Investigating antibacterial activity of biosynthesized silver oxide nanoparticles using Phragmanthera Macrosolen L. leaf extract

Abstract

The crude leaf extract of Phragmanthera macrosolen L. has been utilized for the first time as an effective reducing, capping and stabilizing agent to synthesize silver oxide nanoparticles (Ag₂O NPs) through a green approach. The prepared Ag₂O NPs were analyzed by scanning electron-microscopy (SEM), High Resolution Transmission electron microscope (HR-TEM), X-ray diffractions (XRD), Fourier transforms infrared (FTIR) spectroscopy, energy dispersive spectroscopy (EDS), and Ultra-violet visible spectrometry (UV-Vis). The biosynthesized Ag₂O NPs applied on gram-positive (S. aureus) and gram-negative (E. coli) bacterial types. FTIR spectral peaks indicate that the phytochemicals in the extract are responsible for the formation of Ag₂O nanoparticles. The XRD result shown, poly-crystalline nanoparticles and the average crystalline size calculated was 45.8 nm. UV-Vis analysis shows absorbance existed at 590.5 nm, and the energy band gap calculated through the Tauc relation was 2.1 eV. The SEM images gave a globular and some rod like morphology with diameter of particle obtained between 20.11 and 46.50 nm with some hollow cubic microstructure of Ag₂O NPs which makes it suitable for antimicrobial application. The EDS confirms the elemental composition and existence of Ag and O₂. The HR-TEM images, specific area electron diffraction (SAED), and XRD patterns confirmed the morphology of Ag₂O NPs mean 45.84 nm and polycrystalline nature of the nanoparticles. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 15 and 30 µl respectively for the samples tested. In this study, it was observed that Ag₂O NPs highly sensitive to E. coli than s. aureus. The present study revealed that the possible use of green synthesized Ag₂O NPs as potential antibacterial agent.

Introduction

Nanotechnology is one that is categorized under upcoming fields of study in the recent research area of condensed matter sciences. It shows absolutely novel characteristics, like sizes, structures and morphologies of the particles¹. Metallic oxide nanomaterials have gained specific attentions due to their unique physicochemical properties with great potentials in environment remediation^2,3. Their use ranges from the removal of heavyweight metals, toxic gas-sensing, fabrics, bio-medical application, and photo-catalytic degradations of organic contaminant^4,5. Ag₂O NPs were newly initiated to have special characteristics for application in solar cells, sensor, storage device, medical applications, as well as photo-catalysis^6,7. Ag₂O NPs have been discovered in several investigational studies concentrating on their preparation, analysis techniques, and photo-catalytic performances. Their virtuous optical characteristics as well as narrow energy band gap (1.2-2.4 eV), which permits them to absorb the photovoltaic spectrums. Ag₂O NPs photo-catalyst was also published to experience self-stabilizing procedure during photo-catalytic degradations of organic compound, ensuing in a great photo-catalytic performance⁸.

Synthesis methods of Ag₂O NPs, is one of the further encouraging area of nano technology⁹. Ag₂O NPs have been prepared by through several physical and chemical methods such as chemical reductions^10,11, laser ablation^12,13, microwave irradiation¹⁴ and solvo-thermal. Though chemical as well as physical approaches may efficiently produce pure nanomaterials, but these techniques are quite costly and potentially dangerous to the atmosphere. Chemical root for nanoparticle preparation uses poison chemicals, whereas physical root releases poisonous byproducts, harmful to the environments. In the combustions root, citric-acid, urea, and glycine- are utilized as fuels through nanoparticles synthesis. In current times, the combustions root categorized as solutions combustions synthesis, Conventionally (SHS): condensed phases combustions, and sol gel based combustions^15,16. The foremost challenge of this technique is uncontrollability on phases as well as surface morphologies, but in current few years, certain advancement in phases and surface morphologies control has been perceived¹⁷. Therefore there is appropriate need to use a green synthesis method which is ecologically gentle, eco-friendly methods for nanoparticles fabrications extracting plant’s parts. The basic principles of green root for the fabrications of metallic oxide nanoparticles are reductions of metal complexes in the weak solutions to get metallic colloids dispersion. Hydrazine’s hydrate and sodium borohydride are commonly acknowledged reducing agents, however they are favored due to their toxic soak them through the prepared nanoparticles¹⁸.

Rare reported regarding the green synthesis of Ag₂O NPs through plant leaf extracts. To mention some of them are Lippia-citriodora¹⁹, Ficus-benghalensis²⁰, Telfairia occidentalis²¹, Azadirachta-indica²², Artemisia Herba-Alba²³, Centella Asiatica²⁴, Mentha-pulegium and Ficus carica²⁵, Zephyranthes Rosea²⁶, Rhamnus virgata²⁷, Curcuma zanthorrhiza²⁸, Panicum miliaceum²⁹, Cyathea nilgiriensis Holttum³⁰, and Bacillus paramycoides³¹. The researchers have been paying attention to green-chemistry to increase and protection of environment. The applications of silver oxide nanoparticles are wide, starting from photo-catalysis³², medicinal value, and drug delivery³³. Pharmaceutically, Ag₂O nanoparticles have been used for treating diseases like malaria, cancer treatment, tuberculosis and diabetes^30,34–36. Additionally, silver-based nano particles are extensively applied for their exclusive characteristics in chemical identifying, bio-sensing, photonic activities and opto-electronics³⁷. They have an excessive potential in medical applications like anti-microbial activities³⁸.

