Silvar Nanoparticles Synthesis from Endophytic Curvularia Colbranii from Medicinal Leaf Sample of Acalypha Indica in Srivilliputhur, Tamil Nadu, India

Abstract

Silver nanoparticle synthesis employing endophytic Curvularia colbranii from a leaf sample of Acalypha indica. The color shift from pale yellow to dark brown was used to identify and observe silver nanoparticles. The development of turbity signifies the reduction of silver ions (Ag+) to silver atoms (Ag0), which is followed by the formation of silver nanoparticles (AgNPs). Following the color changes, the solution containing the Curvularia colbranii silver nanoparticles was exposed to UV-visible absorption spectroscopy 24 hours later. The separation of the nanoparticle extract from the pure Curvularia colbranii extract was verified by the appearance of two distinct clean peaks for the Ch-AgNPs nanocomposites at 500 nm and 262 nm. The FTIR Spectra analysis was carried out for the silver nanoparticle sample and has the absorption peak. Morphological characterization at the nanoscale to micrometer scale is accomplished using scanning electron microscopy (SEM). The effective synthesis of silver nanoparticles is ensured by the presence of silver being verified by the silver-specific peaks seen in the EDX spectrum, antibacterial activity of the nanoparticle extract and obtained the zones of inhibition (ZOI) against Escherichia coli, Staphylococcus aureus. The produced silver nanoparticle's antifungal properties were tested against Aspergillus flavus and Aspergillus niger.

Keywords

Introduction

Nanoparticles attracted many scientists working in different disciplines due the opportunity to engineer their properties. The characteristics of nanoparticles are determined by size, shape, composition, crystallinity and morphology. Nanoparticles have distinct properties compared to bulk materials and offer many new developments in the fields of biosensors, biomedicine and nanotechnology. Nanotechnology is used in medicine for diagnosis and as therapeutics for the treatment of diseases and disorders. Nanotechnology is powerful technology with a promise for the design and development of many types of novel products for medical applications on early disease detection, treatment and prevention. It is said that this field is an upcoming area of research in the modern-day material science. Nanorobots are emerging which are helpful in targeting the cancerous cells at the cellular level. Nanotechnology can be used as carriers of drug and can act as nanocarriers. It is known that most of our drugs are incapable of crossing the blood-brain barrier and hence there is always a lesser bioavailability of the drug in the body. Nanoparticles can incorporate drug into them acts as nanocarriers which can cross the blood brain barrier and thus enhancing the bio availability of the drug [1,2].

AgNPs are unique due to their increased stability, biocidal activity, and possible agricultural uses [3,4]. The ability to biosynthesize AgNPs has been shown by a number of fungus, such as Aspergillus niger, Aspergillus ochraceus, and Fusarium oxysporum. This indicates the potential of these biologically produced 5 nanoparticles in a variety of agricultural applications [5]. These fungi are attractive candidates for agricultural and environmental applications because they produce AgNPs with a variety of physical, chemical, and biological characteristics [6]. Specifically, AgNPs showed a variety of inhibitory actions against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, according to Shende et al. Furthermore, AgNPs prevent the growth of fungal toxins made by Alternaria, Aspergillus niger, and Aspergillus flavus [7]. AgNPs' effects on DON production were investigated by Bello et al. [8], who also evaluated how well they worked to inhibit drug resistant strains of Fusarium graminearum. In addition to reducing silver ions to create nanoparticles, biological components such as proteins, polyphenols, and amino acids also stabilize the nanoparticles, avoiding aggregation and boosting their bioavailability and effectiveness in agricultural applications. The biosynthesized silver nanoparticles' tiny size, high surface area, and biocompatibility all contribute to their improved antibacterial and antifungal qualities, which make them a perfect choice for agricultural applications, including the control of fungal diseases like B. cinerea. Traditional medicinal plants like Panax notoginseng have attracted attention in the quest for environmentally friendly and sustainable solutions, both for their pharmacological qualities and for the potential of the endophytic fungi that they are connected with in the production of nanoparticles. Saponins, flavonoids, amino acids, polysaccharides, sterols, volatile oils, polyols, fatty acids, and other active substances are among the bioactive components of Panax notoginseng that contribute to its medicinal value, including its capacity to reduce inflammation, eradicate blood stasis, halt bleeding, and alleviate pain and swelling [9]. Although fungi like Aspergillus species have been shown to synthesize AgNP, nothing is known about the potential of Panax notoginseng endophytic fungi in AgNP biosynthesis [10].

