KR102590739B1 - Selenium nanoparticles formed using Artemisia annua extract and encapsulated with starch and method for manufacturing the same - Google Patents

Abstract

The present invention relates to selenium nanoparticles formed using mugwort extract and encapsulated in starch, and a method for producing the same. The present invention relates to selenium nanoparticles formed using mugwort extract and encapsulated in starch to reduce manufacturing costs and enable ecologically sustainable production. It relates to selenium nanoparticles formed using mugwort extract with improved antibacterial function and encapsulated in starch, and a method for producing the same.

Description

Selenium nanoparticles formed using Artemisia annua extract and encapsulated with starch and method for manufacturing the same}

The present invention relates to selenium nanoparticles formed using mugwort extract and encapsulated in starch, and a method for producing the same. The present invention relates to selenium nanoparticles formed using mugwort extract and encapsulated in starch to reduce manufacturing costs and enable ecologically sustainable production. It relates to selenium nanoparticles formed using mugwort extract with improved antibacterial function and encapsulated in starch, and a method for producing the same.

The exploration of the synthesis of nanoparticles and their applications has fascinated researchers around the world, making nanotechnology a rapidly growing and innovative field of research. NPs with sizes ranging from 1 to 100 nm show improved properties due to their large surface area and quantum effects. It can be applied to various fields such as materials science, environmental research, electronics, energy harvesting, manufacturing industry, machinery industry, textile industry, and food industry. Novel nanomaterials have received increasing attention in recent years due to their usefulness in fields of biology, engineering, agriculture and food safety.

Meanwhile, selenium is a key factor in producing nearly 25 selenoproteins, including the essential antioxidants deiodinase, glutathione peroxidase, and thioredoxin reductase. In the preparation of new nanomaterials, selenium (Se) is the most used metal known since ancient times, and research on the synthesis of selenium nanoparticles (SeNPs) has been increasing in recent years. Selenium nanoparticles (SeNPs) are attracting attention from researchers for different applications, including drug design and drug delivery, which are important aspects of the medical and pharmaceutical industries.

The most efficient protocol is required for the synthesis of nanoparticles with desired properties. Size, shape and stability are greatly affected by manufacturing method, temperature, solvent concentration, and chemicals used as reducing and capping agents.

As a conventional technology related to nanoparticles containing such selenium, Korea Patent Publication No. 10-2019-0014047 (published on February 11, 2019) discloses “antibacterial nanoparticles and method for producing the same,” which is ( a) preparing a copper-tin mixed solution containing an organic solvent containing copper (Cu), tin (Sn), and amine; (b) first heating the prepared copper-tin mixed solution; (c) preparing a copper-tin-selenium mixed solution by adding selenium (Se) to the copper-tin mixed solution that has undergone the heating step; and (d) secondary heating the prepared copper-tin-selenium mixed solution.

However, the conventional “antibacterial nanoparticles and method for producing the same” used chemicals, so the manufacturing cost was high, it was not environmentally friendly, and there were problems in that ecologically sustainable production was difficult.

Republic of Korea Patent Publication No. 10-2019-0014047 (published on February 11, 2019), “Antibacterial nanoparticles and manufacturing method thereof”

In order to solve the above problems, the present invention relates to selenium nanoparticles formed using mugwort extract and encapsulated in starch, and a method for manufacturing the same, which is formed using mugwort extract and encapsulated in starch, thereby lowering the manufacturing cost. The purpose is to provide selenium nanoparticles formed using mugwort extract and encapsulated in starch, which enable ecologically sustainable production and improve antibacterial function, and a manufacturing method thereof.

In order to achieve the above object, the present invention is Artemisia annua ), dried, pulverized, and extracted to produce mugwort extract (S10); A SeNP generation step (S20) of mixing sodium selenite with the mugwort extract prepared in the mugwort extract preparation step (S10) and stirring to generate SeNP; A method for producing selenium nanoparticles formed using mugwort extract and encapsulated in starch is provided, comprising a centrifugation step (S30) of centrifuging and separating the SeNPs generated in the SeNP generation step (S20). .

In addition, the mugwort extract in the mugwort extract manufacturing step (S10) is characterized in that it is obtained by extracting the whole plant of mugwort.

In addition, in the SeNP production step (S20), the mugwort extract and sodium selenite are mixed at a volume ratio of 5:95 to 15:95.

