Introduction

Nanobiotechnology integrates biotechnology and nanotechnology, dealing with applications of nanomaterials in biological sciences1,2,3,4. With the advent of nanobiotechnology, nanostructures' biological and physiochemical properties are tuned to serve the most relevant areas of human welfare, like medicine and agriculture5. Among different types, metallic nanoparticles (MNPs) are one of the widely exploited antimicrobial nanomaterials against phyto- and animal pathogens6,7,8,9,10,11,12. Researchers have recently witnessed a growing interest in synthesizing biocompatible metal-based NPs, utilizing green chemistry and bioinspired fabrication routes13,14,15,16,17,18,19,20,21.

Biological synthesis offers an eco-friendly and cost-effective method for the fabrication of NPs22 and is preferred over conventional methods23. Different biogenic sources for the synthesis of MNPs, like bacteria, plants, algae, fungi, yeasts, and actinomycetes, cause considerable modifications in the properties of their corresponding metals24. Among such sources, plant-based bioinspired fabrication of NPs is one of the preferred approaches24,25. The bioactive compounds in plant extracts can act as potential reducing and capping agents in synthesizing MNPs24,25. Green synthesis of MNPs like silver, copper, gold, iron, titanium, zinc, platinum, palladium, etc., has been extensively investigated26,27,28. However, limited investigations for green synthesis of manganese (Mn) NPs have been reported so far24, despite various applications in catalysis, biomedicine, electronics, electrochemistry, energy, optics, biosensors, food, pharmaceutical, cosmetics, textile industries, etc.24,29.

Metal oxides such as ZnO, CuO, TiO2 and MnO etc. have also a great potential to have excellent antimicrobial activity19. Chamaecostus cuspidatus extract is used to green synthesis CeO2 and ZnO nanoparticles (NPs) and effective antibacterial activities. The anticancer effects of CeO2 and ZnO nanoparticles were investigated in human breast cancer cell lines21. Similarly, Green synthesis is used to prepare Cerium oxide nanoparticles (CeO2 NPs) from Artabotrys hexapetalus leaf extracts. The prepared NPs exhibit excellent antibacterial activity against a variety of bacterial species. The anticancer potential of the compound was studied against the (MCF-7) human breast cancer cell line22. In addition to that, Zinc oxide nanoparticle (ZnO NPs) was prepared utilizing starch in a single step green synthesis and had highly porous, novel hollow multi-sphere in morphology. Because of their morphology and porosity, the synthesized ZnO NPs can be employed in a variety of drug delivery applications19. Mn has been reported as the transition element with the third highest abundance on earth followed by iron and titanium30. Among various 3d transition metal-oxides, Mn-oxides (MnO, MnO2, Mn2O3, Mn3O4, and Mn5O8) have obtained key attention owing to their compositional and structural diversity24,31. Mn-oxides NPs also possess structural adaptability with varying physicochemical qualities32. Mn-oxide NPs have excessive potential for sustainable-nanotechnology research and innovation24,30. Mn-oxides can have applications in optoelectronics, magnetic storage devices, imaging contrast agents, magnetic materials, drug delivery, catalysis, wastewater treatment, solar cells, etc.24.

Sclerotinia sclerotiorum is a necrotrophic phytopathogen that harbors a broad host range and causes stem rot disease in different crops including soybean, oilseed rape, sunflower, tomato, etc., resulting huge losses of agricultural produce33. On the other hand, C. gloeosporioides follows the hemibiotrophic infection mode and is known for causing anthracnose in fruits like papaya, mango, avocado, apple, guava, banana, papaya, cashews, grapes, pitaya, etc., resulting in serious postharvest losses34,35. Although chemical fungicides have been utilized for their control, their indiscriminate utilization has serious environmental consequences that necessitate the search for novel and eco-friendly alternatives36. E. faecalis is known to colonize the human intestine, and its occurrence in aquatic bodies implies fecal contamination37. These bacteria have been reported as multidrug-resistance microbial pathogens associated with hospital-acquired infections37,38.