MATERIALS AND METHODS

Sample collection

The medicinal herb was collected from Srivilliputhur (9°30'48.6" N, 77°37'52.46" E), where it originated. Acalypha Indicais the plant sample. After being gathered, samples were processed within 24 hours after being put in individual sterile zip-lock bags. The isolation procedure was carried out using fresh plant leaves and stems to reduce the possibility of contamination.

Acalypha indica

Acalypha Indicais an herbaceous annual that has catkin-like inflorescences with cup-shaped involucres surrounding the minute flowers. It is mainly known for its root being attractive to domestic cats, and for its various medicinal uses. Acalypha indica, known as Indian copperleaf, has traditional uses for respiratory ailments, skin conditions, digestive disorders, and wound healing. The plant has shown potential in research for antibacterial and antifungal activities. Common remedies include using the leaves for coughs, skin diseases like eczema, and to expel intestinal worms. The roots are sometimes used as a purgative and for treating conditions like jaundice and liver infections.

Surface sterilization

After being taken out, the medicinal plant samples' leaves were put in a laminar air flow chamber to be surface sterilized. The samples were rinsed for three minutes with a 5% sodium hypochlorite solution, rinsed for five minutes with distilled water up to two times, and then rinsed for three minutes with 70% ethanol before being rinsed for five minutes with sterile distilled water up to two times. After being surface sterilized, the samples were put on sterile tissue paper to remove any remaining water or moisture. Following a two-minute treatment with 1% mercuric chloride, the samples were rinsed three times with sterile distilled water. Lastly, samples were cleaned one more for two minutes using 79% ethanol and then rinsed again for two minutes each time using sterile distilled water. A laminar air flow chamber was used for each of these procedures.

Sample inoculation

The inoculation medium was prepared using potato dextrose agar. After being prepared, these media were autoclaved at 121 degrees Celsius for 20 minutes to disinfect them. As the medium was being transferred to a sterile Petri plate, antibiotics such as amoxicillin and tetracycline were introduced at a temperature that was comfortable for the palm. After the

Lactophenol cotton blue mounting

A loopful culture was picked up with the help of a sterile inoculation loop and semipermanent slides were prepared using lacto phenol cotton blue. The slides were gently heated in a spirit lamp so as to release the air bubbles, if any present inside cover glass. The excess stain was removed by using tissue paper and the cover glass was sealed with white nail polish.

Morphological identification of fungi

Lacto phenol and Lacto phenol cotton blue stain (Hi Media laboratories private limited) were used as the staining solution. Slides prepared were sealed with DPX mountant. Identification of the fungal species was done using “The Genera of Hyphomycetes from soil” by (Barron et al., 1972); Compendium of soil fungi by [11].

Optimization of silver nanoparticles

To improve the synthesis of silver nanoparticles, fungal biomass concentrations 20 ml were optimized. The mycelial free extract was mixed with doses of silver nitrate (2 mM) in 80 25 ml and incubated for up to 48 hours, the colour changes from transparent to dark brown [12]. 

UV-VIS Spectroscopy

By tracking the surface plasmon resonance (SPR) band, which is normally seen for silver nanoparticles at about 420 nm, UV-Vis spectroscopy is a popular method for verifying the creation of silver nanoparticles. A distinctive absorption peak at 420 nm signifies the development of silver nanoparticles when the reaction mixture is examined using a UV-Vis spectrophotometer following nanoparticle synthesis [13] The method which useful for tracking the reduction process and confirming that there are nanoparticles present in the solution.

Fourier transform infrared (FTIR) spectroscopy

The functional groups in the fungal extract that are in charge of stabilizing and reducing the silver nanoparticles are found using FTIR spectroscopy. FTIR is used to identify the presence of particular functional groups, such as hydroxyl, carbonyl, or amide groups, after the nanoparticles have been synthesized and purified. In order to facilitate the reduction of silver ions into nanoparticles, these functional groups frequently interact with them. Understanding the function of fungal metabolites in the synthesis and stability of nanoparticles is made easier by FTIR analysis [14].

Energy dispersive X-Ray spectroscopy (EDX) and Scanning electron microscopy (SEM)

These are used to examine the size, shape, and surface morphology of silver nanoparticles. Following synthesis, a tiny sample of the suspension of nanoparticles is put on a conductive surface and examined under an electron microscope. High-resolution SEM pictures offer important information about the size and distribution of the nanoparticles. The elemental makeup of the produced nanoparticles is ascertained concurrently using EDX analysis. The effective synthesis of silver nanoparticles is ensured by the presence of silver being verified by the silver-specific peaks seen in the EDX spectrum [15].

