1 Introduction

Guava, sorghum, citrus and pearl millet are important crops because of their broad application in the manufacturing of animal feed and nourishment for human being. Guava has a high dietary fibre content, which helps reduce the risk of type 2 diabetes and relieve constipation. Sorghum is grown for animal feed and fodder as well as human nourishment and beverage. Citrus fruits are excellent source of vitamins required by human body and pearl millet significantly contributes to animal feed and human diet due to high level of fats, proteins, fibres and minerals. The primary challenge to the growing of these crops is the instability of yield resulting from pathogenic microorganisms. Anthracnose of guava caused by Colletotrichum gloesporioides and anthracnose of sorghum caused by Colletotrichum graminicola are the most catastrophic fungal diseases leads to loss in crop production. Citrus canker by Xanthomonas axonopodis and stem rot of Pearl millet caused by Klebsiella aerogenes are the worst bacterial diseases that leads to poor crop yield [1,2,3,4]. Nowadays, the majority of plant disease control strategies rely on hazardous pesticides and fungicides that may be harmful to the environment and public health [5].

The most recent strategy for managing plant diseases involves the use of NPs, which has the potential to be highly successful. Nanoparticles have been employed as antimicrobial agents against crop pathogens and have potential biological uses in biosensing and medication delivery [6]. Recently, there have been findings showing the possible use of nanoparticles in agricultural farming for managing plant diseases. Toxic reacting substances are used in chemical-based nanoparticle production as reducers, having a negative impact on ecosystems, particularly beneficial organisms [7]. However, nanoparticles made from biological sources are more economical and environmentally benign, because the synthesis process does not require harsh chemicals, high temperature and pressure. Current technologies, mainly nanotechnology, can recycle biodegradable trash into materials for human utilization [8].

Researchers are looking for ecological routes for nanotechnology by employing biodegradable trash as a raw material for nanomaterials preparation. Recently, there has been a growing interest among academics to investigate the medicinal properties of fruit waste, including peel and husk [9, 10]. Large amount of fruit wastes are produced by the food processing and wholesale markets. Also, because these organic wastes are very perishable, they provide a significant risk of environmental contamination [11]. By effectively using these fruit wastes, greenhouse gas emissions and carbon footprint may be decreased, which is beneficial for reaching sustainable development goals. Since many fruit wastes possess a large number of biologically active compounds, they are commonly experimented for bio-fabrication of nanomaterials due to their affordability and safe handling [12]. Among all nanomaterials, silver nanoparticles (AgNPs) have been studied most extensively and frequently because of their enormous biomedical applications.

Pomegranate (Punica granatum L.) peel is one of these fruit waste and possess essential nutrients and phytochemicals including proteins, polysaccharides, vitamins, phenolics and enzymes that may act as facilitating agent in the biosynthesis of nanomaterials. However, some phenolic compounds have been connected to the bioreduction of metal salts into 0 valence metal nanoparticles [13,14,15]. Pomegranate peel, rich source of polyphenolic phytochemicals, has been shown to exhibit significant antimicrobial and antioxidant activities [16].

Antimicrobial action of Pomegranate peel mediated AgNPs have been checked previously against Listeria monocytogenes, S. aureus, C. albicans, E. coli, Pseudomonas aeruginosa (ATCC 27584), Salmonella typhi (ATCC 14028), Proteus vulgaris (ATCC 8427), Staphylococcus aureus (ATCC 29213), Klebsiella pneumonia and Staphylococcus epidermidis (MTCC 3615), B. subtilis, R. oryzae, E. faecalis, A. flavus and Alternaria solani [17,18,19,20,21,22]. However, research on the production of AgNPs utilizing pomegranate peel for the evaluation of antibacterial activity against K. aerogenes, X. axonopodis, C. graminicola, and C. gloesporioides has not been extensively researched up till now. Fernandes et al. 2018 presented pomegranate peel mediated biosynthesis of AgNPs and assessed antimicrobial action against S. aureus and C. albicans with MIC of 67.50 µg/mL and 68.75 µg/mL respectively [18]. Farouk et al. 2023 synthesized AgNPs utilizing pomegranate peel and assessed antimicrobial potential against B. subtilis, R. oryzae, S. typhi, E. coli, E. faecalis and A. flavus with MIC (µg/mL) of 25, 250, 50, 250, 75, 125 respectively [21]. Mostafa et al. 2021 reported pomegranate and orange peel mediated AgNPs against A. solani with zone of inhibition of 16.14 mm and 12.52 mm respectively [22]. Sahoo et al. 2022 presented AgNPs synthesis utilizing Pomegranate peel for evaluation of DPPH assay. 500 µg/mL of concentrationof synthesized AgNPs exhibited 25.78% free radical scavenging activity [23].