To combat such underlying issues, present study focused on developing nanoformulation comprised of Pd-doped Mn3O4 NPs through the green chemistry route as a potential tool to offer a broad spectrum of antimicrobial activity.

In the current study, green synthesis of Mn3O4 NPs was completed by using Syzygium aromaticum bud extract as a potential reducing and capping agent. Adding novelty to this work, attempts were made to modify the structural attributes of Mn3O4 NPs through Pd doping to prioritize their multifaced applications in sectors such as agriculture, environment and medicine. To the best of our knowledge and available literature, this is the first report on the bioinspired fabrication of nanocorn-like Pd-doped Mn3O4 NPs using ASAB extract.

This present work investigated the antimicrobial potential of bioinspired Pd-doped Mn3O4 NPs against S. sclerotiorum, C. gloeosporioides, and E. faecalis.

Experimental

Materials and methods

All the chemicals used in the present study (such as manganese chloride tetrahydrate, palladium chloride, sodium hydroxide, dextrose, agar, hydrochloric acid, potassium hydroxide, sodium chloride, yeast extract, beef extract, and peptone) were of analytical grade and utilized without any further purifications. The solutions and reagents were prepared in double-distilled water (DDW).

Collection of plant material

The evenly looking dried flower buds of S. aromaticum were obtained from the nearby local market of Meerut (Uttar Pradesh, India). The taxonomical evaluation was performed by Prof. Vijai Malik, Head, Department of Botany, CCS University, Meerut (UP) (letter reference no. Bot/PB/380). Plant material was procured as per applicable Institutional, International, and National guidelines. The specimens were deposited in the University Herbarium at the Department of Botany (Accession no. Bot. 26 V2L4).

Preparation of S. aromaticum bud extract

The procured flower buds were thoroughly washed with distilled water to remove dirt particles and dried at 40 °C for 48 h. The dried flower buds were homogenized into a fine powdered form and stored in an air-tight container until used. To prepare the extract, powdered flower buds were macerated in DDW (1:10, w/v) at 60 °C for 2 h. After cooling at room temperature, the aqueous solution of phytoextract was filtered using Whatman filter paper number 1 at stored at 4 °C temperature until use.

Green synthesis of pure and Pd-doped Mn3O4 nanoparticles

The NPs were synthesized using a sol–gel method39, assisted by the addition of phytoextract as a potential source of capping agent (Fig. 1). Briefly, 2% (w/v) PdCl2 was added to the aqueous solution of MnCl2.4H2O (49 mM) followed by drop-wise addition of 10 ml aqueous Syzygium aromaticum bud extract under continuous stirring at 650 rpm at 90 °C for one hour. After getting the light brown color of the solution, 25 mM aqueous solution of NaOH was added drop-wise to adjust the pH. The alkali-mediated synthesis provides rapid precipitation leading to the mixture of manganese dihydroxide (Mn (OH)2) and manganese trihydroxide (Mn (OH)3)40,41. The appearance of a dark brownish-black precipitate indicated the formation of Pd-doped Mn3O4 NPs. The precipitate was collected using centrifugation at 5000 rpm for 10 min. The recovered precipitate gave multiple washings with ethanol and DDW to remove impurities, then dried at 150 °C for 3.5 h in a hot air oven. The finally dried residue was transformed into fine powder through mechanical grinding. The obtained Mn3O4 NPs were stored in an airtight bottle for further characterization. Except for the dopant addition, all other synthesis steps for pure Mn3O4 NPs were the same as those mentioned for Pd-doped Mn3O4 NPs. The key reaction steps in synthesizing Mn3O4 NPs are mentioned below40.

