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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Pomegranate Rind-assisted Green Synthesis of ZnO Nanoparticles: Characterization, Antioxidant Activity and Antibacterial Potential against Bacterial Fish Disease

P
Prathisha Rajamani1,*
C
Chrisolite Bagthasingh1,*
S
Sivasankar Panchavarnam1
V
V. Rani2
R
Rajendran Shalini3
K
K.S. Vijay Amirtharaj4
E
Evangelin Paripoorana David1
1Department of Fish Pathology and Health Management, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi-628 008, Tamil Nadu, India.
2Dr. M.G.R. Fisheries College and Research Institute, Thalainayeru-614 712, Tamil Nadu, India.
3Department of Fish Quality Assurance and Management, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi-628 008, Tamil Nadu, India.
4Kanyakumari Ganapathipuram Centre for Sustainable Aquaculture, Directorate of Sustainable Aquaculture, Ganapathipuram-629 502, Kanyakumari, Tamil Nadu, India.

ABSTRACT

Background: Nanoparticles have gained attention in animal health for their applications as feed additives, antimicrobials, vaccine adjuvants and immunostimulants. This study aims to explore their potential as an eco-friendly alternative to conventional antibiotics in aquaculture.

Methods: In this study, zinc oxide nanoparticles were synthesized from pomegranate rind (P-ZnO NPs), characterized using UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Field Emission Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (FESEM-EDS) and their antibacterial and antioxidant activities were evaluated by well diffusion and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assays.

Result: P-ZnO NPs exhibited a spherical morphology with an average size of 84.15 nm, strong antioxidant activity (IC50 = 98.48±0.89 µg/mL) and significant antibacterial effects against Photobacterium damselae subsp. damselae (MIC = 6.25 µg/mL), P. damselae subsp. piscicida and Aeromonas hydrophila (MIC = 3.12 µg/mL). These results highlight pomegranate rind mediated ZnO nanoparticles as a green and effective approach for controlling major aquaculture pathogens.

INTRODUCTION

Nanotechnology emerged as a significant sector in science during the early 21st century, with numerous applications in the agriculture, environment, biomedicine and food safety particularly as food additives and antimicrobials (Kavanashree et al., 2024; Altammar, 2023). In aquaculture, the increased use of antimicrobials has led to the development of antimicrobial resistant genes in fish bacterial pathogens. This indiscriminate use not only contributes to antimicrobial resistance (AMR) but also results in the accumulation of antibiotic residues in fish (Chen et al., 2020), leaching of antibiotic residues into the environment which increase their presence in agricultural runoff, ultimately contributing to the development of antimicrobial resistance in human pathogens highlighting the broader impact of AMR in aquaculture (Miller and Harbottle, 2018).
       
Green synthesized nanoparticles are a promising alternative to antibiotics, especially plant-based methods are favored for their eco-friendliness and added antioxidant and antibacterial properties (Anjali et al., 2025; Sudeepta and Siddhartha, 2024; Keshari et al., 2020). Zinc, which is generally regarded as safe, is extensively used as an antimicrobial agent at both micro and nanoscale. When reduced to the nanoscale, its antimicrobial activity is enhanced due to its high surface area to volume ratio and unique bactericidal mechanisms. These include disruption of the bacterial cell membrane, release of Zn2+ ions and the formation of reactive oxygen species (ROS) (Jalal et al., 2010; Seil and Webster, 2012). Aquaculture, being a vital sector in the food industry, is expected to surpass wild catch production by 2028. However, this sector continues to face challenges due to the prevalence of bacterial pathogens (Miranda et al., 2016). In this context, green synthesized zinc oxide (ZnO) nanoparticles offer a promising alternative to antibiotics, with demonstrated antibacterial efficacy and low toxicity to human cells (Sirelkhatim et al., 2015).
       
Several in vitro studies have demonstrated the antibacterial activity of ZnO nanoparticles against both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria such as Salmonella paratyphi, Vibrio cholerae and Escherichia coli with the synthesized nanoparticles using Solanum nigrum (Ramesh et al., 2015). Many studies have documented the antibacterial effecet of ZnO-NPs against fish pathogens such as Aermonas hydrophila, Edwardseilla tarda, Flavobacterium branchiophilum, Citrobacter spp., Staphylococcus aureus, Vibrio species, Bacillus cereus and Pseudomonas aeruginosa (Shaalan et al., 2017). Pomegranate rind, a bio-waste product of the fruit, contains various bioactive compounds with proven antibacterial properties  (Siddiqui et al., 2024). In this study, zinc oxide nanoparticles were synthesized using pomegranate rind extract (P-ZnO NPs) and their potential antibacterial effects against major fish pathogenic bacterial species (marine and freshwater origin) were investigated.

