Abstract*

F1000Research

2046-1402

F1000 Research Limited

London, UK

10.12688/f1000research.173222.2

Research Article

Articles

Eco-friendly Biosynthesis of Silver Oxide Nanoparticles Using Boswellia carterii with Antibacterial Applications

[version 2; peer review: 2 approved with reservations, 3 not approved]

Alani

Ban M. A.

Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing a 1 K. Taha

Sarah

Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Software Supervision Validation Visualization b 2 Jabbar Kttafah

Anfal

Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Software Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing 3 M Aziz

Leqaa

Conceptualization Data Curation Investigation Methodology Project Administration Software Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-8309-1926 1 1Medicine, University of Fallujah, Fallujah, Al Anbar Governorate, Iraq 2Collage of Dentistry, AL-Iraqia University, Baghdad, Baghdad, Iraq 3Institute of Medical Technology, Middle Technical University, Baghdad, Baghdad, Iraq

a Banscince82@uofallujah.edu.iq b Sarah.Kh.taha@aliraqia.edu.iq

No competing interests were disclosed.

6 4 2026

2025

1487

24 3 2026

2026

This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract* Background

Frankincense (Boswellia carterii) a natural resin rich in biologically active compounds, especially boswellic acids, which contribute to its anti-inflammatory and therapeutic properties. Plant extracts have become valuable in the green synthesis of metal nanoparticles because they act as safe, low-cost and stabilizing agents, offering an environmentally friendly alternative to conventional chemical methods.

Methods

In this study, silver oxide nanoparticles were prepared using an aqueous extract of Frankincense. The extract was obtained by dissolving 1 g of crude resin in 100 mL of deionized water, heating at 50°C, and filtering to remove insoluble material. A 1 mM solution of silver nitrate was prepared by dissolving 0.015 g of AgNO₃ in 100 mL of water. The silver solution was added gradually to the warm extract under continuous stirring, and the mixture was heated up to 140°C for 50 minutes until a dark brown color appeared, indicating nanoparticle formation. The product was then centrifuged, washed several times, and left to dry. Structural characterization was carried out using X-ray diffraction, and the crystallite size was calculated using the Scherrer equation. Particle morphology was examined by scanning electron microscopy.

Results

The antibacterial activity of the synthesized nanoparticles was evaluated against Pseudomonas aeruginosa and Streptococcus species at concentrations of 60%, 80%, and 100%. The XRD results confirmed the formation of silver oxide with an orthorhombic crystalline structure corresponding to standard reference data, with an average crystallite size of 80.6 nm. SEM images revealed that the particles were relatively uniform in distribution, with diameters ranging from 55.84 to 82.17 nm. Antibacterial testing showed clear inhibition zones measuring between 14.5 and 23 mm for both bacterial strains, with a stronger effect observed against Streptococcus.

Conclusion

These findings demonstrate that Boswellia carterii extract can serve as an effective natural agent for the green synthesis of silver oxide nanoparticles, producing particles with suitable structural characteristics and notable antibacterial activity.

Biosynthesis Silver oxide Nanoparticles Boswellia caretter

The author(s) declared that no grants were involved in supporting this work.

Revised Amendments from Version 1

The revised version of this article incorporates several clarifications and methodological refinements implemented in response to reviewer comments, with the aim of improving the transparency, accuracy, and overall scientific rigor of the study. The role of the plant extract as a negative control in the green synthesis process has been explicitly clarified to confirm that the observed antimicrobial activity is attributable to the synthesized nanomaterial rather than to intrinsic components of the extract. Additional methodological explanations have been introduced regarding the experimental design, including the rationale for not performing independent pre-synthesis testing of silver nitrate and the purification procedures applied to minimize the potential contribution of residual ionic silver. These clarifications help delineate the interpretation of the antibacterial activity associated with the post-synthesis nanosystem. The description of the antimicrobial assay has also been expanded to clarify the replication strategy, measurement precision, and the expected variability associated with the agar diffusion method, emphasizing its semi-quantitative nature and the consistency of the observed concentration-dependent trends. Furthermore, supplementary characterization information has been included by presenting the UV–Vis spectrum obtained during nanoparticle preparation as supporting evidence of nanoparticle formation, while maintaining X-ray diffraction (XRD) analysis as the primary technique for crystalline phase identification. Terminology describing the synthesized material has been standardized throughout the manuscript to ensure consistency with the structural characterization results. Finally, minor linguistic and editorial revisions have been implemented to enhance clarity, and a statement regarding the availability of raw experimental data and characterization files upon reasonable request has been added to further strengthen research transparency.

Introduction

Frankincense, an aromatic resin, is extracted from dried liquids (also known as olibanum), and is extracted from Boswellia trees. Boswellic acids (BAs) extracted from Boswellia trees are essential in in vivo and in vitro studies of their bioactive components. Boswellic acids provide the high degree of anti-inflammatory properties of frankincense, due to its biomechanical and molecular properties. Due to their lipophilic nature, their ability to bind or dissolve liquids or fats reduces their solubility in aqueous environments, as well as their bioavailability. Frankincense also contains a group of pentacyclic terpenic acids known as lipoic acids. ^{1,
2}

Plant exudates have been used as capping and reducing agents in the green synthesis of silver, gold and platinum nanoparticles. ^{3–
5} These natural polymers stabilize the nanoparticles, which are non-toxic to cells and potentially useful for molecular imaging, biomedical diagnosis, and safe drug delivery. Silver nanocomplexes are promising materials and can be used as coating materials in food packaging and biomedical engineering, where biocompatibility and antibacterial activity are required. ^{6,
7} The regulation of the mean particle size and the distribution of particle size during synthesis of the silver nanoparticle with hydroxyl functionalized water soluble polymers is also facilitated. ⁸

Gum olibanum is chosen as a biopolymer for silver oxide nanoparticles synthesis because it is available, nontoxic and low cost and has medicinal applicability. In this context, we have established an easy, biosynthetic procedure for the synthesis of silver oxide nanoparticles using gum olibanum, a natural plant polymer that is both renewable and biodegradable, as the stabilizing and reducing agent. The aim of the present work was the synthesis and characterization of silver oxide nanoparticles. For potential biological applications, we have also shown antibacterial activity on the produced nanoparticles towards the Gram-positive and Gram-negative bacteria.

One of the more frequent causes of infections is Pseudomonas aeruginosa in a medical facility. These infections are linked to substantial morbidity and medical costs, particularly when It takes longer to receive the right antibiotic treatment. The selection of antibiotics It is difficult to treat patients with P. aeruginosa infections because of the infection’s innate resistance to numerous commercially available capable of antibiotics. Multidrug-resistant strains are prevalent, and often require treatment with novel or “last resort” agents. ⁹

Gram-positive, spherical, non-motile, and spore-forming streptococcus bacteria are frequently seen in chains. They are categorized as beta-hemolytic (full lysis, like S. pyogenes), alpha-hemolytic (partial lysis, like S. pneumoniae and the viridans group), and gamma-hemolytic (non-lysis, like some Enterococcus) based on their capacity to lyse blood on blood agar. They can also be categorized into groups (A, B, C, etc.) using the Lancefield approach.