$${\text{MnCl}}_{{2}} \cdot {\text{4H}}_{{2}} {\text{O}} \rightleftharpoons {\text{Mn}}^{{{2} + }} + {\text{2Cl}}^{ - } + {\text{4H}}_{{2}} {\text{O}}$$
(1)
$${\text{2 Mn}}^{ + } \overset {{\text{NaOH}}} \rightleftharpoons {\text{Mn }}\left( {{\text{OH}}} \right)_{{2}} + {\text{ Mn }}\left( {{\text{OH}}} \right)_{{3}}$$
(2)
$${\text{Mn }}\left( {{\text{OH}}} \right)_{{2}} + {\text{ Mn}}\left( {{\text{OH}}} \right)_{{3}} \mathop{\longrightarrow}\limits^{{{\text{Calcination}}}}{\text{Mn}}_{{3}} {\text{O}}_{{4}} + {\text{4H}}_{{2}} {\text{O}}$$
(3)
Figure 1
figure 1

Schematic of the green synthesis of pure and Pd-doped Mn3O4 NPs.

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Characterization of synthesized nanoparticles

XRD analysis was performed to study the crystallinity and phases of prepared NPs (Bruker AXS, D8 Advance). The phase transitions and chemical composition of synthesized NPs were examined by Raman spectroscopy. The morphology and elemental composition were studied by FESEM (FEI, Quanta 200F), and EDAX, respectively. FTIR analysis conducted on ASAB extract and synthesized NPs to investigate the role of bioactive compounds in the development of NPs. The zeta potential and structural properties were determined using Zetasizer (Malvern Nano ZS) and HRTEM/SAED, respectively. XPS analysis was conducted to study the composition, and oxidation states of Pd-doped Mn3O4 NPs. The optical properties of prepared NPs were determined by UV–Visible spectroscopy.

Antimicrobial activity of nanoparticles

The antifungal activity of NPs was tested against S. sclerotiorum and C. gloeosporioides using the poisoned food technique. The fungal cultures were procured from Indian Type Culture Collection (ITCC), Division of Plant Pathology, Indian Agriculture Research Institute, New Delhi, India. The potato dextrose agar (PDA) media was prepared for fungal growth with following composition: dextrose (2% w/v), potato starch (0.4% w/v), and agar (1.5% w/v). The pH of 5.6 ± 0.2 was adjusted using 0.1N KOH/0.1N HCl. The synthesized NPs were dispersed in PDA media to get the desired concentrations (up to 1000 ppm). The media containing NPs was poured into a Petri plate. At the next step, a ~ 8 mm piece of actively growing mycelia from 5 to 8 days old pure cultures of S. sclerotiorum and C. gloeosporioides were placed in the middle of each plate and incubated for five days at ~ 25 ± 2 °C and ~ 28 ± 2 °C temperature, respectively. The media plates without NPs treatment served as the negative control, and plates with 2 mg/ml of carbendazim + mancozeb were designated as the positive control. The % of growth inhibition was calculated using the below formula:

$$\frac{{\left( {{\text{Growth}}\;{\text{in}}\;{\text{Control}}\;{\text{plates}} - {\text{Growth}}\;{\text{in}}\;{\text{treatment}}\;{\text{plates}}} \right)}}{{{\text{Growth}}\;{\text{in}}\;{\text{Control}}\;{\text{plates}}}}{ } \times { }100$$
(4)

The antibacterial activity of NPs was determined using agar disc diffusion assay (ADDA). The pure culture of E. faecalis was inoculated to freshly prepared nutrient broth media (NBM) and maintained at 37 °C for ~ 18 h. The composition of NBM was Yeast extract (0.2% w/v), Beef extract (0.1% w/v), Peptone (0.5% w/v), NaCl (0.5% w/v), and pH ~ 7.4 ± 0.2. 2% (w/v) agar was added to the nutrient broth for preparing solid media plates (SPM). The pure culture of E. faecalis at the active log phase was uniformly spread on SPM. The sterile filter paper discs of about 6 mm diameter, each dipped in various concentrations of NPs (0 to 1000 ppm), were placed on SPM. The DDW-dipped discs were served as a negative control. The Petri plates were placed in an incubator overnight, and the ZOI was measured in millimetre. All the experiments on antimicrobial activity were performed in triplicates under aseptic conditions in a laminar airflow chamber. The nutrient media, glassware, and other utilities were autoclaved at 121 °C for 15 min at 15 psi pressure before use to maintain aseptic conditions throughout the assay.

