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• Published: 03 June 2020

Green route to synthesize Zinc Oxide Nanoparticles using leaf extracts of Cassia fistula and Melia azadarach and their antibacterial potential

• Minha Naseer 1 ,
• Usman Aslam   ORCID: orcid.org/0000-0001-8145-8360 2 ,
• Bushra Khalid 3 , 4 &
• Bin Chen   ORCID: orcid.org/0000-0002-0925-9209 5 , 6  

Scientific Reports volume  10 , Article number:  9055 ( 2020 ) Cite this article

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• Antimicrobial resistance
• Nanoparticles

Development of plant based nanoparticles has many advantages over conventional physico-chemical methods and has various applications in medicine and biology. In present study, zinc oxide (ZnO) nanoparticles (NPs) were synthesized using leaf extracts of two medicinal plants Cassia fistula and Melia azadarach . 0.01 M zinc acetate dihydrate was used as a precursor in leaf extracts of respective plants for NPs synthesis. The structural and optical properties of NPs were investigated by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscope (SEM), ultraviolet-visible spectrophotometer (UV-Vis) and dynamic light scattering (DLS). The antibacterial potential of ZnO NPs was examined by paper disc diffusion method against two clinical strains of Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) based on the zone of inhibition and minimal inhibitory indices (MIC). Change in color of the reaction mixture from brown to white indicated the formation of ZnO NPs. UV peaks at 320 nm and 324 nm, and XRD pattern matching that of JCPDS card for ZnO confirmed the presence of pure ZnO NPs. FTIR further confirmed the presence of bioactive functional groups involved in the reduction of bulk zinc acetate to ZnO NPs. SEM analysis displayed the shape of NPs to be spherical whereas DLS showed their size range from 3 to 68 nm. The C. fistula and M. azadarach mediated ZnO NPs showed strong antimicrobial activity against clinical pathogens compared to standard drugs, suggesting that plant based synthesis of NPs can be an excellent strategy to develop versatile and eco-friendly biomedical products.

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Introduction.

Plant mediated synthesis of nanoparticles (NPs) is a revolutionary technique that has wide range of applications in agriculture, food industry and medicine. NPs synthesized via conventional methods have limited uses in clinical domain due to their toxicity. Due to the physio-chemical properties of plant based NPs, this method also offer an added advantage of increased life span of NPs that overcome the limitations of conventional chemical and physical methods of NPs synthesis 1 , 2 , 3 . Plants possess rich genetic variability with respect to number of biomolecules and metabolites like proteins, vitamins, coenzymes based intermediates, phenols, flavonoids and carbohydrates. These plant metabolites contain hydroxyl, carbonyl, and amine functional groups that react with metal ions and reduce their size into nano range. More specifically, flavonoids contain several functional groups and it is believed that -OH group of flavonoids is mainly considered responsible for the reduction of metal ions into NPs 4 . These molecules not only help in bioreduction of the ions to the nano scale size, but they also play a pivotal role in the capping of the nanoparticles which is important for stability and biocompatibility 5 . Reducing agents such as phenolic compounds, sterols and alkaloids can reduce metal ions into NPs in a single reaction 6 .

The type and nature of the metal used for NPs biosynthesis mainly determines the NPs end use industry. Several metals such as silver (Ag), copper (Cu), gold (Au) and many others have been widely used for the biosynthesis of NPs using plant extracts of various plant species 7 , 8 , 9 . However their higher toxicity to animals and humans pose a serious limitation for use in medical industry. ZnO is an inorganic compound which occurs rarely in nature. It is generally found in crystalline form. Naturally occurring ZnO has manganese impurities that give it a typical red or orange color appearance 10 . When purified, ZnO appears as white crystalline powder which is nearly insoluble in water. Due to their low toxicity and size dependent properties, ZnO NPs have been widely used for various applications in textiles, cosmetics, diagnostics and even in micro-electronics. Because ZnO is generally recognized as safe (GRAS) and exhibits antimicrobial properties, ZnO NPs hold greater potential to treat infectious diseases in humans and animals 11 .

ZnO has been found to be potentially useful and efficient than other metals for biosynthesis of NPs for clinical purposes. Several studies have demonstrated the synthesis of ZnO NPs using different plant extracts. For example, flower extract of the medicinal plant Cassia auriculata 12 and leaf extract of Hibiscus rosasinensi 13 were used as reducing agents for zinc nitrate to synthesize ZnO NPs.

Plant type or source species from which plant extract used for NPs synthesis also affects the size of NPs. For example, when Olea europea leaf extract was used to synthesize ZnO nano sheets, it ranged from 18–30 nm in size 14 . However, when Aloe barbadensis 15 and Ocimum tenuiflorum 11 were used as reducing agent for the green synthesis of ZnO nanoparticles, the average nanoparticle sizes were 25–40 nm and 13.86 nm respectively. Recently various reports have also demonstrated the antimicrobial activity of ZnO NPs. For example, ZnO NPs synthesized by using leaf extracts of Passiflora caerulea , Scadoxus multiflorus and Camellia sinensis showed strong antimicrobial efficacy against Klebsiella pneumonia , Aspergillus spp., and Staphylococcus aureus and Pseudomonas aeruginosa respectively 16 , 17 , 18 , suggesting that medicinal plant extract mediated synthesis of ZnO NPs can be very useful for medical industry.

Cassia fistula commonly known as Golden Shower or Amaltas is a deciduous tree with medicinal importance, native to Pakistan and India and found as an exotic species in Egypt, Australia, Ghana, Mexico, and Zimbabwe. It belongs to the family Fabaceae . It produces shiny green leaves which are about 30–40 cm long, pinnate in shape and arranged in alternate fashion on the terminal branches 19 . Leaves of C. fistula contain a wide variety of antioxidants for example; terpenoids, flavonoids, alkaloids, phenolic compounds, tannins, saponins, anthocyanosides, carbohydrates, proteins, steroids, cardiac glycosides and phlobatannins 20 .

Similarly, Melia azadarach commonly known as Cape Lilac and locally as Bakain belongs to the family Meliaceae . It is native to Southeast Asia and found naturally in most of the tropical and subtropical countries. This plant is locally famous for its anti-microbial, anti-inflammatory and anti-cancer activities and often used to treat stomach pains and parasitic infections. It produces dense array of dark green leaves which are short stalked and arranged in alternate pattern on terminal branches. Fruits are yellow colored, smooth and fleshy berries. M. azadarach is naturally enriched in phytochemicals. It is endowed with alkaloids, sterols, glycosides, flavonoids, limonoids, fixed oil and fats, phenolic compounds, tannins, saponins, gum and mucilages, triterpenes, azadirachitin, nimbin, melianoninol, melianol, meliandiol, vanillin, meliacin, quercertin and rutin 21 . Due to the presence of diverse array of these phytochemicals and medicinal properties, C. fistula and M. azedarach hold greater potential for efficient biosynthesis of NPs that can be useful to treat clinical pathogens.