The most significant species are S. pneumoniae, which causes pneumonia, otitis media, meningitis, and sinusitis; S. pyogenes (group A), which causes pharyngitis, scarlet fever, skin infections, rheumatic fever, and glomerulonephritis; S. agalactiae (group B), which causes infections in neonates; and viridans streptococci, which are naturally found in the mouth and can cause endocarditis. Culture and assays such the optochin test (for S. pneumoniae), CAMP test (for group B), and bacitracin susceptibility (for group A) are used to make the diagnosis. Although penicillin or its derivatives constitute the mainstay of treatment, some species—especially S. pneumoniae—may exhibit resistance, necessitating the use of cephalosporins or macrolides as alternatives. ¹⁰

The novelty of this research is achieved through the environmentally friendly greening of silver oxide nanoparticles using Boswellia carterii extract as a natural and safe reducing agent. These particles were tested for their antibacterial activity against Pseudomonas aeruginosa and Streptococcus, bacteria, demonstrating promising antibacterial results, suggesting their potential as safe alternatives to conventional antibiotics.

This research aims to prepare silver oxide nanoparticles using Boswellia carterii extract in an environmentally friendly manner, and study their antibacterial activity against Pseudomonas aeruginosa and Streptococcus.

Experimental part

Silver oxide nanoparticles were prepared using a 1 mM Boswellia caretterii extract. 0.015 g of silver nitrate (AgNO ₃) was dissolved in 100 ml of deionized water to obtain a 1 mM solution. The Boswellia cartterii extract was prepared by dissolving 1 g of crude Boswellia cartterii in 100 ml of deionized water. The mixture was then heated to 50°C for 10 min using a magnetic stirrer to facilitate the extraction of the active compounds. The solution was then filtered through filter paper to obtain the pure Boswellia cartterii extract. The resulting extract was placed on a magnetic stirrer and heated to 50°C. The silver nitrate solution was then gradually added to the extract under continuous stirring. The temperature of the mixture was gradually increased, taking care not to exceed 140°C, for 50 min until the solution turned dark brown, indicating the synthesis of silver oxide nanoparticles. After the reaction was complete, the resulting solution was cooled and then separated using centrifugation at 10,000 rpm for 15 min. The resulting precipitate was rinsed with deionized water several times to remove residual contaminants and then allowed to dry at room temperature. ^{11,
12}

Particle size and crystallite size were calculated from the results of scanning electron microscope (SEM) and x-Ray diffraction respectively. The dimensions of the nanocrystals are calculated using the Scherrer equation ( D = Kλ / β cos θ ), where X-ray diffraction (XRD) data are used by determining the apex angle and measuring its width at half height (FWHM). The tests were done in thin film laboratory at University of Tehran.

Results and discussion

This research studies the resulting patterns through X-ray diffraction testing, and determined the particle size through SEM testing. The effect of the produced silver oxide nanoparticles on two types of bacteria, namely Pseudomonas and Streptococci, was also evaluated.

The goal of this study was to confirm the crystalline phase and assess the biological activity rather than perform a comprehensive physical characterization. The crystal size was estimated using the Scherrer equation, a well-established technique for determining the crystalline structure of nanomaterials, and the crystalline phase and its conformation to a common reference were directly identified by XRD analysis. ^{13,
14} The nanostructure and particle distribution were also validated by SEM.

XRD remained the final method for verifying the crystalline phase, while UV-Vis analysis was carried out as a preliminary test to deduce nanoparticle formation from the distinctive absorption band. The UV-Vis spectrum acquired during preparation is displayed in the accompanying image.

When clear XRD data are available and consistent with the reference, techniques like FTIR, DLS, or Zeta potential are useful tools for characterizing surface properties and stability, but they are not necessary for verifying the crystalline phase. it’s possible to do it in the future to determine crystal phase more deeply. ¹⁵

From Figure 1. We noticed that two absorption bands in the UV-Vis spectrum, at 371 and 425 nm, show that silver oxide nanoparticles are going through energy gap transitions. ^{15,
16} Even though UV-Vis is not a reliable way to find out what the crystalline phase is, these results are in line with what has been reported in the literature about how silver oxide nanoparticles behave optically. Conversely, metallic silver oxide particles typically exhibit a characteristic surface plasmonic resonance band within the 400–450 nm range. ¹⁷ So, XRD is still the best way to confirm the crystalline phase, and UV-V is analysis gives more information.

Figure 1. UV-Vis spectrum silver oxide nanoparticles prepared by Boswellia extract. The result of the X-ray examination presents the diffraction pattern

The result of the X-ray examination presents the diffraction pattern as shown in Figure 2. After comparison with the standard cards, it was found that the produced material, which is silver oxide, has a polycrystalline structure and is related to diffraction from the crystal planes of the orthorhombic system, which was determined through the standard values (JCPDS 84-1547) as listed in Table 1.

Figure 2. X-ray pattern of AgO nanoparticles.

Table 1. Results that obtained from X-ray diffraction examination.

2θ (Deg.)	FWHM (Deg.)	d _hkl Exp.(Å)	C.S (nm)	d _hkl Std.	hkl	Card No.
19.576	0.09	4.5311	89.6	4.53484	(101)	JCPDS 84-1547
21.5602	0.1002	4.1184	80.7	4.12177	(110)	JCPDS 84-1547
24.1907	0.0738	3.6762	110.1	3.6792	(111)	JCPDS 84-1547
29.5228	0.1968	3.0232	41.7	3.02572	(200)	JCPDS 84-1547
31.826	0.0946	2.8095	87.3	2.81182	(002)	JCPDS 84-1547
32.756	0.2952	2.7318	28.0	2.73408	(112)	JCPDS 84-1547
35.5914	0.3936	2.5204	21.2	2.52251	(201)	JCPDS 84-1547
38.2102	0.492	2.3535	17.1	2.35543	(102)	JCPDS 84-1547
39.2521	0.3936	2.2934	21.4	2.29528	(210)	JCPDS 84-1547
40.258	0.492	2.2384	17.2	2.24022	(211)	JCPDS 84-1547
43.589	0.3936	2.0747	21.7	2.07644	(202)	JCPDS 84-1547
46.3751	0.492	1.9564	17.6	1.95797	(212)	JCPDS 84-1547
47.9244	0.0096	1.8967	905.6	1.89823	(220)	JCPDS 84-1547
49.91	0.3936	1.8258	22.3	1.82727	(310)	JCPDS 84-1547
53.8383	0.5904	1.7014	15.1	1.70285	(113)	JCPDS 84-1547
55.2077	0.7872	1.6624	11.4	1.66381	(311)	JCPDS 84-1547
58.626	0.09	1.5734	101.2	1.57469	(222)	JCPDS 84-1547
62.6666	0.295	1.4813	31.5	1.48253	(400)	JCPDS 84-1547
64.8764	1.1808	1.4361	8.0	1.43727	(321)	JCPDS 84-1547
67.5144	0.2465	1.3862	38.8	1.38739	(420)	JCPDS 84-1547
70.3283	0.2072	1.3375	46.9	1.33862	(402)	JCPDS 84-1547
75.224	0.0945	1.2621	106.1	1.26319	(213)	JCPDS 84-1547
77.6092	0.7872	1.2292	13.0	1.23022	(421)	JCPDS 84-1547

