Preparation of Nanoparticles of Selenium / Zinc Oxide Reductase in an Environmentally Friendly Green Synthesis Method and Studying
Their Effect on Staphylococcus Aureus
Worod Anedi Al-ebadi , Hiyam Abdul ridha Al-awad , Abbas Matrood Bashi University of Kerbala/collage of education for pure sciences
University of Kerbala/collage of education for pure sciences Departement of anastasi and intensive care/ Altuff college _iraq Abstract :
The current study included testing the two nanocomposites (curcumin / zinc oxide Nps) (curcumin / selenium Nps) on Staphylococcus aureus bacteria which isolated from urine, nose and wounds from several hospitals in Babilon Governorate (Al-Musayab General Hospital and al-Turky Hospital And al-jamhori Hospital) after it was diagnosed by manual and biochemical methods. Two nanocomposites, namely (curcumin / zinc oxide Nps) and (curcumin / selenium Nps) were prepared and charecterised by: UV-Visible spectroscopy and Fourier-transform infrared spectroscopy, infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), and evaluation of the inhibitory efficacy of nano composites and free curcumin in addition to tow types of antibiotics (vancomycin, ciprofloxacin). It was highly effective against the studied bacteria species. The MIC test was performed by broth dilution method, and it was found that the minimum inhibitory concentration of curcumin is 250 and 500 µg /ml. And the MIC for cur / zno NPs was 1000. Whereas, the MIC for cur / se NPs was 125 µg /ml. The results also indicated an increase in the effect of nanocomposites on the bacteria compared to the free curcumin, and this increase resulted from the synergistic action between the two components of the nanocomposite. The results of cur / zno Nps are close to those of the antibiotic vancomycin. The results of the cur / se Nps nanocomposite are close to those of the antibiotic ciprofloxacin for staphylococcus aureus
Introduction
Infectious diseases resulting from infection with microorganisms in general and bacteria in particular are the most prevalent diseases around the world and cause millions of deaths each year (Singh, Krishnamurthy, & Mathew, 2014) and there are many bacterial species that are capable of causing diseases to humans and various living organisms as a result. Because they have a number of structural, chemical or genetic characteristics that can constitute a pathogen for humans and other organisms, and these factors can be called together as virulence factors that help the bacterial cell cause damage in the host's body, such as producing toxins and a number of other enzymes.( Murray et al.,2020).
The excessive and repeated use of antibiotics has lead to appearance of new resistant generation and strains which represented a global problem at present (Kariminik and Nazoori, 2018). ).
S. aureus bacteria is a symbiotic bacteria that is present on the bodies of 30% of people, but it can transform into pathological bacteria on the skin, wounds and internal tissues, and it can also be more life-threatening causing a number of systemic infections such as pneumonia, endocarditis. Heart endocarditis, septicemia, and infection (UTI) urinary tract, and this bacteria is one of the most common causes of infection in hospitals around the world and the increase in resistance to antibiotics, especially methicillin, which became known as MRSA. Which is the main factor of infection in hospitals and local communities and has become a major disease problem worldwide in recent years (Stark, 2013).
From this context, it can be said that there are two main challenges that health organizations and treatment centers face in the world today: the spread of infection in hospitals and health centers around the world, its rapid transmission from one country to another, and the emergence of new strains of antibiotic resistance bacteria (Niakan et al., 2019). Therefore, studies are heading to solve this problem by searching for alternatives such as nanomaterials for some metals and their oxides, which have appeared as a promising alternative to antibiotics in the last few years (Kariminik and Nazoori, 2018) Also, parts of some plants and their alcoholic and aqueous extracts were used as antidotes and treatments for cases of bacterial infections in general, as a result of the effective compounds they contain with therapeutic properties (Lawhavinit et al., 2010). Turmeric is one of the spices obtained from the Curcuma longa L. plant and one of the plants widely used in the medical field because it
contains phenolic compounds and antioxidants, which can make its use available to treat fungal bacterial infections, fight cancer and analgesic pain. Ulcerative treatment (Altunatmaz et al., 2016).
that the effectiveness of curcumin against microbiology appears on two different levels, it can be used to prevent Wounds and treatment of pathogenic bacteria contaminating them, and it can also be used as a protective material for food preserved against various pathogens, and it has been evaluated by the expert committee of the FDA and the World Health Organization WHO as a natural food additive extracted from the plant and it can be manufactured chemically However, when chemically manufactured, it cannot be added to food (Altunatmaz et al., 2016).