MATERIALS AND METHODS

Green synthesis and characterization of nanoparticles
 
The nanoparticles were synthesized following Fouladi-Fard et al. (2022); Naiel et al., (2022) and Tilahun et al., (2023)  with slight modifications. P-ZnO NPs were characterized by UV–Vis spectrophotometer (280-800 nm), FTIR (JASCO FT/IR-6600 Type A spectrometer ,Tokyo, Japan; Serial No: B049361790) (Velmurugan et al., 2024), XRD (XPERT-PRO diffractometer, PANalytical, Netherlands) and crystallite size was calculated using the Debye–Scherrer equation (Esther and Kumar, 2024). Morphology and particle size and elemental composition were examined by FESEM-EDS (TESCAN MIRA3 XMU FE-SEM, Czech Republic) (Abazari et al., 2023).
 
Antioxidant assay
 
The antioxidant activity of P-ZnO NPs was evaluated using the DPPH assay (Sisco Research Laboratories Pvt. Ltd.) following Hameed et al., (2023). Different concentrations of nanoparticles (10-750 µg/mL) and Vitamin C (1 mg/mL, standard) were mixed with DPPH solution (800 µg/mL in methanol) to a final volume of 1.5 mL and incubated in the dark. Absorbance was measured at 517 nm and the percentage scavenging activity was calculated as follows:


Where,
A0= control absorbance.
A1= Sample absorbance.
Results were compared with Vitamin C (Baliyan et al., 2022; Boopathi et al., 2024).

Well diffusion assay
 
Three pathogenic bacterial strains; Photobacterium damselae subsp. damselae (Pdd), Photobacterium damselae subsp. piscicida (Pdp) and Aeromonas hydrophila (Ah) maintained at the Department of Fish Pathology and Health Management, Fisheries College and Research Institute (FCandRI), Thoothukudi were used in this study. These isolates were subsequently inoculated in fresh TSB and incubated overnight. The bacterial density was standardized to 0.5 McFarland standard (approximately 1.2 × 107 cfu/ml) and the suspensions were evenly swabbed onto Mueller-Hinton Agar (MHA-HIMEDA) plates. Wells of approximately 6 mm diameter were made using a sterile cork borer. P-ZnO NPs were prepared in dimethyl sulfoxide (DMSO) at concentrations of 20, 40, 60 and 80 µg/mL and added to the wells. The plates were incubated at 37°C for 24 hours, after which the zones of inhibition were measured in millimeters (Balouiri et al., 2016).
 
MIC and MBC determination
 
The MIC and MBC of P-ZnO NPs were determined by broth microdilution following CLSI (2018) guidelines (Kakian et al., 2024). A stock solution (25 µg/mL) in Mueller Hinton Broth (MHB) was serially diluted (25-0.195 µg/mL) in 96-well plates containing bacterial suspensions (~10w  cfu/ml). Controls included sterility (MHB only) and growth (MHB with inoculum). Plates were incubated at 37°C for 24 h and MIC was recorded as the lowest concentration without visible turbidity. For MBC, 50 µL from clear wells were spread on MHA and incubated at 37°C for 24 h; the lowest concentration showing no colonies was taken as the MBC.