Using the microwave irradiation approach, we demonstrate the environmentally friendly manufacture of nanoparticles based on earlier studies. The primary advantage of this process over traditional heating is that the reactants instantly absorb the internal temperature increase [24], leading to the extremely quick synthesis of nanoparticles in a little amount of time. Therefore, The novelty of microwave-assisted green synthesis of AgNPs lies in its ability to synergize the advantages of rapid, controlled microwave heating with the eco-friendliness of green synthesis, resulting in a method that is faster, more efficient, and environmentally sustainable [25].

In this research work, AgNPs were biosynthesized utilizing pomegranate peel extract as reducing and stabilizing agent via green approach. The biosynthesized AgNPs were subjected to different analytical techniques (UV–Vis spectroscopy, DLS, FTIR, XRD, HRTEM and FESEM) for characterization. Morever, the antimicrobial action of synthesized AgNPs was evaluated against K. aerogenes, X. axonopodis, C. graminicola, and C. gloesporioides. Additionally, the antioxidant potential of green synthesized AgNPs was assessed vis ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate), DPPH (2,2-diphenyl-1-picrylhydrazyl), total antioxidant and FRAP (ferric ion reducing antioxidant power) assays. Cytotoxicity assay using African green monkey kidney (Vero) cells was also performed.

2 Materials and methods

2.1 Plant material and chemicals

Pomegranate fruits were harvested from Chaudhary Charan Singh Haryana Agricultural University’s research farm in Hisar. The strains of K. aerogenes, X. axonopodis, C. graminicola, and C. gloesporioides were acquired from and antimicrobial activity was performed in Department of Plant Pathology, College of Agriculture, CCS HAU Hisar. African green monkey cells were acquired from and cytotoxicity assay was performed in ICAR-National Research Centre on Equines, Hisar. All the utilized analytical grade chemicals were acquired from Himedia Private Limited.

2.2 Plant extract preparation

Fresh Pomegranate fruits were picked and rinsed with distilled water. Fruits were peeled off, peels were shade dried at room temperature and grounded into fine powder. Then 5 g of powder was agitated with 250 mL of deionised water and heated up to 60 °C for 40 min. Whatman No. 1 filter paper was utilized for extract filtration before centrifuging it for 20 min at 5000 rpm. The resulting pomegranate peel extract (PPE) was then kept at 4 °C for further experimentation.

2.3 Quantitative analysis

2.3.1 Total phenolics

Using gallic acid as standard phenolic, the Folin–Ciocalteu procedure was employed to estimate the total phenolics of aqueous PPE [26]. Folin–Ciocalteu reagent (1 mL) and 20% Na2CO3 (2 ml) were added to 1 mL of PPE. Distilled water was poured to mark the final volume up to 10 mL. The absorbance at 730 nm was noted after this combination was left in dark for 90 min. Blank was prepared similarly, except that the proper solvent was used in place of the extract.

2.3.2 Total flavonoids

Using catechin as standard flavonoid, the AlCl3 colorimetric assay was employed to quantify the total flavonoids [27]. To 1 mL of PPE, 4 mL of distilled water and 0.3 mL of 5% NaNO2 were mixed.0.3 mL of 10% AlCl3 was mixed after 5 min. Immediately, 2 mL of NaOH (1 M) was poured and volume was increased with distilled water up to 10 mL. Blank was prepared similarly, except that the proper solvent was used in place of the extract. Then absorbance at 510 nm was noted down.