Green chemistry grounded approaches have been stated in the preparation of nano particles in current years, amongst the numerous chemical and physical methods of nano particle production³⁹. Plant body extracts have been applied to serve as reducing, capping, and stabilizing agents in the preparation of nanoparticles. These extracts do not contain any laboratory-prepared chemical mixtures on their surfaces, making them non-toxic to humans and the environment^40,41. Ag₂O is an excellent semi-conducting material, widely used in sensors, solar cells, fuel cells, and corrosion catalysis⁴². The exceeding reports provide information roughly the significances of the Ag₂O NPs as well as its performance researches on biosynthesis for bio-medical and agricultural solicitations. However green synthesis of Ag₂O NPs from traditional medicinal plants were rarely reported. In our knowledge there is no report on synthesis of Ag₂O NPs using the novel Phragmanthera macrosolen leaf extract; without using any other chemical reagents as reducing agent. Current work displays green preparation of Ag₂O NPs using Phragmanthera macrosolen leaf extracts, characterizations and antibacterial activities. Mistletoe, a common name used generally for woody shoot parasites, belongs to the order Santalales, it includes Loranthaceae, Santalaceae, and Misodendraceae families⁴³. The novel plant mistletoe of Albizia Gummifera, which is known as Phragmanthera macrosolen from Loranthaceae family selected for this work due to its availability and traditionally usable for different medical values in the research location conducted. In this study, Ag₂O NPs were synthesized by novel approach using Phragmanthera macrosolen and its antibacterial activity against gram negative bacteria (E. coli) and gram-positive bacteria (S. aureus) were investigated.

Materials and methods

Materials

Silver-nitrate (AgNO₃)-99% was bought from Sigma Aldrich company, through Indian Institute of Technology, Hyderabad. All chemicals as well as substances utilized were of extremely pure analytical grades. A leaf of mistletoes of Albizia Gummifera (Phragmanthera macrosolen L.) was gathered from Dambi Dollo University, and Dambi Dollo Town, Oromia, Ethiopia. The plant we have used in this report was cultivated in Dambi Dollo University research center, Oromia, Ethiopia. This study complies with relevant international, national, institutional and legislative guidelines.

Phyto-chemical screening

Phyto-chemical screening with chemicals to test the content of bioactive compounds in 80% ethanol extracts of Phragmanthera macrosolen L. leaf. The Phyto-chemical test contains the bioactive compounds by reagents tests of 80% ethanol rigorous extracts from Phragmanthera macrosolen L. leaf plants. The bioactive compounds were tested following previously reported methods with changing the leaves with Phragmanthera macrosolen L^44–48.

Flavonoid tests

Leaf extracts of Phragmanthera macrosolen L. with a concentration of 1000 mg/L were transferred into test tubes in a volume of 0.5mL. Subsequently, they were liquefied in 2 mL of hot 50% methanol solvent. Following this, the mixture was combined with magnesium metal and a few drops of concentrated hydrochloric acid. The automatically observed orange-colored solution indicated the presence of flavonoids in the Phragmanthera macrosolen L. leaf.

Alkaloid tests

Leaf extracts of Phragmanthera macrosolen L. with a concentration of 1000 mg/L were dispensed into test tubes at a volume of 0.5 mL. Subsequently, 0.5 mL of 2% hydrochloric acid was added to the solutions, and the resulting mixtures were divided among three test tubes. Test tube 1 received an additional 0.5 mL of dilute acid solution for comparison, test tube 2 was supplemented with 2–3 drops of Dragendorff reagent, and test tube 3 was treated with 2–3 drops of Mayer reagent. A positive indication for alkaloids was confirmed if an orange precipitate formed in test tube 2 and a yellowish-white deposit formed in test tube 3.

Sesquiterpenoid tests

Leaf extracts of Phragmanthera macrosolen L. with a concentration of 1000 mg/L were introduced into the test tube at a volume of 0.5 mL. Subsequently, diethyl ether was added, and the mixture was shaken. The ether layer was then separated and evaporated using a vaporizer cup positioned above a water bath. The resulting filtrate was combined with 10% vanillin in H₂SO₄. The absence of a color change in the solution indicated the non-existence of Sesquiterpenoid in the Phragmanthera macrosolen L. leaf.