Additionally, in the SeNP production step (S20), the stirring of mugwort extract and sodium selenite is performed at 10 to 40°C for 2 to 8 hours.

In addition, the centrifugation step (S30) is characterized in that the SeNPs generated in the SeNP generation step (S20) are separated by centrifugation at 11,000 to 13,000 xg for 30 minutes.

In addition, the centrifugation step (S30) is characterized in that it is performed twice.

In addition, the present invention is a product formed using a mugwort extract and encapsulated in starch, which is obtained by centrifuging and separating the SeNPs produced by adding sodium selenite to the mugwort extract extracted by drying and pulverizing mugwort and stirring it. Selenium nanoparticles are provided.

In addition, the mugwort extract is characterized in that it is obtained by extracting the whole plant of mugwort.

In addition, the mugwort extract and sodium selenite are characterized in that they are mixed at a volume ratio of 5:95 to 15:95.

Additionally, the stirring of the mugwort extract and sodium selenite was performed at 10 to 40°C for 2 to 8 hours.

In addition, the SeNPs were separated by centrifugation at 11,000 to 13,000 xg for 30 minutes.

In addition, the SeNPs are characterized by being centrifuged twice.

The selenium nanoparticles formed using mugwort extract and encapsulated in starch according to the present invention and the manufacturing method thereof are formed using mugwort extract and encapsulated in starch, thereby lowering manufacturing costs and enabling ecologically sustainable production. , to improve antibacterial function.

Figure 1 is a UV-visible spectroscopic analysis graph for A.annua and green synthesized AaSeNPs and StAaSeNPs.
Figure 2 is a graph showing FTIR analysis of A.annua and green synthetic AaSeNP and StaaSeNP.
Figure 3 is an X-ray diffraction spectrum (XRD) graph of StaaSeNPs.
Figure 4 shows photographs and graphs showing TEM images of AaSeNPs (a, b) and StAaSeNPs (d, e) at different magnifications and EDS analysis of AaSeNPs (c) and StAaSeNPs (f).
Figure 5 shows the average particle size (a) and zeta potential (b) of AaSeNPs. Graph showing the average particle size (c) and zeta potential of StaaSeNPs.
Figure 6 is a TEM image of cell internalization of AaSeNP and StAaSeNP treated with Bacillus cereus.
Figure 7 is a TEM image of cell internalization of Salmonella enterica treated with AaSeNPs and StaaSeNPs.
Figure 8 shows the effect of cell viability on NIH-3T3 cells treated with A.annua, AaSeNPs, and StAaSeNPs in a WST-based cytotoxicity assay (a). Photo and graph showing morphological changes (b) according to concentration of A.annua, AaSeNPs, and StAaSeNPs treated NIH-3T3 cells.

The following detailed description of the present invention is an embodiment in which the present invention can be practiced and refers to the accompanying drawings, which are shown as examples of the corresponding embodiments. These embodiments are described in sufficient detail to enable any person skilled in the art to practice the invention. It should be understood that the various embodiments of the invention are different from one another but are not necessarily mutually exclusive. For example, specific shapes, structures and characteristics described herein may be implemented in one embodiment without departing from the spirit and scope of the invention. Additionally, it should be understood that the location or arrangement of individual components within each described embodiment may be changed without departing from the spirit and scope of the invention.

Accordingly, the detailed description set forth below is not intended to be taken in a limiting sense, and the scope of the invention is limited only by the appended claims together with all equivalents to what those claims would assert if properly described. Similar reference numbers in the drawings refer to identical or similar functions across various aspects.

The terms used in the present invention are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person working in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.

In the present invention, when a part “includes” a certain component, this means that, unless specifically stated to the contrary, it does not exclude other components but may further include other components.

Hereinafter, with reference to the attached drawings, the manufacturing method of selenium nanoparticles formed using mugwort extract and encapsulated in starch according to the present invention will be described in detail.

The method for manufacturing selenium nanoparticles formed using mugwort extract and encapsulated in starch is Artemisia . annua ), dried, pulverized, and extracted to produce mugwort extract (S10); A SeNP generation step (S20) of mixing sodium selenite with the mugwort extract prepared in the mugwort extract preparation step (S10) and stirring to generate SeNP; and a centrifugation step (S30) of separating the SeNPs generated in the SeNP generation step (S20) by centrifugation, and adding a dispersion of starch dissolved in sodium hydroxide to the SeNPs obtained in the centrifugation step (S30) and stirring the starch. Includes an encapsulation step (S40).