RESULTS AND DISCUSSION

Green synthesis and characterization of the nanoparticles
 
The formation of nanoparticles was initially indicated by a visible color change in the reaction mixture (Fig 1). A transition from brown to a pale yellow precipitate suggested the successful synthesis of nanoparticles. The colloidal solution containing P-ZnO NPs exhibited a prominent peak at 375 nm (Fig 2). FTIR showed prominent many absorption bands (Fig 3). The broad band at 3506.06 cm{ ¹ corresponds to O-H stretching vibrations, indicating the presence of phenolic compounds. The peaks at 2923.59 cm-1 and 2853.24 cm-1 are attributed to the asymmetric and symmetric stretching vibrations of -CH2 groups indicating the presence of aliphatic compounds. The absorption at 2358.57 cm-1 is associated with O=C=O stretching vibrations. The band at 1745 cm-1 corresponds to the C=O stretching of carboxyl functional groups. Peaks at 1630.55 cm-1 and 1457.30 cm-1 are indicative of N–H bending vibrations. The strong band at 1114.80 cm-1 is assigned with the possible metal oxygen (Zn-O) bonding, resulting in the formation of ZnO. The bands at 988.42 cm-1, 886.27 cm-1 and 792.28 cm-1 correspond to C-H bending vibrations of aldehydes or aromatic compounds. The sharp band at 618.52 cm-1 is due C-Cl stretching vibrations. The bands at 518.50 cm-1 and 464.93 cm-1 fall within the characteristic range of Zn-O stretching vibrations, confirming the successful synthesis of ZnO nanoparticles. The XRD diffraction profile of the synthesized nanoparticles exhibited distinct peak positions at 2θ values of 32.18°, 34.84°, 36.64°, 47.96°, 57.00°, 63.26°, 66.76°, 68.37°, 69.66°, 72.93° and 77.33°. These peaks were indexed to the (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) in good agreement with the standard diffraction data reported in JCPDS file No. 36-1451 (Fig 4). The average crystallite size of the ZnO nanoparticles was found to be approximately 24.4 nm. The SEM images revealed spherical-shaped nanoparticles with an average particle size of 82.15 nm (ImageJ.exe software) (Fig 5). The EDS spectrum showed zinc, oxygen and carbon weight percentage as 73.53% 18.65% and 7.82% respectively. The EDS peaks were observed between 1 and 10 keV, with a prominent zinc peak at approximately 1 keV and another at 8.6 keV, along with an oxygen peak around 0.5 keV (Fig 5).

Fig 1: (A) Represents the crude pomegranate rind extract after the addition of zinc nitrate hexaydrate (B) Shows the color change following P-ZnO NPs synthesis, with the precipitated nanoparticles settled at the bottom.



Fig 2: UV absorption spectra of the P-ZnO NPs.



Fig 3: FTIR of the P-ZnO NPs.



Fig 4: X-ray diffraction (XRD) pattern of P-ZnO NPs and standard JCPDS card No. 36-1451.



Fig 5: (A) FESEM image of P-ZnO NPs shows well-dispersed spherical nanoparticles (B) Average particle size of 32.4 nm, (C) EDS of P-ZnO NPs confirms the elemental composition: Zn 73.53%, O-18.65% and C-7.82%.


 
Antioxidant activity
 
DPPH assay revealed dose-dependent increase in scavenging activity with increasing concentrations of nanoparticles (10, 50, 150, 250, 350, 500 and 750 µg/mL). The IC50 value of the synthesized nanoparticles was 98.48±0.89 µg/mL, whereas the IC50 value for Vitamin C was 2.33±1.06 µg/ mL (Fig 6).

Fig 6: DPPH radical scavenging assay of the P-ZnO NPs (A) and Vitamin C (B).


 
Antibacterial activity
 
The antibacterial efficacy of P-ZnO NPs was assessed against three pathogenic bacteria Pdd, Pdp and Ah using the agar well diffusion assay and microbroth dilution assay. Wells of 6 mm diameter were created on Mueller-Hinton Agar (MHA) plates, into which P-ZnO NPs at concentrations of 20, 40, 60 and 80 µg/ml were applied. Cefixime was used as a positive control for Pdd and Pdp and ofloxacin for Ah. The synthesized P-ZnO NPs exhibited clear dose-dependent antibacterial activity against all three pathogens in well diffusion assay (Table 1, Fig 7) and MIC and MBC values were also calculated (Table 2).

Table 1: Mean Zone of Inhibition (mm±SD) for different bacteria at varying P-ZnO NPs concentrations.



Fig 7: (A) and (B) ZOI for Pdd, Pdp and Ah against P-ZnO NPs.



Table 2: MIC and MBC values of P-ZnO-NPs against aquaculture pathogens.


       
Nanoparticles are recognized as potent therapeutic and prophylactic agents due to their nanoscale size (Wang et al., 2016). Their antibacterial action involves ROS generation, membrane disruption and inhibition of electron transport or efflux pumps (Aflakian et al., 2023). In this study, ZnO nanoparticles were green-synthesized from pomegranate rind waste rich in bioactive compounds with antibacterial properties (Alexandre et al., 2019; Chen et al., 2020; Siddiqui et al., 2024) providing a sustainable route for nanoparticle production (Suresh et al., 2015).
       
