Green production of biologically active Ag and Ag–Cu nanoparticles from Prosopis cineraria pod waste extract and their application in epoxidation

The main focus of the current research is bio-nano-technologically produced nanoparticles (NPs) utilizing waste materials. There is a need for developing advanced technology to reduce waste in an eco-friendly way. Therefore, presently the discarded aqueous portion of Prosopis cineraria pod was used after boiling, to synthesize Ag and Ag–Cu NPs. FT-IR spectra illustrated the presence of phenyl propenoids and flavonoids displaying capping as well as reducing properties. TEM and SEM imaging exhibited an average size of Ag NPs (14 nm) and Ag–Cu NPs (27 nm). The crystallinity nature was confirmed by XRD, and the Cu in Ag–Cu NPs was validated through energy-dispersive X-ray analysis. According to the antimicrobial data, Saccharomyces cerevisiae displayed a zone of inhibition (ZOI) of 42.85% (Ag NPs) and 33.98% (Ag–Cu NPs) at lower concentrations (0.0321 mg/ml), while Bacillus subtilis was found most susceptible (85% ZOI) to Ag NPs at 0.5 mg/ml concentration. Further, these NPs (Ag and Ag–Cu) were utilized in the epoxidation of alkene moieties. Ag NPs showed lower conversion (65%), while Ag–Cu NPs were very active for epoxidation of linalool (93% conversion), suggesting the presence of Cu-facilitated epoxidation. To the best of our knowledge for the first time, aqueous waste is applied to prepare green NPs that can be used as antimicrobial agents and in the synthesis of platform chemicals (epoxide) for industrial aspects. These inexpensive ways of producing green NPs have been utilized several times and have found potential applications in nano-medicine, therapeutics, and modification of monoterpenoids to fine fragrance.


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Green production of biologically active Ag and Ag-Cu… The aim of this research work is to employ a green capping agent. The P. cineraria pod waste water extract is found beneficial due to its wide spectrum of natural molecules (flavonoids and polyphenols), which avoid particle overgrowth and aggregation during NPs synthesis [23]. From the present work, the NPs synthesized from P. cineraria waste water are further used in the epoxidation of monoterpenes like linalool and limonene to give an industrially relevant pharmacological precursor. The cyclized and epoxide products were highly important in various drug syntheses [24,25]. Therefore, this study focused on exploring waste materials to produce NPs possessing anti-microbial activities. In addition, these NPs selectively oxidize the monoterpenes to monoterpene epoxides economically for various commercial applications.

Materials and Methods
AgNO 3 , Cu (CH 3 COO) 2 , NaOH, H 2 O 2 , acetonitrile (CH 3 CN), and acetic acid (AcOOH) were purchased from Merck, Mumbai, India. All cell culture media were supplied by Hi-Media, Mumbai, India. The double-distilled water was utilized for the synthesis of Ag and Ag-Cu NPs in the present study.

Prosopis cineraria pod extract preparation
To prepare the crude extract, P. cineraria pods (10 g) were thoroughly rinsed and washed. The pods were boiled for 2 h at 80 °C in 100 ml of deionized water. The extract solution was filtered and then stored at 4 °C for the biosynthesis of Ag and Ag-Cu NPs.

Green synthesis of Ag and Ag-Cu NPs
To synthesize Ag-Cu NPs, 10 ml of P. cineraria extract, 25 ml of an aqueous copper acetate solution (2 mM) and 75 ml of an aqueous AgNO 3 solution (10 mM) were added simultaneously under stirring for 3 h at 90 °C. Light brown to black colour change indicated the formation of the bimetallic Ag-Cu NPs. The precipitate was purified by centrifuging at 10,000 rpm for 30 min at 25 °C. The centrifuged particles were then washed three times with ethanol to follow double-distilled water and dried at 120 °C in a vacuum oven in order to get the final nanopowder. Cu NPs and Ag NPs were also synthesized separately in order to endorse the higher impact of bimetallic Ag-Cu NPs on antimicrobial and cytotoxic potential over monometallic nanomaterials. Cu NPs were synthesized by adding 100 ml of aqueous Cu(CH 3 COO) 2 solution after mixing with 10 ml of P. cineraria extract.