First, the mugwort extract preparation step (S10) is performed.

In the mugwort extract production step (S10), mugwort extract is prepared by drying, grinding, and extracting mugwort.

The mugwort (Artemisia annua) is a perennial herb that grows to a height of about 1 m in a sunny place. The entire mugwort is covered with spiderweb-like hairs, and its underground stems grow sideways. The leaves of the mugwort grow alternately on the stem, and the stem leaves are oval-shaped and deeply split like feathers. The flowers of the above mugwort bloom in panicles from midsummer to fall. The flower clusters of the above mugwort have several flowers piled up inside the involucre. The young shoots of the above mugwort are edible and the mature ones are used medicinally. Similar plants include mugwort, mugwort, mugwort, and mountain mugwort.

The mugwort extract in the mugwort extract preparation step (S10) is appropriately obtained by extracting the whole plant of mugwort with water or alcohol having 1 to 4 carbon atoms.

Next, the SeNP generation step (S20) is performed.

In the SeNP generation step (S20), sodium selenite is mixed with the mugwort extract prepared in the mugwort extract preparation step (S10) and stirred to generate SeNPs.

The sodium selenite (Na ₂ SeO ₃ ) is a salt obtained by reacting sodium hydroxide with selenium oxide. It is a white to light off-white, odorless crystalline powder. It is a food additive used in health functional foods for nutritional supplementation.

Meanwhile, in the SeNP generation step (S20), the mugwort extract and sodium selenite are appropriately mixed at a volume ratio of 5:95 to 15:95. Most appropriately, it would be preferable to mix the mugwort extract and sodium selenite in a volume ratio of 10:90 in the SeNP production step (S20).

Meanwhile, in the SeNP generation step (S20), it is appropriate to stir the mugwort extract and sodium selenite at 10 to 40°C for 2 to 8 hours. Most appropriately, in the SeNP production step (S20), it would be preferable to stir the mugwort extract and sodium selenite at 20°C for 6 hours.

Next, a centrifugation step (S30) is performed.

In the centrifugation step (S30), a process of centrifuging and separating the SeNPs generated in the SeNP generation step (S20) is performed.

Meanwhile, in the centrifugation step (S30), it is appropriate to separate the SeNPs generated in the SeNP generation step (S20) by centrifuging them at 11,000 to 13,000 xg for 20 to 40 minutes. Most appropriately, it would be desirable to separate the SeNPs generated in the SeNP generation step (S20) by centrifugation at 12000xg for 30 minutes.

In addition, it is appropriate that the centrifugation step (S30) is performed through steps 1 to 3. Most appropriately, it would be desirable to perform it in two steps.

The mugwort extract manufacturing step (S10); SeNP generation step (S20); After performing the centrifugation step (S30), selenium nanoparticles formed using the mugwort extract according to the present invention and encapsulated in starch are produced.

Next, the starch encapsulation step (S40) is performed.

In the starch encapsulation step (S40), a dispersion of starch dissolved in sodium hydroxide is added to the SeNPs obtained in the centrifugation step (S30) and stirred.

At this time, the dispersion may be 5 to 15 mg of starch dissolved in 10 to 30 mL of sodium hydroxide. More specifically, the dispersion may be 10 mg of starch dissolved in 20 mL of sodium hydroxide. Additionally, it is preferable to stir at 65°C and 250 rpm until a clear solution is obtained.

Meanwhile, the SeNP and the dispersion solution obtained by dissolving starch in sodium hydroxide may be mixed at a volume ratio of 20:10.

Meanwhile, the manufacturing method according to the present invention may further include performing a starch encapsulation step (S40) and then centrifuging at 150,000 to obtain dried nanoparticles (S45).

Meanwhile, the present invention provides selenium nanoparticles formed using mugwort extract and encapsulated in starch.

Below, with reference to the attached drawings, selenium nanoparticles formed using mugwort extract according to the present invention and encapsulated in starch will be described in detail.

Selenium nanoparticles formed using mugwort extract and encapsulated in starch according to the present invention are separated by centrifuging the SeNP (Selenium nanoparticles) produced by adding sodium selenite to the mugwort extract extracted by drying and pulverizing mugwort and stirring. The obtained product is characterized in that it is formed by mixing starch with a dispersion dissolved in sodium hydroxide.

