Bioengineered synthesis of phytochemical-adorned green silver oxide (Ag2O) nanoparticles via Mentha pulegium and Ficus carica extracts with high antioxidant, antibacterial, and antifungal activities

Silver oxide nanoparticles have various biomedical and pharmaceutical applications. However, conventional nanofabrication of Ag2O is associated with the use of toxic chemicals and organic solvents. To circumvent this hurdle, herein silver oxide quantum dots (Ag2O-QDs) were synthesized quickly (3 min) via the use of ultrasonic irradiation and plant-extract. Additionally, due to ultrasonic irradiation's effect on cell-wall destruction and augmentation of extraction efficiency, ultrasonic was also used in the preparation of Mentha pulegium and Ficus carica extracts (10 min, r.t) as natural eco-friendly reducing/capping agents. The UV–Vis result indicated a broad absorption peak at 400–500 nm. TEM/SEM analysis showed that ultrasound introduced a uniform spherical particle and significantly reduced particle size compared to the conventional heating method (∼ 9 nm vs. ∼ 100 nm). Silver and oxygen elements were found in the bio-synthesized Ag2O by EDS. The FTIR and phenol/flavonoid tests revealed the presence of phenol and flavonoid associated with the nanoparticles. Moreover, nanoparticles exhibited antioxidant/antibacterial/antifungal activities. The MIC and MBC results showed the Ag2O QDs synthesized with M. pulegium extract have the highest antibacterial activity against E. coli (MBC = MIC:15.6 ppm), which were significantly different from uncoated nanoparticles (MBC = MIC:500 ppm). The data reflects the role of phyto-synthesized Ag2O-QDs using ultrasonic-irradiation to develop versatile and green biomedical products.

Ultrasound-assisted extraction (UAE). The fruits of F. carica and M. pulegium leaves were washed using distilled water to remove dust particles and any other impurities. They were then dried at 50 °C for 12 h. Then, they were powdered by a mechanical grinder and stored.
To prepare M. pulegium leaves extract (MPE) and F. carica fruit extract (FCE), 10 g of previously prepared powders was added to 80 mL of deionized water. The mixture was then placed in the ultrasonic device (40 kHz) at room temperature for 10 min, and finally, the crude extract was filtered through whatman paper.
Chemical and green ultrasound-assisted synthesis (UAS) of Ag 2 O QDs. The UAS/phyto-synthesized Ag 2 O quantum dots (Ag 2 O QDs) were prepared by bio-reducing AgNO 3 under ultrasonic irradiation (WUC-D, Korea) in the presence of aqueous M. pulegium and F. carica extracts 22 . To do this, 12.5 mL of aqueous extract was mixed with 25 mL of 26 × 10 -4 mol L −1 AgNO 3 solution, and the mixture was immersed in the ultrasonic bath (40 kHz, r.t) until no further color change was observed in the solution. It was confirmed that the phyto-reduction of Ag + ions to Ag 0 takes place completely within 3 min by varying the initial color of the reaction mixture. Then, the suspension was centrifuged (10 min, 6000 rpm) and washed with double distilled water (DDW). A vacuum dryer was used to dry the precipitated Ag 2 O QDs (45 °C, 24 h). As a result, Ag 2 O QDs were synthesized from F. carica extract (UAS/FCE-Ag 2 O QDs) as well as M. pulegium extract (UAS/MPE-Ag 2 O QDs) using the UAS method.