Here, we report a simple and eco-friendly method of ZnO NPs synthesis from the plant extracts of C. fistula and M. azedarach as reducing agents and zinc acetate as precursor for their comparative analysis of antimicrobial potential. This research will increase the potential of usage of plant based NPs in biomedical industry.

Results and Discussion

Optical analysis of zno nps formation.

Adding zinc acetate dihydrate in leaf extracts of C. fistula and M. azedarach leads to physio-chemical changes in the aqueous solution. The most prominent of which is change in the colour of the reaction mixture that can be observed within few minutes. This was considered as an initial signature to formation of NPs. In present study, change of color from yellow to light brown and red to off-white indicated the formation of ZnO NPs in leaf extracts of C. fistula and M. azedarach , respectively. Flavonoides and phenolic compounds are thought to be responsible for Zn ions to ZnO NPs. In a period of few hours, the colour of the solution stopped changing further suggesting the complete bioreduction of ZnO salt into NPs. A clear illustration of change in color of the reaction mixtures due to formation of ZnO NPs has been shown in Fig.  1(A,B) . These results were consistent with the previous reports of color changes in plant based synthesis of ZnO NPs 22 . Temperature is considered an important contributing factor in synthesis of good sized nanoparticles. It is also well established that higher the temperature of reaction process of NPs synthesis, the smaller the size of the NPs 23 , 24 . Therefore, we use a relatively higher temperature of 70 °C for incubating the reactants that leads to the production of very small sized ZnO NPs.

Optical analysis of ZnO NPs. ( A,B ) Color changes indicating formation of ZnO NPs. A) Cassia fistula mediated ZnO NPs. ( B ) Melia azadarach mediated ZnO NPs. ( C,D ) UV-visible absorption spectrum confirming presence of ZnO NPs. ( A ) Cassia fistula mediated ZnO NPs. ( B ) Melia azadarach mediated ZnO NPs.

The synthesis of ZnO NPs was further examined by UV spectrophotometry. Figure  1(C,D) shows the UV peaks recorded by the spectrophotometer. The maximum absorption peak for ZnO NPs synthesized via C. fistula was recorded at 320 nm and with that of M. azadarach at 324 nm that further verified the formation of ZnO NPs. Firstly, these results satisfy standard ZnO absorption pattern because all oxide materials have wide band gaps and tend to have shorter wavelengths. Moreover, if the material is of nanoscale, it tends to have further shorter wavelengths. This notion support the results observed for ZnO NPs here 25 .

Surface morphology of ZnO NPs

The presence of nanoparticles and examination of their structural properties were confirmed by X-ray diffractrometer. C. fistula and M. azedarach associated ZnO NPs showed peaks with 2θ values identified at 31.841°, 34.507°, 36.324°, 47.592°, 56.634°, 66.426°, 67.983°, 69.091°, and 76.987° which are indexed as (100), (002), (101), (102), (110), (103), (112), (201) and (202) planes (Fig.  2A,B ). These peaks were in accordance with those of data card (JCPDS-36-1451). Average crystal size calculated using the Scherrer’s equation ( \(Dp\,of\,ZnO\,NPs=(0.9(1.5406)/0.63(\cos \,36)\) came out to be around 2.72 nm for both C. fistula and M. azedarach associated ZnO NPs that is comparable with the size of good quality NPs in existing reports 26 .

( A,B ) XRD pattern indicating presence of ZnO peaks. ( A ) Cassia fistula mediated ZnO NPs. ( B ) Melia azadarach mediated ZnO NPs. ( C,D ) FTIR pattern indicating the functional groups involved in ZnO NPs synthesis. ( C ) Cassia fistula mediated ZnO NPs. ( D ) Melia azadarach mediated ZnO NPs.

To identify the functional groups associated with the ZnO NPs formation, FTIR spectrometry was performed. Spectral peaks at 683–500 cm −1 and 698–505 cm −1 proposed the formation of ZnO nanoparticles in C. fistula and M. azedarach extracts, respectively (Fig.  2C,D ). Absence of peaks in the region of 3500 and 2500 cm −1 indicated no characteristic OH and N-H stretching of aldehydes. The bands at 1600–1510 cm −1 correspond to amide I and amide II regions arising due to carbonyl stretching in proteins and that of 1400 to 1000 cm −1 correspond to methylene from the proteins in the solution and C-N stretching vibrations of amine. Peaks from 1460–1410 cm −1 suggested C-C stretching vibration of alcohol, carboxylic acid, ether and ester and bands at 946–769 cm −1 demonstrated presence of carboxylic acid and aromatic C-H bending. Although, many changes were not observed at these frequencies but all peaks showed a shift to lower frequency and a decrease in intensity on binding with the nanoparticles. This trend of free carbonyl and NH 2 groups from proteins and amino acid residues indicates that they have ability to bind to a metal and that the proteins could possibly form a layer around the metal for preventing agglomeration and thereby stabilizing the nanoparticles. It is revealed from the FTIR spectra that in fact, the protein molecules present in the leaf extract possibly cause the reduction of metal ions which is in agreement with the previous reports 27 . These findings suggest that not only the OH group of flavonoids but also the protein molecules and their functional groups play important role in bioreduction of salts and capping of NPs.

Dynamic Light Scattering (DLS) measurements showed the average diameters of C. fistula and M. azadarach mediated ZnO NPs (Fig.  3 ). Average diameters of ZnO NPs synthesized from C. fistula and M. azadarach were 68.1 nm and 3.62 nm, respectively. The results demonstrated that the particles synthesized were ultrafine i.e. less than 100 nm in diameter. It clearly depicts that M. azadarach extract was more efficient than C. fistula for synthesizing smaller NPs. It may be attributed to the presence of more variety of phytochemicals in M. azadarach when compared to C. fistula . As it has already been mentioned in the introduction section that M. azadarech possesses complete set of phytochemicals that can be the reason behind higher efficacy of this plant as a reducing agent when compared to C. fistula . In addition, DLS analysis demonstrated that the NPs formed had fairly well-defined dimensions 28 . Smaller the size of the NPs, higher the surface area, thus higher the antimicrobial activity. Generally, bacterial cellular membranes have nanometer size. If the nanoparticles are smaller in size than cell membrane pores, there is more possibility of crossing the cell membrane barrier and thus inhibiting the bacterial growth 29 .