Meanwhile, the mugwort extract is preferably obtained by extracting the whole plant of mugwort. Most appropriately, the mugwort extract is preferably obtained by extracting the whole plant of mugwort with water or alcohol having 1 to 4 carbon atoms.

Additionally, it is appropriate that the mugwort extract and sodium selenite are mixed at a volume ratio of 5:95 to 15:95. Most appropriately, the mugwort extract and sodium selenite are preferably mixed at a volume ratio of 10:90.

Additionally, it is appropriate that the stirring of the mugwort extract and sodium selenite was performed at 10 to 40°C for 2 to 8 hours. Most appropriately, the stirring of the mugwort extract and sodium selenite is preferably performed at 20°C for 6 hours.

Additionally, it is appropriate that the SeNPs were separated by centrifugation at 11,000 to 13,000 xg for 20 to 40 minutes. Most appropriately, SeNPs are preferably separated by centrifugation at 12000xg for 30 minutes.

Additionally, it is appropriate that the SeNPs have been centrifuged 1 to 3 times. Most appropriately, it would be desirable to centrifuge twice.

Meanwhile, selenium nanoparticles formed using mugwort extract and encapsulated in starch are stirred by adding a dispersion to the obtained SeNPs. The dispersion is preferably one in which starch is dissolved in sodium hydroxide. More specifically, the dispersion may be 10 mg of starch dissolved in 20 mL of sodium hydroxide. Additionally, it is preferred that the solution is stirred at 250 rpm at 65°C until a clear solution is obtained. The SeNP and the dispersion solution obtained by dissolving starch in sodium hydroxide may be mixed at a volume ratio of 20:10.

Meanwhile, the manufacturing method according to the present invention may further include performing a starch encapsulation step (S40) and then centrifuging at 150,000 to obtain dried nanoparticles (S45).

Hereinafter, through the following experimental examples, the effect of selenium nanoparticles formed using mugwort extract according to the present invention and encapsulated in starch will be confirmed in detail.

1. Materials and Methods

Precursors sodium selenite, starch, tetracycline hydrochloride, RPMI medium, fetal bovine serum, antibiotic solution, and phosphate-buffered saline were purchased from Sigma Aldrich (Korea). Mueller Hinton broth (MHB), agar powder, and sodium hydroxide were obtained from Difco Laboratories (Fisher Scientific, Republic of Korea).

Example One. Mugwort extract Selenium nanoparticles used ( AaSeNPs )Manufacture of

Mugwort was purchased from the medicinal market in Seoul, Korea, and was certified by a botanist from the Department of Biomedical Convergence at Kangwon National University. Mugwort was dried, chopped, ground into fine powder, and used for biosynthesis of selenium nanoparticles.

Selenium nanoparticles (AaSeNPs) were prepared using the mugwort extract of Example 1 according to the following manufacturing process.

Mugwort extract manufacturing step (S10): Artemisia annua ) whole plant was dried, ground, and extracted with purified water to prepare mugwort extract.

SeNP generation step (S20): Mix sodium selenite with the mugwort extract prepared in the mugwort extract preparation step (S10) at a volume ratio of 5:95 to 15:95 and stir at 10 to 40°C for 2 to 8 hours. SeNPs were produced.

Centrifugation step (S30): SeNPs generated in the SeNP generation step (S20) were centrifuged at 11,000 to 13,000 xg for 20 to 40 minutes, and this was repeated twice to produce a solution using mugwort extract. Selenium nanoparticles (AaSeNPs) were prepared.

Example 2. Mugwort extract Preparation of selenium nanoparticles formed using and encapsulated in starch.

Selenium nanoparticles formed using the mugwort extract of Example 1 and encapsulated in starch were prepared according to the following manufacturing process.

Mugwort extract manufacturing step (S10): Artemisia annua ) whole plant was dried, ground, and extracted with purified water to prepare mugwort extract.

SeNP generation step (S20): Mix sodium selenite with the mugwort extract prepared in the mugwort extract preparation step (S10) at a volume ratio of 5:95 to 15:95 and stir at 10 to 40°C for 2 to 8 hours. SeNPs were produced.