Nanoparticles are defined as tiny particles of at least one dimension with a size ranging from 1- 100 nm (Christian et al., 2008). Nanoparticles have unique properties in electronic, magnetic and chemical stimulation, they are characterized by high stability and little reactivity, and they have biocompatibility, they also lack toxicity and come in many forms, and this is what makes them widely applied in various fields of biomedicine such as diagnosis, treatment of cancers and Other diseases, drug manufacturing, and gene delivery, and some metal nanoparticles have anti-viral, bacterial, fungi, and some tumor properties (Vetchinkina et al., 2018).
Selenium nanoparticles are more attractive to researchers compared to other minerals, due to its low toxicity compared to organic and inorganic nanoparticles, as inorganic nanoparticles are more toxic than organic, and selenium nanoparticles have important biological properties as it is characterized by its small size, which facilitates its absorption by cells. It can also be used in the non-oxidation state of Se0, which is less toxic compared to other oxidation states (Se + IV, Se + VI), although it is unstable and may become ineffective with Rapidly, but addressing this problem by coating it with a substance expressed in nano-vehicles such as chitosan (CS) (Hosnedlova et al., 2018). Nanoparticle selenium is a good choice for use as an anti-microbial growth due to its unique phenotypic and chemical properties.
Using it as a preservative by coating the metal cans used to preserve food with it (Rajan et al., 2019). It has been shown that it binds to the cell envelope by means of the chemisorption mechanism to penetrate the cells via lipopolysaccharides and through the change in the composition of the three enzymes: preprolipoprotein diacylglyceryl transferase, lipoprotein signal apolipoprotein N- acyltransferase and peptidase which play a pivotal role in maintaining cell life (Guisbiers et al., 2016)
ZnO-NPs is an antimicrobial agent for many pathogenic and food-spoiling microorganisms, and ZnO-NPs is characterized by its wide applications as an anti-microbial agent compared to nanoparticles of other metals. Of reactive oxygenation and disruption of cell membranes as a result of accumulation of ZnO-NPs (Souza et al., 2019). And zinc oxide nanoparticles have the ability to inhibit the growth of Gram negative and Gram positive bacteria (Ugaili et al., 2014).
Materials and Methods
preparation of Cur/ZnO nanoparticle was done by following the method described by (Bashi et al. 2013) with some modification in preparing ,1 gram of curcumin was added to a solution of zinc oxide (resulting from dissolving 1 g of zinc oxide in 50 ml of distilled water). The mixture was stirred by a magnetic stirrer at room temperature for 24 hours and then the mixture was placed in the vibrating incubator at a temperature of 40 ° C for 18 hours.The color of the solution changed from pale yellow to reddish yellow. The precipitate was separated by a centrifuge and then washed with distilled water to remove the ions several times, and then the residue was dried in an electric oven at a temperature of 50
° C, and then grinded with good ceramic mortar to obtain a fine powder and it was stored in the refrigerator until it was used in the study.and to prepare Cur/Se nanoparticle1gram of curcumin was added to 2 ml of selenium solution (prepared by dissolving 0.263 grams (0.01 mol) in 100 ml of distilled water) and then placed on a magnetic stirrer at a temperature of 40 ° C. The color of the solution changes from pale yellow to reddish yellow, then the permeability of the solution is measured in the Spectrophotometer, after that the solution is placed in the shaker incubator for a period of 24 hours, and the residue is separated by a centrifuge and finally washed with distilled water Several times after that it was dried at a temperature of 50 ° C, then grinded well to obtain a fine powder and stored in the refrigerator until it was used in the study.
To characterize the Cur/ZnO,Cur/Se nanoparticle four exams was used .first ,UV-Visible Spectroscopy, were Curcumin / selenium nanoparticles characterized by dissolving 500 mg of the
prepared substance in 4 ml of distilled water, then subjected to ultrasound for 15 minutes, and then measured in an ultraviolet spectrometer, as both visual examination and absorbance measurements were detected. The apparatus was cleared by means of Distilled water was then screened with a wavelength range of 200-800nm. This test is used to confirm the formation of curcumin / selenium nanoparticles. second, X-ray Diffraction Analysis, this exam was used to explains the difference in layer thickness before before and after the intercalation process by using Brack's law nλ = 2dSinƟ to get the value of the layer thickness
Where:
n: the order of the crystal plane from which the X-ray diffraction is.