ANTIMICROBIAL ACTIVITY

The agar well diffusion method is used to assess the produced silver nanoparticles' antibacterial efficacy. This technique involves inoculating nutrient agar plates with bacterial strains including Staphylococcus aureus and Escherichia coli, the positive control as amoxicillin. Where in antifungal activity Aspergillus niger and Aspergillus flavus, the positive control as fluconazole. Different concentrations of the silver nanoparticle solution are added to wells that have been made in the agar. The zone of inhibition surrounding each well is measured to assess the antibacterial impact after the plates are incubated for 24 hours at 37°C. Greater antibacterial activity is indicated by larger inhibition zones. The potential of silver nanoparticles as antibacterial agents is demonstrated by this assay [16].

ANTIOXIDANT ACTIVITY DPPH(2,2-diphenyl-1-picrylhydrazyl)

A common method for assessing the antioxidant capacity of artificial silver nanoparticles is the DPPH assay. The assay's basic idea is based on the reduction of the DPPH radical, a stable free radical that, when reduced, turns from purple to yellow. A 0.1 mM DPPH solution is combined with various doses of the silver nanoparticle solution to conduct the assay. A UV-Vis spectrophotometer is used to measure the drop in absorbance at 517 nm after the reaction mixture has been incubated in the dark for 30 minutes [16]. 

SCAVENGING ASSAY FOR HYDROGEN PEROXIDE (H2O?)

A popular method for evaluating silver nanoparticles' capacity to neutralize reactive oxygen species (ROS) like hydrogen peroxide is the Hydrogen Peroxide (H2O?) scavenging assay. The ability of nanoparticles to scavenge hydrogen peroxide, a highly reactive chemical that can induce oxidative stress, is a sign of their antioxidant potential. This assay is carried out by mixing different quantities of silver nanoparticle solutions with a set concentration of H2O? solution (typically 10 mM). A UV-Vis spectrophotometer is used to measure the solution's absorbance at 230 nm following 10–30 minutes of room temperature incubation [16]. 

RESULTS

Endophytic microfungal isolation

The endophytic fungi Curvularia Colbranii  were isolated from the medicinal plant which is Acalypha Indica(Fig.1,2), after 7 days of incubation, the endophytic fungi recorded from plant sample in PDA plate. Lacto phenol cotton blue dye and paraffin oil were used to identify the morphology of fungi.

Fig.1. Medicinal plant leaf Acalypha Indicafor endophytic micro-fungi isolation

Fig.2. Inoculation of Acalypha Indicaleaf sample on PDA after surface sterilization

Curvularia Colbranii  

After 7 days at 25 °C, colonies on PDA with a diameter of around 5 cm, Curvularia Colbranii  developed into a blackish brown colony with brown spores. Curvularia Colbranii  was observed under a microscope as aerial mycelium white, olivaceous black, and border fimbriate. Hyphae are smooth, septate, subhyaline, and up to 3 μm wide. Conidiophores are smooth, septate, upright, flexuous, geniculate, and consistently pale brown to brown (Fig.3,4,5).

Fig.3. Pure culture of endophytic micro-fungi from Acalypha Indicaleaf sample isolates

       

 

Fig.4. Structure of Curvularia Colbranii  under 40X of microscope

Fig.5. Pure culture of Curvularia Colbranii  in PDA method

Curvularia Colbranii , an endophytic fungus, has been chosen for the synthesis of bioactive chemicals and secondary metabolites. By inoculating the isolated endophytic fungus in the production medium in the 500 ml Erlenmeyer flask, bioactive substances were produced. Samples were then cultured for up to 20 days at room temperature with a light source. Following incubation, Whatman filter paper 1 was used to separate the broth and mycelia mat. The extracellular extracts were used for further analysis.

Synthesis of silver nanoparticles from endophytic fungi

Optimization of silver nanoparticles

When Curvularia Colbranii  cell-free filtrate was challenged with 1 mM silver nitrate, the formation of silver nanoparticles occurred. The formation of silver nanoparticles was greatly aided by the 1 mM concentration; it turned from yellow to dark brown (Fig.6).

Fig.6. Optimization of silver nanoparticles from water extract of endophytic fungi Curvularia Colbranii

According to reports, the possible enzymatic pathways for silver bio-reduction include enzymes such as hydrogenase, nitrate reductase, and reductase.