Characterization of NPs
The NPs were characterized using SEM, TEM, XRD, FT-IR, and UV-Vis analysis. A UV-Vis spectrometer (model number UV-245 Shimadzu) was used to visualize the surface plasmon peak of the Ag-Cu NPs. A quartz cell with a path length of 1 cm was loaded with a few drops of fresh solution containing biosynthesized Ag-Cu NPs, and the cell was scanned in the wavelength range of 200-700 nm. The XRD diffraction pattern of the composites was recorded through Rigaku diffractometer (Rigaku, Japan) by using Cu K 〈 radiation at 40 kV and 130 mA in the scanning angle of 5-50° at 0.05° scanning speed. The diffraction patterns are presented with the 2θ Vs. intensity. FT-IR spectra of samples were acquired using Nicolet 138 400D-Impact FT-IR spectrophotometer. The method used was the KBr pellet method, and the region analysed was at 4000-500 cm −1 with a resolution of 4 cm −1 . After applying 10,000 hydraulic pressures for 2-3 min, the dry KBr of 200 mg and the sample of 5 mg were well mixed to create pellets. Each sample was scanned 200 times, and the wavenumber against transmittance (%) was used for recording the spectra. The QUANTA-250, FEI (Netherlands) SEM equipment was utilized in order to conduct morphological study of the NPs. Samples (50 mg) have been coated with gold by Sputter Coater for 50 s (Model 147 No: Q150 TES frocoram Technology, UK). Sputtering with Ag was used to create layers on the NPs. The instrument was kept at 20 kV for determining the particle size and shape of the NPs. TEM was performed by using an electron microscope with a filament as a source of electrons operated at 300 kV. TEM imaging and mapping were carried out by using field emission gun-transmission electron microscope 300 kV (TEM 300 kV), FEI, Tecnai G2, and F30.

Antimicrobial activity
Kirby Bauer's disc diffusion method was used to observe the antimicrobial activity [26]. Fresh inoculum (100 μL of 10 7 cfu) of Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis was spread onto the Mueller-Hinton agar plates and dilution of 10 μl stock solution of Ag and Ag-Cu NPs, gentamycin (standard), and water (negative control) was impregnated into a disk and placed in the Petri plate. After incubation for 24 h at 37 °C, the inhibition zone was observed and calculated by comparing it with the negative control.

Epoxidation reaction
The catalytic tests were carried out in a three-necked glass flask equipped with a condenser and immersed in a temperature-controlled water bath. Typically, epoxidation of monoterpenes was employed by both NPs using an environmental-friendly oxidant H 2 O 2 to investigate the catalytic activity. For this, linalool was taken as a substrate in order to optimize the reaction condition of alkene epoxidation and reacted with H 2 O 2 in the presence of NPs. The effect of Ag NPs and Ag-Cu NPs in linalool epoxidation was studied in the following operating conditions as linalool: H 2 O 2 (1:2), 20 ml acetonitrile, 0.1% AcOOH at 50 °C. The reaction products were monitored by GC-FID and GC-MS. The final products were extracted in diethyl ether, and the NPs were separated out through filtration, washed numerous times, and calcined at 400 °C for 4 h.
where Ccal o = initial concentration, and Ccal t = concentration at time t. Product selectivity (Si) is calculated as:

GC-FID and GC/MS analysis
The conversion (epoxidation) of monoterpenoids and product selectivity were determined by the use of GC-FID and GC/MS techniques. Here the CP 3800 (Varian Associates Inc) gas chromatography system coupled with flame ionization detector (FID) was used. The column used in the analysis was Elite-5 MS capillary column (30 m, 0.25 mm, 0.25 μm, PerkinElmer make). The oven temperature was programmed at a range of 60 °C to 240 °C, at a rate of 3 °C/min, with a hold time of 6 min at 240 °C. The temperatures of the injector and detector were maintained at 250 °C and 260 °C, respectively. GC/MS analyses were carried out using Clarus 680 gas chromatography system coupled with Clorus SQ 8C Quadrupole mass spectrometer (PerkinElmer make) using the above-mentioned column. Operating conditions was as follows: helium was utilized as carrier gas (99.99%), the flow rate: 1 mL/min, split ratio: 1:100, mass range (m/z): 50 to 500, EI: 70 eV, inter-scan delay: 0.01 s, scan time: 0.8 s, ion source temperature: 280 °C and inlet line temperature: 280 °C. Compounds were identified by using their mass spectra and compared the results with the available software database Wiley and NIST libraries.