λ: The wavelength of the X-ray used (copper tube).
Ɵ: the diffraction angle of the X-ray.
d: the vertical crystal distance between two parallel planes.
Third, the scanning electron microscope (SEM) was used to determine The surface morphology of the two nanocomposites ,which done by dispersed and dried NPs were placed over an aluminum heel containing a double-stick conductive carbon tape and coated with gold under an atmosphere of argon using a spray coating. Fourth , the Fourier-transform infrared spectroscopy (FTIR) which is a unique technology that allows to perform biochemical analysis of biological samples, functional properties, and structural information of a compound. The plant extract has distinct peaks in the FTIR spectrum, indicating the presence or absence of functional groups.
The infrared spectrum of each compound of the free nanocomposites under study as well as the free form therapeutic materials as well as the nanocomposites were studied after loading the treatment on them, as a tablet of each of these compounds was made with potassium bromide (KBr) after being well ground and measured. The infrared spectrum in the range of wave numbers (400-4000) cm-1. All visible beams were fixed as most of the main beams were diagnosed.
After the nanocomposites was examined ,the Minimum Inhibitory Concentration (M.I.C.) was determined by broth dilution method as following:
1.same quantities of bacteria were grown in a liquid medium, which is Muller Hinton Broth, in sterile test tubes with the number of concentrations required to be prepared from the antagonist (the number of bacteria is 105 - cells / ml).
2. Adding increased concentrations, as the first tube (No. 1) contains the concentration (zero) of the nanomaterial, then it is followed by the second tube (No. 2) that contains the lowest concentrations of the nanomaterial, followed by the third tube (No. 3) that contains the concentration of twice as much It is present in tube No. 2 and so on for the rest of the tubes. Concentrations of (62.5-2)mg were used.
3.All tubes are incubated at a temperature suitable for the studied bacteria. The tubes are examined after incubation to identify any of them that contain growth as an indication of turbidity. The clear tubes indicate no growth due to the effectiveness of the antagonist.
4.The first clear tube that comes after a series of cloudy tubes is tested so that the concentration of the nanoparticle in it is the MIC.
The last step in this work is determination the Antibacterial Activity of Cur/ZnO, Cur/se NPs by Disk Diffusion Assay as Morello et al., (2006) mentioned and according to the following steps:
1. The bacterial isolates were activated on a nutrient agar and were incubated in the incubator at 37 ° C for 24 hours.
2. A small amount of the growing bacteria colonies were transferred by the Loop to tubes containing the physiological saline solution and mixed well. The turbidity of the growth was compared with the turbidity of the standard McFarland standard 0.5: turbidity solution.
3. 20 μl of the bacterial suspension was placed by a micropipette, then spread by a glass diffuser (L- shape) on the surface of the Muller-Hinton agar homogeneously, after which the dishes were left to dry at room temperature for a period of (10-15) minutes. .
4. Sterile tablets were prepared and saturated with serial concentrations of nanocomposites (8,6,4,2 mg / ml) by means of a micropipette, then these tablets were transferred with sterile forceps to the dishes, then the dishes were incubated at a temperature of 37 ° C for 24 hours. As for the antibiotic tablets, they were transferred directly to the plates and incubated in the same way.
5. The results were read by measuring the diameters of the damping zones in mm around each disc.
results
UV-Visible Spectroscopy for cur/se Nps show that The change in the color of the prepared solution from yellow to reddish orange is due to the plasmon resonance peak, which lies between the 300 nm-450 nm (Snigdha, Sugathan, & Mathew, 2017). The examination is used to show the type of plasmon on the surface of the nanomaterial, When the wavelength nm is plotted on the x-axis versus the absorption on the y-axis, we see the height of the plasmon as in Figure (1). The emergence of the high peak is the spectral pattern that occurs due to the excitation of the local surface plasmons that cause strong scattering of light by an electric field of a specific wavelength where the resonance phenomenon occurs (Deepa & Ganesan, 2013). The rise starts from (300 nm) and is in the form of a parabolic oval from the top and has a peak representing the highest absorption located at (450 nm) and then falls after. The oval shape with its head up and its base down is evidence of a nanomaterial that begins to be affected when exposed to visible ultraviolet rays. The apparent beams that were observed in the nanocomposites of the main compounds (curcumin + selenium) clearly indicate that the interaction between the two components occurred and the formation of a stable nanocomposite.