2.4 Synthesis of pomegranae peel extract AgNPs (PPE-AgNPs)

To synthesize PPE-AgNPs, 30 µL of PPE was combined with 1 mM AgNO3 (10 mL) solution. In an Anton Parr GmbH-Monowave 300 microwave reactor, the synthesis process was carried out for 6 min at 35 °C, 600 rpm stirring speed and 420 W of power. The resulting PPE-AgNPs were lyophilized for further examination after being centrifuged for 30 min at 11,000 rpm and 20 °C and repeatedly washed with deionized water.

2.5 Characterisation of PPE-AgNPs

The Shimadzu spectrophotometer (Model UV 1900) was used to execute UV–visible spectroscopic analysis in the 350–550 nm wavelength range. Using a DLS instrument (Malvern ZETASIZER PRO), the zeta potential, PDI (polydispersity index) and hydrodynamic size were evaluated. The surface morphology and elemental mapping (both qualitative and quantitative evaluation) were assessed with FESEM equipped with EDX detector. The chemical interactions within PPE and PPE-AgNPs were assessed throughPerkin Elmer FTIR Spectrophotometer.The XRD investigation was evaluated using the Miniflex II desktop X-ray diffractometer, which is outfitted with Ni-filtered Cu-Kα radiation (λ = 1.5418 A°) in the 10°–90° 2θ range. JEM/2100 PLUS HRTEM model was used to achieve the spatial resolution at atomic level.

2.6 Antimicrobial action of PPE-AgNPs

The antibacterial action of PPE and PPE-AgNPs against K. aerogenes and X. axonopodis were assessed in Nutrient Agar (NA) medium using the disc diffusion technique [28]. For a whole day, sterilized paper discs were soaked in solutions containing PPE (50 µg/mL), PPE-AgNPs (50 µg/mL) and Streptomycine sulphate (100 µg/mL). Using a swab, the pure grown bacteria were spread on the agar plates. Afterwards, soaked discs were moved to the middle of the plates and were cultured for 24 h at 27 °C to obtain the final outcomes.

Using food poison approach, antifungal action of PPE and PPE-AgNPs against C. gloesporioides and C. graminicola were assessed in Potato Dextrose Agar (PDA) media [29]. A 4 mm mycelium disc was cut from the outer edges of a 5-day-old fungus and placed in the center of PDA plates that had been seeded with PPE (50 µg/mL), PPE-AgNPs (50 µg/mL), and Nystatin (100 µg/mL). Nystatin was applied as standard antifungal agent. Using deionised water as control, PDA plates underwent the same procedure. The plates were incubated for 5 days at 27 °C. The diameter of the mycelia colonies was measured, and the following equation was applied to calculate the % inhibition:

$$ \% \text{ Inhibition }=1-T/C\times 100$$

where T and C are the colonial diameters of sample and control.

The minimum inhibition concentration (MIC) was determined by visually observing the growth of microbes in diluted solutions of PPE, PPE-AgNPs, Streptomycin sulphate and Nystatin. Initially, diluted solutions were mixed with broth media in flasks and each flask was then mixed with spore suspension having 2 × 105 spores/mL [30]. At 27 °C, bacterial and fungal cultures were incubated for 24 h and 5 days respectively. The microbesʹ growth was visually observed to determine the MIC value.

2.7 Antioxidant potential of PPE-AgNPs

2.7.1 ABTS assay

In ABTS assay, 5 mL ammonium persulfate (2.45 mM) and 10 mL ABTS (7 mm) were mixed and stored in dark for 12–16 h. An absorbance of 0.700 ± 0.002 at 734 nm was obtained by mixing methanol to the above ABTS solution. 2 mL ABTS solution was alloyed with 1 mL of each sample (PPE, PPE-AgNPs) at different concentrations (1–10 µg/mL) [31]. BHA was processed as standard.

$$\% ABTS\, free \,radical \,scavenging \,activity = \left[1-\frac{S(abs)}{C(abs)} \right]\times 100$$

where S(abs)and C(Abs) are the absorbance of sample and control respectively.

