Eco-Friendly One Pot Synthesis of Zinc Oxide Nanoparticles Using Catkin Extract of Piper longum : In Vitro Antibacterial, Antioxidant and Antibioﬁlm Potential against Multi Drug Resistant Enteroaggregative E. coli †

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Introduction
Antimicrobial resistance (AMR) has been emerged as a nagging public health menace globally. The failure of conventional antibiotic therapies, along with the drastic decline in the antibiotic discovery pipeline, could jeopardize socioeconomic development and may even lead to untoward scenarios [1]. Of late, an unusual rise of drug resistance among EAEC strains has been recognized worldwide; hence, the AMR research paradigm should be shifted towards novel alternative tactics.
Recently, nanotechnology employing ZnO NPs have attained huge recognition due to their unique physicochemical features and tremendous scope for application in the field of biomedicine. The green route of NP synthesis has replaced the conventional physical and chemical methods, as they produce unintended effects such as potential health hazards and environmental pollutions [2].
Piper longum (Family Piperaceae), commonly known as "long-pepper" or "Pippali", is a well-known perineal shrub widely distributed in the tropical and subtropical world, including India. The extract of Piper longum is a rich source of various bio active phytoconstituents, including alkaloids, flavonoids, steroids and esters, and possesses excellent antibacterial, antioxidant and anti-inflammatory properties [3]. The synthesis of AgNPs using catkin extract of P. longum was previously documented [4], but not ZnO NPs. Considering these facts, the present study attempted to synthesize ZnO NPs using P. longum catkin extract and, further, to estimate their in vitro antimicrobial, antioxidant and antibiofilm activity against MDR-EAEC strains.

Bacterial Strains
The characterized MDR field strains of EAEC (E1; E2; E3) retained in the laboratory repository of College of Veterinary and Animal Sciences, Pookode were reaffirmed using antimicrobial susceptibility testing [5] and PCR assays [6]. The quality control strain used for the antimicrobial susceptibility testing in this study was E. coli ATCC 25922.

Preparation of Piper longum Catkin Extract
The powdered catkin of P. longum (10 g) was added to 100 mL of nanopure water, which was then heated to 60 • C for 1 h; using Whatman No. 1 filter paper, the solid particulates were removed, and the filtrate was stored at 4 • C until the synthesis of ZnO NPs [7].

Synthesis of ZnO NPs
The aqueous extract of P. longum catkin and zinc acetate dihydrate (0.10 M) solution was used for the green synthesis of ZnO NPs. In short, P. longum catkin filtrate (20 mL) was mixed with zinc acetate dihydrate solution (80 mL) for 2 h at 60 • C using a magnetic stirrer (300 rpm). The colour of the reaction mixture changed from colourless to light brown, demonstrating the formation of ZnO NPs. To remove impurities, the ZnO NPs were washed three times with methanol, followed by nanopure water. Finally, the ZnO NPs were air dried overnight at 80 • C and stored at 4 • C until further use [7].

Characterization of Green Synthesized ZnO NPs
The characterization of green synthesized ZnO NPs was accomplished via UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential thermal analysis (DTA), powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). To begin with, the ZnO NPs, dissolved in ultrapure water (1 mg/mL), were scanned using a UV-Vis spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) within a range of 250 to 450 nm. In order to assess the functional groups present on the green synthesized ZnO NPs, FTIR analysis was carried out at a resolution of 4 cm −1 within the range of 4000 to 400 cm −1 (Perkin Elmer C94012, Waltham, MA, USA). Approximately 12.473 mg of samples were heated at a rate of 40 • C per min in a nitrogen atmosphere between 40 and 1300 • C to accomplish the TGA-DTA of ZnO NPs (Perkin Elmer STA 600, Waltham, MA, USA).
The structural properties of the green synthesized ZnO NPs were investigated by means of PXRD (Bruker D8 Advance, Billerica, MA, USA) operated with a scanning step size of 0.02λ (λ = 1.54060 Å) at CuKα radiation using 40 KeV and 40 mA. The high-resolution TEM (HR-TEM; JEM 2100, Jeol, Tokyo, Japan) and SEM (Jeol 6390LV, Tokyo, Japan) analysis of the samples was used to perform the morphological analysis of ZnO NPs [7].