DLS indicating average size of ZnO NPs. ( A ) Cassia fistula mediated ZnO NPs. ( B ) Melia azadarach mediated ZnO NPs.

Figure  4 shows Scanning Electron Microscopy (SEM) images of ZnO NPs synthesized from leaf extracts of C. fistula and M. azadarach . The images were recorded at magnification of 10 µm, 1 µm and 100 nm. Topographical view shows that nanoparticles are more or less spherical in nature, clustered together and surface of the aggregates seems to be rough 30 . SEM images also revealed that NPs derived from both plants are entirely pure and it can be concluded that both the plants have tremendous capability to synthesize ZnO NPs. Shape of NPs plays very crucial role in the effectivity against pathogens. Because spherical NPs tend to be very potent during antibacterial activity owing to their ability to easily penetrate into the cell wall of pathogens 31 , therefore, ZnO NPs syntheized from these two plant species can be of great importance in treating clinical pathogens.

SEM images of ZnO particles showing their morphology at three different resolutions. ( A–C ): Scanning Electron Micrographs of Cassia fistula mediated Zno NPs. ( A ) SEM of Zno NPs captured at 500× magnification. ( B ) SEM of Zno NPs captured at 16,000× magnification. ( C ) SEM of Zno NPs captured at 65,000× magnification. ( D–F ): Scanning Electron Micrographs of Melia azadarach mediated Zno NPs. ( D ) SEM of Zno NPs captured at 800× magnification. ( E ) SEM of Zno NPs captured at 8,000× magnification. ( F ) SEM of Zno NPs captured at 30,000× magnification. Red dotted circles in ( C,F ) indicate the NPs circumference.

Antibacterial activity of ZnO NPs

The bactericidal activities of C. fistula and M. azadarach mediated ZnO NPs were tested against two main clinical pathogens; a (Gram-negative pathogen) E. coli and b (Gram-positive pathogen) S. aureus . Figure  5 illustrates zones of inhibition of E. coli and S. aureus against standard drugs and biosynthesed ZnO NPS at concentrations ranging from 50 µg/mL (10 µL) to 1000 µg/mL (200 µL). The mean values of zone of inhibition (mm) of three replicates are presented in (Table  1 ). Comparison between standard antibiotics and biosynthesed NPs showed strong antibacterial effect of NPs as compared to standard drugs (Table  2 ). In E. coli , zone of inhibition of standard drugs ranged from 15–20 mm while that of ZnO NPs was 16–40 mm. S. aureus was resistant to a variety of standard drugs and zone of inhibition for rest of the standard drugs was ranged from 4–13 mm while that of ZnO NPs was 14–37 mm in range. (Table  3 ) shows zones of inhibition of various standard drugs and standard drug potency according to WHO standards.

Resistance level of two clinical pathogens against (i) standard drugs, (ii) ZnO NPs 10 µL, 50 µL and (iii) ZnO NPs 100 µL, 200 µL. ( A ) Inhibition zones of ZnO NPs against E. coli growth. ( B ) Inhibition zones of ZnO NPs against S. aureus growth. Lower panel in both part A and B illustrates the labelling of petri plates.

Both the E. coli and S. aureus showed minimum inhibitory concentration (MIC) at 10 µL for the synthesized ZnO NPs. Furthermore, as the concentration of NPs increased so did the zone of inhibition. It is evident from the recordeded images and statistical data that zone of inhibition of C. fistula mediated ZnO NPs was more significant against E. coli ( ∼ 44 mm) as compared to S. aureus (Fig.  5 , Table  2 ). The mild inhibitory effect of C. fistula mediated ZnO NPs on S. aureus when compared to E. coli can be attributed to the differences in membrane strutures of Gram-positive and Gram-negative bacteria. The most disntinctive feature of Gram-positive bacterium is the thickness of cell wall due to the prescence of peptidoglycan layer. It has also been reported that ZnO NPs may damge bacterial cell membrane resulting lysis of intracellular contents and ultimately proved to be lethal for the bacterial cell 32 . Lower efficacy of C. fistula mediated ZnO NPs against S. aureus compared to the Gram-negative species might be due to the resistance of cell wall in Gram-positve species 33 . By contrast, the zone of inhibition of M. azadarach mediated ZnO NPs was compareable against both the pathogens. However, it is important to note that the zone of inhibition of M. azadarch mediated ZnO NPs was significantly greater in comparison to C. fistula mediated ZnO NPs against S. aureus (Fig.  5 , Table  2 ). These results suggest that the use of M. azadarch mediated synthesis of ZnO NPs can be more efficient against Gram-positive pathogens like S. aureus . This might be due to the presence of higher number of phenolic compounds and rare secondary metabolites such as nimbinene, meliacin, quercertin and rutin in M. azadarch .

As a schematic layout of this whole study, a model has been given in Fig.  6 that shows the graphical representation of the synthesis of ZnO NPs using leaf extarcts of C. fistula and M. azadarach as reducing agents and zinc acetate as a precursor salt.

Schematic model of ZnO NP synthesis from the leaf extracts of Cassia fistula and Melia azedarach and their antibacterial activity analysis.

Leaf extracts of C. fistula and M. azadarach showed excellent potential as reducing agents in the formation of NPs. Structural and optical studies conducted using UV, FTIR, XRD, DLS and SEM analysis confirmed the formation of efficient ZnO NPs. Antibacterial analysis revealed that ZnO NPs synthesized from leaf extracts exhibited significant capability of inhibition against the clinical pathogens when compared to traditional drugs. Moreover, some plant extratcs are more effective than that of others in synthesizing NPs and biological activities due to their diverse biochemical compositions. In conclusion, synthesis of NPs using extratcs of medicinal plants can have useful medicinal applciations in treatment of numerous human infectious pathogens. However, further studies will be required to validate the efficacy of these NPs in medical applications and their capacity to overcome the risks associated with conventional drugs.

Synthesis of Nanoparticles

All the glassware were autoclaved before use. To prepare leaf extract, fresh leaves of C. fistula and M. azadarach were thoroughly washed with tap water followed by distilled water (d.H 2 O) to remove any contamination. The leaves were air dried for a week at room temperature ( ∼ 37°C). About 5 g of leaves from each of C. fistula and M. azadarach were ground to fine powder with the help of pestle and mortar. This powder was mixed in 500 mL of d.H 2 O and then heated at 70°C for 30 minutes. The mixture was filtered first by muslin cloth and then using Whatman filter paper No.1. As a result, pale yellow and red colored solutions were obtained as leaf extracts of C. fistula and M. azadarach respectively which were stored at 4 °C.