FWHM: Full Width at Half Maximum of the diffraction peak, Θ: Bragg angle (half of the peak position, in radians), C.S: Crystallite size, d: Interplanar spacing.

The average crystal size of silver oxide nanocrystals was also calculated, and the average crystal size was 80.6 nm.

Figure 3 shows the scanning electron microscope image at 60.00 kx and 80.00 kx magnifications. From Figure 3a, which is obtained using 60.00 kx magnification, we notice that the silver oxide nanoparticles are relatively homogeneously distributed in most areas.

Figure 3. Scanning electron microscope image; (a) at 60.00 kx, (b) at 80.00 kx.

From Figure 3b which is obtained using 80.00 kx magnification, which shows fine details, allowing us to determine the diameters of these particles, we notice that the diameters of the nanoparticles range between 55.84 and 82.17 nm. These sizes are suitable for medical and environmental applications. Nanoparticles of this size have a large surface area relative to their volume, which increases their interaction with other molecules, allowing them to easily bind drugs or biosensors. It also increases their ability to absorb contaminants or catalyze chemical reactions. Furthermore, particles smaller than ~100 nanometers can easily enter cells without being quickly eliminated from the body, which is important for drug delivery. ¹⁸

After studying the structural properties of silver oxide nanoparticles produced by the Green Synthesis method, the effect of these manufactured particles on two types of bacteria, namely Pseudomonas aeruginosa and Streptococci, was studied.

Different concentrations were used for the prepared samples, and these concentrations were 60%, 80%, and 100%. When using all the mentioned concentrations separately, an antibacterial effect against Pseudomonas aeruginosa and Streptococci was appeared. The diameter of the inhibition zone ranged between 14.5 mm and 23 mm, as shown in Figure 3. The plant extract was used as a negative control, a crucial step in the green synthesis studies, to ensure that the biological activity was not attributable to the extract's components themselves. The extract showed no inhibition zone under the same testing conditions. See Figure 4.

Since the goal of the study was to assess the biological activity of the nanomaterial following preparation and purification, no independent testing of silver nitrate was carried out before synthesis. The antibacterial activity of silver ions is known to be concentration-dependent, ^{19,
20} and the release of silver ions may be responsible for some of the effects ascribed to the nanoparticles. ²¹ Before the bacteriological tests, the prepared material was thus subjected to multiple cycles of centrifugation and washing in order to minimize any remaining ions. As a result, the outcomes show the biological activity of the post-synthesis nanosystem. Using of samples prepared from silver oxide nanoparticles against Pseudomonas aeruginosa bacteria. The diameters of the inhibition zones ranged from 14.5 mm at a concentration of 60% to 18 mm at a concentration of 100%, as shown in Table 2. From above presented results, we conclude the high effectiveness of silver oxide nanoparticles against highly virulent bacteria. This result is better than the results obtained by Gheni and Odaa. ²⁴ This means diameter of inhibition zone increases with increasing concentration. ^{22,
23}

Figure 4. Effect of Boswellia extract nanoparticles against : A- Pseudomonas aeruginosa bacteria. B- Streptococcus bacteria. Figure 5. Effect of AgO nanoparticles against; (a) Pseudomonas aeruginosa bacteria, (b) Streptococcus bacteria.

Table 2. Diameter of inhibition zone of AgO nanoparticles.

Sol. Concentration %	Diameter (mm) pseudomonas	Diameter (mm) streptococci
100%	18	23
80%	17	19
60%	14.5	17.5

While when using samples prepared from silver oxide nanoparticles against Streptococcus bacteria, the diameters of the inhibition zones ranged between 17.5 mm at a concentration of 60% and up to 23 mm at a concentration of 100%, as shown in Table 2. From above presented results, we conclude the high effectiveness of silver oxide nanoparticles against highly virulent bacteria. This result is better than the results obtained by Gheni and Odaa. ²⁴

The optical and structural properties of nanomaterials undergo substantial modifications as a result of their enormous surface area-to-volume ratio rise, which can approach millions of times. The majority of bacterial cell membranes have nanoscale holes or pores. Nanostructures must successfully penetrate and stabilize these membranes in order to affect bacterial growth and the essential processes of the cells. ^{25,
26}

The mechanism of action of nanoparticles, which researchers employ in a variety of ways to stop bacterial development, is essentially the same: the particles’ positive charges interact with the negative charges on the surface of the bacterial cell. As a consequence of this contact, particles accumulate on the cell membrane, changing structure and properties of the membrane. This cripples the cell, and it becomes unable to perform life-sustaining functions such as respiration, permeability and electron transport. ^{27–
29}

The way nanoparticles work, which researchers have harnessed in a range of techniques to halt bacterial growth, is fundamentally the same: Positive charges in the particles interact with negative charges on the surface of the bacterial cell. A particle can be deposited on the outer part of the cell membrane by this contact and influence the chemical and physical property of the membrane. This ruins the functionality of the cell, inhibiting vital activities such as respiration, permeability, and electron transport. ^{30,
31}

Studies indicate that nanoparticles with diameters ranging from 1 to 100 nanometers possess unique physical and chemical properties, such as increased surface area to volume ratio, which enhances their interaction with other molecules. This allows them to easily interact with drugs or biosensors and increases their ability to absorb contaminants or catalyze chemical reactions. Furthermore, particles smaller than 100 nanometers can easily enter cells without being rapidly eliminated from the body, which is important for drug delivery. ^{33,
34}

The novelty of this research lies in its adoption of an environmentally friendly method for preparing silver oxide nanoparticles using Boswellia carterii extract as a natural reducing and stabilizing agent. This method is safe and economical compared to traditional chemical methods. The use of this plant to produce silver oxide nanoparticles represents a new step in this field, as it has not previously been widely employed for this purpose. The importance of the study also lies in testing the effectiveness of these nanoparticles against two types of medically and clinically important bacteria, Pseudomonas aeruginosa (Gram-negative) and Streptococcus, (Gram-positive), which are known for their resistance to antibiotics. The results showed a promising ability of these particles to inhibit bacterial growth, suggesting their potential use as alternatives or complements to traditional antibiotics.