Results and discussion
In the current study, P. cineraria's pod waste water extract was first time utilized to make Ag and Ag-Cu NPs. The waste water showed high reducing and capping abilities. The overall process of NPs synthesis is given in Scheme 1. During the traditional way of dish preparation from sangri (P. cineraria) different types of waste waters were generated and from this waste water further Ag and Ag-Cu NPs were prepared which is listed in Table 1 and Fig. 1. As shown in Table 1 the washed and boiled P. cineraria waste water yielded the maximum precipitate, i.e. 458 mg (Ag NPs) and 710 mg (Ag-Cu NPs). Before synthesis, both NP solutions are light yellow but turned brown after 2 h, with olive-green precipitates formed, indicating the successful formation of NPs ( Fig. 1). Pod extract of P. cineraria could be used in NPs synthesis as it contained several plant metabolites such as flavonoids and phenolics [5]. These properties minimized as well as stabilized the metals to prevent particle agglomeration [27]. Table 2 shows a comparative study Scheme 1 Process of Ag and Ag-Cu NPs synthesis from P. cineraria 1 3 Green production of biologically active Ag and Ag-Cu… of reported NPs synthesis using natural products with the present prepared Ag NPs and Ag-Cu NPs.

UV-Vis analysis
UV-Vis spectra in the range of 300-700 nm confirmed the formation of NPs with broad single SPR (surface plasmon resonance) peaks ( Fig. 2A). A single SPR peak around 420 nm matches the λ max of Ag NPs, which was in correspondence with the report of Manjari et al. [28]. For bimetallic NPs, if the metals combine to form alloys, a single SPR peak was observed, whereas a core shell-like structure was displayed with two distinct peaks. In the current study, only a single SPR peak around 426 nm for Ag-Cu has suggested the formation of the alloy-structured NPs [16].  Table 1), B2 and C: Formation of NPs (brown (2) and olive-green precipitates (1))

XRD analysis
The XRD diffractogram for both NPs is shown in Fig. 2B with 2θ values between 10° and 90°. Figure 2B shows the scattering peaks of 38° (111), 44° (200), 64° (220), 77° (311), which correspond to the standard FCC structure of Ag NPs [20,29]. Due to Ag-Cu NPs containing low amounts of Cu, hence, no distinct peak of Cu was observed except a minor shoulder peak at 43°, which corresponded to the Cu NPs [29,30]. The characteristic peaks in both the Ag and Ag-Cu NPs confirmed the formation of Cu-Ag NPs without significant oxides [31]. Furthermore, the peaks shift in case of Ag-Cu NPs in comparison with Ag NPs is revealed by the presence of Cu and its proper incorporation in the synthesized NPs.

FT-IR analysis
FT-IR spectra showed the functional groups in both NPs (Fig. 2C). was gauche skeletal C-C stretching. The band at 2853 cm −1 was assigned to CH 2 symmetric stretching. Further, the λ max at 1358 cm −1 was of a CH 2 wagging band [32], while 820 cm −1 was due to symmetric C-O stretching [33].

SEM-EDAX analysis
During morphological study of the synthesized NPs, it was found that both the SEM images (Ag NPs and Ag-Cu NPs) are spherical in shape, as shown in Fig. 3A and Fig. 3B. It may be due to the nature of capping agents present in the pod extract. The only difference in both is that the agglomeration of particles is a bit more in the case of Ag-Cu NPs in comparison with Ag NPs, which also confirmed the presence of good Cu infusion along with the Ag particles. These findings in the morphology are also supported by TEM results. The elemental composition of both NPs was assessed from the EDX spectrum as shown in Fig. 3A 1 and Fig. 3B 1 , where the signal of Ag from the Ag NPs, and Ag and Cu from the Ag-Cu NPs spectrum gets confirmed and also displayed the successful formation of Ag NPs as well as Ag-Cu NPs. Green production of biologically active Ag and Ag-Cu…

TEM analysis
The images illustrated the spherical shape and the dispersed nature of NPs ( Fig. 3C and Fig. 3D); here, the spherical shape is due to phytochemicals that reduce particle size and act as a capping agent [20]. Ag NPs (Fig. 3C) have fewer particles than Ag-Cu NPs (Fig. 3D), indicating a good infusion of Cu metal in Ag-Cu NPs. In Fig. 3C, the Ag NPs were uniformly monodispersed with a diameter of 11.9-15.1 nm with a 14-nm average particle size. In Fig. 3D, the Ag-Cu NPs were polydisperse with a diameter of 22.9-31.4 nm and have a 27-nm average particle size. The capping of the produced NPs was confirmed by the scum surrounding them, as shown in Fig. 3C and 3D.

NPs formation using natural products
The preparations of NPs from different natural products are reported as listed in  [35]. They observed that the extract acted as a good bioreductant and capping agent with an average particle size of 15 nm. Its enhanced reduction properties were due to the presence of high amounts of flavonoids, alkaloids, and terpenoids in Origanum vulgare extract. Interestingly, the Ag NPs had displayed significant antimicrobial activity against selective bacterial and fungal strains. In particular, P. cineraria leaf extract was reported on Ag and Cu NPs preparation and studied their antimicrobial and anticancer activities [1]. But in the present work, for the first-time bimetallic NPs is prepared from the waste aqueous extract of P. cineraria pod, and it is used as a catalyst for epoxidation reaction. In addition, Ag and Ag-Cu NPs are also displaying significant antimicrobial activity. However, in the present work, for the first time, bimetallic NPs are prepared from the aqueous waste extract of P. cineraria pod, and it is used as a catalyst for the epoxidation reaction. In addition, Ag and Ag-Cu NPs are also displaying significant antimicrobial activity.