0.01 M zinc acetate dihydrate (Zn (C 2 H 3 O 2 ) 2 .2H 2 O) solution was prepared in d.H 2 O. For synthesis of ZnO nanoparticles, 95 mL of 0.01 M zinc acetate dihydrate (Zn (C 2 H 3 O 2 ) 2 .2H 2 O) solution was mixed separately with 5 mL plant extract of each of C. fistula and M. azadarach in individual 250 mL flasks. These mixtures were incubated at 70°C for 1 hour with continuous shaking at 150 rpm. This led to the settlement of bio-reduced salt at the bottom of the flask which appeared as white precipitate. The supernatant was decanted and powdery precipitate was transferred to 1.5 mL centrifuge tubes. Both the samples were subjected to washing with d.H 2 O by centrifugation at 3000 rpm for 30 minutes. Washing step was repeated thrice to ensure removal of impurities 22 .

Characterization of NPs

Optical Spectroscopy . To measure the optical parameters, ZnO synthesized nanoparticles were dispersed in d.H 2 O. The absorption spectrum of synthesized NPs was measured using UV–VIS-NIR spectrophotometer (UV-1601, Shimadzu, Japan) in wavelength range between 200–800 nm. The d.H 2 O was used as a reference. Energy gap or band gap was calculated using the following equation

where Eg is the bulk band expressed in eV. Lambda ( 𝜆 ) is peak absorbance wavelength in nm. Therefore, the energy gap for ZnO ranges from 4.27–3.87 eV 34 .

FTIR Analysis . The surface chemistry of NPs was analyzed by FTIR spectroscopy. The functional groups attached to the surface of NPs were detected in the range of 4000–400 nm. The samples were prepared by dispersing the ZnO NPs uniformly in a matrix of dry KBr which was then compressed to form a transparent disc. KBr pellet was used as a standard 35 .

XRD Analysis . X-ray diffractrometer (PAN analytical X-Pert PRO) was used to study the surface morphology, size and crystalline nature of ZnO NPs. The diffraction pattern was obtained using CuKα radiation with wavelength of λ = 1.541 A°. A thin film of the sample was made by putting a small amount of sample on a glass plate for XRD studies. The scanning was done in 2θ value range of 4° to 80° at 0.02 min −1 and 1 second time constant. The instrument was operated at a current of 30 mA and voltage of 40 kV. Scherrer’s equation was used to calculate the average grain size of synthesized NPs which is as under

where D represents the crystallite size, λ stands for the wavelength (1.5406 Å for Cu Kα), β symbolizes the full-width at half-maximum (FWHM) of main intensity peak after subtraction of the equipment broadening and θ is used as a diffraction angle in radians.

DLS Analysis . The particle size distribution of the samples was obtained through Particle Size Analyzer (Zetasizer Ver. 7.11 Malvern). The liquid samples of ZnO NPs was diluted ten times using Milli-Q water, centrifuged and then transferred to cuvette for analysis. The zeta potential of ZnO NPS was determined in water as dispersant.

SEM Imaging . The samples of ZnO NPs were dispersed in methanol (evaporating solvent) at a concentration of 1 mg/20 mL. A single drop of aqueous solution of ZnO NPs was placed on the carbon coated grid to prepare a thin film. Extra solution was removed with the help of blotting paper and the grid was allowed to dry under mercury lamp for around five minutes. The morphological measurements of the ZnO NPs samples were recorded with field emission scanning electron microscope (JEOL, Model: JSM-7600F) in the range of 0.1 nm to 10,000 nm. The data collected from all techniques was analyzed in Origin software version 9.1.

Antimicrobial analysis

To check the bactericidal potential of the NPs, pure cultures of Escherichia coli (EPEC-A (P16), and Staphylococcus aureus [(MRSA belonging to clonal complex 8 (CC8) and sequence type 239 (ST239)] were obtained from the Department of Microbiology, Pakistan Institute of Medical Sciences (PIMS), Islamabad. Disc diffusion method was used to carry out the antibacterial assay of NPs on Muller Hinton Agar (MHA) medium containing petri plates. Contamination test was carried out by incubating the plates over night at room temperature. After confirmation of no contamination, bacterial cultures were streaked on to these MHA plates.

Stock solution of NPs was prepared in d.H 2 O at a concentration of 5 mg/mL. Further, four working dilutions i.e. 50 µg/mL (10 µL), 250 µg/mL (50 µL), 500 µg/mL (100 µL) and 1000 µg/mL (200 µL) were made to find out minimum inhibitory concentration (MIC). The Minimum Inhibitory Concentration (MIC) of the ZnO NPs was determined based on batch cultures containing varying concentrations of ZnO NPs in suspension (10–200 µg/mL). Bacterial concentrations were determined by measuring optical density (OD) at 600 nm.

To examine the bactericidal effect of NPs on clinical strains, approximately 10 8 CFU of each strain was cultured on nutrient agar plates. Following disc diffusion method, the sterile discs were dipped in ZnO nanoparticles solution at varying concentrations from 50 µg/mL to 1000 µg/mL. Discs were placed onto the MHA plates and incubated at 37 °C. Control samples were prepared by placing standard medicine discs onto MHA plates containing bacterial isolates. Standard medicines used for E. coli were Ceftazidime, Imipenem, Cefoperazone, Amoxicillin, and Cefixime, whereas, Erythromycin, Gentamycin, Vancomycin, Chloramphenicol, Lanzolid were used for S. aureus . Mean values of inhibitory zone diameter were recorded in three experimental repeats. The average values of inhibition zones were calculated as Mean ± Standard Deviations. The data was statistically analyzed using Origin software version 9.1 36 .

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Acknowledgements

This work was supported by the International Partnership Program of the Chinese Academy of Sciences [grant number 134111KYSB20180021), the National Natural Science Foundation of China [grant numbers 41590871], and the International Science & Technology Cooperation Program of China [grant number 2013DFG22820]. The authors highly acknowldege the techinal support for this research from the Department of Microbiology, Pakistan Institute of Medical Sciences (PIMS), Islamabad and Pakistan Institute of Nuclear Science and Technology (PINSTECH), Islamabad, Pakistan.