Centrifugation step (S30): SeNPs generated in the SeNP generation step (S20) were centrifuged at 11,000 to 13,000 xg for 20 to 40 minutes, and this was repeated twice to produce a solution using mugwort extract. Selenium nanoparticles (AaSeNPs) were prepared.

Starch encapsulation step (S40): A dispersion of starch dissolved in sodium hydroxide is added to the selenium nanoparticles (AaSeNPs) obtained in the centrifugation step (S30) and stirred to form selenium nano-sized particles formed using mugwort extract and encapsulated in starch. Particles (StAaSeNPs) were formed.

Step of obtaining dried nanoparticles (S45): After performing the starch encapsulation step (S40), further performing the step of centrifuging at 150,000 to obtain dried nanoparticles (S45), Selenium nanoparticles (StAaSeNPs) formed using the extract and encapsulated in starch were obtained.

Comparative example 1. Mugwort extract (A. Annua Extracts )Manufacture of

For comparative experiments, mugwort extract (A.Annua Extracts) of Comparative Example 1 was prepared.

Mugwort extract manufacturing step (S10): Artemisia annua ) whole plant was dried, ground, and extracted with purified water to prepare mugwort extract.

2. Experimental method

2.1 of SeNPs Check characteristics

The synthesized SeNPs were characterized by electronic spectra recorded on a UV-visible spectrophotometer (Optizen 2120UV, Korea) in the wavelength range of 200–600 nm. The functional groups of bioreductants present in the extracts were detected with the KBr pellet technique on a Fourier transform infrared spectrophotometer (FT-IR, PerkinElmer, UK) at room temperature. The 16-scan spectrum was run in the wavenumber range 500-4000 cm ^-1 at a resolution of 4 cm ^-1 . XRD analysis established the structure of the nanoparticles using an X'Pert Pro MPD (PANalytical, USA) operated at 45 kV, 40 mA current, and CuKα radiation. Zeta potential particle size analysis (Malvern Panalytical, UK) was used to document the average particle size, polydispersity index and zeta potential of the NPs. The surface morphology of the prepared SeNPs was examined using a transmission electron microscope (JEM-2100 JEOL TEM, Japan) at 90 kV, and the elemental composition of the NPs was confirmed using EDS analysis.

2.2 Confirmation of antibacterial effect

The area of inhibition and MIC measurement methods were used to screen the antibacterial activity of the NPs. Pathogenic bacteria used in the study were Bacillus cereus (ATCC 14579), Staphylococcus aureus (ATCC 19095), Escherichia coli (ATCC 43888), and Salmonella enterica (ATCC 14028). The zone of inhibition test was performed according to a slightly modified method from a previous study. 20 μL of each bacterial sample was inoculated into 10 mL of Muller Hinton broth and kept in a shaking incubator for 24 hours and allowed to reach a growth of approximately 109 CFU/mL. Samples were then spread onto MHA agar plates using a Drigalski spatula. Wells were created on the surface of the solid medium using a sterile bore into which various concentrations of samples (12.5, 25, 50, 100 μg/mL) and tetracycline (20 μg/mL) were added. The plate was kept in an incubator at 37°C for 24 hours, and the diameter of the incubation area was measured in millimeters. The experiment was performed in triplicate.

A microdilution method was adopted to screen the minimum inhibitory concentration (MIC) of AaSeNPs and StaAaSeNPs. First, 100 μL of MHA medium was placed in a 96-well plate. Then, 10 μL of each well-grown bacterial sample (109 CFU/mL) was seeded into the wells, followed by different concentrations of NPs (5–100 μg/mL). Untreated bacterial samples were considered negative controls. The plate was stored in an incubator at 37°C. Samples were collected at regular intervals and NP-mediated MICs were recorded by measuring OD at 600 nm. Based on preliminary studies, Bacillus cereus and Salmonella enterica were selected to investigate cellular and ultrastructural damage using TEM analysis. Bacterial samples were collected after overnight incubation of AaSeNPs and StAaSeNPs. The standard procedure of Matias et al. (2003) was used for TEM analysis.