Figure 1: UV-vis spectroscopy of the cur-se Nps nanocomposite
from the X-ray Diffraction Analysis for cur/se nps The spectrum in Figure (2) shows that the peak before 30 ° are indicative of selenium, which means that curcumin has reduced selenium to a nanocomposite and its beams appeared in a clear and sharp form, which are 19.72 °, 25.13 °, 36.33 °, 44.44 ° and with a crystalline distance of 674.80, 1000.00 , 53.91 and 55.07 nm respectively.
And from the X-ray Diffraction Analysis for cur/zno Nps We note that the spectrum in Figure (3) is zinc oxide, and curcumin was not clear between the layers and did not give crystals, perhaps because it is squeezed horizontally, so it does not give height to the crystals, but it is very clear with respect to zinc oxide, which is at the peak 34.73 °, 47.81 °, 52.33 °, 56.71 °, 62.33 °, 67.36 °,. With crystal
distances of 1000.00, 221.75, 16.52, 213.27, 536.37, and 375.84 nm respectively, there is one peak that indicates curcumin at 26.23 ° and a crystalline distance of 38.14 nm.
the second exam was Scanning Election Microscope for cur/zno Nps and it was clearly shown In figure (4), the appearance of ZnO as a hexagonal crystal in Image No. (1) was enlarged in Image No.
(2) and appeared in the form of layers of zinc oxide, perhaps jammed between curcumin, but not completely clear. It was also enlarged in Image No. (3) and granules appeared to us. Zinc oxide is clear on the surface, and in picture No. (4) one molecule was taken and confined between two lines and its diameter was calculated, which is at d1 it is 61.32 nm and at d2 it is 26.49 nm and at d3 it is 17.37 nm is an indication of the occurrence of a reduction due to the presence of zinc oxide particles of different sizes, which It means that curcumin has reduced zinc oxide (ZnO) to molecules of different sizes.
Fig. No. (5) shows the Scanning Election Microscope for cur/se nps , Image No. (1) shows the layers of precipitated selenium reduced in the form of clear crystals. In pictures No. (2) the layers were enlarged and above the layers granules or balls of nanoparticles appeared. In Image No. (3) they have clusters.
The balls are in the shape of a cauliflower, in picture No. (4) certain balls were taken and limited between two lines and the measurements in d1 were 45.59 nm and d2 equal to 24.60 nm and at d3 equal to 31.23 nm indication p is that the reduction is not homogeneous and it shows molecular sizes between (24-45 nm) This indicates that the reduction is either in the form of selenium metal or selenium oxide (selenium dioxide(
Figure (4) the cur-zno Nps nanocomposite under a scanning electron microscope (SEM(
Figure (2) X-ray diffraction (XRD) spectrum of selenium nanoparticles (seNPs) formed by curcumin Figure (3) X-ray diffraction (XRD)
spectrum for zinc oxide (ZnO) formed by curcumin
4 3 2 1
Figure (5): the cur-se Nps nanocomposite under a scanning electron microscope (SEM).
The next examination that the samples underwent was The ftir technique which is one of the important techniques for viewing the effective groups on the surface, and from these groups we infer the process of manufacturing nanocomposites, in the selenium spectrum as shown in Figure (6) We see the existing beams which are 3439 cm-1 low intensity beam (2922, 2854, 2360,2342) cm-1, and these beams are due to the synthesis of nanocomposites from selenium nanoparticles that have been reduced with curcumin juice; Where a process of stabilizing and reduction occurred by the action of the compounds present in the curcumin juice, as the compounds that act on the reduction act first and then those that fix the nanoparticles..
The beams (1457, 1636, 1734) cm-1 are attributed to the vibration of the O-H bond resulting either from water or from alcohol. As for the amino NH groups, the bond elongation is 3439 cm -1.
Elsewhere, the beam (1734) cm-1 means the presence of O-H and. N-H beams at (2800-2900) cm-1 are attributed to the compounds containing the C-H bond. The beam at (2360) cm-1 is attributed to the carbonyl COO bond, the beam at (2842) cm-1 is specific to the amine group of NH. The beams at (1457,1057) cm-1 are due to the absorption of the C-H bond. The last beam, between (600-400) cm-1, is attributed to the formation of the nanocomposite of selenium, which is either selenium oxide or selenium dioxide.