According to the literature 23 , the Ag 2 O nanoparticles were also chemically synthesized (Chem-syn/Ag 2 O NPs). First, the aqueous solution of 5 × 10 -3 mol L −1 AgNO 3 (80 mL) was prepared and stirred with a magnetic stirrer at 60 °C. Adding 20 mL of NaOH (aq) solution (25 × 10 -3 mol L −1 ) immediately produced a gray-yellow suspension containing a large amount of silver hydroxide (AgOH) precipitate. Since AgOH particles are thermodynamically unstable, they are chemically turned into Ag 2 O particles 23  www.nature.com/scientificreports/ ogy, and elemental analysis of the quantum dots were then identified by scanning electron microscopy/energydispersive X-ray spectroscopy (SEM-EDS, FEI ESEM Quanta 200, EDS Silicon Drift 2017, USA). In addition, identification of phase purity and structural information of the sample was obtained using X-ray diffraction (XRD) (PHILIPS, PW1730, Netherlands) diffractometer with Cu Kα source (λ = 1.54056 Å). Microplate reader BioTek (Epoch 2) was used to read biological data.
Total phenolic and flavonoid content. The total phenol content in the samples was assessed using the Folin-Ciocalteu colorimetric assay 24 , with some modifications. In this method, Folin-Ciocalteu's phenol reagent (125 µL) and Na 2 CO 3 solution (100 µL, 7.5% w/v) were first added to each sample (25 µL). The mixture was then incubated (25 °C, 2 h, and dark). The absorbance of all samples was measured at 760 nm. Gallic acid was used as a standard, and the results were expressed as gallic acid equivalents (µg GAE/mg sample). The samples' total flavonoid content was quantified using an aluminium chloride colorimetric method as previously reported 24 . Firstly, aliquots of 25 μL of each sample, DDW (100 μL), and sodium nitrite (7.5 μL, 5% w/v NaNO 2 ) solution were mixed. The resulting mixture was incubated for 6 min. Then, aluminium chloride (7.5 μL, 10% w/v AlCl 3 ), 100 μL of sodium hydroxide (4% w/v NaOH) solution, and DDW (10 μL) were added to the mixture and kept in the dark (r.t, 15 min). Finally, the absorbance of the mixture was recorded at 510 nm using a UV-Vis spectrophotometer. Quercetin was used as a standard, and the results were expressed as quercetin equivalents (µg QU/mg sample) 25 . The total phenol and flavonoid content was quantified five times for each sample.
Antioxidant activities. DPPH assay. According to the literature 24 , the scavenging activity of the samples against the DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical was determined with some modifications. 50 μL of each sample was mixed with 200 μL of DPPH methanolic solution (0.0788 mg/mL) and set aside in the dark (30 min, r.t). Ascorbic acid was used as a positive control. The ability to scavenge DPPH radical was measured at 517 nm, as follows: Where A 0 and A 1 are the absorbance intensities of the control and the sample, respectively. DPPH assay was repeated five times for each sample.
ABTS assay. The ABTS radical scavenging activity of the samples was evaluated using the previously reported method 26 . Firstly, ABTS (aq) (7 mM) solution and potassium persulfate solution (2.45 mM) were mixed and then left to dark (r.t, 24 h), to generate radical cation (ABTS ⋅+ ). The ABTS radical cation is stable at room temperature in the dark for more than 2 days. The solution was diluted with PBS to obtain a final absorbance of 0.700 ± 0.02 at 734 nm and equilibrated at 30 °C. Then, 290 μL of ABTS solution was treated with 10 μL of each sample. The chemical changes were monitored by a colorimetric method at 734 nm. The ABTS radical-scavenging activity was calculated according to Eq. (1). ABTS assay was repeated 5 times for each sample. The Disc Diffusion Method (DDM), was used to evaluate the in vitro antibacterial potential of aqueous extracts and Ag 2 O QDs against bacteria strains, according to the literature 28 . In summary, 0.1 mL of each organism was first dispersed using a sterile swab on the Muller Hinton Agar medium. After preparing different concentrations (12.5, 25, and 50 mg/mL) of the samples, the filter paper discs (diameter: 6 mm) were loaded with the different concentrations of samples and placed on the plates. After incubation (37 °C, 24 h), the diameter of the inhibition zone (mm) surrounding the disc was used to evaluate the samples' antimicrobial activity.