2.3 Confirmation of cytotoxicity

The biocompatibility and non-toxic properties of AaSeNPs and StaaSeNPs were studied in non-cancerous NIH-3T3 cell line. Cell lines were procured from the Korea Cell Line Bank, Korea. Cells were grown in RPMI medium supplemented with 10% fetal bovine serum and 0.5% antibiotic solution in a humidified CO atmosphere (5%). Cells were allowed to reach 80-90% confluency. Afterwards, cells (100 μL) were cultured in a 96-well plate. At the end of the 24-hour incubation, the cells were treated with 10 μL of different concentrations of AaSeNPs/StAaSeNPs and incubated overnight in a humidified CO2 atmosphere. Then, 100 μL of WST solution was added to all wells. After incubation for 4 hours, absorbance was measured at 450 nm with a microplate reader (Optizen 2120UV, Republic of Korea). Percentage of cell viability was calculated using methods described elsewhere.

3. Results and Discussion

In general, green synthesis of nanoparticles uses secondary metabolites as reducing agents to replace harmful chemicals. A wide range of natural materials, including plant extracts and their secondary metabolites, have been used to achieve long-term measurements for nanoparticle synthesis. However, to the best of our knowledge, there are few reports on the synthesis of SeNPs using plant material from Artemisia annua. In the current attempt, we fabricated SeNPs from plant materials and confirmed their antibacterial activity.

The UV-visible absorption spectrum of the AaSeNPs solution showed a peak at 278.51 nm, which was attributed to the surface plasmon resonance (SPR) of SeNPs (Figure 1). This observation is consistent with previous findings. In this study, SeNPs were produced by incorporating sodium sulfite into plant material of A. annua. The color shift in A. annua may occur due to reduction of selenium ions to their elemental form, consistent with the excitation of SPR. Plant extracts are thought to produce various biomolecules that act as reducing and capping agents to produce compact and shape-controlled NPs, which was confirmed by FTIR analysis.

The FTIR spectrum of the aqueous extract of A. annua shows prominent bands at 3290.93 (OH stretching; polyhydroxy compounds), 2916.63 & 2851.29 (CH stretching; alkane compounds), 1729.60 (C=O stretching; keto compounds), 1634.65 (C=C). showed a peak. stretching, alkenes), 1542.66 (NO stretching, nitro compounds), 1442.13 & 1317.63 (OH bending, carboxylic and phenolic compounds), 1236.48 (CN stretching, amines) and 1031.54 cm-1 (CO, carboxylic acids). The FT-IR spectrum of green synthesized SeNPs showed a strong stretching vibration at 3190.18 (O-H stretching). 2923.35 (C-H stretching of alkanes); 1790.63 (overtones of C-H bending of aromatics); 1716.88 (C=O stretching, keto compounds), 1581.91 (N-O stretching, nitro compounds) 1352.41 (C-H bending); 1234.41 (C-N stretching; amine), 1140.10 & 1033.86 (C-O; carboxylic acid) and 827.69 (C=C bending) cm-1. However, the absorption peak of AaSeNP at 3190.18 cm-1 shifted to 3243.89 cm-1 in the IR spectrum of StAaSeNP. The peak at 1654.17 was assigned to the stretching vibration of the C=O band (amide I) in starch molecules and water molecules absorbed in the amorphous region. These results indicate the existence of various functional groups that can be responsible for both reduction and stabilization of SeNPs. In particular, hydroxyl groups play an important role in reducing selenium ions to their elemental form (Figure 2).

The crystalline properties of StaaSeNPs were analyzed by XRD analysis (Figure 3). Peaks were seen at 2ta values of 26.16°, 31.64°, 37.82°, 40.56°, 50.92°, 51.16°, 54.36°, and 55.08°. 60.92°, 65.08°, 69.09° and 77.29°corresponds to planes (100), (101), (110), (102), (111), (200), (201), (112), (202) , (210), (113) and (301). These results are from the Joint Committee on Powder Diffraction Standards (JCPDS) file no. 06-0362. The XRD pattern showed that the synthesized StaaSeNPs were crystalline. The sharp peaks in the The XRD results are consistent with the study of Shin et al. (2021), who showed that SeNPs obtained from methanol extract of Cirsium setidens were crystalline.