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Minha Naseer

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Naseer, M., Aslam, U., Khalid, B. et al. Green route to synthesize Zinc Oxide Nanoparticles using leaf extracts of Cassia fistula and Melia azadarach and their antibacterial potential. Sci Rep 10 , 9055 (2020). https://doi.org/10.1038/s41598-020-65949-3

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Green synthesis of Silver-iron-zinc oxides nanocomposite via Embelia schimperia leaf extract for photo-degradation of antibiotic drug from pharmaceutical wastewater

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• Published: 31 August 2024
• Volume 14 , article number  210 , ( 2024 )

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• Defar Getahun Gizachew 1 ,
• Edo Begna Jiru 1 ,
• Tsigab Tekle’Ab 1 ,
• Yigezu Mekonnen Bayisa 1 &
• Tafere Aga Bullo 1  

The co-precipitation approach is used in the current study to create an environmentally friendly Ag/Fe/ZnO nanocomposites utilizing an aqueous leaf extract of Embelia schimperia . The synthesized nanocomposite was characterized using Fourier-transform infrared, UV, X-ray, UV–vis, DLS, TGA, and SEM to determine its functional group, structure, bandgap energy, size distribution, a mass of loss, and energy gain or loss, and morphological structure, respectively. The bioactive components of Embelia schimperia , synthesized Ag/Fe/ZnO NCs and degradation of Amoxicillin via photocatalyst were assessed. The response surface methodology of central composite design (CCD) was used to examine and optimize the effects of three independent variables on the degradation of Amoxicillin under visible light. According to the experimental findings, the maximum photocatalytic degradation efficiency was achieved at green synthesized Ag/Fe/ZnO NCs dosage of 100 mg, a concentration of Amoxicillin of 30 mg/L and a radiation time of 180 min. Their findings show that Embelia schimperia extract-derived Ag/Fe/ZnO nanocomposites is a promising alternative for degradation of pharmaceuticals contamination of wastewater via photocatalytic under the given conditions.

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Introduction

Water is very useful for all humans, animals, plants, and the whole ecosystem. However, to use the water keeping, the quality of this water is very important. Nowadays, water is contaminated by different contaminates: Natural, physical, chemical, and biological contaminants such as salt, sand dust, organic, inorganic, bacteria, protozoa, viruses, and parasite contaminants (Pandit & Kumar 2019 ; Sharma et al. 2018 ). Chemical contaminants are among the most abundant pollutant in water sources (Bozorg-Haddad et al. 2021 ; Khan et al. 2020 ). Pharmaceutical waste is one of the major worrying emerging contaminants, which arise from pharmaceutical industries that are biologically active compounds that daily life used (Bayisa et al. 2021 ; Costa et al. 2019 ; Kharissova et al. 2019 ). When the effluent from Pharmaceuticals waste is discharged into the water stream, that bring significant risks posed, leading to genotoxic, mutagenic, and eco-toxicological effects on plants, animals, humans, microbial, living things, and others (Samal et al. 2022 ). This pharmaceutical wastewater contained different organic and inorganic pollutants, which contaminate water (Aththanayaka et al. 2022 ). To treat this pharmaceutical wastewater, different methods and strategies are there from different sources of raw material (Khan et al. 2020 ). According to different scholars stated treatments of pharmaceutical wastewater by green synthesized metal oxide nanocomposites (MONCs), which is better than another, are in terms of cost, time, efficiency, and safety (Afolalu et al. 2022 ; Bullo & Bayisa 2022 ; Khan et al. 2020 ; Kharissova et al. 2019 ). Among other magnetic metals, Ag, Fe, and Zn get great attention due to their unique optoelectronic properties, shapes, sizes, and large specific surface areas (Immanuel et al. 2019 ). Moreover, Ag is easily produced because of its chemical inertness, durability to surface oxidation, better shape control, size, and relative stability when compared to other alloys (Ishak et al. 2019 ; Samal et al. 2022 ) and induce oxidative stress and cellular toxicity by producing reactive oxygen species (ROS). In addition, more ROS can be produced by the ZnO and Ag nanocomposite, which can also have a cumulative influence on photocatalysis, antibacterial, and anticancer properties. Likewise, because of its superparamagnetic and distinctive physicochemical characteristics, iron nanoparticles are a versatile remediation material. They also have good electron-donating qualities and are quite reactive in water at ambient conditions.

The green synthesis methods include the synthesis of nanomaterials from the plant, bacterial, fungal species, algae, and yeast (Salem & Fouda 2021 ). Green synthesis methods of nanocomposite from the plant are most appropriate than bacterial, yeast, and fungi (Singh et al. 2018 ). Plant is a better source of nanocomposite as a reducing agent than bacteria, yeast, fungi, and algae in terms of efficiency, reusability, ecofriendly and cost (Aththanayaka et al. 2022 ; Jeevanandam et al. 2022 ). Thus, in this study, Ag–Fe–ZnO nanocomposite was synthesized using Embelia schimperia leaf as a reducing agent and AgNO 3 , Fe 2 NO 3 , ZnNO 3 as the precursor. The synthesized nanocomposite was used for the treatment of pharmaceutical wastewater.

Material and methods

Materials and reagents.

The silver nitrate (AgNO3, 99%), zinc acetate hexahydrate (Zn(CH3OO) 2 .2H 2 O, 98%, extra pure), and iron chloride (FeCl 3 , 99%), produced by Loba Chemicals Pvt. Ltd in India, was purchased from Atomic Educational Materials Supply PLC in Addis Ababa, Ethiopia. Embelia schimperia leaf was acquired from Jimma Institute of Technology Campus, Southwestern Oromia, Ethiopia. All the other chemicals were analytical reagent grade and bought from Al-Chem-Supply Kirkos Ltd. in Addis Ababa, Ethiopia. All reagents used in this study were pure analytical grade.

Preparation of plant extract

Embelia schimperia leaves were collected from the Jimma Institute of Technology campus and washed with distilled water to remove sludge and dust. The collected raw materials (leaves) were air-dried for a week and grinded with a mortar and pestled to small sizes. It was then kept in a clean, dry place and ready for extraction. After one week, 20 g of crushed dried leaves and 200 ml of deionized water were added to a 500-ml conical flask and mixed vigorously. The mixture was then heated at 70 °C for 45 min in a water bath, and then cooled to room temperature and filtered the extract with filter paper. The filtrate was used as a reducing and stabilizing agent. The filtrate was stored at 4 °C for further use (Alwhibi et al. 2021 ; Biswal et al. 2020 ).

Qualitative phytochemical screening of Embelia schimperia leaf extract

The presence of alkaloids, glycosides, steroids, saponins, flavonoids, terpenoids, and tannins, among other things, was detected by a distinctive color shift in extracts using established techniques (Bayisa & Bultum 2022 ).

Test for Alkaloids To determine the alkaloid presence in leaf extract, 5 ml drops of Wagner’s and Dragendorff’s reagents were added to different 5 ml of the filtrate. Then, the color changed was observed (Bayisa & Bultum 2022 ).

Flavonoid Test The flavonoid presence in leaf extract was determined by taking 4 drops of concentrate HCl and magnesium ribbon, and added to 5 ml leaf extract (Bayisa & Bullo 2021 ).