Antimicrobial activity
Three bacterial strains, i.e. E. coli, B. subtilis, and S. cerevisiae, were examined with a 0.0038-0.5 mg/ml concentration range of Ag and Ag-Cu NPs using the disc diffusion method (Fig. 4A-F). Ag NPs have a higher inhibitory impact than Ag-Cu NPs at all concentrations. B. subtilis is the most susceptible to Ag NPs at 0.5 mg/ml concentration with a maximum ZOI of 85% area (Fig. 5A), whereas E. coli and S. cerevisiae exhibited 73% (Fig. 5C) and 71% area (Fig. 5B), respectively. Ag-Cu NPs (0.5 mg/ml) is inhibited 60% of B. subtilis, 20% of E. coli, and 50% area of S. cerevisiae (Fig. 5A-C). At lower concentrations, Ag NPs (0.0321 mg/ml) are inhibited 42.85% area of S. cerevisiae (Fig. 5B), while Ag-Cu NPs (0.0156 mg/ml) is inhibited it by 33.98% area (Fig. 5B). Similar trends are observed in Fig. 5D for displaying the energy kinetics of NPs against selected bacteria at 24 h and 48 h. The increasing order of potency of Ag NPs at high concentrations against microbes was B. subtilis > S. cerevisiae > E. coli. Ag NPs were more effective than Ag-Cu NPs against test microorganisms. Previous studies revealed that plant-derived metal NPs were effective against several pathogens and also played a key role in drug delivery [36,37]. Feng et al. reported that reactive oxygen species which trigger cell self-destruction were prepared by Ag NPs disintegrate into Ag + ion, inactivating the respiratory enzymes [38].

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Green production of biologically active Ag and Ag-Cu…
Further, the effect of oxidant loading on linalool epoxidation was studied using Ag-Cu NPs. As above, the ratio of substrate to oxidant loading (1:2) was effective for almost complete conversion with good selectivity. So, this ratio of substrate to oxidant loading at different concentrations is listed in Fig. 6B3, where an increase in linalool conversion percentage is observed on increasing the load from (1:1) to (1:2) (70-93%) after 210 min of reaction time, but the product selectivity is slightly comparable. While in case of 1:3 oxidant loading, the conversion percentage was nearly the same at 120 min, but the product selectivity was altered. Product 1a selectivity is decreased to 38%, and products 1c and 1d selectivity are increased to 20% and 25%, respectively (Fig. 6B6).
Also, the epoxidation of other monoterpenes like limonene, geraniol, α-pinene, and citronellol is studied using Ag-Cu NPs (Table 3). From the results, limonene is oxidized to produce limonene monoxide with 98% selectivity. Limonene oxide has numerous uses in the polymer sector and has been explored as a technique for introducing CO 2 into polycarbonate production [40]. Similarly, α-pinene has a double bond inside the ring, which is preferably transformed to α-pinene epoxide as compared to the β-pinene [25]. These epoxides have ample use as a flavouring agent due to their enhanced water solubility, lower volatility, and organoleptically superior profile [41].

Conclusions
P. cineraria pod's waste water extract was utilized for the synthesis of Ag and Ag-Cu NPs. In this study, the characterization of NPs revealed their crystalline nature, stability, capping properties, and average sizes of Ag NPs (14 nm) and Ag-Cu NPs (27 nm). Ag NPs showed high inhibitory activity (85%) against B. subtilis. Also, the epoxidation of linalool (monoterpenes) was achieved (93% conversion) using Ag-Cu NPs. Similarly, monoterpenes such as limonene, α-pinene, geraniol, and citronellol were transformed through epoxidation (81-93%) to demonstrate the importance of utilizing these green synthesized NPs for the production of commercial important pharmaceutical active ingredients (API) and fine fragrances. The prepared Ag and Ag-Cu NPs are greener in nature and have ample scope for being used as antimicrobial agents with excellent catalytic properties for the selective oxidation. Ag-Cu NPs successfully modified the model monoterpenoids such as linalool limonene, geraniol, α-pinene, and citronellol to their corresponding epoxides for use as drug intermediates, natural polymers, as well as flavouring sectors.