2.7.2 DPPH assay

In DPPH assay, 0.1 mM DPPH solution (1 mL) was alloyed with 2 mL of diverse concentrations (1–10 µg/mL) of each sample (PPE, PPE-AgNPs) and kept in dark at room temperature for 30 min [32]. At 517 nm, absorbance was recorded using UV–Vis spectrophotometer. BHA was processed similarly as a model antioxidant. %DPPH free radical scavenging activity was gigured using the equation,

$$\%DPPH\, free \,radical \,scavenging \,activity = \left[1-\frac{S(abs)}{C(abs)} \right]\times 100$$

where S(abs) and C(abs) are the absorbance of sample and control respectively.

2.7.3 Total antioxidant assay

Total antioxidant assay was examined through phosphomolybdenum method [33]. Ammonium molybdate (4 mM), sodium phosphate (28 mM) and H2SO4 (0.6 M) were combined in equal proportion to produce phosphomolybdenum reagent. 0.3 mL of different concentrations (1–10 µg/mL) of PPE and PPE-AgNPs were mixed with 3 mL of prepared reagent. Resulting mixture was stored at 95 °C in dark for 90 min and then absorbance at 695 nm was recorded. Ascorbic acid was processed similarly as standard antioxidant.

2.7.4 FRAP assay

In FRAP assay, 300 mM acetate buffer, 10 mM TPTZ and 20 mM FeCl3.6H2O were combined in 10:1:1 (v/v/v) ratioto prepare FRAP reagent [34]. 1 mL of various doses (1–10 µg/mL) of PPE and PPE-AgNPs were combined with FRAP reagent (3 mL) and incubated at 37 °C for 10 min. The FeSO4.7H2O was processed as standard antioxidant. At 593 nm, absorbance was recorded utilizing UV–Vis spectrophotometer.

2.8 Cytotoxicity assay

To conduct the cytotoxicity assay, African green monkey kidney (Vero) cells were treated in 96-well culture plates for 96 h with serial dilutions (2500, 500, 100, 20, 5, 1 µg/mL) of PPE and PPE-AgNPs. DMEM (Dulbecco’s Modified Eagle’s Medium) was used for serial dilution of samples. DMSO was applied as vehicle control. After adding 10 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye of 10 µg/mL concentration to each well and letting it sit for 4 h, DMSO (100 µL) was added to facilitate the formazan crystals dispersion [35]. Finally, the Thermo Multiscan GO microplate reader was employed to record the absorbance at 560 nm.

3 Results and discussion

3.1 Quantitative analysis

The quantitative analysis of phytochemicals is depicted in Table 1. Total phenolics and total flavonoids in PPE were quantified to be 289.93 ± 1.01 mg GAE/g and 114.70 ± 0.83 mg CE/g respectively. Rani et al. 2024 reported 244.11 mg GAE/g total phenolics in methanol and 196.47 mg GAE/g in acetone extract of Pomegranate peel. Total flavonoids were found to be 106.50 mg CE/g in methanol extract and 63.35 mg CE/g in acetone extract [36].

Table 1 Quantitative analysis of aqueous PPE
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3.2 Synthesis of PPE-AgNPs

Current study highlights the biosynthesis of PPE-AgNPs using PPE as reducing, capping and stabilizing agent. The specific effects of phytochemicals depend on their chemical structure, concentration, and the conditions under which the synthesis is carried out. Understanding the role of phytochemicals in this process not only contributes to the optimization of AgNP synthesis but also opens up opportunities for tailoring nanoparticles with specific properties for targeted applications [37]. Ag+ is reduced to Ag0 by various phytochemicals detected in Pomegranate peel, including tannins, anthocyanins, phenolic acids, anthocyanins and flavonoids. Punicalagin, ellagic acid and gallocatechin are the major components of PPE, responsible for the synthesis of PPE-AgNPs [38, 39]. The reduction and capping mechanism via punicalagin is illustrated below (Fig. 1). Due to the reduction potential difference, phytochemical compounds get oxidised and Ag+ get reduced. Change in extract colour from pale yellow to brown confirmed the formation of PPE-AgNPs. Furthermore, PPE-AgNPs formation was confirmed through analytical techniques.