Each concentration was replicated twice under identical incubation conditions, and the diameters of the inhibition zones were measured with a digital measuring foot calibrated to ±0.1 mm. Given the nature of diffusion in the solid medium and the technique for visually identifying the edge of the inhibition zone, the variation between replicates did not surpass ±0.5 mm, falling within the expected limits of uncertainty for the agar diffusion test. This degree of variation is within the typical systematic error of the test and does not suggest significant biological variability.

Given that the agar diffusion test is a semi-quantitative test that illustrates the overall trend of antimicrobial activity rather than being a precise quantitative analytical tool, inferential statistical analysis would not change the scientific interpretation of the results because the differences between replicates were within the bounds of measurement accuracy and because the upward trend with increasing concentration was distinct and consistent. ^{35,
36}

Conclusion

This study successfully produced silver oxide nanoparticles bioactively using Boswellia caretteri extract. The particle sizes ranged between 55.84 and 82.17 nm, with an average size of 80.6 nm. The results demonstrated the clear effectiveness of these particles against the bacteria under study, with their effect on Streptococcus bacteria being higher than on Pseudomonas aeruginosa bacteria. These results suggest the potential for relying on environmentally friendly bio-based methods to produce biocompatible nanomaterials that can be used in medical applications. Further studies are recommended to more precisely elucidate the mechanism of action and ensure their safety before clinical use.

Data availability

No datasets were created or analyzed during the current study. All data relied upon in this article are derived from previously published studies, which have been cited and included in the references.

Acknowledgements

We thank everyone who helped and contributed to the completion of this research.

References 1

Rashan

: Boswellia gum resin and essential oils: Potential health benefits− An evidence based review. International Journal of Nutrition, Pharmacology, Neurological Diseases. 2019;9(2):53–71. 10.4103/ijnpnd.ijnpnd_11_19

Nwachukwu

: Frankincense: Biological Activities and Therapeutic Properties. Graduate Institute of Health Sciences, Near East University, Nicosia, Turkish Republic of North Cyprus;2020. Master’s Thesis.

Sen

Sr Das

Banerji

: Isolation and structure of a 4-O-methyl-glucuronoarabino-galactan from Boswellia serrata. Carbohydr. Res. 1992;223:321–327. 10.1016/0008-6215(92)80031-U

Leung

TC-Y

Wong

Xie

: Green synthesis of silver nanoparticles using biopolymers, carboxymethylated-curdlan and fucoidan. Mater. Chem. Phys. 2010;121(3):402–405. 10.1016/j.matchemphys.2010.02.026

Pal

Esumi

Pal

: Preparation of nanosized gold particles in a biopolymer using UV photoactivation. J. Colloid Interface Sci. 2005;288(2):396–401. 15927605

10.1016/j.jcis.2005.03.048

Kora

Sashidhar

Arunachalam

: Gum kondagogu (Cochlospermum gossypium): a template for the green synthesis and stabilization of silver nanoparticles with antibacterial application. Carbohydr. Polym. 2010;82(3):670–679. 10.1016/j.carbpol.2010.05.034

Vinod

Saravanan

Sreedhar

: A facile synthesis and characterization of Ag, Au and Pt nanoparticles using a natural hydrocolloid gum kondagogu (Cochlospermum gossypium). Colloids Surf. B: Biointerfaces. 2011;83(2):291–298. 21185161

10.1016/j.colsurfb.2010.11.035

Kora

Sashidhar

Arunachalam

: Aqueous extract of gum olibanum (Boswellia serrata): a reductant and stabilizer for the biosynthesis of antibacterial silver nanoparticles. Process Biochem. 2012;47(10):1516–1520. 10.1016/j.procbio.2012.06.004

Lodise

Jr : Pseudomonas aeruginosa. Clin. Infect. Dis. 2016;63:e61–e111.

Patterson

: Streptococcus. Baron’s Med Microbiol. B. S , editor. Galveston: University of Texas Medical Branch; 4th ed. 1996.

Abdalameer

Khalaph

Ali

: Ag/AgO nanoparticles: Green synthesis and investigation of their bacterial inhibition effects. Mater. Today Proc. 2021;45:5788–5792. 10.1016/j.matpr.2021.03.166

Ismail

Ali

Abdul Hussien

: Green synthesis and characterization of CdO nanoparticles from Crocus sativus: a promising material for hydrogen gas storage. Energy Storage. 2024;6(1):e534. 10.1002/est2.534

Cullity

Stock

: Elements of X-ray Diffraction. 3rded Upper Saddle River: Prentice Hall.2001.

Klug

Alexander

: X-ray Diffraction Procedures. 2nded New York: Wiley,1974.

Mourdikoudis

Pallares

Thanh

NTK

: Characterization techniques for nanoparticles: comparison and complementarity upon studying nanoparticle properties. Nanoscale. 2018;10(27):12871–12934. 10.1039/C8NR02278J

Arbuj

Mulik

Amalnerkar

: Preparation, characterization and optical properties of Ag2O nanoparticles. Mater Sci Eng B. 2011;176(16):1173–1179.

Bhosale

Bhanage

: Silver oxide nanoparticles: synthesis and characterization. Mater Chem Phys. 2015;155:114–120.

Kelly

Coronado

Zhao

: The optical properties of metal nanoparticles: the influence of size, shape, and dielectric environment. J Phys Chem B. 2003;107(3):668–677. 10.1021/jp026731y

Rai

Yadav

Gade

: Silver nanoparticles as a new generation of antimicrobials. Biotechnol Adv. 2009;27(1):76–83. 10.1016/j.biotechadv.2008.09.002

Morones

Elechiguerra

Camacho

: The bactericidal effect of silver nanoparticles. Nanotechnology. 2005;16(10):2346–2353. 10.1088/0957-4484/16/10/059

Xiu

Zhang

Puppala

: Negligible particle-specific antibacterial activity of silver nanoparticles. Nano Lett. 2012;12(8):4271–4275. 10.1021/nl301934w

Paliwal

: Nanomedicine, Nanotheranostics and Nanobiotechnology: Fundamentals and Applications. CRC Press;2025.