Characterization confirmed the successful synthesis of P-ZnO NPs with UV-Vis absorption peak at 375 nm (Sharma et al., 2025). The FTIR spectrum of P-ZnO NPs revealed the presence of various functional groups in the reduction and stabilization of nanoparticles (Jayachandran et al., 2021; Lakshmi et al., 2024; Monika et al., 2024; Golzarnezhad et al., 2025; Krithika et al., 2021). SEM images showed spherical nanoparticles with an average size of 82.15 nm, while EDS confirmed elemental composition (Zn: 73.53%, O: 18.65%, C: 7.82%) (Saravanan et al., 2025), where carbon likely originated from phytochemical capping agents (Jayappa et al., 2020). The P-ZnO NPs exhibited strong antioxidant activity with an IC50 value of 98.48±0.89 µg/mL. Comparable studies with pomegranate rind-mediated ZnO NPs reported an IC50 of 124 µg/mL in barley seed germination assays (Shaban et al., 2024), suggesting that the lower IC50 in the present study reflects a comparatively higher antioxidant capacity.
       
Pdd is a marine opportunistic pathogen with zoonotic significance, infecting diverse fish species, while Pdp, derived from Pdd through gene loss, is linked to major outbreaks (Baseggio et al., 2022; Andreoni and Magnani, 2014). Ah is a virulent facultative anaerobe in carps with zoonotic potential (Semwal et al., 2023). Well diffusion assays showed dose-dependent inhibition, with AH most sensitive (20.0±0.0 mm at 80 µg/mL), followed by Pdd (18.0±0.0 mm) and Pdp (16.0±0.0 mm). MIC/MBC assays confirmed efficacy, with values of 6.25/12.5 µg/mL for Pdd and 3.12/6.25 µg/mL for both Pdp and Ah (Dube, 2024). These results are comparable to previous studies where CuO nanoparticles (100 µg/mL) produced inhibition zones of 17-21 mm against Pseudomonas fluorescens, Vibrio parahaemolyticus and Flavobacterium branchiophilum (Kumar et al., 2015). Similarly, Gum Arabic-silver nanoparticles inhibited A. hydrophila and P. aeruginosa with MIC values of 1.625 and 3.25 µg/mL, respectively. MIC and MBC assays further supported the antibacterial efficacy of P-ZnO NPs. Pdd showed MIC and MBC values of 6.25 and 12.5 µg/mL, respectively, while both Pdp and Ah exhibited MIC of 3.12 µg/mL and MBC of 6.25 µg/mL. Several studies have reported nanoparticles effectiveness against diverse fish pathogens including Vibrio harveyi, Edwardsiella tarda, Flavobacterium branchiophilum, Citrobacter spp., Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa (Shaalan et al., 2017). The rising concern of antibiotic resistance in aquaculture is notable, as tetracycline use in fish farms has been linked to resistant strains. For instance, tetracycline, widely used in fish farms, has been linked to resistant bacterial strains (Shaalan et al., 2017; Tuševljak et al., 2013). Also resistance in fish pathogens such as Aeromonas salmonicida, Photobacterium, Yersinia ruckeri, Vibrio spp., Listeria, Pseudomonas and Edwardsiella poses risks of resistant gene transfer to human pathogens (Sørum, 2008; Swain et al., 2014). Thus Pomegranate rind mediated ZnO nanoparticles (P-ZnO NPs) offer an ecofriendly and sustainable antibacterial alternative in aquaculture.

CONCLUSION

These findings demonstrate the strong antioxidant (IC50 = 98.48±0.89 µg/mL) and bactericidal activity of P-ZnO NPs at low concentrations, with MIC values of 3.12-6.25 µg/mL against marine (Pdd and Pdp) and freshwater (Ah) pathogens. Hence, eco-friendly green-synthesized nanoparticles are gaining prominence in aquaculture as sustainable alternatives to conventional antimicrobials.

ACKNOWLEDGEMENT

The authors acknowledge the financial support received from PMMSY-ICAR, National Bureau of Fish Genetic Resources, under the NSPAAD Sub Project No. 19. We are also grateful to Dr. J. Jayalalithaa Fisheries University for providing the necessary research facilities to conduct this work. This study constitutes a part of the doctoral thesis of the first author.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent/ethical approval
 
This study did not involve any live animals or human participants. All experiments were performed using in vitro antibacterial and antioxidant assays and hence no ethical approval was required.
 