Fig. 1
figure 1

Mechanism of PPE mediated Ag+ reduction to PPE-AgNPs

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3.3 Characterisation of PPE-AgNPs

UV–Vis spectroscopy is an important analytical technique for the initial confirmation of synthesized nanoparticles. SPR (surface plasmon resonance) band at 428 nm confirmed the formation of PPE-AgNPs (Fig. 2). Dielectric constant, shape, size and nature of capping agents affect the absorption study of synthesized PPE-AgNPs [40]. Absorption spectra of PPE-AgNPs revealed that SPR band of AgNPs lies in 350–550 nm range [41].

Fig. 2
figure 2

UV–Vis absorption spectra of PPE and PPE-AgNPs

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DLS study investigated PDI, zeta potential and hydrodynamic diameter of PPE-AgNPs (Fig. 3). PSA reflects hydrodynamic diameter of 71.90 nm, PDI value of 0.298 and zeta potential of − 14.41 mV. Phenolic compounds present in PPE were responsible for the negative zeta potential of PPE-AgNPs [42]. The monodispersed nature of the biosynthesized AgNPS is shown by the sharpness of the particle size distribution peak and the PDI value [43]. In actuality, it ascertained AgNPs' hydrodynamic diameter, which was connected to the different phenolic components on their surface [44].

Fig. 3
figure 3

DLS study of PPE-AgNPs

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To spot out the active functional groups reasonable for stabilizing PPE-AgNPs synthesised using PPE, an FTIR study was conducted. FTIR data displays many distinct and visible peaks in the observable scale, ranging from 4000 to 650 cm−1 (Fig. 4). According to Table 2, FTIR results revealed that PPE and PPE-AgNPs exhibit similar peaks with just slight positional variations. An additional peak at 1655.44 cm−1 is ascribed to –C=O of oxidized phenolic components formed during PPE-AgNPs synthesis. These structural modifications suggested that the reduction and stabilization of silver nanoparticles is caused by the coordination of the silver ions with the phenolic compounds in the plant extracts. FTIR studies revealed that phenolic group has a better potential to bind metal ions [45].The results were in perfect agreement with earlier research that found comparable maxima for the AgNPs prepared from the leaf extract of Trigonella foenum-graceum L. [46].

Fig. 4
figure 4

FTIR study of PPE and PPE-AgNPs

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Table 2 FTIR study of PPE and PPE-AgNPs
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XRD analysis was performed to determine the crystalline character of PPE-AgNPs [Fig. 5]. The X-Ray Diffraction pattern matches the COD (Crystallographic Open Database ID No. 1509146), which shows that the planes [111], [200], [220], [311], and [222] have diffraction peaks that emerge at 2θ values of 38.08°, 44.02°, 64.47°, 77.38°, and 81.53°, respectively. The crystalline (FCC) character of PPE-AgNPs is indicated by these observed peaks. Utilizing Debye–Scherrer's Equation, the average particle size was determined to be 19.62 nm [47].

Fig. 5
figure 5

XRD analysis of PPE-AgNPs

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HRTEM study analysed the spherical shape of PPE-AgNPs with mean size of 6.43 nm [Fig. 6]. The SAED pattern showed the circular rings with d-spacing values 2.359, 2.042, 1.447, 1.231, 1.182 related to [111], [200], [220], [311], [222] planes respectively. The d-spacing values closely interrelated with XRD pattern indicating the crystalline nature of PPE-AgNPs.