In Vitro Antibacterial Efficacy of ZnO NPs
The antimicrobial potency of the green synthesized ZnO NPs against the field strains of MDR-EAEC was assessed in vitro by evaluating the MIC and MBC values employing the micro broth dilution method [4].

In Vitro Stability Assays of ZnO NPs
The stability of the green synthesized ZnO NPs using the catkin extract of P. longum was evaluated by subjecting them to high-end temperatures (70 • C and 90 • C), proteases (trypsin, lysozyme, and proteinase-K), physiological concentrations of cationic salts (150 mM NaCl and 2 mM MgCl2) and varying pH (4.0, 6.0, and 8.0) [8].

In Vitro Safety Assays of ZnO NPs
A haemolytic assay, employing chicken erythrocytes, as well as a cytotoxicity assay using human epithelial embryonic kidney (HEK) cell lines were performed, and effect on beneficial gut lactobacilli (L. acidophilus MTCC 10,307 and L. plantarum MTCC 5690) was determined to investigate the in vitro safety of green synthesized ZnO NPs [8].

In Vitro Antioxidant Activity of ZnO NPs
By using the reducing power assay and the 2,2 -azinobis (3-ethylbenzothiazoline-6sulfonic acid) (ABTS•+)-free radical scavenging test, the in vitro antioxidant property of green synthesized ZnO NPs was evaluated [9].

In Vitro Antibiofilm Efficacy of ZnO NPs
By using the crystal violet staining technique in 96-well microtiter plates at 24 and 48 h, the in vitro antibiofilm efficacy of green synthesized ZnO NPs against the investigated strains of MDR-EAEC was evaluated [10].

Green Synthesis of ZnO NPs
Currently, the physical and chemical methods of nanoparticle synthesis are gradually being swapped for green routes employing natural and eco-friendly resources, owing to the release of toxic chemicals and complexities in synthesis conditions [2]. Being a rich source of pharmacologically active secondary compounds, P. longum has been widely used in ayurvedic preparations for various ailments [11].
The synthesis of ZnO NPs was accomplished by employing the P. longum catkin extract, which reduced the aqueous solution of zinc acetate dihydrate (0.10 M; 1:4 ratio) to ZnO NPs under vigorous stirring at 60 • C. After 2 h, the colour of the solution changed from colourless to brown, with the appearance of a brown-coloured precipitate at the bottom clearly indicating the formation of ZnO NPs.
The occurrence of various functional groups, as well as the stabilizing and capping properties of the aqueous P. longum catkin extract, plays a crucial role in the synthesis of ZnO NPs. In addition, the excitation of surface plasmon resonance (SPR) and reduction of Zn ions by the extract might have led to the colour change during ZnO NP synthesis [7].

Characterization of Green Synthesized ZnO NPs
The characterisation data were presented in our earlier report [7]. To begin with, the synthesis of ZnO NPs was confirmed by performing UV-Vis spectroscopy. The obtained green synthesised ZnO NPs showed a progressive SPR band flanked by 320 nm to 350 nm with a maximum absorption peak at 340 nm [7], which was in accordance with previous literatures [12,13]. The FTIR peaks observed (3640 cm −1 , 2850 cm −1 , 2100 cm −1 , 1739 cm −1 , 1490 cm −1 , 870 cm −1 , 915 cm −1 and 620 cm −1 ) indicated the presence of phytochemical components associated with the biosynthesized ZnO NPs in the extract [7]. Moreover, the TGA data demonstrated an initial weight loss of about 6% from 40 • C to 100 • C, which was corroborated by the DTG graph, with an exothermic peak seen at 200 • C. The weight of the green synthesized ZnO NPs had been steadily decreasing as a result of this rise in annealing temperature. Additionally, annealing temperatures between 900 • C and 1300 • C showed satisfactory thermal stability [7].
The PXRD graph obtained was juxtaposed with the Joint Committee on Powder Diffraction Standards (JCPDS) standard powder diffraction card of ZnO (File No. 36-1451) [14]. The intense and sharp peaks revealed the hexagonal wurtzite crystalline structure of the green synthesized ZnO NPs [7].
Moreover, the SEM image revealed primarily hexagonal shaped ZnO NPs and few in the form of cubes. Additionally, the TEM images confirmed the hexagonal shaped ZnO NPs, thereby supporting the results of SEM [7]. The presence of reducing agents in the catkin extract could be the main reason for the agglomeration and morphological differences of the biosynthesized ZnO NPs [2].