The minimum inhibitory concentration (MIC) value is the lowest concentration of the sample that inhibits the visible growth of bacteria 29 . This assay was performed according to the previous study 30 . In this method, different concentrations of extracts as well as Ag 2 O QDs samples (0.488 to 1000 ppm) were mixed and inoculated with Mueller-Hinton broth which was exposed to different test organism suspensions. After an incubation period (37 °C, 24 h), the concentration of the last well that shows no macroscopic growth defined the MIC value.
According to the literature 30 , the minimum bactericidal concentration (MBC) was directly determined from MIC wells and by sub-culturing diluted sample solutions (with no turbidity or growth) onto Muller Hinton Agar. After incubation (37 °C, 24 h), the minimum concentration of the sample which can kill the bacteria on this solid medium was reported as the MBC values.
Antifungal activity. The antifungal activity of the extracts and Ag 2 O quantum dots was evaluated using a disc diffusion technique, as previously reported 31 , with slight modifications. To perform this test, two different www.nature.com/scientificreports/ fungi, including Aspergillus oryzae and Candida albicans, were selected. Then, the filter paper discs were placed on the plates after loading with the different concentrations of samples (12.50, 25, and 50 mg/mL) and were then incubated (29 °C, 72 h). The inhibition zone (mm) was measured and recorded as the antifungal activity of the samples.

Result and discussion
Characterization of Ag 2 O QDs. In this study, Ag 2 O QDs were synthesized with ultrasonic irradiation, plant extract, and silver nitrate solution as a metal salt precursor (Fig. 1A). The M. pulegium and F. carica extracts were employed as reducing, capping, and stabilizing agents. Ultrasound irradiation has several advantages in the extraction process, including reducing solvent consumption, increasing plant cell wall destruction, and mass transferring active components (e.g., phenolic compounds) into solution 21 . In addition, the sonosynthesis method led to the very rapid formation of monodisperse and fine Ag 2 O QDs. The reduction process was initially assessed visually by changing the color and turbidity of the solutions containing Ag + ions and FCE or MPE, after ultrasound irradiation. It was confirmed that the phyto-reduction of Ag + ions to Ag 0 takes place completely by varying the color of the reaction mixture.
A UV-Vis analysis was used to investigate the optical behavior and preliminary characterization of QDs. Figure 1B,C exhibits the spectra of the green synthesized Ag 2 O QDs obtained from the corresponding extracts under ultrasound irradiation within the range of 350-500 nm. The Ag 2 O QDs biosynthesized by MPE and FCE led to a broad absorption band between 400 and 500 nm. This confirms the existence of Ag 2 O QDs in the mixture and is in line with earlier reports [32][33][34][35] .
Since the stability of quantum dots is very critical, due to the adhesion of metabolites in plant extracts to the surface of quantum dots, they were used for in situ bio-capping and the stability of Ag 2 O NPs 34,36 . Given that the stabilizing or capping agents attached to the Ag 2 O QDs' surface change the FTIR spectrum, it provides useful information about the surface chemistry of QDs ( Fig. 2A-D) 37,38 . The control spectrum (FCE) reflects the complex nature of biological materials ( Fig. 2A). In this spectrum, the strong peak at 3392 cm −1 is due to the stretching vibration of the OH groups. Furthermore, the absorption peaks at 2931, 1624, 1406, and 1065 cm −1 www.nature.com/scientificreports/ can be attributed to the stretching vibration of the C-H methyl/methylene bond, C=O of ester or carboxylic acid, C-O bond of ester/ether, and the C-N/C-O of aliphatic amines or alcohol/phenol, respectively 39,40 . The presence of these functional groups on nanoparticles' surfaces increases their stability and biological efficiency 41 . As shown in Fig. 2B, after the reaction of the FCE with silver ions and the formation of particulates, there was a shift in the peaks. These shifts indicate the binding of extract phytochemicals as coating and stabilizing agents to the surface of the quantum dots 38,39 . It is known that biological components reduce metal salts through their functional groups and form nanoparticles 39,40 .