Animal ethics
 
No live animals were used in this study. All experiments were carried out using bacterial cultures only.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

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    Pomegranate Rind-assisted Green Synthesis of ZnO Nanoparticles: Characterization, Antioxidant Activity and Antibacterial Potential against Bacterial Fish Disease

    P
    Prathisha Rajamani1,*
    C
    Chrisolite Bagthasingh1,*
    S
    Sivasankar Panchavarnam1
    V
    V. Rani2
    R
    Rajendran Shalini3
    K
    K.S. Vijay Amirtharaj4
    E
    Evangelin Paripoorana David1
    1Department of Fish Pathology and Health Management, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi-628 008, Tamil Nadu, India.
    2Dr. M.G.R. Fisheries College and Research Institute, Thalainayeru-614 712, Tamil Nadu, India.
    3Department of Fish Quality Assurance and Management, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi-628 008, Tamil Nadu, India.
    4Kanyakumari Ganapathipuram Centre for Sustainable Aquaculture, Directorate of Sustainable Aquaculture, Ganapathipuram-629 502, Kanyakumari, Tamil Nadu, India.

    ABSTRACT

    Background: Nanoparticles have gained attention in animal health for their applications as feed additives, antimicrobials, vaccine adjuvants and immunostimulants. This study aims to explore their potential as an eco-friendly alternative to conventional antibiotics in aquaculture.

    Methods: In this study, zinc oxide nanoparticles were synthesized from pomegranate rind (P-ZnO NPs), characterized using UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Field Emission Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (FESEM-EDS) and their antibacterial and antioxidant activities were evaluated by well diffusion and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assays.

    Result: P-ZnO NPs exhibited a spherical morphology with an average size of 84.15 nm, strong antioxidant activity (IC50 = 98.48±0.89 µg/mL) and significant antibacterial effects against Photobacterium damselae subsp. damselae (MIC = 6.25 µg/mL), P. damselae subsp. piscicida and Aeromonas hydrophila (MIC = 3.12 µg/mL). These results highlight pomegranate rind mediated ZnO nanoparticles as a green and effective approach for controlling major aquaculture pathogens.

    INTRODUCTION

    Nanotechnology emerged as a significant sector in science during the early 21st century, with numerous applications in the agriculture, environment, biomedicine and food safety particularly as food additives and antimicrobials (Kavanashree et al., 2024; Altammar, 2023). In aquaculture, the increased use of antimicrobials has led to the development of antimicrobial resistant genes in fish bacterial pathogens. This indiscriminate use not only contributes to antimicrobial resistance (AMR) but also results in the accumulation of antibiotic residues in fish (Chen et al., 2020), leaching of antibiotic residues into the environment which increase their presence in agricultural runoff, ultimately contributing to the development of antimicrobial resistance in human pathogens highlighting the broader impact of AMR in aquaculture (Miller and Harbottle, 2018).
           
    Green synthesized nanoparticles are a promising alternative to antibiotics, especially plant-based methods are favored for their eco-friendliness and added antioxidant and antibacterial properties (Anjali et al., 2025; Sudeepta and Siddhartha, 2024; Keshari et al., 2020). Zinc, which is generally regarded as safe, is extensively used as an antimicrobial agent at both micro and nanoscale. When reduced to the nanoscale, its antimicrobial activity is enhanced due to its high surface area to volume ratio and unique bactericidal mechanisms. These include disruption of the bacterial cell membrane, release of Zn2+ ions and the formation of reactive oxygen species (ROS) (Jalal et al., 2010; Seil and Webster, 2012). Aquaculture, being a vital sector in the food industry, is expected to surpass wild catch production by 2028. However, this sector continues to face challenges due to the prevalence of bacterial pathogens (Miranda et al., 2016). In this context, green synthesized zinc oxide (ZnO) nanoparticles offer a promising alternative to antibiotics, with demonstrated antibacterial efficacy and low toxicity to human cells (Sirelkhatim et al., 2015).
           
    Several in vitro studies have demonstrated the antibacterial activity of ZnO nanoparticles against both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria such as Salmonella paratyphi, Vibrio cholerae and Escherichia coli with the synthesized nanoparticles using Solanum nigrum (Ramesh et al., 2015). Many studies have documented the antibacterial effecet of ZnO-NPs against fish pathogens such as Aermonas hydrophila, Edwardseilla tarda, Flavobacterium branchiophilum, Citrobacter spp., Staphylococcus aureus, Vibrio species, Bacillus cereus and Pseudomonas aeruginosa (Shaalan et al., 2017). Pomegranate rind, a bio-waste product of the fruit, contains various bioactive compounds with proven antibacterial properties  (Siddiqui et al., 2024). In this study, zinc oxide nanoparticles were synthesized using pomegranate rind extract (P-ZnO NPs) and their potential antibacterial effects against major fish pathogenic bacterial species (marine and freshwater origin) were investigated.