The peaks corresponding to the expansion and vibration of the bonds in NH2, COOH, CH2, O-H indicate the presence of reducing groups that have reduced and fixed selenium molecules in the form of a nanocomposite, as three-dimensional particles were formed (Sampath et al., 2013).
Secondary metabolites are the main agents of nanoparticle biosynthesis, and plant extracts contain phenol, alcohol, amine, carboxylic acid, alkaloids and terpenoids responsible for reducing and stabilizing nanoparticles (Jha et al.). al., 2009).
in cur/zno spectrum in Figure (7) many packages, 3400,2800,2900, 1600, 1450, 1200 cm-1 that are attributed to the hydroxyl (OH), amine (NH2) and carboxylate (COOH) groups, meaning we have a mixture of hydrocarbon compounds, amino acids or acids. Carboxylic works on the process of reduction and fixation, the bundles returning to the curcumin juice are visible between the layers of zinc, the bundle between 400-600 cm-1 is due to zinc oxide, an indication of the presence of these compounds between the layers of zinc oxide.
Figure (6): FT-IR spectrum of nano-selenium formed by curcumin.
2 1 4 3
Figure (7): FT-IR spectrum of nano-zinc oxide formed by curcumin.
50 bacterial isolates were collected from S. aureus bacteria. These isolates included urine samples and swabs from the nose and wounds were collected from al-Turky Hospital in Babylon, Musayyib General Hospital and al-jamhory Hospital, After transplantation on blood agar and cuituring at 37 ° C for 24 hours, circular, convex, luminous colonies of yellow-golden color were obtained.
As for the microscopic examination, the bacteria appeared in the form of Gram-positive cocci, clustered in clusters, and this is evidence that they belong to the genus Staphylococcus (Quinn et al., 2004).When performing the catalase test, it was found that the bacteria produce this enzyme, as they break down hydrogen peroxide (H2O2) and convert it into water and oxygen, and this is evidenced by the appearance of gas bubbles when the bacteria are placed on a drop of hydrogen peroxide. Also, the isolates fermented mantol when planted on it, which is considered one of the diagnostic media for S.
aureus bacteria. And when the isolates were subjected to coagulation examination, it gave a positive result, which indicates that it is S. aureus bacteria.
After characterization of the bacteria the Minimum Inhibitory Concentration (M.I.C.) was measured by using 6 serial concentrations of nanocomposites µg / ml (2,1,500,250,125,62.5) by the broth dilution method, the MIC of free curcumin was 250 µg / ml. One study reported that the MIC of curcumin in its single form ranges from 125-250 µg / ml Another study reported that the MIC of curcumin on staphylococcus aureus was 219,217 µg / ml (Gunes et al. ,2013). The MIC for cur / zno NPs was 1000 µg / ml. Previous studies indicated that the ratio of MIC to cur / zno NPs on staphylococcus bacteria is 1000 µg / ml (Reddy et al., 2007, Perera et al., 2020). While the MIC for cur / se NPs was 125 µg / ml. Boroumand et al.,(2019) indicated that the MIC on staphylococcus aureus was 125 when using Ascorbic acid as a reducing agent for selenium instead of curcumin.
The results in Table (1) indicate that free curcumin gave excellent inhibition at a concentration of 2 mg / ml, which was 13.7 mm. One of the researches gave similar results on S. aureus bacteria, which were 14.6 ± 2, 13.3 ± 1.5 mm (Oves et al., 2020). As for Perera et al. , (2020), their results gave inhibition diameters of 8.1 ± 0.4 and 8.8 ± 0.2 mm when using 5 g / mL of curcumin on the same bacteria, the results of S. aureus were consistent with those reached by others as it is One of the bacteria most affected by curcumin (Teow et al., 2016). As for cur / zno, it gave an inhibition diameter of 8.1 mm. the diameter of the inhibition zone was 15.4 and 17 mm on staphylococcaus aureus when using 5 mg / ml(perera et al.,2020). Selenium gave an excellent inhibition rate of 11.6 mm, and this is the highest inhibition rate among the materials used in our research
Table (1): shows mean ± sd, LSD inhibition diameters for curcumin and cur / zno Nps, cur / se Nps, VA30, CIP10 in staphylococccus aureus.
