  N. Yeh, C. H. Wu, T.C. Cheng, Light-emitting diodes-their potential in biomedical ap-plications, Renew. Sust. Energ. Rev. 14 (2010) 2161-2166.  G.A. Plaza, J. Chojniak, I.M. Banat, Biosurfactant mediated biosynthesis of selected metallic nanoparticles, Int. J. Mol. Sci. 15 (2014) 13720-13737.

It is an object of the present invention to provide a method for producing nanoparticles using spectral extracts.

It is an object of the present invention to provide nanoparticles produced by the process according to the invention.

Another object of the present invention is to provide an antimicrobial or disinfecting composition comprising nanoparticles prepared by the method according to the present invention as an active ingredient.

The present invention comprises the steps of mixing the spectral ( Dryopteris crassirhizoma ) extract and an aqueous metal solution;

Irradiating the mixed mixture to prepare a reaction product; And

Separating the nanoparticles from the reaction product; provides a method for producing nanoparticles using an extract containing spectral.

The spectral extract is heated by mixing the spectral powder with purified water; And

Filtering the heated mixture to separate the liquid extract; may be prepared.

The spectral extract may be to extract from the crow root.

The metal aqueous solution may be prepared by dissolving a metal precursor in an aqueous solution.

The metal precursor may be AgNO ₃ .

In the step of mixing the spectral extract and the metal aqueous solution, the spectral extract and the metal aqueous solution may be mixed in a volume ratio (v / v) of 1 to 2: 8 to 9.

The light irradiation may be to irradiate direct sunlight or LED for 15 to 45 minutes.

The LED may be any one selected from the group consisting of blue, red, green or white.

The LED may be irradiated with 10 to 15V.

Nanoparticles prepared using the spectral extract may be 5 to 60 nm in size.

Nanoparticles prepared using the spectral extract may have an antimicrobial effect.

The present invention provides nanoparticles prepared by the method according to the present invention.

The present invention provides an antimicrobial or disinfecting composition comprising nanoparticles prepared by the method according to the present invention as an active ingredient.

Nanoparticles prepared using the extract according to the present invention shows a size of 5 to 60 nm and exhibits good antimicrobial activity against harmful bacteria, it can be applied as an antibacterial and disinfectant.

1 is a diagram showing the results of an ultraviolet / visible spectrophotometer to confirm the optimal pH of silver nanoparticle production for each light source; (a) sunlight, (b) blue LEDs, (c) red LEDs, (d) green LEDs, (e) white LEDs, (f) no irradiation, and (g) optimal pH.
FIG. 2 shows ultraviolet / visible spectrophotometer results for identifying optimal DCRE concentrations of silver nanoparticle generation for each light source; FIG. (a) sunlight, (b) blue LEDs, (c) red LEDs, (d) green LEDs, (e) white LEDs, (f) no irradiation, and (g) optimal DCRE concentrations.
FIG. 3 is a diagram showing ultraviolet / visible spectrophotometer results for confirming the optimal silver nitrate solution concentration of silver nanoparticle generation for each light source; FIG. (a) sunlight, (b) blue LEDs, (c) red LEDs, (d) green LEDs, (e) white LEDs, (f) no irradiation, and (g) optimal silver nitrate solution concentrations.
4 is a view showing a surface photograph, a single particle enlargement photograph, a crystalline photograph of the silver nanoparticles generated in each light source; (ac) sunlight, (df) green LEDs, (gi) red LEDs, (jl) blue LEDs, (mo) white LEDs.
5 is a view showing the results of the FTIR analysis of the functional group of the silver nanoparticles generated in the DCRE and each light source.
6 is a diagram showing the results of XRD analysis on the crystallinity of the silver nanoparticles generated in each light source.

The present invention comprises the steps of mixing the spectral ( Dryopteris crassirhizoma ) extract and an aqueous metal solution; Irradiating the mixed mixture to prepare a reaction product; And separating nanoparticles from the reaction product.

In one embodiment of the present invention, the spectral extract and the silver nitrate (AgNO ₃ ) solution was mixed and reacted by irradiation with sunlight (direct sunlight) or LED, the reaction product was centrifuged to separate the nanoparticles.