The same results were observed for MPE and Ag 2 O QDs phyto-synthesized from it (Fig. 2C,D). All these peaks show the presence of phytochemicals including flavonoids, polyphenolics, amino acids, etc., which have different functional groups as bio-reducing, bio-capping, and bio-stabilizing agents in the synthesis of UAS/ phyto-synthesized Ag 2 O QDs and prevent aggregation of quantum dots 42 . Total phenol and flavonoid contents. The total phenolic and flavonoid contents in the crude extracts and phyto-synthesized QDs were analyzed (Fig. 3). Some phenols and flavonoids in F. carica 46 and M. pulegium 47 plants are shown in Fig. 4. The biological effects of plant extracts depend on their components, such as phenolic and flavonoid contents, solvent type, polarity, and extraction method 48 . The phenolic compounds are a class of bioactive components in plants that have a wide range of biological properties, including antifungal, antibacterial, anti-inflammatory, antiviral, antiallergic, and antiviral properties 48 .
As shown in Fig. 3, the amount of phenolic compounds in all samples is significant, and the total phenolic contents of samples are in the following order: MPE > UAS/MPE-Ag 2 O QDs, as well as FCE > UAS/FCE-Ag 2 O QDs. As can be seen, extracts showed higher phenol content than extracts containing quantum dots. This can be attributed to the phenolic compounds´ involvement in the reduction of silver ions as well as their absence in the reduction process of molybdenum and tungsten ions in Fulin reagent. In contrast to total phenols, quantum dots-bearing extracts have a higher level of total flavonoids (Fig. 3), possibly as a result of silver ions participating in chelate formation with flavonoids. Therefore, the chelating potential of flavonoids is confirmed by measuring the flavonoid content of quantum dots 49 .
Antioxidant activity. Free radicals produced in the body cause hundreds of diseases, but they are usually controlled by the body's antioxidant defense system. This is because antioxidants neutralize or end the chain reaction started by free radicals 33 . Phenolic compounds have antioxidant activity due to their ability as reducing agents, radical scavengers, and hydrogen donors. Since they can donate hydrogen atoms from their aromatic hydroxyl groups to free radicals and have a resonance effect in their aromatic rings, they are excellent antioxidants 50 . In general, antioxidant activity is often attributed to the total phenol content and sometimes to the synergistic or antagonistic effects of compounds in crude extracts as well as the chemical structures of the compounds 51 . Additionally, phytochemical compounds on the surface of the synthesized QDs can improve www.nature.com/scientificreports/ antioxidant activity 52 . During this study, different assays, including DPPH, ABTS, and ferric-reducing power (FRAP) methods, were employed to determine the antioxidant ability of the plant extracts and their corresponding QDs. The reaction mechanism between these oxidants and antioxidants is shown in Fig. 5 53 . The antioxidant properties of the samples to neutralize the stable free chromogenic radical (2,2-diphenyl-1-picrylhydrazyl) DPPH ⋅54 and the cation radical ABTS ⋅+ (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 26 were evaluated in the DPPH and ABTS assays, respectively. The cation radicals of ABTS ⋅+ are more reactive than DPPH radicals and are used to estimate both the lipophilic and hydrophilic antioxidant activity 55 . According to the DPPH and ABTS results (Fig. 5A,B), the maximum inhibition percentage values occurred in the UAS/FCE-Ag 2 O QDs (56.72% for DPPH and 66.43% for ABTS). Notably, the UAS/FCE-Ag 2 O QDs have better antioxidant activity than FCE. These results are in line with the reports of Maheshwaran et al. 45 and Abdel-Aziz et al. 56 . The obtained antioxidant activity confirmed the inhibition potential of biosynthesized Ag 2 O QDs against reactive oxygen species (ROS) 45 . As seen, the antioxidant activity of UAS/FCE-Ag 2 O QDs does not follow the phenol content trend. Thus, the synergistic activity of other compounds may enhance the antioxidant activity of these extracts, which indicates that phenolic compounds may not be the only or main components resulting in their antioxidant activity 51,57 .