Statistical analysis

All of the experiments were completed in triplicates and recorded data presented as mean ± standard deviation using Microsoft Excel®.

Results and discussion

Green synthesis of nanoparticles and FTIR analysis

Pure and Pd-doped Mn3O4 NPs were synthesized using an aqueous Syzygium aromaticum bud (ASAB) extract. Preliminary phytochemical analysis indicated the presence of phenols (ferric chloride test)42, flavonoids (lead acetate test)43, alkaloids (Wagner test), carbohydrates (fehling's test), and tannins (ferric chloride test)44. The results were in concordance with the findings of Jimoh et al.43, which established the suitability of the tested plant as a potential substrate for developing Phyto inspired nanoparticles45,46,47. Through FTIR analysis, Rajesh et al.45 predicted the role of metabolites present in S. aromaticum bud extract, such as flavonoids, tannins, alkaloids, and carotenoids, in the green synthesis of CuNPs. We have compared the FTIR spectra of ASAB extract, pure and Pd-doped Mn3O4 NPs to validate the capping and stabilizing potential of bioactive compounds present in phytoextract (Fig. 2).

Figure 2
figure 2

FTIR spectra of ASAB (clove) extract, pure, and Pd-doped Mn3O4 NPs.

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FTIR spectrum of ASAB extract revealed a broad band at ~ 3431.62 cm−1, which corresponds to the OH group45, alkyl CH stretching (sp3), and C–O ester group was observed at ~ 2931.95 and ~ 1711.92 cm−1, respectively48. A sharp peak at ~ 1611.99 cm−1 belongs to –C = C aromatic stretching vibrations and C = O stretching vibrations of proteins denoting amide linkages 45. The aromatic groups were indicated at ~ 1511.36 cm−148, while two separate peaks at ~ 1366.60 cm−145 and ~ 1051.25 cm−148 denoted the C–O group. These characteristic FTIR spectral peaks suggest for eugenol presence in ASAB extract48. The peak at ~ 640 cm-1 are attributed to the Mn–O in synthesized nanoparticles49. The peaks common to ASAB extract, pure and Pd-doped Mn3O4 NPs (~ 3450–3400, ~ 1750–1700, ~ 1650–1600, ~ 1400–1350, and ~ 1050 cm-1) depicted the capping potential of bioactive compounds in phytoextract.

Structural analysis

The XRD confirms the crystalline structure of the prepared manganese oxide NPs with two distinct phases, as shown in Fig. 3. The diffraction peaks of Mn3O4 samples at 2θ values of 31.04, 32.36, 36.13, 44.47, 53.83, 58.57, 59.90, and 64.69 matched well with the (200), (103), (211), (220), (312), (321), (224), and (400) crystal planes of Mn3O4 phase respectively50. The above (hkl) planes correspond to the Hausmannite phase of the Mn3O4 crystal structure (JCPDS 24–0734)51. The intensity of XRD peaks was decreased in the case of Pd doping, indicating peak shifting towards lower diffraction angles and crystalline lattice expansion52, suggesting the successful incorporation of Pd in Mn3O4. There is no shifting of XRD peaks in the case of Mn3O4 NPs, indicating that as-synthesized NPs are comprised of the tetragonal Hausmannite phase53. The sharp peaks confirmed the highly crystalline nature of NPs54. There were no other peaks in the XRD pattern that demonstrated the phase purity of the produced NPs55.

Figure 3
figure 3

XRD spectra of pure and Pd-doped Mn3O4 NPs.

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The crystallite size of synthesized NPs were calculated by Scherrer’s formula at a extremely intense peak (Eq. 5), and the values were ~ 32 and ~ 28 nm for pure and Pd-doped Mn3O4 NPs, respectively56,57. The Pd-doped Mn3O4 NPs were smaller than the pure Mn3O4 NPs, which could be owing to the fact that ionic radii of Pd (0.137 nm) are much larger than that of Mn (0.082 nm)58.

$$D = \frac{0.89\lambda }{{\beta cos\theta }}$$
(5)

where D is crystallite size, λ is the wavelength, θ is Bragg’s angle, and β is FWHM.