In Vitro Antibacterial Activity of ZnO NPs
In this study, the anti-bacterial efficacy (MIC and MBC values) of the green synthesized ZnO NPs against the MDR-EAEC test strains was found to be 125 µg/mL and 250 µg/mL, respectively ( Table 1). The relatively lower MIC and MBC values of the ZnO NPs in this study might be due to the lipopolysaccharide present in the Gram-negative bacterial cell wall, which exerted the resistance and subsequent aversion towards NPs [7].

In Vitro Stability Assays of ZnO NPs
The green synthesized ZnO NPs were investigated for their stability when subjected to high-end temperatures (70 • C and 90 • C), proteases (trypsin, lysozyme and proteinase-K), the physiological concentration of cationic salts (150 mM NaCl and 2 mM MgCl 2 ) and pH (4.0, 6.0, and 8.0). All the strains exhibited a constant MIC value when incubated at 70 • C and 90 • C. The MBC values remained unchanged until 5 min at both temperatures; nevertheless, a 2-fold rise in MBC value was observed thereafter (Table 2). Overall, the green synthesized ZnO NPs were found to be variably stable and capable of withstanding high-end temperatures [15]. The residual antimicrobial activity of the green synthesized ZnO NPs on exposure to protease enzymes (trypsin, lysozyme and proteinase-K) at different incubation intervals were also investigated. Remarkably, the MIC values of the green synthesized ZnO NPs were halved on exposure to protease enzymes, and the MBC values remained constant throughout the exposure period, except under certain conditions, where it reduced to half (Table 3). The fate of NPs is greatly affected by their interaction with biological fluids. Hence, in the present study, the stability of the green synthesized ZnO NPs against physiological concentrations of cationic salts were also evaluated. The green synthesized ZnO NPs maintained their antibacterial activity (MIC and MBC values) throughout the incubation period, irrespective of the cationic salts (Table 4). A modification of the physicochemical conditions such as pH and ionic strength of the solution can affect the intrinsic properties of NPs, such as their size, stability, zeta potential, morphology and shape [16]. Therefore, the stability of the green synthesized ZnO NPs at varying pH values (4, 6,8) was estimated by determining their antimicrobial activity against the MDR-EAEC strains. The green synthesized ZnO NPs tested were found to be stable at different pH values, as they retained their antimicrobial activity (MIC and MBC values). Notably, the MIC value of ZnO NPs was reduced to half at pH 8, as demonstrated by their MIC and MBC values (Table 5).

In Vitro Safety Assays of ZnO NPs
The in vitro haemolytic assay is often employed as a versatile tool for carrying out the initial toxicity assessment of a therapeutic compound [17]. In the present study, minimal haemolysis (less than 2%) was noticed for the green synthesized ZnO NPs at 2×, 5× and 10× MIC levels (Table 6). Similarly, the in vitro cytotoxicity effect of the green synthesized ZnO NPs on the viability of HEK cell lines was evaluated using the MTT assay. Overall, the green synthesized ZnO NPs did not exhibit any cytopathic effect at 1×, 5× and 10× MIC levels. Furthermore, the investigation of their effect on beneficial gut microflora is also important as they form a vital part of the body's innate immune system [18]. In this study, L. acidophilus and L. plantarum revealed similar growth patterns in both treatment control (treated with green synthesized ZnO NPs) as well as the untreated control. Moreover, a non-significant (p > 0.05) antimicrobial efficacy was observed for the green synthesized ZnO NPs against the tested strains of L. acidophilus and L. plantarum.