Fig. 6
figure 6

HRTEM study of PPE-AgNPs, a at 100 nm scale; b particle histogram; c SAED at 10 nm−1

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FE-SEM analysis identified the spherical surface morphology of PPE-AgNPs and prticle size varied up to 40 nm with 19.59 nm mean size [Fig. 7]. Silver (Ag), carbon (C), oxygen (O), nitrogen (N), chlorine (Cl), phosphorus (P) and gold (Au) were all confirmed to be present elementally in the electron micrographs by EDX. 80.6% of the entire weight was discovered to be Ag (Table 3). The presence of phytochemicals in PPE which are necessary for the production of PPE-AgNPs is indicated by energy signals for C, O, N, and Cl. A gold peak was seen during the FESEM investigation when Au was being applied to cover the PPE-AgNPs.

Fig. 7
figure 7

FESEM study of PPE-AgNPs, a at 100 nm scale; b particle histogram; c EDX of PPE-AgNPs

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Table 3 EDX elemental mapping of PPE-AgNPs
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3.4 Antimicrobial action of PPE-AgNPs

PPE, PPE-AgNPs and streptomycin sulphate were tested for their antibacterial action against K. aerogenes and X. axonopodis (Fig. 8). Deionized water was applied as control. When tested against K. aerogenes, PPE (50 µg/mL), PPE-AgNPs (50 µg/ml) and streptomycin sulphate (100 µg/mL) demonstrated ZOIs of 11.23 ± 0.20 mm, 14.33 ± 0.08 mm and 13.6 ± 0.18 mm respectively. But against X. axonopodis, PPE (50 µg/mL), PPE-AgNPs (50 µg/mL) and streptomycin sulphate (100 µg/mL) demonstrated ZOI values of 10.10 ± 0.05 mm, 15.30 ± 0.05 mm and 8.5 ± 0.20 mm, respectively (Table 4). For both bacterial strains, PPE-AgNPs exhibited more antibacterial efficacy than PPE and Streptomycin sulphate. PPE-AgNPs exhibited higher antibacterial efficacy against X. axonopodis followed by K. aerogenes. Streptomycin was discovered to be less effective against X. axonopodis and comparable findings were previously published, whereby the impact of several antibiotics and biosynthesized AgNPs against X. axonopodis was assessed [48].

Fig. 8
figure 8

Antibacterial action PPE and PPE-AgNPs against: a Klebsiella aerogenes, b Xanthomonas axonopodis

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Table 4 Antimicrobial action of PPE and PPE-AgNPs, nystatin and streptomycin sulphate with MIC (µg/mL)
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The antifungal action of PPE, PPE-AgNPs and Nystatin were assessed against C. graminicola and C. gloesporioides (Fig. 9). Deionised water and Nystatin were applied as control and standard antimicrobial agent respectively.The mean percentage inhibition against C. graminicola for PPE (50 µg/mL), PPE-AgNPs (50 µg/mL), and Nystatin (100 µg/mL) was 28.65 ± 0.41%, 56.33 ± 0.28%, and 57.37 ± 0.58% respectively (Table 4).On the other hand, the mean percentage inhibition against C. gloesporioides at the same concentration of PPE, PPE-AgNPs and Nystatin was 20.18 ± 0.35%, 76.52 ± 0.33%, and 58.71 ± 0.60%, respectively. For C. graminicola, PPE-AgNPs exhibited much higher antifungal action than PPE but slight lower than Nystatin. However, for C. gloesporioides PPE-AgNPs showed much higher antifungal action than both PPE and Nystatin. PPE-AgNPs showed greater antifungal action against C. gloesporioides than C. graminicola. The shape, size and capped polyphenolics adhered to the surface of biosynthesized AgNPs may be linked to their antifungal action [49].

Fig. 9
figure 9

Antifungal action of PPE and PPE-AgNPs against: a Colletotrichum gloesporioides, b Colletotrichum graminicola

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AgNPs exhibited significant antimicrobial activity through oxidative stress pathway through the induction of ROS (reactive oxygen species), such as superoxide anion,hydrogen peroxide (H2O2) and hydroxyl radical (·OH). Chloroplast, mitochondria and peroxisomes organelles produced ROS upto a favourable level which is essential for normal metabolic functioning [50]. Introduction of AgNPs inhibit the ETC (electron transport chain) of mitochondria and photosynthesis mechanism occurring in chloroplast which results in excessive level of ROS. Excessive level of ROS caused oxidative stress resulting in DNA damage, lipid oxidation and protein carbonylation that leads to cell rupture and cell-lysis [51, 52].