    MATERIALS AND METHODS

    Green synthesis and characterization of nanoparticles
     
    The nanoparticles were synthesized following Fouladi-Fard et al. (2022); Naiel et al., (2022) and Tilahun et al., (2023)  with slight modifications. P-ZnO NPs were characterized by UV–Vis spectrophotometer (280-800 nm), FTIR (JASCO FT/IR-6600 Type A spectrometer ,Tokyo, Japan; Serial No: B049361790) (Velmurugan et al., 2024), XRD (XPERT-PRO diffractometer, PANalytical, Netherlands) and crystallite size was calculated using the Debye–Scherrer equation (Esther and Kumar, 2024). Morphology and particle size and elemental composition were examined by FESEM-EDS (TESCAN MIRA3 XMU FE-SEM, Czech Republic) (Abazari et al., 2023).
     
    Antioxidant assay
     
    The antioxidant activity of P-ZnO NPs was evaluated using the DPPH assay (Sisco Research Laboratories Pvt. Ltd.) following Hameed et al., (2023). Different concentrations of nanoparticles (10-750 µg/mL) and Vitamin C (1 mg/mL, standard) were mixed with DPPH solution (800 µg/mL in methanol) to a final volume of 1.5 mL and incubated in the dark. Absorbance was measured at 517 nm and the percentage scavenging activity was calculated as follows:


    Where,
    A0= control absorbance.
    A1= Sample absorbance.
    Results were compared with Vitamin C (Baliyan et al., 2022; Boopathi et al., 2024).

    Well diffusion assay
     
    Three pathogenic bacterial strains; Photobacterium damselae subsp. damselae (Pdd), Photobacterium damselae subsp. piscicida (Pdp) and Aeromonas hydrophila (Ah) maintained at the Department of Fish Pathology and Health Management, Fisheries College and Research Institute (FCandRI), Thoothukudi were used in this study. These isolates were subsequently inoculated in fresh TSB and incubated overnight. The bacterial density was standardized to 0.5 McFarland standard (approximately 1.2 × 107 cfu/ml) and the suspensions were evenly swabbed onto Mueller-Hinton Agar (MHA-HIMEDA) plates. Wells of approximately 6 mm diameter were made using a sterile cork borer. P-ZnO NPs were prepared in dimethyl sulfoxide (DMSO) at concentrations of 20, 40, 60 and 80 µg/mL and added to the wells. The plates were incubated at 37°C for 24 hours, after which the zones of inhibition were measured in millimeters (Balouiri et al., 2016).
     
    MIC and MBC determination
     
    The MIC and MBC of P-ZnO NPs were determined by broth microdilution following CLSI (2018) guidelines (Kakian et al., 2024). A stock solution (25 µg/mL) in Mueller Hinton Broth (MHB) was serially diluted (25-0.195 µg/mL) in 96-well plates containing bacterial suspensions (~10w  cfu/ml). Controls included sterility (MHB only) and growth (MHB with inoculum). Plates were incubated at 37°C for 24 h and MIC was recorded as the lowest concentration without visible turbidity. For MBC, 50 µL from clear wells were spread on MHA and incubated at 37°C for 24 h; the lowest concentration showing no colonies was taken as the MBC.