Test for Terpenoids To determine the terpenoids presence in leaf extract, 2 ml of chloroform was added to 5 ml of leaf extract and waited for few minutes. Then, 3 ml of concentrate H 2 SO4 was added, and color changed was observed (Bayisa & Bullo 2021 ).

Test steroids 20 gm leaf extract was mixed with 200 ml of distilled water and heated for 45 min. After, the solution was cooled and filtered, 4 ml of chloroform added to 5 ml of leaf extract. Then, 3 ml of concentrate H 2 SO4 was added, and color changed was observed.

Test for Tannin : To determine the tannin presence in leaf extract, 5 ml of leaf extract was mixed with a few drops 5% of ferric chloride solution, and the color changed was observed (Siddiqui et al. 2021 ).

Test for Saponin : 5 ml of extract and 5 ml of distilled water were added and shaken vigorously in a graduated cylinder for 2 min lengthwise. Then, the shacked was waited for 5 min, and its layer foam height indicated the presence of saponins (Yadav & Agarwala 2011 ).

Test for Polyphenol A 3 drops of 3% of ferric chloride and 3 drops of 1% K 3 Fe (CN) 6 were added into 5-ml leaf extract. Then, the solution was shacked little, and color change was observed (Yadav & Agarwala 2011 ).

Test for Glycosides To 2 ml of extract, 3 ml of chloroform and 10% ammonia solution was added. Formation of pink color indicates presence of glycosides. Using Liebermann’s test, crude extract was mixed with each of 2 ml of chloroform and 2 ml of acetic acid. The mixture was cooled in ice. Carefully concentrated H 2 SO 4 was added (Bayisa & Bullo 2021 ).

Green synthesis of Ag/Fe/ZnO nanocomposites

Ag/Fe/ZnO nanocomposite was synthesized by using the hydrothermal process. Firstly, Ag/Fe/ZnO nanocomposite was prepared from 0.1 M AgNO 3 solution, 0.1 M FeCl 3 solution and 0.1 M of Zinc acetated dehydrate were prepared separately. Prepared solutions were mixed together and the solution was stirred constantly. After the completed homogenization of the solution was precipitated was dried in a hot-air oven at 90 °C for 10 h, reduced to a powder, and calcined at 400 °C for 3 h in a muffle furnace to obtain pure Ag/Fe/ZnO nanocomposite (Bayisa et al. 2023 ; Biswal et al. 2020 ).

Nanocomposite characterization

The synthesized Ag–Fe–ZnO Nanocomposite was characterized by its size, shape, surface area, optical properties, and disparity using advanced instruments such as, DLSC, UV–Visible spectroscopy, XRD, FTIR, TGA, SEM, and BET.

Photocatalyst experiment

The photocatalytic effectiveness of Ag/Fe/ZnO NCs against Amoxicillin degradation was studied utilizing a 15 W LED lamp as a light source. Amoxicillin and catalyst were mixed to make a reaction suspension in a 250-ml beaker with a double-layer jacket. The temperature was kept constant at 25 °C throughout the studies. The photocatalytic degradation of Amoxicillin was measured using a 200-mL working solution having an Amoxicillin concentration, and Ag/Fe/ZnO NCs in 250-ml beaker. Then, solution was sonicated and magnetically agitated for 30 min in a dark room to ensure homogeneity. The solution was then irradiated with 15 W LED bulb visible light and constantly swirled with a magnetic stirrer. The sample was then collected every 30 min and centrifuged to separate the catalysts from the liquid before the absorbance measurement. The absorbance of each sample was determined after Amoxicillin was photocatalytically degraded. After the photocatalytic degradation of Amoxicillin, the absorbance of each sample after 30 min interval was measured using UV–visible spectroscopy. The percentage of photocatalytic degradation of those calculated by Eq. ( 1 ).

whereas % D = percentage of photocatalytic degradation.

Co = initial concentration of Amoxicillin.

Ct = concentration at time Amoxicillin.

Experimental design

For this study, Design Expert 13.8.0 software was used for Experimental data analysis (Bayisa & Teso 2021 ; Mekonnen 2021 ). The experimental design selected for this work is two-level three-factor response surface methodology of the central composite design experiment model for the parameter variable Ag/Fe/ZnO NCs dosage, time and dosage of amoxicillin on the photocatalytic degradation of the efficiency.

Result and discussion

Phytochemical analysis.

The phytochemicals found in the leaves are responsible for the formation of Ag/Fe/ZnO NCs. As a result, phytochemical assays on Embelia schimperia leaf extract were performed prior to NC synthesis. The extract was tested the presence of alkaloids, glycosides, steroids, saponins, flavonoids, terpenoids, and tannins among others and identified by characteristic color changes. The test was confirmed the presence of photochemical ingredient as shown in Table  1 .

Characterization of synthesized Ag/Fe/ZnO NCs

The synthesized Ag–Fe–ZnO nanocomposite was characterized by its size, shape, surface area, and disparity using sophisticated instruments UV–Visible spectroscopy, FTIR, XRD, DLCS, and SEM.

UV–Visible spectroscopy

UV–visible spectroscopy is an important technique to determine the formation and stability of metal nanoparticle. The optical properties of synthesized Ag/Fe/ZnO NCs were determined by UV–Visible spectroscopy in a range of 200–800 nm to observe their absorption at the specified wavelength. Therefore, when light source applied to the solution, the amount of transmitted in the solution shows the number of electrons excited from doped NCs. The optical properties of Ag/Fe/ZnO was expressed by UV–Vis spectroscopy as shown in the Fig.  1 depicts that broader absorption spectrum over the range peaks were found in the 375–450 nm regions. The characteristic absorbance peak observed at about 328 nm, 385 nm indicated an ultra-small particles size formation, and 440 nm shifted toward longer wavelengths and broadens with increasing nanoparticle size. The bandgap energy of AgNPs, Fe 2 O 3 NPs, and ZnONPs reported previous was greater than bandgap energy of Ag/Fe/ZnO NCs of current work. These indicated in photocatalytic activity of Ag/Fe/ZnO NCs required minimum amount of energy to exited elector form vacant band to covalent band. The determination of the bandgap energy showed a shift to the visible region in doped samples (2.81 eV). These shows improved efficiency of visible light-harvesting makes Ag/Fe/ZnO NCs suitable candidates for photocatalytic applications. Therefore, the obtained result was confirmed by previous studied (Hoseinpour et al. 2017 ; Kolya & Kang 2022 ; Nhi et al. 2022 ; Rajendran & Sengodan 2017 ).