Characterization of silver nanoparticles UV-vis spectroscopy

A UV–vis absorption spectrophotometer with a wavelength range of 200–800 nm was used to characterize the colloidal solution of nanoparticles. AgNPs were detected by maximum absorbance at 430 nm. The formation of chitosan-capped AgNPs was confirmed by the detection of two distinct clean peaks for the Ch-AgNPs nanocomposites at 500 nm and 262 nm (Fig.7).

Fig.7. UV-VIS Spectroscopy graphical representation of silver nanoparticles in Curvularia Colbranii

Fig.8.FTIR graphical peak representation of silver nanoparticles from endophytic fungi Curvularia Colbranii

The essential functional group peak intensity range of 439.77 cm-1 (C–Br stretch, alkyl halides, strong), 516.92 cm-1 (C-Br stretching, halo compound, strong), 594.08 cm-1 (C-CI stretching, halo compound, strong), 624.94 cm-1 (C-Cl stretching, halo compound, strong), 702.09 cm-1 (C-Cl stretching, halo compound, strong ), 779.24 cm-1(C-Cl stretching, halo compound, strong), 817.82 cm-1 (C-Cl stretching, halo compound, strong), 918.12 cm-1 (C=C bending, Alkenes, strong), 979.84 cm-1(C=C bending, Alkenes, strong), 1056.99 cm-1 (C-F stretching, Fluro compound, strong), 1141.86 cm-1 (C-F stretching, Fluro compound, strong), 1273.02 cm-1 (C-F stretching, Fluro compound, strong), 1381.03 cm-1 (C-H bending, aldehyde, medium), 1512.19 cm-1 (N-O stretching, Nitro compound, strong), 1643.35 cm-1 (C=O stretching, Amides, strong), 1697.36 cm-1 (C-H bending, aromatic compound, weak), 1797.66 cm-1 (C-H bending, aromatic compound, weak), 1921.1 cm-1 (C=C=C stretching, allene, medium), 1990.54 cm-1 (N=C=S stretching, Isothiocyanate, strong), 2314.58 cm-1 (O=C=O stretching, carbon dioxide, strong), 2939.52 cm-1 (O-H stretching, carboxylic acid, strong), 3356.14 cm-1 (O-H stretching, Alcohols, strong)(Table.1).

Fig.9. EDX graphical peak representation of silver nanoparticles from endophytic fungi Curvularia Colbranii

                                

 

Fig.10. a. SEM marked image range from 45nm-100nm             Fig.10. b. SEM image range of 500nm

                           

Fig.10.c.SEM image range of 1µm                    Fig.10.d. SEM image range of 2µm

Fig.10. SEM images of the silver nanoparticles synthesized by endophytic fungi Curvularia Colbranii

Antimicrobial activity

          

 

Fig.11. Antibacterial activity of silver nanoparticles from Curvularia Colbranii  extract against E.coli

Antifungal activity of silver nanoparticles from Curvularia Colbranii  

     

 

Fig.12. Antifungal activity of silver nanoparticles synthesized from endophytic fungi Curvularia Colbranii

There is no inhibition of Aspergillus flavus at any dose. Fluconazole was used to perform the antifungal activity, and the control showed a zone of inhibition of 27 mm (50 µL), 27 mm (100 µL), 27 mm (150 µL), and 28 mm (200 µL). After subtracting the well's diameter using the agar well diffusion method, the zone was measured and computed

          

 

Silver nanoparticles activity                             Ascorbic acid activity

Fig.13. DPPH scavenging activity of silver nanoparticles synthesized from endophytic fungi Curvularia Colbranii

Scavenging assay for hydrogen peroxide (H2O?)

     

 

Silver nanoparticles activity                               Ascorbic acid activity

Fig.14. H2O2 scavenging activity of silver nanoparticles synthesized from endophytic fungi Curvularia Colbranii

UV spectrophotometry was used to record the standard and test sample absorption in H2O2 scavenging activity. In 45% at 10% test sample concentration, a high range of absorption was achieved. Comparing the H2O2 solution to other silver nanoparticle samples and standards, the lowest range of scavenging activity was found to be 30% at 4% concentration and 20% at 5% concentration were tabulated (Table.4)