The surface morphology of NPs at different magnifications was studied by TEM. SeNPs and AaSeNPs showed uniform distribution in a spherical shape (Figures 4a-b & d-e). The stability of StAaSeNPs can be attributed to the presence of starch and plant molecules. Additionally, EDS mapping analysis showed the presence of Se elements in AaSeNPs and StAaSeNPs (Figures 4c & f). These results are consistent with previous studies where elemental Se showed peaks at 1.33, 11.22, and 12.49 keV. The average particle size, polydispersity index (PDI), and zeta potential were analyzed with a particle size analyzer, and the values were 70.81 nm and 0.147 for AaSeNPs, respectively (Figure 5a-d). In comparison, StaaSeNPs were found to have an average particle size of 109.2 nm, a PDI value of 0.149, and a zeta potential of -30.9 eV. The strong negative zeta potential suggests that the NP is stable.

The antibacterial activity of AaSeNPs and StaAaSeNPs was tested against Gram-positive (B.cereus, S.aureus) and Gram-negative (E.coli, S.enterica) pathogenic bacterial strains by agar well diffusion and MIC analysis. The zone of inhibition in the agar well diffusion method indicates that the green synthetic nanoformulation has broad antibacterial activity against the tested bacterial strains. Dose-dependent inhibition was observed in the study (Tables 1 and 2).

Bacterial
Strain AaSeNPs Tetracycline 12.5μg/mL 25μg/mL 50μg/mL 100μg/mL 20μg/mL E. coli 6.2± 7.1± 7.8± 10.2± 22.6± S. enterica 8.6± 12.3± 16.3± 18.7± 25.4± B. cereus 5.8± 6.6± 12.5± 11.2± 29.4± S. aureus 5.3± 6.2± 9.2± 10.3± 20.6±

Bacterial
Strain StAaSeNPs Tetracycline 12.5μg/mL 25μg/mL 50μg/mL 100μg/mL 20μg/mL E. coli 8.6± 9.5± 10.6± 12.7± 25.2± S. enterica 11.3± 15.4± 18.3± 20.2 25.6 B. cereus 6.2± 10.3 12.8± 17.3± 27.5± S. aureus 6.6± 8.1± 10.5± 13.6± 21.3±

At the highest concentration (100 μg/mL), AaSeNPs showed maximum inhibition areas of 18.7 mm, 11.2 mm, 10.3 mm, and 10.2 mm against S. enterica, B. cereus, S. aureus, and E. coli, respectively. StaaSeNPs showed 20.2 mm, 17.3 mm, 13.6 mm and 12.7 mm for bacterial strains in the same order given above. MIC studies were used to investigate the antibacterial activity of nanoparticles in the concentration range of 5–100 μg/mL. The MICs of StaAaSeNPs against B.cereus, S.aureus, E.coli, and S.enterica are 40, 50, 100, and 30 μg/mL, whereas the MICs of AaSeNPs are 10, 40, 100, and 20 μg/mL. Data analysis shows that StAaSeNPs have higher efficacy against Gram-positive bacteria than Gram-negative bacteria.

These findings indicate that the antibacterial activity of nanoparticles may be related to the characteristics of the bacterial species. The presence of abundant pores and a thin peptidoglycan layer in the bacterial wall allows NPs to rapidly penetrate into the nucleus and interfere with transcription and translation processes. Similar results have been reported by many authors. High concentrations of negatively charged nanoparticles can exhibit significant antibacterial activity due to a phenomenon known as molecular crowding. However, the NPs showed significant antibacterial activity against Gram-positive strains of B.cereus and S.aureus. Negatively charged SeNPs had a stronger affinity for Gram-positive bacteria, which could be explained by the absence of negatively charged LPS molecules in the cell wall. Therefore, StaaSeNPs can be considered as a promising candidate for inhibiting bacterial growth based on the results obtained from ZOI and MIC assays.

TEM analysis demonstrated the interaction of NPs with bacterial cell walls. When StaAaSeNPs were added to B. cereus and S. enterica, they were found to adhere to the bacterial cell wall surface. This is consistent with published observations regarding the interactions of proteins in cell membranes when exposed to NPs. This observation is very important because it indicates the damaging effect of StAaSeNPs on bacterial cell membranes. They showed complete loss of cellular integrity and cell membrane rupture, resulting in loss of metabolic control causing homeostatic imbalance, resulting in cellular metabolic discharge and ultimately cell death. TEM analysis results show that the bactericidal activity of StAaSeNPs appears to be mediated by bacterial cell wall lysis (Figures 6 and 7). Because starch molecules are composed of long chains of glucose units, they act as hydrogen bond donors and acceptors. The presence of hydroxyl groups may be linked to strong electrostatic contact between SeNPs and Gram-negative or Gram-positive bacteria, leading to cell wall collapse. When the cell wall collapses, cell balance is disrupted, potentially causing cytoplasmic material, including nucleic acids, to leak out, resulting in cell lysis or necrosis.