Ullah

Kilic

Habib

: Reliable prediction of thermophysical properties of nanofluids for enhanced heat transfer in process industry: a perspective on bridging the gap between experiments, CFD and machine learning. J. Therm. Anal. Calorim. 2023;148(12):5859–5881. 10.1007/s10973-023-12083-7

Taha

Hawar

Sulaiman

: Extracellular biosynthesis of silver nanoparticles from Penicillium italicum and its antioxidant, antimicrobial and cytotoxicity activities. Biotechnol. Lett. 2019;41(8):899–914. 31201601

10.1007/s10529-019-02699-x

Gheni

Odaa

: The Antimicrobial Activity of Silver Nanoparticles Synthesized by Pseudomonas aeruginosa Against UTI Pathogenic Microorganisms. Ibn AL-Haitham Journal For Pure and Applied Sciences. 2024;37(4):119–128. 10.30526/37.4.3373

Ahmadi

Kordestany

: Investigation on silver retention in different organs and oxidative stress enzymes in male broiler fed diet supplemented with powder of nano silver. American-Eurasian Journal of Toxicological Sciences (AEJTS). 2011;3(1):28–35.

Alani

A-AK

Ali

: Use of pepper plant leavesâ aqueous extract in the synthesis of silver nanoparticles and the treatment of some pathogenic microorganisms. J Theor Appl Phys. 2024;18:1–8. 10.57647/j.jtap.2024.si-AICIS23.08

Asharani

Gong

: Toxicity of silver nanoparticles in zebrafish models. Nanotechnology. 2008;19(25):255102. 10.1088/0957-4484/19/25/255102

Razuqi

Muftin

Murbat

: Influence of dielectric-barrier discharge (DBD) cold plasma on water contaminated bacteria. Annual Research & Review in Biology. 2017;14(4):1–9. 10.9734/ARRB/2017/34642

Aziz

Alqaissy

WQM

Alani

: Effect of carbon nano colloid and its synergism with some antibiotics on Klebsiella pneumonia isolated from UTI of pregnant women. Res. J. Biotechnol. 2023;18(1):29–35. 10.25303/1801rjbt29035

Kim

: Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells. Toxicology in vitro. 2009;23(6):1076–1084. 19508889

10.1016/j.tiv.2009.06.001

Roomy

Alani

A-AK

: Studying the Effect of Silver Oxide Nanoparticles Using Laurus Nobilis Leaves in Inhibiting Bacterial Action. J. Nanostruct. 2024;14(4):1058–1066. 10.22052/JNS.2024.04.007

Mazhir

Abdalameer

Yaaqoob

: Bio-synthesis of (Zn/Se) core-shell nanoparticles by micro plasma-jet technique. Int. J. Nanosci. 2022;21(05):2250041. 10.1142/S0219581X22500417

Al-Rawi

Mazhir

: Evaluation of antimicrobial agents of ag–zno core-shell prepared by micro-jet plasma technique. Int. J. Nanosci. 2023;22(05):2350044. 10.1142/S0219581X23500448

Balouiri

Sadiki

Ibnsouda

: Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal. 2016;6(2):71–79.

CLSI . Performance standards for antimicrobial disk susceptibility tests. 13 ^th ed.CLSI standard M02. Wayne, PA: Clinical and Laboratory Standards Institute;2018.

10.5256/f1000research.197624.r479825

Reviewer response for version 2

N.L

Sheeba

1 Referee https://orcid.org/0000-0002-3346-670X 1Sri Paramakalyani College, Alwarkurichi, Tamil Nadu,, India

Competing interests: No competing interests were disclosed.

9 5 2026

2026

This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

recommendation

reject

Reviewer Comments

Journal Title: F1000Research

Manuscript Title: Eco-friendly Biosynthesis of Silver Oxide Nanoparticles Using Boswellia carterii with Antibacterial Applications

Major Comments

1. Insufficient physicochemical characterization of nanoparticles

The manuscript relies mainly on XRD and SEM for nanoparticle characterization. While these techniques confirm crystallinity and morphology, they are insufficient to fully validate the successful green synthesis of AgO nanoparticles. Important characterization techniques such as FTIR, EDX/EDS, DLS, zeta potential analysis, TEM, and XPS are missing. These analyses are essential to confirm elemental composition, surface chemistry, particle size distribution, colloidal stability, and oxidation state.

2. Unclear identification of the silver oxide phase

The authors repeatedly refer to “silver oxide nanoparticles”; however, the exact oxidation state is not clearly established. It remains unclear whether the synthesized material is Ag₂O, AgO, metallic Ag, or a mixed phase. XRD analysis alone, without Rietveld refinement or complementary spectroscopic evidence, cannot conclusively distinguish these phases. The authors should provide stronger evidence for phase purity and use consistent and accurate nomenclature throughout the manuscript.

3. Questionable XRD interpretation and crystallite size calculation

Several concerns are noted in Table 1:

* The reported crystallite size of 905.6 nm at 47.9244° is unrealistic when compared with the reported average size of 80.6 nm.

* The FWHM values appear inconsistent and may not have been corrected for instrumental broadening.

* The parameters used in the Scherrer equation (K value, X-ray wavelength, and unit conversions) are not clearly described.

The XRD analysis should be recalculated carefully, and the authors should provide the diffraction pattern with properly indexed peaks and baseline correction.

4. Lack of appropriate controls in the antibacterial study

The study used only the plant extract as a negative control. However, the observed antibacterial activity may also originate from residual Ag⁺ ions. A proper experimental design should include AgNO₃ as a control, a standard antibiotic as a positive control, and a blank solvent control. Without these controls, the antibacterial activity cannot be attributed solely to the synthesized nanoparticles.

5. Absence of statistical analysis

The antibacterial assay was reportedly performed only in duplicate. Duplicate measurements are insufficient for reliable biological interpretation. At least triplicate independent experiments should be conducted, followed by appropriate statistical analysis (mean ± SD, ANOVA or t-test).

6. Inadequate description of antibacterial methodology

Several important experimental details are missing, including: exact bacterial strains and ATCC numbers, inoculum concentration, culture conditions, well/disc diffusion methodology, nanoparticle concentration in mg/mL, incubation temperature and duration. These details are essential for reproducibility.

7. Mechanistic discussion is too generic

The discussion section mainly contains generalized statements from previous nanoparticle literature rather than interpretation of the present findings. The authors should specifically discuss why Streptococcus exhibited higher sensitivity, the influence of particle size and morphology on antibacterial activity, and comparisons with previously reported Boswellia-mediated Ag/AgO nanoparticles.

8. Novelty claim is overstated

The manuscript repeatedly claims novelty; however, green synthesis of silver-based nanoparticles using plant extracts and their antibacterial evaluation has already been widely reported. The authors should clearly explain what aspect of the present study is genuinely novel compared with the existing literature.

9. Lack of evidence for nanoparticle purity after washing

The manuscript states that repeated centrifugation removed residual ions, but no analytical evidence is provided to support this claim. Techniques such as ICP-OES, conductivity measurements, or elemental analysis would strengthen this conclusion.

10. Language quality and scientific writing require major improvement

The manuscript contains numerous grammatical errors, awkward sentence constructions, repeated statements, and inconsistent terminology, all of which reduce readability. Professional English language editing is strongly recommended.