Bacterial name material
concentration Mean &Std. Deviation
lsd lsd
staphylococccus aureus curcumin
2 mg/ml 13.77±1.17
0.86
Total 13.77±1.17
curcumin/zno NPs 2 mg/ml
8.13±0.97
0.23
4 mg/ml 10.12±1.09
6 mg/ml 12.06±1.05
8 mg/ml 13.87±0.97
Total 11.05±2.37
curcumin/selinium NPs 2 mg/ml
11.60±1.11
0.30
4 mg/ml 14.38±1.32
6 mg/ml 17.35±1.14
8 mg/ml 20.54±1.60
Total 15.97±3.58
VA30 2 mg/ml
17.69±5.83
Total 17.69±5.83
CIP10 2 mg/ml
22.18±6.97
Total 22.18±6.97
Total 2 mg/ml
14.64±6.37 0.59
Fig. (8) Pictures representing the inhibitory diameters of all compounds used in the research on molar Hunton agar. (A) cur / zno Nps ( B) cur / se Nps (c) CIP. + VA. Antibiotic (d) cur on s.aureus.
After comparing the values of the inhibition diameters of the antibiotics vancomycin 30, ciprofloxacin10, it was found that the nanocomposites cur / zno Nps, cur / se Nps were good inhibitors of bacterial growth and showed anti-bacterial activity similar to the activity of the studied antibiotics at certain concentrations. The results of cur / zno Nps were found. It is close to the results of the antibiotic vancomycin. The results for the cur / se Nps nanocomposite are close to those of the antibiotic ciprofloxacin. The percentage of resistant and sensitive isolates can be seen in Table No. (2) and Fig.
No. (9).
Observed N % Q2 p.value
VA sensetive 111 0.74 34.56 0.00
a b c d
resist 39 0.26
CIP
sensetive 114 0.76
131.88 0.00
intermadeat 3 0.02
resist 33 0.22
Table No. (2) represents the rates of sensitivity and resistance of bacteria to antibiotics
Figure (9) represents an illustration of the sensitivity and resistance of bacteria to each antibiotic studied
discussion
It has also been shown that curcumin has an anti-bacterial effect, as it has shown its ability to weaken the bacterial membrane,regardless of its response to the Gram stain, leading to its degradation (Tyagi et al., 2015). We find that the inhibition ratio of cur / zno is higher than curcumin alone. It is worth noting that the preparation of the nanomaterial was using 1 g of curcumin to 1 g of zinc oxide, meaning that the concentration of free curcumin was Weakening the concentration of the cur / zno nanocomposite, this means that there is an increase in the effect of the nanocomposite on bacteria compared to curcumin alone. This increase is the result of the synergistic action between curcumin and zinc oxide.
The study of Perera et al., (2020) indicates that the degree of efficacy of nanomaterials depends on the size. Therefore, nanomaterials give better results than free materials, cur / zno Nps It gave high inhibition results, indicating that there is an additive effect between zinc oxide and curcumin, Therefore, this mixture can be used as an antibacterial and medical drug. Curcumin is hydrophobic and therefore strongly binds to the bacterial membrane and provides a platform to increase the activity and effectiveness of nanocomposites (Snigdha et al., 2017). The main proposed mechanisms for cur / zno Nps include the destruction of bacterial cells through the generation of reactive oxygen species and the release of ions (Zn2). (Sirelkhatim et al. 2015; Augustine et al. 2014). Thus, both curcumin and zinc oxide can act synergistically to lead to an anti-staphylococcus aureus effect (Snigdha et al., 2017). It is possible that zinc oxide nanocomposites have toxic effects at high concentrations, as well as curcumin on the other hand, despite its therapeutic properties, has weak bioavailability (Rasmussen et al. 2010).
Thus, the current study benefited from the best of both substances from Therapeutic effect even at low concentrations, knowing that the use of lower concentrations of these substances reduces or eliminates the toxicity dependent on these concentrations.
Recently, research indicated that selenium nanoparticles give an excellent effect against staphylococcus aureus (Srivastava & Mukhopadhyay, 2015). Nanoparticle selenium prepared by green synthesis and reduced with Ascorbic acid gave inhibitory droplets estimated at 10.75 on staphylococcus aureus, according to Boroumand et al., (2019). It can also be observed in Table No. (1) that there is a direct relationship, i.e. the higher the concentration, the greater the inhibition rate. This was also indicated by Rajeshkumar et al .,(2018).
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