UV–Visible absorption spectra of Ag/Fe/ZnO NCs

Fourier transmissions infrared spectroscopy (FTIR).

The broad/sharp and weak/strong peaks spectra of transmission vs wavenumber on FTIR graph represent the bond of atoms that indicate the functional groups of nanocomposite formation. Figure  2 indicates that the green synthesized Ag/Fe/ZnO NCs peaks were obtained at 425, 564, 699, 878,1429, and 1632 cm −1 wavenumber. A weak short peak at 425 cm −1 appears indicating the bond of Zn–O formation (Afzal et al. 2022 ; Bharathi et al. 2019 ). The medium broad peak at 564 cm −1 indicated the bond of Fe–O formation. At 699 cm −1 , assigned alkyl halides compounds indicate the presence (–C–Cl) from the precursor. 878 cm −1 and at 1442 cm −1 indicate –C = C bond. 1631 cm −1 and 1773 cm −1 peak assigned to C = O and C–O stretching indicate the presence of tertiary amides alcohol and phenols that are used for capping organic molecules in nanoparticles, phenols and amines compounds present in the leaf extract that are responsible for reduction of silver, iron and zinc ion and stability of NCs formed. However, above the 2000 cm −1 , there is no peak indicating free from water and other. This result is the same within previous study (Khan et al. 2018 ; Tolouietabar et al. 2020 ). The peaks represent stretching and bending of atom that indicated the functional groups of nanocomposite formation.

FTIR spectral analysis of Ag/Fe/ZnO nanocomposite

Dynamic light scattering

The DLS measurement is the most technique used to identify size distribution and disparity of synthesized nanocomposite. The result in Fig.  3 shows the average particle size of distribution 42.276 nm Ag/Fe/ZnO of nanocomposite, which observed from the appearance of one peak with a 100% intensity and 4.265 nm standard deviation. In addition, PDI is 0.183, which shows high stability of NCs. On this graph only one peak and the result indicate monodispersed and homogeneity of Ag/Fe/ZnO of nanocomposite. This result was confirmed with previous study (Ayinde et al. 2020 ).

DLS result for Ag-Fe/ZnO NCs

X-Ray diffraction spectroscopy (XRD).

The crystalline nature of the green synthesized Ag–Fe–ZnO NCs were characterized by XRD. The patterns of XRD were recorded using a diffract meter by Cu anode material within radiation (λ = 0.154060 nm) from 10° to 90° angle within tube current was 15 mA and its voltage was 40 kV at 25°C temperature. As it shown in Fig.  4 , the patterns for the Ag/Fe/ZnO NCs sample have distinct strong diffraction peaks at 2θ observed were 28.75°, 32.02°, 34.55°, 38.42°, 40.05°, 44.50°, 45.72°, 56.87°, 64.87°, 75.5° and 77.8° on the plane suggested that the Ag/Fe/ZnO NCs formed was ordered crystalline structure. The average crystallite size of Ag/Fe/ZnO NCs corresponding to the most intense diffraction peak was calculated by the Debye–Scherer formula. The XRD result show the presence and formation of Ag/Fe/ZnO NCs at optimum parameter which have average crystallite size 26.456 nm within cubic crystalline nature that confirm the synthesized nanoparticles were crystalline nature. The obtained result was very close to previously studied (Khorshidi et al. 2016 ; Kumar & Pandey 2017 ).

XRD spectra for Ag/Fe/ZnO NCs

Thermal gravimetric analysis and differential thermal analysis (TGA and DTA)

Figure  5 shows the TGA and DTA results of Ag/Fe/ZnO NCs in the temperature 0–850°C range. The weight loss of Ag/Fe/ZnO NCs was observed from TGA spectrum in 90°C, 380–450, and 750–800°C. This loss is due to evaporation of water molecule and ignition of organic material. However, there is almost no weight loss around 100–380°C and 500–750°C temperature range. DTA spectrum result displays one exothermic peak at 390ºC (Khan et al. 2018 ).

Thermal gravimetric analysis and differential thermal analysis (TGA and DTA) of Ag/Fe/ZnO NCs

Scanning electron microscope (SEM)

The morphology, particle size and shape of green synthesis Ag/Fe/ZnO NCs were investigated by SEM analysis. As it shown in Fig.  6 , the shape of the Ag/Fe/ZnO NCs most likely spherical morphology. However, due to some aggregation of particle the shape is approach to cylindrical morphology. The small size of Ag and Fe-metal coated on the ZnO surface that created Ag–Fe–ZnO NCs rod shape. The result agreed with the size measured by DLS which was 49.19 nm and XRD indicated the average crystal size of 26.456 nm. The SEM image indicate average particle size of Ag/Fe/ZnO NCs is 49.1 nm within 50.1 nm 2 mean surface area that confirm DLS and XRD data.

Photocatalytic degradation of a Amoxicillin using Ag/Fe/ZnONCs

BET surface area analysis

The specific surface area of green synthesis of Ag/Fe/ZnO NCs was investigated by Brunauer–Emmett–Teller (BET) analysis. As it shown in Fig.  7 , the N 2 adsorption/desorption isotherms plotted for Ag/Fe/ZnO NCs, and the surface area was found to be 8.2 m 2 /g. This is indicated that relative pressure (P/P0) from 0.5 to 0.9 increased the surface area of the sample due to the presence of a heterogeneously distributed mesoporous nature of particle. Likewise, a reduction in crystallite size is thought to be the cause of Ag/Fe/ZnO NCs’ increased surface area. Therefore, it implies that Ag/Fe/ZnO NCs adsorb pharmaceutical molecules and other contaminants more strongly because of their larger surface area, which increases the percentage of degradation. In addition to this, the average pore diameter and a total pore volume of Ag/Fe/ZnO NCs is found to be 15.224 nm and of 0.0336 cm 3 /g, respectively. The entire pore volume, average pore diameter, and BET surface area of Ag/Fe/ZnO NCs were seen to have increased, and its mesopore structure was validated by the determined textural parameter values.

BET surface area plot from nitrogen adsorption/desorption isotherms for Ag/Fe/ZnO NCs

Statistical experimental analysis of amoxicillin degradation using synthesized Ag/Fe/ZnO NCs

Table 2 displays the variables that significantly affected the photocatalytic degradation efficiency based on the analysis of variance (ANOVA). This table provided a summary of each model’s significant coefficients of response and responses from respondents were highly significant ( p  < 0.0001), and statistical studies show that the design models provide a good fit to the data. The photocatalytic degradation efficiency using green synthesized of Ag/Fe/ZnO NCs of Embelia schimperia leaf extract indicated by the Model F value of 1253.35, which suggests the model is important. A, B, C, AC, A 2 , B 2 , C 2 , and D 2 were discovered to have a substantial impact on the photocatalytic degradation efficiency.