Carbohydrates tests

In the Fehling’s test, 5 mL of Fehling’s solution was introduced to 0.5 mg of Phragmanthera macrosolen L. extract, followed by boiling in a water bath. The emergence of a yellow or red precipitate signals the existence of reducing power. For the Benedict’s test, 5 mL of Benedict’s solution was applied to 0.5 mg of the extract and subjected to boiling in a water bath. The formation of a yellow precipitate indicates the presence of reducing sugars.

Phlobatannins tests

The lack of a red precipitate formation was considered indicative of the absence of Phlobatannins when an aqueous crude methanol Phragmanthera macrosolen L. leaf extract of the sample was boiled with 5mL of 1% aqueous hydrochloric acid.

Proteins and amino acids tests

For the Biuret test, 0.5 mg of the Phragmanthera macrosolen L. leaf extract was mixed with an equal volume of 40% NaOH solution, and two drops of 1% copper sulfate solution were added. The development of a violet color signifies the presence of protein. In the Ninhydrin test, approximately 0.5 mg of the extract was taken, and 2 drops of freshly prepared 0.2% ninhydrin reagent were added before heating. The emergence of a pink color indicates the presence of proteins, peptides, or amino acids in Phragmanthera macrosolen L. leaf.

Preparation of Phragmanthera macrosolen L. Leaf

Phragmanthera macrosolen L. leaf was collected and cleaned systematically under running distilled water, then wetted carefully with ultra-purified water as well as dried at normal temperatures for seven days. Then the dried leaf was grounded by using a grinder to crush the leaf skins to a fine powder as shown in Fig. 1. The solutions of both leafs broth were prepared separately by taking 3 g leaf powder added to 100 ml deionized water and then boiling for 1 h at temperature of 70°C.

Figure 1.

Green synthesis of Ag₂O NPs using Phragmanthera macrosolen L. leaf extract.

Then the leaf extract were cooled at room temperature for 24 h. Then the leaf extracts were filtered through What-man filter papers, and kept in a fridge-freezer for supplementary uses.

Bio-synthesis of Ag₂O NPs using Phragmanthera macrosolen L. leaf extracts

In the synthesis process, 1.67 g of AgNO₃ was dissolved in 80 ml of distilled water, rather than ethanol, to ensure optimal solubility and appropriate reaction conditions. Once the silver nitrate was completely dissolved, we proceeded by adding 20 ml of plant extract to the solution. The mixture was stirred continuously for 45 min at room temperature to guarantee uniform mixing and facilitate the interaction between the silver nitrate and the bioactive compounds in the extract as shown in Fig. 1. Interestingly, we observed that no additional base, such as NaOH or KOH, was required to initiate precipitation, as it occurred naturally during the reaction process. The pH of the solution was measured to be approximately 3.8, a slightly acidic environment that contributed to the successful formation of Ag₂O NPs. After precipitation formed the samples were washed using ethanol and distilled water several times. Then filtration followed by drying using oven at 90 °C for 4 h. This method, which utilizes the plant extract’s reducing and stabilizing properties, allows for the green synthesis of nanoparticles without the need for harsh chemical agents. Finally, the samples were kept for supplementary characterization and applications.

Characterization techniques

The phytochemical analysis of the sample was analyzed using FTIR spectroscopy. Spectrum were verified to distinguish functional group elaborated in biosynthesized nanoparticle preparation and were attained through a Perkin Elmer FTIR spectro-photometer (Norwalk, CT, USA) at a resolution of 5/cm in a diffuse transmittance manner using potassium bromide (KBr) pellet. FIB-SEM and EDS was utilized to study the superficial morphology, size, and crystalline structure of the silver oxide nanoparticles. HR-TEM and SAED pattern (HR-TEM model, JEOL-2010, Japan) were utilized to check the surface morphology, sizes, and crystalline structures of the Ag₂O NPs. An optical property of material was analyzed using UV-Vis spectroscopy. X-ray diffractions of the nanoparticles were gained using a Rigaku X-ray diffraction meter (XRD) to investigate the crystalline phases of biosynthesized Ag₂O NPs.

Antibacterial assay

Agar well diffusion method

The antibacterial activity of Ag₂O NPs and Phragmanthera macrosolen L. leaf extract was done through an agar well diffusions technique⁴⁹. The instruments that we use for disc diffusion and broth dilution are: Laminar airflow cabinet, Incubator, UV-Vis spectrophotometer, freezer, autoclave and hot air oven. In addition, we use many disposable items like 15 ml tubes, Petri dishes, spreaders, microfuge tubes and tips. The Mueller-Hinton agar (MHA) mediums were equipped for anti-microbial tests since it is the finest mediums for emerging the furthermost pathogenic microorganisms. Two types of microbial bacterial strains: Gram-positive (S. aureus) and Gram-negative (E. coli) were used to investigate the anti-bacterial tests. To stimulates the microorganisms, they were grown-up in a richest medium cultures like as tryptic-soy-agar and nurtured at room temperature over-night for a nice sensitivity test. The microorganism’s suspensions were moistened on the surface of the MHA underneath sterilized situations through the mopping approaches. Afterward the microorganisms desiccated up, made wells in the MHA with diameter of 6 mm. The well was penetrating with the bottom of sterilized indigo micro-pipette tips. The antimicrobial agents (Ag₂O NPs) are introduced into the wells using a micro-pipette. All the Bacterial straining were nurturing in dishes for 24 h. (it should not more than 24 h) at room temperature over-night. Lastly, the positively growing inhibition zones diameters around each well were recorded in millimeter.