Minor Comments

1. The species name Boswellia carterii is inconsistently written throughout the manuscript as: “Boswellia caretter”, “Boswellia cartterii”, “Boswellia caretterii”. Scientific nomenclature should be corrected and standardized throughout the manuscript.

2. In the Abstract, the sentence: “Frankincense ( Boswellia carterii) a natural resin…” requires a comma after the species name.

3. The phrase: “silver oxide nanoparticles are going through energy gap transitions” is scientifically inaccurate and should be rewritten more clearly.

4. Figure captions are insufficiently descriptive. Experimental conditions, magnification details, and scale bars should be clearly included.

5. The UV-Vis discussion contains conceptual inaccuracies. The explanation of plasmon resonance for silver oxide nanoparticles differs from that of metallic silver nanoparticles and should be clarified carefully.

6. References are inconsistently formatted. Some citations contain missing spaces, punctuation errors, and inconsistent journal formatting.

7. The manuscript contains repetitive discussion regarding antibacterial mechanisms of nanoparticles. Redundant content should be reduced.

8. The statement “This result is better than the results obtained by Gheni and Odaa” should be supported with proper numerical comparison and citation context.

9. The SEM analysis should include average particle size along with standard deviation based on measurements from multiple particles.

10. The manuscript alternates between “Streptococcus” and “Streptococci”. One term should be used consistently throughout the manuscript.

11. Units and symbols should be standardized throughout the manuscript. Use “°C” consistently, include spaces between numbers and units, use “mL” instead of “ml”.

12. Some sentences are excessively long and difficult to follow. The manuscript would benefit from careful proofreading and restructuring.

13. The conclusion section is too brief and should include study limitations and future research perspectives.

14. The authors should provide raw characterization data as supplementary material to improve transparency and reproducibility.

15. The manuscript would benefit from a comparison table summarizing previously reported plant-mediated AgO nanoparticle syntheses and their antibacterial efficiencies.

Overall Recommendation

The manuscript addresses a relevant topic in green nanotechnology and antibacterial nanomaterials. However, substantial methodological, analytical, and presentation-related weaknesses remain unresolved. Therefore, the manuscript requires major revision before it can be considered scientifically reliable and suitable for indexing.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Nanoparticles

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

10.5256/f1000research.197624.r474844

Reviewer response for version 2

Patil

Bheemanagouda N

1 Referee https://orcid.org/0000-0001-7214-3906 1Smt. Allum Sumangalamma Memorial College for Womens Ballari, Ballari, Karnataka, India

Competing interests: No competing interests were disclosed.

5 5 2026

2026

recommendation

approve-with-reservations

General comments:

In the manuscript entitled " Eco-friendly Biosynthesis of Silver Oxide Nanoparticles

Using Boswellia carterii with Antibacterial Applications ", the authors report the synthesis of AgONP using green method and characterized using techniques such as XRD, UV-Vis and SEM further toxicity examination of antimicrobial activity was performed. The presented work is needed critical improving the following points.

Major and specific points:

Manuscript should be thoroughly rechecked for language correction, spelling, typographic and formatting errors (etc. superscript, subscript).

The introduction part is so vague.

Plant authentication vochour number and plant photo should be incorporate. Add the collection site with longitude and latitude, Plant name should be italic with author name.

Why you choose this plant..? what are medicinal properties are encoded. This all the points is incorporate in the introduction part.

Visible photo of NPs synthesis photo should add.

The concentration of initial cell concentration should mention in the method section of cytotoxicity assessment.

Silver oxide nanoparticles were prepared using a 1mM Boswellia caretterii extract, mentioned in Experimental part first page, are used the 1mM Boswellia extract clarify.

Phytochemical constitute of plant should incorporate.

Add the statistical analysis, its gives clear data of experimental work.

In reference section add 2026 incorporate the recent articles.

The following publications are recommended to strengthen the background of synthesis of nanoparticles

Antimycobacterial and antibacterial activity of green-synthesized silver and zinc oxide nanoparticles using Diospyros montana L. leaf extract. (refer 1)

Limonia acidissima L. leaf mediated synthesis of silver and zinc oxide nanoparticles and their antibacterial activities, (refer 2)

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Bionanotechnology, Ecotoxicology, Environmental Science, Cytotoxicity

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References 1

: Antimycobacterial and antibacterial activity of green-synthesized silver and zinc oxide nanoparticles using Diospyros montana L. leaf extract. Next Nanotechnology .2025;8: 10.1016/j.nxnano.2025.100248

10.1016/j.nxnano.2025.100248

: Limonia acidissima L. leaf mediated synthesis of silver and zinc oxide nanoparticles and their antibacterial activities. Microbial Pathogenesis .2018;115: 10.1016/j.micpath.2017.12.035 227-232

10.1016/j.micpath.2017.12.035

10.5256/f1000research.197624.r479823

Reviewer response for version 2

Sohag

Md. Shihab Uddin

1 Referee https://orcid.org/0000-0001-7242-7079 1Khwaja Yunus Ali University, Sirajganj, Bangladesh

Competing interests: No competing interests were disclosed.

5 5 2026

2026

recommendation

approve-with-reservations

Your research is interesting and well-intentioned. In this study, you extracted Boswellia carterii resin, formulated it into nanoparticles, and demonstrated enhanced antibacterial activity compared to the crude extract alone. The work has good potential; however, the following comments may help improve the manuscript’s clarity, quality, and overall scientific understanding:

The scientific names of all plants and bacteria should be written in italics consistently throughout the manuscript.

Although your research focuses on a comparative antibacterial study, the background section in the abstract and introduction emphasizes the anti-inflammatory activity of frankincense. Since several studies have already reported the antibacterial activity of Boswellia species, the literature review should be better aligned with the study’s actual focus.

The first paragraph of introduction is well written, but the later part of the introduction lacks coherence. The overall flow of the study is missing. Make a paragraph about phytochemistry in resin parts and their antimicrobial activity based on literature. The hypothesis, justification for conducting this study, and clear aims and objectives should be explicitly stated.

Please maintain a clear and logical research sequence. The nanoparticle section should be introduced first, followed by antibacterial evaluation, consistently across the abstract, methods, and results and discussion sections.

The resolution of all figures is very poor. Please upload high-quality images. If figures are inserted via a Word document, adjust the settings by going to File > Options > Advanced > Image Size and Quality, then select High Fidelity. Upload figures using their original resolution.

Figure 5b is not appropriate, as the extract solution appears to have overflowed from the well. Please ensure proper experimental setup. Additionally, mention the borer cavity size clearly in the figure caption.

A well-structured Materials and Methods section is essential to ensure reproducibility, but this is currently inadequate. In particular, the antibacterial testing method needs to be clearly described, including how different concentrations were prepared.