Surface morphology and elemental analysis

The surface morphology of the pure Mn3O4 NPs in Fig. 4a, b appeared to be rod-like nanostructures. In contrast, Fig. 4d, e showed the surface of Pd doped-Mn3O4 NPs with the likely appearance of nanocorn-like structures. The morphological changes from rod (Mn3O4 NPs) to nanocorn-like nanocorn (Pd-doped Mn3O4 NPs) could be owing to the decoration of Mn3O4 with Pd2+. EDAX analysis revealed Pd, Mn, and O in their suitable stoichiometric proportion, as given in Fig. 4c, f 59.

Figure 4
figure 4

FESEM micrographs, and EDAX spectra of (a, b, c) pure Mn3O4 NPs and (d, e, f) Pd-doped Mn3O4 NPs.

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Optical studies

The absorption spectra of pure and Pd-doped Mn3O4 NPs were recorded at room temperature using a UV–visible spectrophotometer. In absorption spectra, the optical absorption edge was shifted to a higher wavelength region with Pd doping; consequently, the red shift was noticed in Pd-doped Mn3O4 NPs. The energy band gap of developed NPs was calculated by Tauc’s relation (Eq. 6)60.

$$\left( {\alpha {\text{h}}\nu } \right)^{{\text{n}}} = {\text{ B}}\left( {{\text{h}}\nu - {\text{E}}_{{\text{g}}} } \right),$$
(6)

where α, hν, Eg, and B are the absorption coefficient, photon energy, the band gap energy, and constant, respectively. The value of index ‘n’ calculated from Tauc’s Plot was 2 (Fig. 5). The estimated band gap was ~ 3.79 and ~ 3.75 eV for pure and Pd-doped Mn3O4 NPs, respectively, which is in agreement with the previous reports for Mn3O4 NPs61. It was noticed that the band gap decreased in Pd-doped Mn3O4 NPs, compared to pure Mn3O4 NPs. A red shift was observed in the band gap of Pd-doped Mn3O4 NPs. This may be due to intermediate levels forming between the CB and VB of the host Mn3O4 matrix62,63. Pd atoms act as an accepter to decrease the band gap of Mn3O4 NPs64,65. Therefore, variation in the energy band gap of Mn3O4 NPs by Pd doping may have applications in photocatalytic activity66.

Figure 5
figure 5

Tauc’s plot of pure and Pd-doped Mn3O4 NPs.

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Electronic states and elemental composition analysis

The oxidation states of Pd-doped Mn3O4 NPs were determined by XPS analysis67. The survey spectrum (Fig. 6a) revealed the presence of Pd, Mn, and O, confirming their existence in the product, i.e., Pd-doped Mn3O4 NPs. Further analysis of the Pd 3d spectrum showed a doublet feature68, providing evidence of Pd species' presence in the material. The peaks observed at 332.17 eV and 335.56 eV in the Pd 3d5/2 region, along with 340.57 eV and 344.58 eV in the Pd 3d3/2 region, corresponded to Pd (II) and Pd (IV) states68. Moving on to the Mn 2p core-level spectrum, two distinct peaks were observed at binding energies of 654.94 eV, 653.56 eV and 643.53 eV, 642.13 eV for pure and Pd-doped Mn3O4 nanocorns, respectively. These peaks were associated with Mn 2p1/2 and Mn 2p3/2 in Mn3O4 NPs and indicating a spin-orbital splitting of 11.4 eV (Fig. 6b)69,70. Additionally, the O1s spectrum peaked at 529.68 eV for pure Mn3O4 NPs and 532.89 eV for Pd-doped Mn3O4 NPs (Fig. 6d)70. This peak confirmed the presence of oxygen in both materials. The XPS analysis provided conclusive evidence that the prepared manganese oxide material was indeed Pd-doped Mn3O4 NPs, with the oxidation states of Pd (II) and Pd (IV) and specific Mn 2p states characteristic of Mn3O4.