The extract contains many enzymes such as polyphenols, saponins, antioxidants, terpenoids and alkaloids. These were confirmed by FT-IR functional group analysis in one embodiment of the present invention, the typical functional groups are C = C (Alkenyl), C = N (amide), O = H (phenolic and alcohol), NH (amine), It was confirmed that CH and COO- (carboxylic group). Among the enzymes, nitroreductases combine with silver ions in an AgNO ₃ aqueous solution to reduce metals to form silver nanoparticles. In addition, a direct light or LED light source performs a light reducing action on the silver particles formed from AgNO ₃ so that the spectral extract participates in reducing silver ions and promotes their formation.

The spectral extract is heated by mixing the spectral powder with purified water; And separating the liquid extract by filtration of the heated mixture.

In the present invention, " Dryopteris crassirhizoma " is a perennial fern belonging to the Cotton family, and the roots are called spectators or cotton roots, which are harvested from autumn to spring and dried in the spring. Write it as medicine. Efficacy in insect repellent, hemostasis, and uterine contraction. Take it as a pill, pill, or powder as a remedy for roundworms or tapeworms. It grows in all regions of Korea, and is distributed in Japan, Sakhalin, Kuril Islands, and Manchuria.

The spectral extract may be to extract from the crow root (root).

In one embodiment of the present invention, the root stem of the tube is powdered and mixed with distilled water, and then heated and stirred mixture is filtered with filter paper to separate the liquid extract of the root stem of the crowd to prepare an extract.

The metal aqueous solution may be prepared by dissolving a metal precursor in an aqueous solution.

The metal precursor may be AgNO ₃ .

In the step of mixing the spectral extract and the metal aqueous solution may be to mix the spectral extract and the metal aqueous solution in a volume ratio (v / v) of 1 to 2: 8 to 9, preferably a volume ratio of 1: 9 (v / v ) May be mixed.

The light irradiation may be irradiated with direct sunlight or LED for 15 to 45 minutes, preferably for 30 minutes.

The LED may be any one selected from the group consisting of blue, red, green or white.

The LED may be irradiated with 10 to 15V, preferably 12V may be irradiated.

Nanoparticles prepared using the spectral extract may be 5 to 60 nm in size.

Nanoparticles prepared using the spectral extract may be an antimicrobial effect, the antimicrobial effect may be an antimicrobial effect against harmful bacteria.

The harmful bacterium may be Bacillus cereus (KACC-10001) or Pseudomonas aeruginosa (KACC-10186) strain.

In one embodiment of the present invention, the nanoparticles prepared using the spectral extract showed excellent antimicrobial activity against gram-positive or gram-negative bacteria.

The present invention provides nanoparticles prepared by the method according to the present invention.

The present invention provides an antimicrobial or disinfecting composition comprising nanoparticles prepared by the method according to the present invention as an active ingredient.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Preparation of silver nanoparticles using the extract and LED light source

Cut 200 g of rhizome of Dryopteris crassirhizoma into small pieces , wash in distilled water, and dry. Root stem powder was prepared in a uniform size by filtering the sieve of the mesh size. Root stem powder in the tube was mixed with 500 ml of sterilized distilled water and boiled for 30 minutes. Boiled extract was filtered through a filter paper, and the dried rootis extract ( DC ) was stored at 4 ° C.

5 ml of DCRE and 45 ml of 1 mM silver nitrate (AgNO3) solution (99.9%, Daejeong Chemical, Seoul) were mixed to produce sunlight (direct sunlight) or 12V LEDs (blue, red, green, white) (ODTech LED light chamber, Korea), respectively. The reaction was carried out for 30 minutes. The reaction product was centrifuged at 12,000 rpm for 15 minutes to give a solid. The obtained solid was lyophilized and powdered to prepare silver nanoparticles.