In the Results and Discussion section, each experiment should be presented under clear subheadings. The discussion should follow a logical flow, and the final paragraph should ideally propose a possible mechanism of action, supported by relevant literature.

In the result and discussion section, statistical comparison of antibacterial results was found missing. In the method section, add a statistical analysis section, number of replications.

All references have been listed properly except 2; it is better to avoid unpublished thesis books. 50% of references have been cited more than 10 years ago.

Overall, with careful revision and improved organization, this manuscript can be significantly strengthened.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Reviewer Expertise:

Phytochemistry, Medicinal Microbiology, Nanotechnology, Pharmacology, Food and Nutrition

10.5256/f1000research.191018.r453608

Reviewer response for version 1

Nimesh

Surendra

1 Referee 1Central University of Rajasthan, Ajmer, Rajasthan, India

Competing interests: No competing interests were disclosed.

26 2 2026

2026

recommendation

reject

REVIEW COMMENTS

The manuscript titled “Eco-friendly Biosynthesis of Silver Oxide Nanoparticles Using Boswellia carterii with Antibacterial Applications” reports the green synthesis of silver oxide nanoparticles using aqueous extract of Boswellia carterii and evaluates their antibacterial activity against Pseudomonas aeruginosa and Streptococcus species. The topic is relevant to green nanotechnology and antimicrobial nanomaterials.

However, there are several critical points that need to be addressed.

The manuscript lacks a scientific tone or professional English editing. The manuscript contains numerous typographical errors (e.g., “Psedumonas”, “Striptococci”, “caretterii”), repetitions (the mechanism section is duplicated), inconsistent bacterial naming, and informal scientific phrasing. The manuscript must be revised properly before it can be considered for indexing .

The authors must provide a proper materials and methods section with mention of chemicals used, bacterial strains identification (ATCC numbers or clinical isolate source), and instruments used throughout the study.

The introduction section has an extensive taxonomy discussion of Streptococcus, which is not much required as it is not relevant much to the study; rather, the authors must elaborate on the silver oxide nanoparticles and their applications, and provide proper justification of the novelty claim, which is ambiguous throughout the study.

The nanoparticle characterization is insufficient as the manuscript lacks the basic characterization techniques. The authors must include the characterization data for UV-Vis Spectroscopy (preliminary data, which is essential), DLS, Zeta Potential, FTIR, and EDX for confirmation of efficient nanoparticle formation. Without these analyses, the claim of successful green synthesis and stabilization is weakly supported.

The images provided for the SEM results are not clear and appropriate and must be revised. The markings and figure legends are improper and unclear throughout the manuscript. The authors are advised to revise the same for proper justification.

The antibacterial experimental design is inadequate. There is no justification or mention of the type of disc diffusion method used, no mention of inoculum density (e.g., McFarland standard), and no specification of incubation conditions (temperature, duration) is provided. The results reported are of only Disc diffusion, and no MIC/MBC calculation is provided.

The authors are advised to extensively report the antibacterial activity of the synthesized nanoparticles.

The study lacks essential positive (known antibiotic drug), negative (plant extract), and silver nitrate controls in the antibacterial assay. Without these controls, it is not possible to determine whether the observed inhibition is due to the nanoparticles, residual silver ions, or the plant extract itself.

The manuscript lacks statistical analysis. There is no report of the number of replicates or the number of times the experiment is performed, with no statistical evaluation. Antibacterial zone diameters are presented as single values. Without replication and statistical validation, the biological conclusions are not reliable. Statistical analysis must be included, with clearly stated experimental replication (minimum n=3 recommended).

The reported concentrations (60%, 80%, 100%) are not clearly defined in terms of mg/mL or dilution ratios. Details regarding inoculum preparation, incubation conditions, and assay procedure are insufficient for reproducibility.

The manuscript inconsistently refers to silver oxide and silver nanoparticles. The XRD data require clearer indexing and discussion to confirm whether the product is AgO, Ag₂O, or metallic Ag. Extremely inconsistent crystallite sizes are reported (e.g., 905.6 nm for one peak).

Additionally, the reaction conditions (heating aqueous extract to 140°C) require clarification, as water boils at 100°C under atmospheric pressure.

The claim that no datasets were generated is inaccurate. Raw XRD data, SEM images, and antibacterial measurements should be made available to ensure transparency and reproducibility.

In its current form, the manuscript contains significant methodological, analytical, and reporting deficiencies that affect the scientific validity and reproducibility of the work. The lack of appropriate antibacterial controls, absence of statistical analysis, insufficient nanoparticle characterization, ambiguity in phase identification, and unclear methodological details collectively prevent reliable interpretation of the results.

Given these substantial concerns, I regret to recommend rejection of the manuscript at this stage. The study may be reconsidered for indexing only after major experimental revisions, comprehensive data analysis, and thorough improvement in scientific presentation are undertaken.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Nanotechnology

10.5256/f1000research.191018.r453609

Reviewer response for version 1

Yousef

Abdullah

1 Referee https://orcid.org/0000-0002-4519-0034 1Al-Ryada University for Science and Technology, Sadat City, Egypt

Competing interests: No competing interests were disclosed.

16 2 2026

2026

recommendation

reject

This manuscript describes the green synthesis of silver oxide nanoparticles using aqueous extract of Boswellia carterii as a reducing and stabilizing agent. The synthesized nanoparticles were characterized using XRD and SEM, and their antibacterial activity was tested against Pseudomonas aeruginosa and Streptococcus species. The authors report crystallite sizes around 80.6 nm and inhibition zones ranging from 14.5 to 23 mm, suggesting promising antibacterial potential.

The topic is relevant to green nanotechnology and antimicrobial nanomaterials. However, several methodological, analytical, and reporting issues must be addressed before the work can be considered scientifically sound.

Major Comments (Must Be Addressed)

1. Lack of Proper Controls in Antibacterial Testing (Critical)

No mention of:

Positive control (standard antibiotic such as gentamicin or ampicillin)

Negative control (extract alone, AgNO₃ alone)

Solvent control

Without proper controls, it is impossible to determine whether the observed inhibition is due to:

Silver oxide nanoparticles

Residual silver ions

Boswellia extract itself

This is a major methodological flaw and must be corrected.

2. No Statistical Analysis

No mention of:

Number of replicates

Mean ± SD

Statistical comparison between concentrations

Significance testing (ANOVA or t-test)

Reporting inhibition zone diameters without replication or statistical validation makes the biological conclusions weak.

Statistical analysis must be added.

3. Inadequate Characterization of Nanoparticles

Only:

XRD

SEM

Missing critical analyses:

UV–Vis spectroscopy (to confirm nanoparticle formation)

FTIR (to confirm capping by plant extract)

EDX (elemental confirmation of Ag and O)

DLS (size distribution in suspension)

Zeta potential (stability)

For a nanomaterial synthesis study, this characterization is insufficient.