Minimum inhibitory concentration

The testing substances demonstrated solubility in a 10% Dimethylsulfoxide (DMSO) solution. Initial test concentrations were arranged in 96-well plates, with each well receiving 5 µl of a sample containing 10⁸ CFU/ml of bacteria. These plates were incubated at 37℃ for 24 h to observe bacterial growth. The culture potency of each well was recorded at 700 nm, referencing untreated controls. The MIC, representing the lowest nanoparticle concentrations, was identified, showcasing observable destruction of the test bacterial cultures^18,50.

Results and discussion

Phytochemical screening

Phytochemical screening is a qualitative test designed to assess the presence of active compounds in secondary metabolites within plant extracts, as outlined by⁵¹. The analysis involved obtaining samples of 80% ethanol extracts from Phragmanthera macrosolen L. leaf, each placed in a test tube. Subsequently, specific reagents were added corresponding to the identified compound groups, including flavonoids, alkaloids, Sesquiterpenoid Tests, tannins, Carbohydrates Tests, Phlobatannins, Proteins and amino acids. The qualitative analysis of the active compounds in the 80% ethanol extracts of Phragmanthera macrosolen L. plants is detailed in the accompanying Table 1 and FTIR anlysis displayed phytochemical test results through ethanol extract of Phragmanthera macrosolen L. leafs.

Table 1.

Displayed phytochemical test results through ethanol extract of Phragmanthera macrosolen L. leafs.

Compound Groups	Reagent	Color observed	Description
Flavonoids	Mg metal	Precipitate orange	y*
Alkaloids	Dragendorff, Mayer	Orange	y
Sesquiterpenoid	Diethyl ether	No	n
Carbohydrates	Fehling’s	Yellow	y
Phlobatannins	Hydrochloric acid	No	n
Proteins, peptides, and amino acids	Copper sulfate	Pink	y*

Descriptions:.

Symbol(y*): Contains more compounds (solid color)

Symbol(y): contained less compound

Symbol (n): no contained compound (no color formed)

Structural study

The confirmation of crystal structure of the sample was studied utilizing Powder and bulk X-ray diffractometer (Rigaku Ultimate 1 V, Japan) with CuKα radiation sources (λ = 0.15417 nm) in 2θ ranges from 20 to 80°. The peaks intensities were ascertained via the locations of atoms within the lattice plane. The Scherer formula was applied to calculate crystalline size through use of XRD patterns that display line expansion^52,53. In certain, the fine structures of extended x-ray absorptions might be used to detect small particles. Ag₂O NPs made through green approach were found to have XRD in the between 20° and 80° ranges. The XRD graph for Ag₂O NPs as synthesized Fig. 2 displayed intense peaks at 2θ = 26.447, 32.55, 37.764, 46.704, 54.48, 64.92 and 68.20° which can be indexed to the orientation planes (110), (111), (200), (211), (220), (311) and (222) respectively and agree with JCPDS no. 1393^18,54. The result observed from SAED also confirmed as polycrystalline Ag₂O NPs formation⁵⁵. The other observed peaks shown the presence of organic compounds from Phragmanthera macrosolen L. leaf extract which acted for the reductions of silver ion as well as stabilization of resultant nanoparticles^31,56.

Figure 2.

XRD pattern of Ag₂O NPs synthesized through Phragmanthera macrosolen L. leaf extract.

The crystalline size of biosynthesized Ag₂O NPs were calculated using Debye-Scherer formula (Eq. 1) and XRD data plotted^23,57.

The crystallite parameters calculated from XRD and Scherer formula⁵⁸ displayed in Table 2.

1

Table 2.

Crystalline parameters calculated from XRD pattern of green synthesized Ag₂O NPs.

2Thetta (degree)	FWHM (rad)	Crystalline Size(nm)	Dislocation density ( δ)	Micro-strain ()	Lattice parameters (Ǻ)
26.44	0.08873	85.18043	0.000138	0.000377	4.75
32.55	0.15302	48.70405	0.000422	0.000641	4.76
37.76	0.08786	83.61145	0.000143	0.000363	4.76
46.7	0.19124	37.27232	0.00072	0.000766	4.75
54.48	0.39354	17.54022	0.00325	0.001527	4.75
64.92	0.29555	22.16491	0.002035	0.001088	4.76
68.20	0.24412	26.33501	0.001442	0.000882	4.76

Where K is Scherer constant (typically around 0.9, but can vary depending on factors such as crystallite shape),

λ is the wavelength with CuKα radiation sources (λ = 0.15417 nm),

θ is the Bragg diffractions angles (peak positions) recorded from XRD peaks, and.