A positive correlation was observed between antioxidant activity and total phenolic content of MPE and UAS/MPE-Ag 2 O QDs, indicating phenolic compounds have an important role to play in plant antioxidants 51,58 .
The ability of polar and non-polar extracts to reduce Fe (III) to Fe (II) can be considered as the antioxidant ability of the extract. In the ferric reducing antioxidant potential (FRAP) test, the ferric-tripyridyltriazine (Fe III -TPTZ) complex is converted to its ferrous (blue) by antioxidants 59 . According to the FRAP experiment (Fig. 5C), the antioxidant activity results of the samples were found to be in the following order: MPE > UAS/ MPE-Ag 2 O QDs as well as FCE > UAS/FCE-Ag 2 O QDs. Accordingly, the reducing power of the samples also  (Table 1). Each concentration was tested in triplicate. The results showed different levels of antibacterial activity, as well as concentration-dependent antibacterial activity. Furthermore, the UAS/phyto-syn/Ag 2 O-NPs had higher antibacterial activity (inhibition zone) than the UAbased extracts for all tested bacteria (Fig. 6A-E). In addition, UAS/MPE-Ag 2 O QDs showed higher antibacterial activity than those prepared by FCE, and MPE has more antibacterial properties than FCE.
UAS/MPE-Ag 2 O QDs have been found to have the highest antibacterial activity among the studied samples. The samples have a more potent effect on gram-negative bacteria than gram-positive bacteria 60 . This difference in results is due to the cell wall structure of the bacteria and the permeability of samples to their cell wall 43 .
In order to reveal the antibacterial properties of the samples, MIC and MBC tests were conducted. Based on the MIC results, UAS/MPE-Ag 2 O QDs have the maximum antibacterial activities against E. coli and P. aeruginosa at the concentrations of 15.6 and 62.5 ppm, respectively (Table 2). Furthermore, the UAS/phyto-syn/Ag 2 O-NPs coated with phytochemicals of plant extracts exhibit better antibacterial properties than plant extracts and the chem-syn/Ag 2 O-NPs.
A comparison of the inhibitory properties (MIC) of quantum dots synthesized by chemical and green methods shows significant differences: www.nature.com/scientificreports/    Antifungal activity. In vitro antifungal activities of the UAS/phyto-syn/Ag 2 O-QDs and the aqueous extracts were investigated against A. oryzae and C. albicans fungal cultures, as the diameter of the inhibition zone (mm) of growth, using the DDT procedure (Fig. 7). Similar to the results of antimicrobial activities, increased concentrations of QDs and extracts enhanced antifungal activity. The UAS/phyto-syn/Ag 2 O-QDs displayed stronger impacts against all the studied fungi than the extracts. Particularly, the UAS/MPE-Ag 2 O QDs with the maximum susceptibility value in A. oryzae at 50 mg/mL exhibited the highest antifungal activity against all tested fungal strains.
The phyto-syn Ag 2 O QDs at low concentrations, especially UAS/MPE-Ag 2 O QDs showed higher biological activities than the chem-syn NPs and the tested extracts. It may be due to the extremely small size or high available surface area of the Ag 2 O NPs, which enhances contact and friction and facilitates the penetration of Ag 2 O QDs into the cell through the pores of plasma membrane proteins and causes cell death. Additionally, both the synergistic effect and the extract coating, which increase friction between the microorganisms and phytochemicals in plant extracts, increase the antibacterial activity of plant-synthesized QDs 22,61 .