Example 2 Confirmation of Optimum Conditions for the Preparation of Silver Nanoparticles

Silver nanoparticles were prepared by preparing silver nanoparticles in the same manner as in Example 1 at pH 2-10, various DCRE concentrations (1-10 ml) and various silver nitrate solution concentrations (0.1-1.0 mM) for each light source. Optimum conditions were confirmed. In order to evaluate the stability of silver nanoparticles manufactured under various conditions, UV / vis spectrophotometer (UV-vis) using a dual beam operating at 1 nm resolution of silver nanoparticles stored at room temperature for several months. Spectrophotometer (UV-1800-Shimadzu, Japan) was used to analyze within an operating wavelength range of 300 to 800 nm.

As a result, the optimum pH of silver nanoparticle generation for each light source was pH 7, 7 for visible light, pH 5 for LEDs (blue, red), and pH 4 for LEDs (green, white) (FIG. 1). The optimal DCRE concentration of silver nanoparticle generation for each light source was found to be 2 mL in visible light, LEDs (blue, green, white) and 1 mL in LEDs (red) (FIG. 2). The optimal silver nitrate solution concentration of silver nanoparticle generation for each light source was found to be 0.1 mM in all of the sunlight, blue, red, green and white LEDs (FIG. 3).

Example 3 Characterization of Silver Nanoparticles Prepared

The surface morphology and size of silver nanoparticles prepared using DCRE in Example 1 were investigated using TEM / STEM (TEM, JEM-2200FS, JEOL, Japan) operating at 200 kV.

Fourier transform infrared spectroscopy (FTIR) of silver nanoparticles with 4 cm ^-1 resolution of KBr pellets in diffuse reflection mode using a Perkin-Elmer FTIR spectrophotometer (Norwalk, CT, USA) The spectrum was obtained.

X-ray powder diffraction of silver nanoparticles was carried out using an X-ray diffractometer (XRD, Rigaku, Japan) by scanning with a time constant of 2 seconds at 0.04 ° / min in the region of 2θ from 30 ° to 80 °.

As a result, in the HR-TEM analysis, each of the silver nanoparticles showed various shapes, but the arrangement was uniform, and the size was observed to be distributed at 5-60 nm (FIG. 4).

Functional groups for DCRE at 3415.3 cm ^-1 -OH, 2920.8 cm ^-1 in at -NH, 1750.2 cm ^-1 from -CH, 1610.6 cm ^-1 from ^{-C = O, 1400 cm -1 NC} = O, 1136 cm ^-1 to CH ₂ , 1077 cm ^-1 to CO, 823.9 cm ^-1 to CN; It was found to have a functional group form separated from NH; amine in alcohol / phenol, 642.6 cm ⁻¹ . The functional groups of the nanoparticles produced by reacting with DCRE at each of the other light sources are very similar to those of the DCRE, so the functional groups of the silver nanoparticles may be derived from the DCRE. In addition, the main components of the DCRE used in the experiments are known trimeric phlorogolocinol, flavan, terpene, phenolic acid, etc., it was confirmed that the results of the FTIR indicates the functional group of these main components (Fig. 5). In FIG. 5, the white portion of the right (c, f, i, l, o) photo shows the crystal form of each particle, and it can be seen that the crystals of the silver nanoparticles are not the same.

Similarly in the XRD analysis (FIG. 6), the total peaks are represented by (111), (200), (220), and (311), but the peaks of Ag (200) appear slightly different positions in each result. Based on these results, it is thought that there are many kinds of silver nanoparticles produced by the reaction between each light source and the plant extract (JCPDS 04-0783). In the XRD pattern, the size of the nanoparticle crystal was calculated by the following Debye-Scherrer equation. The size of the nanoparticle crystals is 37.6 nm (sunlight), 37.61 nm (green LED), 51.40 nm (red LED), 51.40 nm (blue LED), 51.40 nm (white LED), which correspond to the size observed in TEM analysis. Matched. The lattice parameter (a) for all used light sources, close to the standard lattice parameter for silver (0.4086 nm), is 0.235 nm for the (111) fcc plane using the corresponding value of the interplanar spacing (d-gap) between atoms. Was calculated.