The MIC (µg/mL) values of PPE-AgNPs, PPE and standard drugs varied noticeably. PPE-AgNPs were reported to have MIC (µg/mL) values of 24, 20, 65 and 60 against K. aerogenes, X. axonopodis, C. graminicola and C. gloesporioides respectively (Table 4). PPE and Streptomycin sulphate were shown to be ineffective against bacterial strains up to 32 µg/ml concentration. The MIC values of PPE and Nystatin against fungal strains were similarly found to be greater than those of PPE-AgNPs.

3.5 Antioxidant activity

3.5.1 ABTS assay

ABTS assay indicated that both the PPE and PPE-AgNPs exhibit significant antioxidant activities, with the highest activity observed at 10 µg/mL concentration (Fig. 10). Specifically PPE and PPE-AgNPs achieved maximum scavenging activity of 69.70% and 96.65% respectively at 10 µg/mL concentration. Standard BHA (IC50 4.47 µg/mL) was found to have a higher ABTS scavenging activity than PPE (IC50 7.16 µg/mL) and lesser scavenging activity than PPE-AgNPs (IC50 4.25 µg/mL) as given in Table 5. AgNPs might be used to create formulations that are appropriate for the safe and effective treatment of a range of illnesses since they demonstrated stronger antioxidant properties than plant extract. AgNPs mediated by plants provide a number of benefits over traditional antioxidants, as proven by prior research, including improved stability, increased bioavailability, and target delivery [53]. Gecer, 2021 assessed the dose dependent response of Salvia aethiopis leaves extract to ABTS radical with IC50 4.93 µg/mL. By contributing an electron and a hydrogen atom, the phytoconstituents of GPE significantly increased the capacity to scavenge radicals [54].

Fig. 10
figure 10

Antioxidant activity of PPE and PPE-AgNPs using ABTS assay

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Table 5 Antioxidant activities of PPE and PPE-AgNPs
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3.5.2 DPPH assay

With ascorbic acid serving as a reference, the DPPH scavenging abilities of PPE and PPE-AgNPs were evaluated. The DPPH radical scavenging activity of PPE and PPE-AgNPs was dose-dependent, reaching maximal levels of 61.42% and 82.24% respectively at 10 µg/mL concentration (Fig. 11). PPE-AgNPs shown a substantially higher DPPH activity (IC50 5.22 µg/mL) compared to PPE (IC50 7.98 µg/mL). On the other hand, scavenging activity of PPE and PPE-AgNPs was found to be higher than that of standard ascorbic acid (IC50 16.34 µg/mL). The polyphenolics coated on AgNPs may be responsible for the increased scavenging action of AgNPs. These polyphenolics facilitate quick hydrogen and single electron transfer, therefore stabilizing the DPPH free radical [55]. Saratale et al. 2020 demonstrated the dose-dependent reaction of Grape pomace extract mediated AgNPs to DPPH free radical with an IC50 value 50.50 µg/mL. These findings suggest that plant extracts and AgNPs intercalation might produce powerful antiradical chemicals [56].

Fig. 11
figure 11

Antioxidant activity of PPE and PPE-AgNPs DPPH assay

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3.5.3 Total antioxidant assay

Using an Ascorbic acid standard curve, total antioxidant activity of PPE and PPE-AgNPs were demonstrated. Phosphomolybdenum assay depends on the reduction of Mo (VI) to green colored complex of Phosphomolybdate (V) and the subsequent decrease of green color under acidic conditions [57]. The results were presented as µg AAE/mL of corresponding concentration (Table 5). PPE-AgNPs (86.68 µg AAE/mL) demonstrated higher total antioxidant activity in comparison to aqueous PPE (41.23 µg AAE/mL) at 10 µg/mL concentration, with a dose-dependent effect (Fig. 12). Adherence of polyphenolics to the surface of PPE-AgNPs might be the cause for their improved antioxidant activity. Moond et al. 2023 synthesized AgNPs utilizing Trigonella foenum-graceum L. leaves extract and assessed for biomedical applications. Synthesized AgNPs exhibited 64.36 mg AAE/g total antioxidant activity at 1000 µg/mL concentration [44].