Figure 6
figure 6

(a) The full XPS survey graph of pure and Pd–doped Mn3O4; (b) Pd 3d (c) Mn 2p and (d) O 1 s.

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Transmission electron microscopy analysis

The TEM micrograph of the green synthesized Mn3O4 NPs in Fig. 7a shows that the Mn3O4 NPs were composed of nearly uniform types of particles. The SAED patterns of Mn3O4 NPs in Fig. 7b displayed bright rings with some bright spots, suggesting the high crystallinity of the materials71. Figure 7c represents the high-resolution TEM images of the Pd-doped Mn3O4 NPs, and the magnified calibrated lattice fringes of Mn3O4 NPs for the crystal plane of (103) and (211) revealed an interplanar spacing (d-spacing) of 2.7 and 2.4 Å in Fig. 7d. These planes were also observed in the XRD analysis, and the reduction of the d-spacing of these planes is in good agreement with the shifting of the XRD peaks. Therefore, these findings indicate the successful formation of Pd-doped Mn3O4 NPs, which was also consistent with the XRD patterns72,73.

Figure 7
figure 7

Electron micrographs showing Pd-doped Mn3O4 NPs,: (a) TEM image, (b) SAED pattern, (c) HRTEM image, and (d) interplanar lattice spacing.

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Raman Spectroscopy analysis

Figure 8 presents the Raman spectra of pure and Pd-doped Mn3O4 NPs. Two characteristic peaks at ~ 629 and ~ 630 cm−1 were observed, corresponding to the skeletal vibrations for pure and Pd-doped samples, respectively. The strongest peaks at ~ 629 and ~ 630 cm−1 are consistent with the reported data74 for both materials. These sharp peaks can be assigned to the A1g mode, representing the Mn–O breathing vibration of divalent manganese ions in tetrahedral coordination. This mode is a characteristic feature of Hausmannite75,76. The comparison of the Raman spectra between pure and Pd-doped Mn3O4 NPs reveals similarities in the characteristic peaks, indicating that the introduction of Pd did not significantly alter the skeletal vibrations and Mn–O breathing vibrations in the tetrahedral coordination. The Raman spectra analysis of pure and Pd-doped Mn3O4 NPs confirmed the presence of specific vibrational modes and provides key insights into the structural properties of these materials. The similarities in the Raman spectra between pure and Pd-doped samples suggested that the Pd-doping did not cause significant changes in the observed vibrational features.

Figure 8
figure 8

Raman spectra of pure and Pd-doped Mn3O4 nanoparticles.

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Zeta potential studies

A particle's surface potential substantially impacts its dispersion stability77, which may also influence its bactericidal potential78. Without proper surfactants or capping agents, nanoparticles tend to agglomerate, and their surface area-to-volume ratio decreases due to their increased size79. The zeta potential studies allow us to investigate nanoparticles' surface charge and stability in colloidal solutions80 (Fig. 9). The surface charge of NPs can be influenced by the charged dopants77, which is also observed in the present study. We have obtained highly stable NPs (ZP > 30 mV)81, with recorded values of −33.2 ± 0.404 and −36.6 ± 1.74 mV for pure and Pd-doped Mn3O4, respectively. The NPs with greater ZP values (negative or positive) prevent agglomeration via electrostatic repulsion, hence conferring stability80.

Figure 9
figure 9

Zeta potential of pure and Pd-doped Mn3O4 NPs.