β is the full width at half maximum (FWHM) of the diffraction peaks recorded from XRD patterns.

Moreover, the structural parameters viz.; dislocation density (δ), and micro-strain(ε), lattice parameters were also computed from the XRD data using the formulae below. The dislocation density (δ) was computed using the following formula⁵⁹ and summarized in Table 1:

2

The micro-strain (ε) was computed using the following formula:

3

The lattice parameters/constant (a) and (c) were computed using the following formula:

4

In synthesized materials, the dislocation densities are in direct proportion to the micro-strains and inversely proportion to the crystalline size. The widening of diffraction peak occurs while atoms in crystals unit cell is emigrant from their own sites because of smaller crystalline size as well as the existence of lattice defect that might be measure dislocation densities⁶⁰. The dislocations densities indicate the creations of imperfections and vacancy in the crystals. The smaller dislocation densities of nanoparticles shows the well compacted form of the materials⁶¹. The micro strains and crystalline size are extremely interrelated⁶².

FTIR study

The phytochemical properties and functional groups of the synthesized Phragmanthera macrosolen L. and Ag₂O NPs are analyzed by FTIR spectrometer in the wave number ranges of 400–4000 cm⁻¹, as shown in Figs. 3 and 4 displayed the FTIR spectrums of Ag₂O NPs displayed a shift in bands associated to Phragmanthera macrosolen L. leaf extract and the possible mechanism of reduction of silver ion to form Ag₂O NPs. The phytochemical groups’ alteration caused by addition of extract to AgNO₃ to prepare Ag₂O NPs. Phragmanthera macrosolen L. leaf extract displays several peaks of foremost phytochemical observed at 3351.58, 2861.19, 1475.37, 1147.31, 1027.61, 850 and 538 cm⁻¹ which are categorized in Phenol rings groups, methyl group, C-C = C groups C-O, C-N stretch and -C-H bend correspondingly as shown in Table 3. These bioactive compounds might be involved in reducing the Ag⁺ to Ag⁰. The wide peak observed at 3351.58 cm⁻¹ shows the high amount of existence of phenol rings which acted as reducing Ag⁺ to Ag⁰ in preparation of Ag₂O NPs. This phenol rings which contains flavonoids, alkaloids, tannins and essential oils⁶³ which can donate electron to the Ag⁺ and reduces it to Ag⁰; while Ag₂O NPs shows a shifts in the frequencies at 3467.91, 1610.44, 1364, 1021.44, 714, 595 and 500 cm⁻¹. All sharp and widening peaks agree to the enlarging vibrations of the hydroxyl groups (H-bonded-O-H-stretch), O-H meandering vibrations of an adsorbed water molecules on the surfaces of Ag₂O NPs, that might be needed for anti-microbial tests, and feeble extending vibrations, C = C correspondingly. Additionally, Ag₂O NPs shown Ag-O bending modes of vibrations at (500) cm⁻¹, approving metal-oxygen attachment formations⁶⁴.

Figure 3.

FTIR spectra of Phragmanthera macrosolen L. leaf powder and Ag₂O NPs powder synthesized by Phragmanthera macrosolen L. leaf.

Figure 4.

The possible mechanism of reduction of silver ions to form Ag₂O NPs by bioactive compounds.

Table 3.

The phytochemical analysis Phragmanthera Macrosolen L. leaf and Ag₂O NPs from FTIR analysis.

FTIR study of Phragmanthera macrosolen L.						Ref.
S. No.	Frequency (cm -1 )			Bonds	Functional-groups	63–66
1	3351.58			O-H stretching	Phenols
2	2861.19			C-H bond	Alkane
3	1475.34			C-C = C Asymmetric Stretch	Aromatic Rings
4	1147.31			C–O	stretching assigned to the flavonoid groups
5	1027.61			C-N stretch	Aliphatic amines
6	850			= C-H bend
7	538			C–Cl	stretching of alkyl halides
FTIR of Ag 2 O NPs
1		3467.91	O-H		Stretching of the alcohol, phenol group
2		1610.44	N-H		bending group of 10 amines)
3		1364	C–N		The stretching vibration of amide I bands proteins
4		1021.44	C-N stretch		Aliphatic amines
5		714	-C = C–H: C–H		bending of alkynes
6		595	-C = C–H: C–H
7		840 − 500	Ag-O

UV-Visible absorption spectroscopy

The optical property of Ag₂O NPs was determined by UV-Vis spectrophotometer (PerkinElmer, Lambda 365, and Thailand). The reactions displayed initially, a whitish green color was gained and after two minutes, automatically the color changed to brownish black to confirm the reduction of silver ion. Uv-vis spectrum showed as Fig. 5 an absorption peak at 430 nm, confirming the synthesis of Ag₂O NPs. The wide higher absorption observed in the visible range of around 590.5 nm, which should be significantly activated in the visible light spectrum^56,67. To develop the programs in determination of the energy band gap by through the Tauc relation pilot used the data as an input. In this study we utilized wave length (λ) and absorption (A) which are attained from UV-Vis analysis, types of electron transitions (n), and (t) for the thickness of the sample. The Tauc relation pilot is initially presented by Tauch in 19s when he determined the band gap of amorphous Ge by through the data of absorptions spectra. This technique is grounded on assumptions that the energy is depending on absorption coefficients α⁶⁸. It is then more modified by Davis as well as Mott for semi-conductor materials. They originate that optical absorption is based on the variance between photonic energy and energy band gap. The Tauc relation for semi-conductor material is expressed by Eq. (4)⁶⁹.