    RESULTS AND DISCUSSION

    Green synthesis and characterization of the nanoparticles
     
    The formation of nanoparticles was initially indicated by a visible color change in the reaction mixture (Fig 1). A transition from brown to a pale yellow precipitate suggested the successful synthesis of nanoparticles. The colloidal solution containing P-ZnO NPs exhibited a prominent peak at 375 nm (Fig 2). FTIR showed prominent many absorption bands (Fig 3). The broad band at 3506.06 cm{ ¹ corresponds to O-H stretching vibrations, indicating the presence of phenolic compounds. The peaks at 2923.59 cm-1 and 2853.24 cm-1 are attributed to the asymmetric and symmetric stretching vibrations of -CH2 groups indicating the presence of aliphatic compounds. The absorption at 2358.57 cm-1 is associated with O=C=O stretching vibrations. The band at 1745 cm-1 corresponds to the C=O stretching of carboxyl functional groups. Peaks at 1630.55 cm-1 and 1457.30 cm-1 are indicative of N–H bending vibrations. The strong band at 1114.80 cm-1 is assigned with the possible metal oxygen (Zn-O) bonding, resulting in the formation of ZnO. The bands at 988.42 cm-1, 886.27 cm-1 and 792.28 cm-1 correspond to C-H bending vibrations of aldehydes or aromatic compounds. The sharp band at 618.52 cm-1 is due C-Cl stretching vibrations. The bands at 518.50 cm-1 and 464.93 cm-1 fall within the characteristic range of Zn-O stretching vibrations, confirming the successful synthesis of ZnO nanoparticles. The XRD diffraction profile of the synthesized nanoparticles exhibited distinct peak positions at 2θ values of 32.18°, 34.84°, 36.64°, 47.96°, 57.00°, 63.26°, 66.76°, 68.37°, 69.66°, 72.93° and 77.33°. These peaks were indexed to the (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) in good agreement with the standard diffraction data reported in JCPDS file No. 36-1451 (Fig 4). The average crystallite size of the ZnO nanoparticles was found to be approximately 24.4 nm. The SEM images revealed spherical-shaped nanoparticles with an average particle size of 82.15 nm (ImageJ.exe software) (Fig 5). The EDS spectrum showed zinc, oxygen and carbon weight percentage as 73.53% 18.65% and 7.82% respectively. The EDS peaks were observed between 1 and 10 keV, with a prominent zinc peak at approximately 1 keV and another at 8.6 keV, along with an oxygen peak around 0.5 keV (Fig 5).

    Fig 1: (A) Represents the crude pomegranate rind extract after the addition of zinc nitrate hexaydrate (B) Shows the color change following P-ZnO NPs synthesis, with the precipitated nanoparticles settled at the bottom.



    Fig 2: UV absorption spectra of the P-ZnO NPs.



    Fig 3: FTIR of the P-ZnO NPs.



    Fig 4: X-ray diffraction (XRD) pattern of P-ZnO NPs and standard JCPDS card No. 36-1451.



    Fig 5: (A) FESEM image of P-ZnO NPs shows well-dispersed spherical nanoparticles (B) Average particle size of 32.4 nm, (C) EDS of P-ZnO NPs confirms the elemental composition: Zn 73.53%, O-18.65% and C-7.82%.


     
    Antioxidant activity
     
    DPPH assay revealed dose-dependent increase in scavenging activity with increasing concentrations of nanoparticles (10, 50, 150, 250, 350, 500 and 750 µg/mL). The IC50 value of the synthesized nanoparticles was 98.48±0.89 µg/mL, whereas the IC50 value for Vitamin C was 2.33±1.06 µg/ mL (Fig 6).

    Fig 6: DPPH radical scavenging assay of the P-ZnO NPs (A) and Vitamin C (B).


     
    Antibacterial activity
     
    The antibacterial efficacy of P-ZnO NPs was assessed against three pathogenic bacteria Pdd, Pdp and Ah using the agar well diffusion assay and microbroth dilution assay. Wells of 6 mm diameter were created on Mueller-Hinton Agar (MHA) plates, into which P-ZnO NPs at concentrations of 20, 40, 60 and 80 µg/ml were applied. Cefixime was used as a positive control for Pdd and Pdp and ofloxacin for Ah. The synthesized P-ZnO NPs exhibited clear dose-dependent antibacterial activity against all three pathogens in well diffusion assay (Table 1, Fig 7) and MIC and MBC values were also calculated (Table 2).

    Table 1: Mean Zone of Inhibition (mm±SD) for different bacteria at varying P-ZnO NPs concentrations.



    Fig 7: (A) and (B) ZOI for Pdd, Pdp and Ah against P-ZnO NPs.



    Table 2: MIC and MBC values of P-ZnO-NPs against aquaculture pathogens.


           
    Nanoparticles are recognized as potent therapeutic and prophylactic agents due to their nanoscale size (Wang et al., 2016). Their antibacterial action involves ROS generation, membrane disruption and inhibition of electron transport or efflux pumps (Aflakian et al., 2023). In this study, ZnO nanoparticles were green-synthesized from pomegranate rind waste rich in bioactive compounds with antibacterial properties (Alexandre et al., 2019; Chen et al., 2020; Siddiqui et al., 2024) providing a sustainable route for nanoparticle production (Suresh et al., 2015).
           