DISCUSSION

Endophytes are organisms that live in healthy, living plant tissues without harming their host plants. Plants are protected from illness by endophytic fungi through colonization sites, antibiotic production, nutritional competition with pathogens, and induction of resistance mechanisms. The different fungal species, endophytic fungi are an important and extremely diverse group of endophytes and endophytic fungi are a good source of novel bioactive substances. Singh et al., [17] reported that the host plant Raphanus sativus L. were isolated Alternaria sp. Nees and synthesized silver nanoparticles at the absorbance peak of 426 nm and in range of 4-30 nm reported that, it encounters the antibacterial (Bacillus subtilis, Escherichia coli, Serratia marcescens) and in terms of biomedical applications, metallic nanoparticles such as those made of gold, silver, iron, zinc, and metal oxide have demonstrated significant promise Bhattacharya and Mukherjee, and Hirst et al., [18,19]  reported that numerous studies have shown that metal nanoparticles have bioactive qualities; for example, gold and cerium oxide nanoparticles can be used to treat tumors and reduce inflammation, respectively [20]. In presence studies silver nanoparticles isolated from an endophytic fungi Curvularia Colbranii  in the absorbance peak of 262-500 nm. Devi and Joshi, [ 21] reported that, the bio reduction of silver ions to silver nanoparticles indicated by a change in color of the mycelium free filtrate treated with a 1 mM silver nitrate solution over time. Aliquots (1 mL) were sampled at various time points to track the silver nanoparticles that developed in the mycelium-free fungal filtrate and UV-visible spectrophotometer was used to quantify absorption at a resolution of 1 nm in the 200–800 nm range. In the presence studies characterization of silver nanoparticles, the UV–vis absorption spectrophotometers were carried out, the colloidal solution of nanoparticles was characterized using a wavelength range of 200– 800 nm. The highest absorbance at 430 nm was used to identify AgNPs. The identification of two separate clean peaks for the Ch-AgNPs Nano composites at 500 nm and 262 nm verified the synthesis of chitosan-capped AgNPs.

Seetharaman et al., [22] reported that, the functional groups that might serve as capping and reducing agents during the synthesis of PoAgNPs were identified using FT-IR analysis. The intense peaks in the FT-IR spectra are located at 2923 cm−1, 2854 cm−1, 1648 cm−1, 1384 cm−1, and 1040 cm−1. A peak at 3424 cm−1 represents the protein’s N–H amide, whereas a peak at 2923 cm−1 represents the C–H stretch of the protein’s methylene groups. The C–N stretching vibrations of the aromatic amines were responsible for the peak at 1384 cm−1, whereas the –CO of the proteins' amide I band may have contributed to the peak at 1648 cm−1.Similarly in the presence studies of FTIR analysis of water extract of silver nanoparticles recorded the functional group peak intensity range were 1643.35 cm−1 as amides (C=O stretching), 2939.52 cm−1 as alkanes, methylene groups (C-H stretching), 1381.03 cm−1 as. Sunkar and Valli Nachiyar, [23] reported that quartz cell at a resolution of 1 nm from 250 to 800 nm, the color change from pale white to brown was visually observed after the AgNPs formed, and this was further verified by the sharp peaks provided by the AgNPs in the visible region from the UV-vis spectrum of the reacting solution. The research on the size, shape, and distribution of nanoparticles was carried out using a Hitachi S-4500 Scanning Electron Microscope (SEM) and Transmission Electron Microscopy (TEM) examination utilizing a TEM, JEM-1200EX, JEOL Ltd., Japan. Using an FTIR Nicolet Avatar 660 (Nicolet, USA), the likely biomolecules involved in the production and stability of nanoparticles were captured by FTIR spectra. In presence studies the SEM analysis of AgNPs showed the surface topography of the generated particles, found that the particles were spherical, uniformly produced, uniformly distributed, and least-aggregated with diameter of 500 nm- 5μm and the size range lesser than 100 nm. EDX analysis was used to assess the components of the nanoparticles both quantitatively and qualitatively. The prominent peak observed at 3 keV, which is a characteristic absorption for AgNPs due to surface plasmon resonance, suggested the presence of silver. The elemental analysis revealed that Ag was the predominant element in the AgNPs, with carbon (C) accounting for 8.38 weight percent, silver (Ag) dominating at 35.59 weight percent, and chlorine (Cl) accounting for 7.29 weight percent.