Fig. 12
figure 12

Total antioxidant assay of PPE and PPE-AgNPs

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3.5.4 FRAP assay

The ability of antioxidants to promote the conversion of Fe3+ ions to Fe2+ in the presence of TPTZ was assessed using the FRAP assay. This led to the creation of an intensely blue Fe2+-TPTZ complex, exhibited maximum absorbance at 593 nm [58]. Ferric reducing potential of PPE and PPE-AgNPs improved with increasing concentration, demonstrated utilizing standard curve of FeSO4.7H2O. The findings were expressed as mM Fe(II)/mL for each corresponding concentration. PPE-AgNPs (1.93 mM Fe(II)/mL) showed higher ferric reducing potential as compared to PPE (1.41 mM Fe(II)/mL) at 10 µg/mL concentration (Fig. 13). Erenler et al. 2021 synthesized Tegetes erecta leaves extract mediated AgNPs and assessed for their biomedical applications. Synthesized AgNPs exhibited FRAP activity with 2.79 µmol/mg of extract [59].

Fig. 13
figure 13

Antioxidant activity of PPE and PPE-AgNPs using FRAP assay

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3.6 Cytotoxicity assay

Evaluating the cytotoxicity of AgNPs is essential for ensuring their safe use in medical, consumer, and industrial products. It helps protect human health, comply with regulatory standards, understand environmental impacts, optimize nanoparticle design, and contribute to scientific knowledge. In this study, PPE and PPE-AgNPs at varying doses were applied to African green monkey kidney (Vero) cells using the MTT assay. This study showed that PPE-AgNPs had no detrimental effect on chosen vero cells, with a cell viability of 83.32% at 100 µg/mL of dose. Cytotoxicity of PPE-AgNPs was found dose dependent and greater than PPE (Fig. 14). Biosynthesized silver nanoparticles (AgNPs) have distinct advantages over chemically synthesized AgNPs due to the presence of phytochemicals on their surface. These phytochemicals play a crucial role in preventing agglomeration and enhancing the medicinal properties of the nanoparticles. In contrast, chemically synthesized AgNPs are more prone to aggregation in biological systems, leading to unselective cytotoxicity [60]. Our results are in consistent with previous research on toxicity of AgNPs to living cells, demonstrated that toxicity impacts caused by nanoparticles are dose dependent [61].

Fig. 14
figure 14

Cytotoxicity assay of PPE and PPE-AgNPs

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4 Conclusion

The biological technique for the production of nanoparticles was shown to be simple, economical, and environment benign. Silver nanoparticles with potential broad spectrum antimicrobial and antioxidant properties have been produced using the aqueous extract of pomegranate peel. Analytical techniques revealed the monodispersity, crystallinity and spherical shape of synthesized nanoparticles. Synthesized nanoparticles have antibacterial activity against K. aerogenes and X. axonopodis which causes Citrus canker and stem rot of Pearl Millet respectively and antifungal activity against C. graminicola and C. gloesporioides which causes Anthracnose of Sorghum and Anthracnose of Guava respectively. Synthesized nanoparticles possess significant antioxidant activities (ABTS, DPPH, total antioxidant, FRAP) which further suggest their biomedical application.The biosafety investigation found that the doses of AgNPs commonly used for antioxidant and antibacterial activities did not show any appreciable toxicological consequences. These ecologically produced AgNPs have the potential to enhance agricultural productivity, enhance hospital management, and enable pharmaceutical companies to produce huge quantities of their products. They are a viable replacement for conventional fungicides and antibiotics.