5

Figure 5.

(a) Uv-Vis spectrum and (b). Tauc relation energy band gap of Ag₂O NPs by P. macrosolen L. leaf extract.

Where h is Plank’s constant, v is photonic frequencies, Eg is energy band gap, C is proportionality constant, n is a factor which depending on electrons transition properties, and α is absorption coefficients⁷⁰. The values of γ are ½ and 2 for direct as well as indirect energy band gap transitions, separately. The absorbance coefficient is calculated as Eq. (6)⁷¹

6

Where A is absorptions and l is the thickness of the material in centimeters. The energy band gap by using Tauc relation is worked by plotting the values of (α hv)^γ verses hυ, as shown in Fig. 4 then by taking the extrapolations in the linear area crossways the energies axis in the consistent graphs. The inter-section with energy axis is the estimate of the respective energy band gap⁷².

The energy gap calculated is 2.1 eV it confirms that the biosynthesized energy gap with direct transition. The obtained band gap is suitable for antimicrobial activity. Tauc pilot energy band gap of Ag₂O NPs was depicted in Fig. 5 (a) and (b) shown absorbance spectrums and Tauc pilot energy gap calculation. The calculated direct band gap (Eg) of Ag₂O NPs (2.1 eV) was found to be modified than the band gap value of bulk Ag₂O NPs. This modified in the band gap is attributed to the influence of native defects, which generate localized electronic states within the energy gap. Additionally, the band gap value significantly influences the photo-catalytic performance^72–75. Consequently, this enhancement in absorption capabilities contributes to the improved photo-catalytic activity of Ag₂O NPs across these spectral regions.

The band gap of a material plays a crucial role in determining its light absorption characteristics and influences the wavelength at which antibacterial agents operate most effectively. In comparison, a wider band gap agent like bulk proves beneficial primarily at shorter wavelengths when compared to nanosized nanoparticles. Exciton binding energy, indicative of stability against thermal dissociation of exciton, becomes significant. A narrower band gap results in more free electrons and holes with sufficient lifetime, fostering the photo-generation of reactive oxygen species (ROS) on the nanoparticle’s surface from adsorbed oxygen. Experimental findings emphasize the significant antibacterial performance of Ag₂O NPs at room temperature, owing to its narrower band gap^76,77.

SEM and EDS analysis

FIB-SEM metaphors of Ag₂O NPs have displayed in Fig. 6(a)-(c) and (d) which showed that surface morphology of the Ag₂O NPs are spherical and rod like of various sizes (40–80 nm) and mean particle size of Ag₂O NPs was determined to be 54.5 nm which also confirmed by XRD and HR-TEM results ⁷⁸. The FIB-SEM images shown agglomerations of individual Ag₂O NPs. A nearer looking at the agglomerated sphere and broken rod structures of Ag₂O NPs also displayed⁷⁹. The EDS analysis approved out the existence of silver and oxygen in the sample using the spectrum peak taken as displayed in the Fig. 7. The elemental compositions study showed wide signal from silver regions and feebler signals from oxygen⁸⁰. Furthermore, it is known from EDS study that the regular silver and oxygen amount in the sample was 72.07% and 27.93 by weight respectively.

Figure 6.

(a)-(c). FIB-SEM images of Ag₂O NPs taken on different magnification and position and (d). Particle size distribution.

Figure 7.

EDS Elemental Analysis of Ag₂O NPs using Phragmanthera macrosolen L. leaf extract.

TEM analysis

The wave-like behavior of electrons tunneling through Ag₂O NPs is depicted in the HR-TEM images presented in Fig. 8(a) and (b). These images unveil the spherical distribution of Ag₂O NPs within the powder sample, with observed sizes ranging from 20 nm to 80 nm. The particle size distribution graph illustrates a mean diameter of 45.87 nm for the Ag₂O NPs which also confirmed by XRD and FIB-SEM results Fig. 8(c). Additionally, Fig. 8(d) demonstrates the poly-crystalline nature of the Ag₂O NPs, confirming this characteristic through the presence of bright circular spots in the obtained SAED patterns⁸¹.

Figure 8.

HR-TEM image (a) and (b) at different magnification (c) SAED image and (d) histogram particle size distribution analysis of biosynthesized Ag₂O NPs using Phragmanthera macrosolen L. leaf extract.