Bruna  et al., [24] reported that, different levels of antibacterial activity were demonstrated by the Tp-AgNPs as their concentration increased. According to our findings, AgNPs have strong antibacterial properties. Tp-AgNPs, at a minimum inhibitory concentration of 16.12 μg.mL−1 for Gram-positive pathogens and 13.98 μg.mL−1 for Gram-negative pathogens, effectively suppressed their development. Additionally, Tp-AgNPs exhibited a larger zone of inhibition relative to positive antibiotic control vancomycin against S. aureus (9 mm), P. aeruginosa (13 mm), and E. coli (11 mm). TP-AgNPs did not exhibit the same level of antibiotic-like antibacterial activity against B. cereus (11 mm) and S. enterica (11 mm). In presence studies, for the antibacterial activity of AgNPs, the zone of inhibition against Escherichia coli was measured using silver nanoparticles derived from Curvularia Colbranii . There was no inhibition in 100 μL, 150 μL, or 200 μL, although the largest zone of inhibition was 16 mm (50 μL). Staphylococcus is not suppressed at any concentration. The antibacterial activity was tested using amoxicillin since the control exhibited a 26 mm (50 μL) zone of inhibition. After subtracting the well's diameter, the zone was measured and calculated using the agar well diffusion method.

Balakumaran et al., [25] reported that, for the first time, plant fungal infections were tested for the antifungal properties of mycosynthesized silver nanoparticles. At a dosage of 1 mg/mL, silver nanoparticles shown strong antifungal action against Colletotrichum sp. (12.63 mm), R. solani (12.03 mm), and C. lunata (11.23 mm). The Fusarium sp. (9.37 mm) showed the least amount of activity. With an average inhibitory zone of 4.1–4.6 mm against all investigated plant diseases, silver nitrate, on the other hand, demonstrated the lowest antifungal activity. According to Gajbhiye et al. (2009), fluconazole's antifungal activity was found to be higher when silver nanoparticles were present than when fluconazole was used alone. Accordingly, silver nanoparticles by themselves or in combination with other substances may be employed as potent antifungal agents to combat dangerous phytopathogenic fungi [26]. In presence studies, the antifungal activity for my work done in the synthesized AgNPs from the aqueous extract of Curvularia Colbranii , the largest zones of inhibition against Aspergillus niger were 16 mm (50 μL), 16 mm (100 μL), 12 mm (150 μL), and 15 mm (200 μL). Aspergillus flavus is not inhibited by any dosage. The control exhibited a zone of inhibition of 27 mm (50 μL), 27 mm (100 μL), 27 mm (150 μL), and 28 mm (200 μL) when fluconazole was used to perform the antifungal activity. The zone was measured and calculated after the well's diameter was subtracted using the agar well diffusion method.

Gupta et al., [27] reported that, the artificially produced DPPH free radicals were used to test POAgNPs' antioxidant capacity. With an EC50 value of 9.034 ± 0.449 μg/mL, the results recorded that POAgNPs substantially reduced the free radicals of DPPH. As the quantity of POAgNPs increased, the spectrophotometrically observation at 517 nm revealed a decrease in absorbance, which is explained by the scavenging of free radicals. The finding implies that even at lower concentrations, POAgNPs may have had some effect in scavenging free radicals. With an EC50 value of 10.5 ± 0.265 μg/mL, POAgNPs were shown to have a scavenging activity that was extremely similar to that of the well-known antioxidant agent ascorbic acid. In presence studies, the antioxidant scavenging capacity of silver nanoparticles made from Curvularia Colbranii  extract was evaluated using DPPH. The absorption of the test and reference samples in the DPPH scavenging activity was measured using a colorimeter. A wide range of absorption was attained at 40% at 5% test sample concentration. The DPPH solution's moderate range of 58 scavenging activity was 7.69% at 3% concentration and 8.33% at 1% concentration when compared to other silver nanoparticle samples and standards. Gupta et al., [27] reported that, the assay used in the investigation demonstrated POAgNPs' strong scavenging capabilities. Using FeCl3-EDTA-H2O2, hydroxyl radicals were chemically generated, and the scavenging of radicals by POAgNPs was evaluated spectrophotometrically. The scavenging ability of POAgNPs against hydroxyl radicals was depending on concentration. Free radical scavenging potential as a percentage was used to compute the activity, and the EC50 value fell within the significant range of 34.094 ± 1.944 μg/mL. 19.64 ± 0.988 μg/mL was the measured EC50 value for the positive control. In presence studies, Silver nanoparticles derived from Curvularia Colbranii  extract were ability to scavenge antioxidants using H2O2. UV spectrophotometry was used to record the standard and test sample absorption in H2O2 scavenging activity, 45% at 1% test sample concentration, a high range of absorption was achieved. Comparing the H2O2 solution to other silver nanoparticle samples and standards, the lowest range of scavenging activity was found to be 30% at 4% concentration and 29% at 5% concentration.

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