D = Cλ / β cos θ

D: crystal size of silver nanoparticles

λ: wavelength of X-ray source (1.54056A) used in XRD

β: full width at half maximum (FWHM) of diffraction peaks in radians

C: Scherrer constant (0.9-1)

θ: Bragg angle (in radians)

Example 4 Antibacterial Activity Assay

Using the silver nanoparticles prepared in Example 1, the antimicrobial activity against representative Gram-positive bacteria and Gram-negative bacteria Bacillus cereus (KACC-10001) and Pseudomonas aeruginosa (KACC-10186) was confirmed by the well diffusion method. It was.

Cultures of B. cereus and P. aeruginosa strains were subcultured in a Muller-Hinton medium using a rotary shaker at 35 ° C., 200 rpm. Each strain was evenly spread over the plate using a sterile 'L' bar. Sterile straws were used to form wells of 6 mm on Fuller-Hinton medium. Silver nanoparticles (50, 100, 150, 200, 250 μg) prepared using each light source were introduced into the wells and incubated at 35 ° C. for 24 hours. After 24 hours the diameter of the clear zone in the medium was measured and its average value expressed in mm.

As a result, as shown in Table 1, when tested against Gram-positive bacteria ( B. cereus ) using 250 μg of silver nanoparticles produced in LED (green), it showed the highest antibacterial activity by producing an average of 10 mm clear zone. When tested on Gram negative bacteria ( P. aeruginosa ) using 250 μg of silver nanoparticles generated in LED (red), it was confirmed that the highest antimicrobial activity was generated by generating a clear zone of 7 mm on average.

Samples (AgNPs)
B. cereus P. aeruginosa 50 μg 100 μg 150 μg 200 μg 250 μg 50 μg 100 μg 150 μg 200 μg 250 μg Extract (mL) - - - - - - - - - - White LED - 1 ± 1.2 3 ± 1.2 5 ± 1.8 6 ± 1.6 - - 1 ± 0.2 3 ± 1.33 5 ± 1.3 Green LED 1 ± 1.1 2 ± 1.2 3 ± 1.3 8 ± 0.8 10 ± 1.9 - 1 ± 1.2 2 ± 1.6 4 ± 1.4 6 ± 1.2 Red LED 1 ± 0.9 2 ± 1.5 3 ± 1.1 6 ± 0.6 8 ± 0.3 - 1 ± 1.5 3 ± 1.1 6 ± 0.4 7 ± 0.1 Blue LED - 1 ± 1.1 5 ± 1.8 7 ± 1.2 7 ± 1.6 - - 2 ± 1.9 3 ± 2.6 5 ± 1.9 Sunlight - 1 ± 2.1 3 ± 2.4 5 ± 1.2 6 ± 1.4 - - 2 ± 0.8 5 ± 1.6 5 ± 2.4

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Claims (6)

Mixing the root stem extract (Dryopteris crassirhizoma rhizome extract (DCRE)) and 0.1 mM silver nitrate (AgNO 3) in water;
Irradiating the mixed mixture to prepare a reaction product; And
Separating the silver nanoparticles from the reaction product;
The prepared silver nanoparticles comprise functional groups of trimer phlorogolocinol, flavan, terpene and phenolic acid
The light irradiation is a step of irradiating a red or green LED for 30 minutes,
The size of the prepared silver nanoparticles is 37.61nm ~ 51.40nm manufacturing method of the nanoparticles using the spectral extract, characterized in that. The method of claim 1, wherein the spectral extract
Heating the mixed powder with purified water; And
Separating the liquid extract by filtering the heated mixture; Method for producing nanoparticles using a spectral extract characterized in that it comprises a. The method of claim 1, wherein in the step of mixing the root extract of the stem and the aqueous solution of silver nitrate, the extract of the audience characterized in that the mixed root stem extract and the aqueous solution of silver nitrate in a volume ratio (v / v) of 1 to 2: 8 to 9 Method for producing nanoparticles using. The method of claim 1, wherein the LED is irradiated with a 10 to 15 V method for producing nanoparticles using a spectral extract. The method of claim 1, wherein the silver nanoparticles prepared using the spectral extract has a size of 5 to 60 nm. The method of claim 1, wherein the silver nanoparticles prepared using the spectral extract has an antimicrobial effect.

Images