Antibacterial activity of Ag2O NPs

The synthesized Ag₂O NPs using Phragmanthera macrosolen L. leaf extracts demonstrated notable antibacterial effects, with inhibition zones varying depending on the concentration and volume of the tested sample solution. The antibacterial activity was evaluated against two bacterial strains, E. coli (gram-negative) and S. aureus (gram-positive), using different concentrations of Ag₂O NPs (20 mg/ml and 10 mg/ml). Each concentration was tested with sample volumes of 3.75, 7.5, 15, 30, 45, and 60 µl.

The inhibition zones for E. coli increased with the volume and concentration of the Ag₂O NPs^30,82. For 20 mg/ml samples, the inhibition zones were 40 mm, 37 mm, 35 mm, and 34 mm for 60, 45, 30, and 15 µl, respectively, with no inhibition observed for 3.75 and 7.5 µl. For 10 mg/ml, the inhibition zones were 28 mm, 26 mm, 23 mm, and 22 mm at the corresponding sample volumes, with no inhibition detected for 3.75 and 7.5 µl. This data is represented in Fig. 9(a-h) and Table 4(a) and (b), illustrating the results for E. coli and S. aureus.

Figure 9.

Inhibition zones of Antibacterial activity of Ag₂O NPs against E. coli and S. aureus by the disc diffusion method at different concentrations.

Table 4.

Antibacterial activity of biosynthesized Ag₂O NPs via Phragmanthera Macrosolen L. leaf for.

(a) 20 mg/ml of the samples and
Bacterial strain	Zone of Inhibition(mm)
	3.75µl	7.5µl	15µl	30µl	45µl	60µl
E.coli	0	0	34	35	37	40
S. aureus	0	0	27	29	31	30
Control	-	-	-	-	-	-
(b) 10 mg/ml of samples
Bacterial strain	Zone of Inhibition(mm)
	3.75µl	7.5µl	15µl	30µl	45µl	60µl
E.coli	0	0	22	23	26	29
S. aureus	0	0	20	24	25	28
Control	-	-	-	-	-	-

Similarly, for S. aureus, the 20 mg/ml Ag₂O NPs resulted in inhibition zones of 30 mm, 29 mm, 26 mm, and 24 mm for 60, 45, 30, and 15 µl, with no inhibition for 7.5 and 3.75 µl. The 10 mg/ml sample showed inhibition zones of 28 mm, 25 mm, 24 mm, and 20 mm. The results indicate a clear trend of increased antibacterial activity with higher sample concentrations and volumes⁸³.

The Ag₂O NPs exhibited greater antibacterial activity against E. coli than S. aureus, with the highest inhibition zone of 40 mm observed for 60 µl of the 20 mg/ml Ag₂O NPs solution against E. coli. The highest inhibition zone for S. aureus was 30 mm at the same concentration and volume. At the lower concentration (10 mg/ml), the highest inhibition zones for E. coli and S. aureus were 29 mm and 28 mm, respectively⁸⁴.

The superior antibacterial activity of Ag₂O NPs can be attributed to their ability to generate reactive oxygen species (ROS), which cause oxidative stress and damage to bacterial proteins and DNA^25,85. The interaction of Ag₂O NPs with sulfur and phosphorus in bacterial DNA disrupts cell replication, leading to cell death⁸⁶. This mechanism is likely responsible for the observed antibacterial effects, particularly against E. coli, as depicted in Figs. 9 and 10, and Fig. 11.

Figure 10.

Comparison of zones of inhibition (ZOI) of E. coli and S. aureus at different concentrations.

Figure 11.

Possible mechanism of antibacterial activity of Ag₂O NPs against bacterial strains.

The antibacterial efficacy of green-synthesized Ag₂O NPs was compared with other metal oxides synthesized by various methods, as shown in Table 5. Metal oxides such as ZnO, TiO₂, CuO, and Fe₃O₄ have been widely studied for their antibacterial properties. However, the inhibition zones observed in these metal oxides are generally lower than those exhibited by the green-synthesized Ag₂O NPs in this study. For instance, ZnO NPs synthesized through sol-gel methods typically produce inhibition zones in the range of 15–20 mm against E. coli and S. aureus⁸⁷, while CuO NPs, prepared via hydrothermal techniques, display zones between 18 and 22 mm⁸⁸. In contrast, the green-synthesized Ag₂O NPs in the current study achieved inhibition zones as high as 40 mm against E. coli and 30 mm against S. aureus, highlighting their superior antibacterial activity. The enhanced antibacterial performance of the green-synthesized Ag₂O NPs can be attributed to the environmentally friendly synthesis process, which utilizes plant extracts that act as reducing and stabilizing agents. This method likely produces nanoparticles with a more reactive surface, thereby increasing their interaction with bacterial cells. Furthermore, the smaller particle size and the production of reactive oxygen species (ROS) by Ag₂O NPs contribute to their higher antibacterial efficacy³³. In comparison, other metal oxides, though effective, often show reduced inhibition zones, possibly due to their larger particle sizes or the lack of ROS generation⁸⁹. Thus, the green synthesis of Ag₂O NPs offers a promising alternative, not only for its eco-friendly approach but also for its enhanced antibacterial potential.