In Vitro Antioxidant Activity of ZnO NPs
Antioxidants play a vital role in the functioning of all the biological systems due to their importance in scavenging the noxious free radicals produced in the body, and thus preventing oxidative stress. Hence, the antioxidant scavenging activity of the green synthesized ZnO NPs was evaluated by employing ABTS and reducing power assay, keeping ascorbic acid as standard. In this study, a dose-dependent increase in their antioxidant properties was revealed with ABTS ( Figure 1a) and reducing power assays (Figure 1b) of the green synthesized ZnO NPs, suggesting an enhanced ability to scavenge free radicals.
Overall, the green synthesized ZnO NPs did not exhibit any cytopathic effect at 1×, 5× and 10× MIC levels. Furthermore, the investigation of their effect on beneficial gut microflora is also important as they form a vital part of the body's innate immune system [18]. In this study, L. acidophilus and L. plantarum revealed similar growth patterns in both treatment control (treated with green synthesized ZnO NPs) as well as the untreated control. Moreover, a non-significant (p > 0.05) antimicrobial efficacy was observed for the green synthesized ZnO NPs against the tested strains of L. acidophilus and L. plantarum.

In Vitro Antioxidant Activity of ZnO NPs
Antioxidants play a vital role in the functioning of all the biological systems due to their importance in scavenging the noxious free radicals produced in the body, and thus preventing oxidative stress. Hence, the antioxidant scavenging activity of the green synthesized ZnO NPs was evaluated by employing ABTS and reducing power assay, keeping ascorbic acid as standard. In this study, a dose-dependent increase in their antioxidant properties was revealed with ABTS ( Figure 1a) and reducing power assays (Figure 1b) of the green synthesized ZnO NPs, suggesting an enhanced ability to scavenge free radicals.

In Vitro Antibiofilm Activity of ZnO NPs
Owing to the potential antibacterial property of ZnO NPs, they could be employed as a versatile tool for the treatment of bacterial biofilms. In this study, we investigated the ability of the green synthesized ZnO NPs to inhibit the biofilm-forming ability of MDR-EAEC isolates at both 24 and 48 h, employing crystal violet staining assay. All the MDR-EAEC isolates treated with the green synthesized ZnO NPs exhibited a significant antibiofilm effect after 24 h and 48 h (p < 0.001) relative to their respective controls ( Figure 2).

In Vitro Antibiofilm Activity of ZnO NPs
Owing to the potential antibacterial property of ZnO NPs, they could be employed as a versatile tool for the treatment of bacterial biofilms. In this study, we investigated the ability of the green synthesized ZnO NPs to inhibit the biofilm-forming ability of MDR-EAEC isolates at both 24 and 48 h, employing crystal violet staining assay. All the MDR-EAEC isolates treated with the green synthesized ZnO NPs exhibited a significant antibiofilm effect after 24 h and 48 h (p < 0.001) relative to their respective controls (Figure 2).

Conclusions
In short, we successfully synthesized ZnO NPs employing the aqueous extract of Piper longum catkin. The green synthesized ZnO NPs were characterized by spectroscopy, XRD, TGA and electron microscopy. The antibacterial activity of the ZnO NPs was exhibited using the micro broth dilution technique against MDR-EAEC strains. The in vitro assays revealed that the synthesized NPS were variably stable, safe and possess excellent antioxidant and antibiofilm activity. Overall, the study demonstrated an eco-friendly and safe approach for NP synthesis which further highlights the significance of these particles as a suitable candidate for treating drug resistant pathogens. However, in vivo clinical studies should also be performed to validate the application of ZnO NPs in suitable target hosts.

Conclusions
In short, we successfully synthesized ZnO NPs employing the aqueous extract of Piper longum catkin. The green synthesized ZnO NPs were characterized by spectroscopy, XRD, TGA and electron microscopy. The antibacterial activity of the ZnO NPs was exhibited using the micro broth dilution technique against MDR-EAEC strains. The in vitro assays revealed that the synthesized NPS were variably stable, safe and possess excellent antioxidant and antibiofilm activity. Overall, the study demonstrated an eco-friendly and safe approach for NP synthesis which further highlights the significance of these particles as a suitable candidate for treating drug resistant pathogens. However, in vivo clinical studies should also be performed to validate the application of ZnO NPs in suitable target hosts.