A comparison was made between the conditions of extraction and synthesis of phyto-Ag 2 O QDs, and those obtained from previously published methods (Table 3). As shown by the results, not only the extraction time was reduced; but also the synthesis of the Ag 2 O QDs with better biological activity (antioxidant, antibacterial, and antifungal) was performed much faster, easier, and with minimal use of toxic chemicals in one step, and under completely green conditions than other Ag 2 O nanoparticles previously reported (Table 3). Ultrasonic irradiation produced pure and extremely fine nanoparticles (quantum dots) with monodispersity in shape and www.nature.com/scientificreports/ size. Since the properties of nanoparticles depend on their size, it is very imperative to produce monodisperse and pure quantum dots (QDs). Ag 2 O nanoparticles' antibacterial properties are due to electrical changes that occur during their interaction with bacterial membranes and increase surface reactivity 64 . There is no clear mechanism for the penetration of Ag 2 O nanoparticles, but bacteria exposed to Ag 2 O NPs showed both morphological changes on the bacterial membrane and disruption of the transport mechanism, which led to significant membrane permeability increases 64,65 . It seems that Ag 2 O NPs, after penetrating into the bacteria, interact strongly with molecules containing phosphorus and sulfur, such as DNA (Fig. 8A). Then damaged DNA loses its ability to replicate, and thus the cell cycle halts at the G2/M phase 64 . Due to the inhibition of ATP synthesis and the production of reactive oxygen species (ROS), cells are exposed to oxidative stress, resulting in the induction of apoptosis. Furthermore, these nanoparticles, after penetrating into bacteria, inactivate bacterial enzymes by releasing ionic (Ag + ) and atomic (Ag 0 ) silver clusters and causing cell death by producing hydrogen peroxide and other free radicals 64 . Silver oxide nanoparticles penetrate the cell wall and cytoplasmic membrane by binding to lipids and proteins, leading to cell lysis and toxic effects inside the cell (Fig. 8) 66 . The mechanisms of antifungal activity of Ag 2 O NPs can be attributed to the production of reactive oxygen species (Fig. 8B). Consequently, these nanoparticles negatively affect the expression of the oxidative enzyme, thereby causing the inability to handle the resulting stress 66 .
Additionally, these nanoparticles can have antifungal properties by disturbing the antioxidant endogenic process and unsettling the pathogen's internal environment. They can also alter homeostatic redox reactions and oxidative stress, leading to an imbalance in osmotic pressure, membrane destruction, and ultimately cell death 66 . These nanoparticles enhance their antifungal effect by inhibiting the enzymatic activity of various enzymes, such as the activity of transferase in lecithin or ATPase in P-glycoprotein 66 . They arrest the fungal cell cycle by increasing the percentage of cells in G2/M phase and decreasing them significantly in G1 phase 64 .

Conclusions
This is the first report on fast, eco-friendly, cheap, and completely green synthesis of Ag 2 O QDs using ultrasonic (in both plant extraction and synthesis stages) and plant extracts (F. carica fruits and M. pulegium leaves). Their antibacterial, antifungal, and antioxidant activities were also examined, and compared with conventional phytosynthesized Ag 2 O-NPs (with heating and without ultrasound) and chemically synthesized Ag 2 O nanoparticles (plant-uncoated nanoparticles). The use of ultrasonic in synthesis resulted in rapid formation (3 min) of much smaller monodisperse particles (~ 9 nm) than with conventional methods (~ 100 nm). In addition, its use in extraction enhanced both plant cell wall destruction and mass transfer of bioactive compounds into solution.
The biological activities results showed that they have good antioxidant, antifungal, and antibacterial properties. As measured by MIC/MBC tests, their antibacterial activities were higher than those of chemically synthesized Ag 2 O-NPs (uncoated), and only small amounts (ppm) of these nanoparticles can inhibit the growth of selected bacteria or kill bacteria (15.62-1000 ppm). Consequently, as the bacteria/fungi studied result in infections and fungal diseases in humans, UAS/phyto-syn/Ag 2 O-QDs are ideal agents for controlling these diseases as well as other pharmaceutical applications. Moreover, their antioxidant properties enable them to neutralize free radicals produced in the body, which contribute to hundreds of dangerous and various diseases. Therefore, they can have industrial and biomedical applications.

Data availability
All data generated or analysed during this study are included in this published article.