To use these NPs in pharmacological applications, we investigated their biocompatibility in the noncancerous NIH-3T3 cell line ( Figure 8 ). HEK-293 cells treated with NPs showed improved biocompatibility and substantially retained and maintained cell viability at the tested concentrations when compared to controls. AaSeNPs and StAaSeNPs had little effect on the viability of HEK-293 cells at 100 μg/mL, and the percentage of viable cells was between 97.56 and 84.36% (Figure 8). These findings follow previous reports that the concentration of NPs did not affect cell growth of noncancerous NIH3T3 cells. Our findings are consistent with earlier studies reporting that proteins and metabolites of biosynthesized selenium nanoparticles can increase stability and lower toxicity. Taken together, our findings clearly depicted that green synthetic nanoparticles are non-cytotoxic to normal cells and can be effectively used as antibacterial agents.

4. Conclusion

In this study, SeNPs were biosynthesized using Artemisia annua, and subsequently the surface of biological SeNPs was functionally modified with starch. Both types of nanoparticles were tested for antibacterial activity and biocompatibility. AaSeNPs and StAaSeNPs differed significantly in antibacterial and biocompatibility efficacy. Based on this, it can be inferred that the most influential factor determining the antibacterial activity of SeNPs is surface chemistry. The surface chemistry of NPs varies depending on particle size, and more research is needed to properly define the threshold for specific antibacterial processes.

Accordingly, the present invention provides selenium formed using mugwort extract and encapsulated in starch to reduce manufacturing costs, enable ecologically sustainable production, and improve antibacterial function. It is stated that nanoparticles and their manufacturing method have been developed.

Although the present invention has been described with the accompanying drawings, this is only one example among various embodiments including the gist of the present invention, and is intended to enable those skilled in the art to easily implement the present invention. For this purpose, it is clear that the present invention is not limited to the embodiments described above. Accordingly, the scope of protection of the present invention should be interpreted in accordance with the following claims, and all technical ideas within the equivalent scope by change, substitution, substitution, etc. without departing from the gist of the present invention are subject to the rights of the present invention. will be included In addition, it is clarified that some of the configurations in the drawings are provided in an exaggerated or reduced form compared to the actual figure for the purpose of explaining the configuration more clearly.

(S10): Mugwort extract manufacturing step
(S20): SeNP generation step
(S30): Centrifugation step

Claims (12)

A mugwort extract production step (S10) in which mugwort extract is prepared by drying and pulverizing the whole plant of mugwort ( Artemisia annua ) and extracting it with purified water;
Sodium selenite was mixed with the mugwort extract prepared in the mugwort extract preparation step (S10) at a volume ratio of 5:95 to 15:95 and stirred at 10 to 40°C for 2 to 8 hours to produce SeNPs. Step (S20);
A centrifugation step (S30) of repeatedly centrifuging the SeNPs generated in the SeNP generation step (S20) twice for 20 to 40 minutes at 11,000 to 13,000 xg to produce selenium nanoparticles (AaSeNPs) using mugwort extract; and,
A dispersion of starch dissolved in sodium hydroxide is added to the selenium nanoparticles (AaSeNPs) obtained in the centrifugation step (S30) and stirred to form selenium nanoparticles (StAaSeNPs) formed using mugwort extract and encapsulated in starch. A method for producing selenium nanoparticles formed using mugwort extract and encapsulated in starch, comprising a starch encapsulation step (S40).
delete delete delete delete delete Dry and grind the whole plant of Artemisia annua, mix it with mugwort extract extracted with purified water at a volume ratio of 5:95 ~ 15:95, stir at 10 ~ 40℃ for 2 ~ 8 hours, and stir at 11000 ~ 13000xg for 20 ~ 40 hours. Selenium nanoparticles (AaSeNPs) using mugwort extract, obtained by repeated centrifugation for 2 minutes;
A dispersion of starch dissolved in sodium hydroxide was added and stirred; Selenium nanoparticles (StAaSeNPs) formed using mugwort extract and encapsulated in starch.
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