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Antifungal activity

The developed NPs showed mycelium growth inhibition in a dose-dependent manner. In the case of Mn3O4, NPs, we have observed maximum antifungal activity at 1000-ppm concentration with over 50% inhibitions in the growth of S. sclerotiorum and C. gloeosporioides was recorded at 500 ppm dose (Fig. 10). Overall, S. sclerotiorum exhibited higher sensitivity to the Mn3O4 NPs treatment than C. gloeosporioides. The inhibition of mycelial growth in the case of Pd-doped Mn3O4 NPs was higher than pure Mn3O4 NPs against both fungal strains (Fig. 11). This could be due to the significant modification in structural properties of Mn3O4 NPs as a result of Pd doping, such as the reduction in crystallite size and nanocorn-like morphology. At 1000 ppm concentration, Pd-doped Mn3O4 NPs caused ~ 92% and ~ 72% growth inhibition of S. sclerotiorum and C. gloeosporioides, respectively. Interestingly, C. gloeosporioides was more sensitive to Pd-doped Mn3O4 NPs treatment, specifically at lower doses, and showed ~ 65%, ~ 23%, and ~ 10% higher inhibition compared to pure Mn3O4 NPs at 500, 100, and 10 ppm concentration, respectively. The Pd-doped Mn3O4 NPs at 1000 ppm showed antifungal activity comparable to those of positive control (2 mg/ml of carbendazim + mancozeb; commercial chemical grade fungicide formulation). Hence, doping Mn3O4 NPs with Pd favoured their antifungal potential66, which is highly explicitly recommended in the case of C. gloeosporioides to decrease the effective dose. Overall, the bioinspired fabrication of nanocorn-like Pd doped Mn3O4 NPs can be used as an effective antifungal nano-pesticide against different necrotrophic and hemibiotrophic phytopathogens, known for causing enormous loss to agricultural food crops globally.

Figure 10
figure 10

Effect of (a) pure Mn3O4 NPs, and (b) Pd- doped Mn3O4 NPs on growth of S. sclerotiorum, and C. gloeosporioides.

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Figure 11
figure 11

Antifungal activity of pure and Pd-doped Mn3O4 NPs against (a) S. sclerotiorum, and (b) C. gloeosporioides.

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Antibacterial activity

The antibacterial activity of pure and Pd-doped Mn3O4 NPs was also investigated against E. faecalis to establish their broad spectrum of antimicrobial potential, in terms of pathogen diversity, i.e., phytopathogens and animal pathogens. As stated earlier, E. faecalis is a well-known human pathogen known for hospital acquired infections. This bacterium colonizes the intestine of animals including humans, and its presence in waterbodies is an indicative of fecal contamination. The resistance of E. faecalis against various antibiotics necessitated the search of novel materials possessing significant antibacterial potential against such nosocomial pathogens. In the present work, both pure and Pd-doped Mn3O4 NPs showed dose-dependent increment in ZOI (Fig. 12). The ZOI values showed effect of Pd doping on improving antibacterial activity of Mn3O4 NPs. When compared to pure Mn3O4 NPs, Pd doping showed ~ 14%, ~ 17%, and ~ 16% higher ZOI values at 50, 100, and 200 ppm doses of Mn3O4 NPs respectively.

Figure 12
figure 12

Antibacterial activity of pure and Pd-doped Mn3O4 NPs against E. faecalis.

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Mechanism of antimicrobial activity of Pd-doped Mn3O4 NPs

The plausible routes of inducing antimicrobial activity by Pd-doped Mn3O4 NPs are illustrated in Fig. 1336,66,82. The NPs in fungal cells usually gain entry via diffusion and endocytosis83 and may cause growth inhibition through multiple actions such as DNA damage, protein denaturation, breakdown of the cell wall and cell membrane, ROS-mediated lipid peroxidation, ribosome disassembly, denaturation of enzymes, perforations in the cell wall and cell membrane, mitochondrial damage, release of cytochrome-c from mitochondria to cytosol, and increase levels of metacaspase and promotes cell death36. Similar effects have been proposed in the case of bacterial cells where NPs can cause protein and enzyme denaturation, damage to chromosomal and plasmid DNA, ribosomal depolymerization, interference in ETC, the release of cellular contents, disruption of the cell membrane, etc.84,85.