4. Ambiguity Between Silver Oxide and Silver Nanoparticles

The manuscript repeatedly alternates between:

Silver oxide nanoparticles

Silver nanoparticles

XRD data claim orthorhombic AgO (JCPDS 84-1547), but:

No full diffraction pattern shown

No peak indexing table

No phase purity confirmation

The authors must clarify:

Is the product Ag₂O, AgO, or metallic Ag?

Provide indexed peaks with hkl values.

5. Data Availability Statement Is Incorrect

The manuscript states:

"No datasets were created or analyzed during the current study."

This is scientifically incorrect.

The study clearly generated:

XRD data

SEM images

Antibacterial measurements

Raw data must be provided or deposited.

6. Language and Scientific Writing Issues

Numerous:

Grammatical errors

Repetitions (mechanism section repeated twice)

Typographical errors (e.g., Psedumonas, Striptococci, caretterii)

Inconsistent naming of bacteria

Extensive English editing is required.

Minor Comments

Provide bacterial strain numbers (ATCC or clinical isolate IDs).

Clarify incubation conditions for antibacterial assay.

Specify agar diffusion method (Kirby–Bauer?).

Indicate nanoparticle concentration in mg/mL.

Remove redundant explanation of Streptococcus taxonomy (not essential).

Correct plant species spelling throughout.

Scientific Strengths

Environmentally friendly approach.

Use of natural biopolymer.

SEM size within nanoscale.

Demonstrated antibacterial activity.

Recommendation

The manuscript requires major revisions before it can be considered scientifically sound.

The following points must be addressed:

Add proper antibacterial controls

Include statistical analysis

Provide raw data

Clarify nanoparticle phase

Improve characterization

Correct language

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Are all the source data underlying the results available to ensure full reproducibility?

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

ecause:There are mandatory scientific flaws (not cosmetic issues).Antibacterial testing lacks controls.No statistical validation.Insufficient characterization.Raw data unavailable.Phase identity (AgO vs AgNPs) unclear.These are core validity issues, not minor or moderate revisions.

Aziz

Leqaa

Microbioligy, University of Fallujah, Al-Fallujah, Al Anbar Governorate, Iraq

Competing interests: The authors declare that there are no conflicts of interest. This has been stated in the research.

13 3 2026

Dear Esteemed reviewer, on behalf of the research team . I would like to thank you for your valuable guidance in presenting the research with the highest scientific value and in the best possible way. Therefore, I would like to clarify some points you raised

The plant extract was used as a negative control, a crucial step in the green synthesis studies, to ensure that the biological activity was not attributable to the extract's components themselves. The extract showed no inhibition zone under the same testing conditions.

References:

Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of antimicrobials. Biotechnol Adv. 2009;27(1):76–83.

Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramírez JT, et al. The bactericidal effect of silver nanoparticles. Nanotechnology. 2005;16(10):2346–53.

Xiu ZM, Zhang QB, Puppala HL, Colvin VL, Alvarez PJ. Negligible particle-specific antibacterial activity of silver nanoparticles. Nano Lett. 2012;12(8):4271–5.

2. Each concentration was replicated twice under identical incubation conditions, and the diameters of the inhibition zones were measured with a digital measuring foot calibrated to ±0.1 mm. Given the nature of diffusion in the solid medium and the technique for visually identifying the edge of the inhibition zone, the variation between replicates did not surpass ±0.5 mm, falling within the expected limits of uncertainty for the agar diffusion test. This degree of variation is within the typical systematic error of the test and does not suggest significant biological variability.

Since there were no notable morphological differences between the plates that would require additional comparative documentation, and because the recorded differences were small and within the previously stated standard error margin of measurement, original photographs of the replicates were not kept for visual documentation. Analysis was based on digital measurements taken at the time of the experiment.

References

Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal. 2016;6(2):71–79.

CLSI. Performance standards for antimicrobial disk susceptibility tests. 13 ^th ed. CLSI standard M02. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.

3. The goal of this study was to confirm the crystalline phase and assess the biological activity rather than perform a comprehensive physical characterization. The crystal size was estimated using the Scherrer equation, a well-established technique for determining the crystalline structure of nanomaterials, and the crystalline phase and its conformation to a common reference were directly identified by XRD analysis (1,2).

. The nanostructure and particle distribution were also validated by SEM.

XRD remained the final method for verifying the crystalline phase, while UV-Vis analysis was carried out as a preliminary test to deduce nanoparticle formation from the distinctive absorption band. However, because it was a supporting preliminary procedure, it was left out of the first version. The UV-Vis spectrum acquired during preparation is displayed in the accompanying image.

Two absorption bands in the UV-Vis spectrum, at 371 and 425 nm, show that silver oxide nanoparticles are going through energy gap transitions (4,5).Even though UV-Vis is not a reliable way to find out what the crystalline phase is, these results are in line with what has been reported in the literature about how silver oxide nanoparticles behave optically. Conversely, metallic silver particles typically exhibit a characteristic surface plasmonic resonance band within the 400–450 nm range (6). So, XRD is still the best way to confirm the crystalline phase, and UV-Vis analysis gives more information.

References

Cullity BD, Stock SR. Elements of X-ray Diffraction. 3rd ed. Upper Saddle River: Prentice Hall; 2001.

Klug HP, Alexander LE. X-ray Diffraction Procedures. 2nd ed. New York: Wiley; 1974.

Mourdikoudis S, Pallares RM, Thanh NTK. Characterization techniques for nanoparticles: comparison and complementarity upon studying nanoparticle properties. Nanoscale. 2018;10(27):12871–12934.

Arbuj SS, Mulik UP, Amalnerkar DP. Preparation, characterization and optical properties of Ag2O nanoparticles. Mater Sci Eng B. 2011;176(16):1173–1179.

Bhosale MA, Bhanage BM. Silver oxide nanoparticles: synthesis and characterization. Mater Chem Phys. 2015;155:114–120.

Kelly KL, Coronado E, Zhao LL, Schatz GC. The optical properties of metal nanoparticles: the influence of size, shape, and dielectric environment. J Phys Chem B. 2003;107(3):668–677.

4. The JCPDS card (84-1547) says that the XRD data that fits the orthorhombic phase shows that the material made is silver oxide. The term "silver particles" was used in the text because the terminology was not consistent, not because there was any doubt about the phase identification.

5. On reasonable request, the corresponding author will provide the original SEM micrographs, raw XRD data files, and inhibition zone measurement datasets produced during this investigation.

6.The linguistic problems identified are editorial in nature and relate to the coordination and consistency of scientific nomenclature. It has no impact on the scientific validity of the study, its methodology, or the interpretation of the data.

We appreciate the insightful observations and scientific evaluation that contributed to enhancing the clarity and quality of the study.