    Characterization confirmed the successful synthesis of P-ZnO NPs with UV-Vis absorption peak at 375 nm (Sharma et al., 2025). The FTIR spectrum of P-ZnO NPs revealed the presence of various functional groups in the reduction and stabilization of nanoparticles (Jayachandran et al., 2021; Lakshmi et al., 2024; Monika et al., 2024; Golzarnezhad et al., 2025; Krithika et al., 2021). SEM images showed spherical nanoparticles with an average size of 82.15 nm, while EDS confirmed elemental composition (Zn: 73.53%, O: 18.65%, C: 7.82%) (Saravanan et al., 2025), where carbon likely originated from phytochemical capping agents (Jayappa et al., 2020). The P-ZnO NPs exhibited strong antioxidant activity with an IC50 value of 98.48±0.89 µg/mL. Comparable studies with pomegranate rind-mediated ZnO NPs reported an IC50 of 124 µg/mL in barley seed germination assays (Shaban et al., 2024), suggesting that the lower IC50 in the present study reflects a comparatively higher antioxidant capacity.
           
    Pdd     is a marine opportunistic pathogen with zoonotic significance, infecting diverse fish species, while Pdp, derived from Pdd through gene loss, is linked to major outbreaks (Baseggio et al., 2022; Andreoni and Magnani, 2014). Ah is a virulent facultative anaerobe in carps with zoonotic potential (Semwal et al., 2023). Well diffusion assays showed dose-dependent inhibition, with AH most sensitive (20.0±0.0 mm at 80 µg/mL), followed by Pdd (18.0±0.0 mm) and Pdp (16.0±0.0 mm). MIC/MBC assays confirmed efficacy, with values of 6.25/12.5 µg/mL for Pdd and 3.12/6.25 µg/mL for both Pdp and Ah (Dube, 2024). These results are comparable to previous studies where CuO nanoparticles (100 µg/mL) produced inhibition zones of 17-21 mm against Pseudomonas fluorescens, Vibrio parahaemolyticus and Flavobacterium branchiophilum (Kumar et al., 2015). Similarly, Gum Arabic-silver nanoparticles inhibited A. hydrophila and P. aeruginosa with MIC values of 1.625 and 3.25 µg/mL, respectively. MIC and MBC assays further supported the antibacterial efficacy of P-ZnO NPs. Pdd showed MIC and MBC values of 6.25 and 12.5 µg/mL, respectively, while both Pdp and Ah exhibited MIC of 3.12 µg/mL and MBC of 6.25 µg/mL. Several studies have reported nanoparticles effectiveness against diverse fish pathogens including Vibrio harveyi, Edwardsiella tarda, Flavobacterium branchiophilum, Citrobacter spp., Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa (Shaalan et al., 2017). The rising concern of antibiotic resistance in aquaculture is notable, as tetracycline use in fish farms has been linked to resistant strains. For instance, tetracycline, widely used in fish farms, has been linked to resistant bacterial strains (Shaalan et al., 2017; Tuševljak et al., 2013). Also resistance in fish pathogens such as Aeromonas salmonicida, Photobacterium, Yersinia ruckeri, Vibrio spp., Listeria, Pseudomonas and Edwardsiella poses risks of resistant gene transfer to human pathogens (Sørum, 2008; Swain et al., 2014). Thus Pomegranate rind mediated ZnO nanoparticles (P-ZnO NPs) offer an ecofriendly and sustainable antibacterial alternative in aquaculture.

    CONCLUSION

    These findings demonstrate the strong antioxidant (IC50 = 98.48±0.89 µg/mL) and bactericidal activity of P-ZnO NPs at low concentrations, with MIC values of 3.12-6.25 µg/mL against marine (Pdd and Pdp) and freshwater (Ah) pathogens. Hence, eco-friendly green-synthesized nanoparticles are gaining prominence in aquaculture as sustainable alternatives to conventional antimicrobials.

    ACKNOWLEDGEMENT

    The authors acknowledge the financial support received from PMMSY-ICAR, National Bureau of Fish Genetic Resources, under the NSPAAD Sub Project No. 19. We are also grateful to Dr. J. Jayalalithaa Fisheries University for providing the necessary research facilities to conduct this work. This study constitutes a part of the doctoral thesis of the first author.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent/ethical approval
     
    This study did not involve any live animals or human participants. All experiments were performed using in vitro antibacterial and antioxidant assays and hence no ethical approval was required.
     
    Animal ethics
     
    No live animals were used in this study. All experiments were carried out using bacterial cultures only.

    CONFLICT OF INTEREST

    The authors declare no conflicts of interest.

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