Table 5.

Comparisons of metal oxides and the green synthesized Ag₂O NPs.

Metal Oxide NPs	Synthesis Method	Bacterial Strains	Inhibition Zone(mm)	References
CuO NPs	Green synthesis	E. coli and S. aureus	35 and 0	90
CuO NPs	Laser Production	E. coli and S. aureus	16.5 and 15	91
Fe2O3 NPs	Biosynthesis	E. coli and S. aureus	10 and 8	92
TiO2NPs	Sol-gel	E. Coli, K. pneumoniae and B. subtilis	12.50, 18.75 and 18.75	93
MgO NPs	Ultrasonic-Assisted Sol-Gel Method	S. aureus, E. coli and C. albicans	19.3, 16.1 and 15.2	94
NiO NPs	photolysis method	Klebsiella and E. coli	14 and 16	95
MnO2NPs	Green synthesis	S. aureus, E. coli and P. aeruginosa	22, 18 and 15	96
CeO2NPs	Biosynthesis	S. aureus and E. coli.	22 and 19	97
Ag2O NPs	Green synthesis	S. aureus and E. coli	30 and 40	This Work

Minimum inhibitory concentration (MIC) for the biosynthesized Ag2O NPs

The Minimum Inhibitory Concentration (MIC) method was employed to assess the inhibitory effect of Ag₂O NPs on pathogenic organisms, demonstrating their adverse impact. Serial dilutions ranging from 60 to 3.75 µg/ml were deliberately applied, determining the minimum concentration at which antibacterial properties were still evident. The concentrations for S. aureus and E. coli were found to be 15 and 30 µg respectively for both 10 g/ml and 20 mg/ml. The results indicate that Ag₂O NPs biosynthesized from Phragmanthera macrosolen L. leaf extract effectively inhibit the growth of certain bacteria at lower concentrations. All experiments were conducted in triplicate, and error bars in the form of standard deviation are presented in the data⁴². MIC as well as MBC is generally utilized to estimate the capability of antibacterial agents. After testing, it could be decided that as synthesized Ag₂O NPs gave the MIC of 15 µg/ml and 30 µg/ml much lesser than that of commercially existing particles which gave the MIC of about 800 µg/ml, while the MBC was 30 µg/ml powerfully evidencing the advantages of the quite little using dosage⁹⁸.

Conclusion

Silver oxide (Ag₂O) nanoparticles were successfully synthesized using a green method involving the crude extract of Phragmanthera macrosolen L. which is cost effective and eco-friendly. The prepared Ag₂O nanoparticles characterized through techniques such as SEM, HR-TEM, XRD, FTIR Spectroscopy, EDS, and UV-Vis Spectrometry. All characterization made reveals that the Ag₂O NPs are suitable for antibacterial activity. The nanosized biosynthesized Ag₂O nanoparticles, derived from the leaves of Phragmanthera macrosolen L., exhibited notable antibacterial activity, particularly against strains of S. aureus and E. coli. The investigation into antimicrobial efficacy indicated that Ag₂O nanoparticles displayed superior effectiveness against these bacterial strains than those synthesized by chemical and physical techniques. The bioactive compounds available in crude leaf extracts of Phragmanthera macrosolen L. used as reducing, capping and stabilizing agents during formation of Ag₂O NPs by donating ions. These findings suggest that biosynthesized Ag₂O NPs might serve as highly potent antimicrobial agents against pathogens, making them potentially valuable for various medicinal applications.

Author contributions

Conceptualization, A, S, SR. D, R. K, R. D, N. M and N. N.; Data curation, A, S, SR. D, R. K, R. D, N. M and N. N.; Analysis and Validation, A, S, SR. D, LT. J, R. K, R. D, N. M and N. N.; Formal analysis, A, S, SR. D, LT. J, R. K, R. D, N. M and N. N; Investigation, A, S, SR. D, R. K, R. D, N. M and N. N.; Methodology, A, S, SR. D, R. K, R. D, N. M and N. N.; Project administration, K.R and SR. D. Resources, A, S, SR. D, R. K, R. D, N. M and N. N.; Software, A, S, SR. D, R. K, R. D, N. M and N. N., Supervision, K. R. and SR. D; Validation, A, S, SR. D, R. K, R. D, N. M and N. N.; Visualization, A, S, SR. D, R. K, R. D, N. M and N. N.; Writing—original draft, A, S, SR. D, R. K, R. D, N. M and N. N., Data Visualization, Editing and Rewriting, A, S, SR. D, LT. J, R. K, R. D, N. M and N. N.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on request.

Declarations

Competing interests

The authors declare no competing interests.