Figure 13
figure 13

Schematic representation highligting plausible mechanisms involved in antimicrobial action of Pd-doped Mn3O4 NPs (1. breakdown of cell wall and cell membrane; 2. perforations in cell wall and cell membrane; 3. mitochondrial damage; 4. release of cytochrome-c from mitochondria to cytosol, increase levels of metacaspase and promotes cell death; 5. chromosomal DNA damage; 6. ribosome disassembly; 7. Protein damage; 8. release of cellular contents; 9. denaturation of enzymes; 10. plasmid DNA damage; 11. disruption of cell membrane; 12. perforation in cell membrane; 13. interference in ETC and damage to protein-efflux pump; 14. destruction of membranous proteins; CB = conduction band, VB = valance band; ROS: reactive oxygen species).

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In general, NPs have direct and indirect effects on microbial cells. The direct damage occurs via the electrostatic interaction of NPs with cell membrane resulting in membrane depolarization and loss of membrane integrity leading to the disruption in ETC and cell lysis39,86. The indirect damage to microbial cells is reported via ROS generation (Eqs. 7, 8, 9, 10, 11, 12)39,87. The doping in pure nanomaterial leads to lattice defects (alters band gap), causing overlapping of Fermi levels, variation in cellular redox potential, promotes ROS generation (Fig. 14) and can impart enhanced antimicrobial properties39, which was observed in the present study as well, where Pd-doped Mn3O4 NPs showed higher antifungal and antibacterial activity compared to pure NPs. In addition, doping can improve the binding capacity and cellular internalization ability of NPs. NPs generate ROS outside the cellular environment or can produce it inside the cell due to interference in ETC39. The oxygen molecules that are not reduced in the water get oxidized into free radicals (such as superoxide anion, singlet oxygen, or hydroxyl radicals) in mitochondria (Eqs. 7, 8, 9, 10, 11, 12)39,88. ROS causes alteration in protein structures, oxidative stress, lipid peroxidation, and DNA damage88.

$${\text{Pd-doped }} {\text{Mn}}_{{3}} {\text{O}}_{{4}} + {\text{ hv}} \to {\text{e}}^{ - } + {\text{h}}^{ + }$$
(7)
$${\text{h}}^{ + } {\text{H}}_{{2}} {\text{O}} \to {}^{ \bullet }{\text{OH}} + {\text{H}}^{ + }$$
(8)
$${\text{e}}^{ - } + {\text{ O}}_{{2}} \to {}^{ \bullet }{\text{O}}_{2}^{ - }$$
(9)
$${}^{ \bullet }{\text{O}}_{{2}}^{ - } + {\text{ H}}^{ + } \to {\text{ HO}}_{{2}}^{ \bullet }$$
(10)
$${\text{HO}}_{2}^{ \bullet } + {\text{ H}}^{ + } + {\text{ e}}^{ - } \to {\text{H}}_{{2}} {\text{O}}_{{2}}$$
(11)
$${\text{H}}_{{2}} {\text{O}}_{{2}} + {\text{e}}^{ - } \to {}^{ \bullet }{\text{OH }} + {\text{ HO}}^{ - }$$
(12)
Figure 14
figure 14

Effect of doping on Fermi levels leading to ROS generation (VL = vacuum level, CB = conduction band, VB = valance band) (conceptualized and redrawn from39).

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Conclusion

The present investigation demonstrates a successful green chemistry approach to synthesis pure and Pd-doped Mn3O4 NPs via utilizing an aqueous extract of S. aromaticum buds. Adding Pd in Mn3O4 resulted in significant changes to their structural attributes, including morphology, crystallite size, and lattice defects. The Pd-doped Mn3O4 NPs exhibited antimicrobial activity in a dose-dependent manner and provides higher inhibitory effects than pure Mn3O4 NPs against S. sclerotiorum, C. gloeosporioides, and E. faecalis. The outcome of this study provides a novel, cost-effective method to develop Pd-doped Mn3O4 based nanomaterials for highly effective antimicrobial applications against tested microbial pathogens. This breakthrough opens up new possibilities in the area of green nanotechnology to develop sustainable and multifaceted antimicrobial agents.