Influence of process parameter on the amoxicillin photocatalytic degradation efficiency

The result of process parameter, Ag/Fe/ZnO NCs dosage, Amoxicillin dosage and time on the amoxicillin Photocatalytic Degradation efficiency was analyzed using central composite design experiment as shown in Table  3 . Result depicts that minimum efficiency of 72.18% was obtained at 75 mg of Ag/Fe/ZnO NCs dosage, 40 mg/L of Amoxicillin dosage and 120 min of time. In contrast, the maximum photocatalytic degradation efficiency of 98.5% was obtained at 100 mg of Ag/Fe/ZnO NCs dosage, 30 mg/L of Amoxicillin dosage and 180 min of time. The optimization solutions for maximum yield are shown in Table  3 by using categorical factors.

As can be seen in Fig.  8 a–c, the effects Ag/Fe/ZnO NCs dosage, Amoxicillin dosage and time on the amoxicillin photocatalytic degradation efficiency were studied. According to the curve in Fig.  8 a, the photocatalytic degradation efficiency increased noticeably from 72.5 to 98.5% as the Ag/Fe/ZnO NCs dosage from 50 to 100 mg. Above 75 mg, there has been a modest increase in degradation efficiency, showing that using high Ag/Fe/ZnO NCs dosage has little impact. The effective Ag/Fe/ZnO NCs dosage is 75 mg with a slight rise in degradation efficiency, according to the experimental results of the photocatalytic amoxicillin degradation as a function of Ag/Fe/ZnO NCs dosage analysis. Figure  8 b illustrates the effect of Amoxicillin dosage on the photocatalytic amoxicillin degradation in addition to the effect of Ag/Fe/ZnO NCs dosage, showing that the maximum efficiency was achieved at 30 mg/L of amoxicillin concentration, which was 98.5%, and that it significantly decreased to 89% as the concentration of Amoxicillin dosage was increased to 50 mg/L. This was due to photocatalyst’s reduced active sites for the oxidation of the amoxicillin molecule. Higher starting concentrations of amoxicillin resulted in more reactants and reaction intermediates being absorbed at the photocatalyst level, which meant that there was not enough hydroxyl radical available to decompose amoxicillin at higher concentrations (Bayisa et al. 2023 ; Kumar & Pandey 2017 ). When its concentrations increased, its rate of decomposition was decreased.

Effects of a Ag/Fe/ZnO NCs dosage, b Amoxicillin dosage and c radiation time on the amoxicillin photocatalytic degradation efficiency

Thus, it is evident to say that variations in the concentration of Amoxicillin beyond 50 mg/L instigated changes in the photocatalytic degradation efficiency of amoxicillin to vary, showing that using the concentration of amoxicillin has significant effect on the degradation effect. Additionally, the effect’s reaction time was studied since the photocatalytic efficiency varies with their values. Figure  8 c revealed that the Amoxicillin degradation increased (about 72.5 to 98.5%) with increasing radiation time (60, 120, and 180 min). Nonetheless, the reaction’s initial 60 min saw the maximum degrading efficiency, after which its upward trend constant. This may be explained by the quick breakdown of Amoxicillin contamination in the 60 min prior to the reactivity of free radicals produced by the applied electron excitation. Herein, Embelia schimperia leaf extract was subjected to acidic and slightly basic conditions in order to analyze the development of Ag/Fe/ZnO nanocomposites. This might be explained by the fact that some Embelia schimperia leaf extract phytocompounds release OH ions when they are oxidized in the presence of metal precursor ions (Bayisa & Bultum 2022 ).

Amoxicillin photocatalytic degradation

The photocatalytic degradation of Amoxicillin took place for 150 min applying 15 LED light source to active catalytic surface by formation electron and hole. As shown in Fig.  9 , the initial concentration of Amoxicillin is relatively high (0.79) at 0 min, and there was some degradation in the dark case for 30 min. However, after 30 min of 15 W LED light used, the concentration/absorbance of Amoxicillin reduced and then gradually decreased, and as time moved to the final 150 min, the absorbance and concentration decreased. This drop in concentration/absorbance is due to the catalytic surface receiving light/photon energy from a 15 W LED source, where electrons escape from the valance band to the covalent band as negative charge and holes as positive charge, which is responsible for the degradation of the Amoxicillin. When the interaction period is departed, the electron and holes regenerate. When contact time increased, the reformation of electron and hole regenerates. As the result, the Amoxicillin degraded to non-toxic carbon dioxide and water molecule. Therefore, the efficiency of Ag/Fe/ZnO NCs calculated is 98.5% for degradation of Amoxicillin drugs.

UV–visible data for removal of Ciprofloxacin using Ag/Fe/ZnO NCs

In this study, green Ag/Fe/ZnO NCs from Embelia schimperia leaf extract were successfully produced for Photocatalytic Degradation of amoxicillin using a straightforward co-precipitation technique. The synthesized Ag/Fe/ZnO photocatalyst was characterized and validated by FTIR, XRD, UV–vis, DLS, TGA, SEM and DTA analysis. Using a response surface methodology of central composite design, the impact of green synthesized Ag/Fe/ZnO NCs dosage, Amoxicillin dosage and radiation time on the photocatalytic degradation of Amoxicillin was examined. Thus, the natural Embelia schimperia leaf extract played a significant role in the increasing of photocatalytic properties at the Ag–Fe–ZnO nanocomposite, which leads to photocatalyst’s reduced active sites for the oxidation of the amoxicillin molecule. Approximately 98.5% of Amoxicillin has been eliminated after 180 min in the presence of Ag–Fe–ZnO under visible light.

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Acknowledgements

We would like to acknowledge the FTIR, XRD analysis and UV–vis spectrum platform at the Faculty of Material Science and Engineering, and the school of Chemical Engineering Laboratory staff members at Jimma Institute of Technology for their knowledge-sharing and technical support. This work was financially supported by the Jimma Institute of Technology Center of Excellence-CRGE RESOURCE CART (Climate Resilient Green Economy Resource Centre for Advanced Research and Training-Linking Energy with Water and Agriculture).

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Gizachew, D.G., Jiru, E.B., Tekle’Ab, T. et al. Green synthesis of Silver-iron-zinc oxides nanocomposite via Embelia schimperia leaf extract for photo-degradation of antibiotic drug from pharmaceutical wastewater. Appl Water Sci 14 , 210 (2024). https://doi.org/10.1007/s13201-024-02272-6

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Phytochemical fabrication of ZnO nanoparticles and their antibacterial and anti-biofilm activity

Hussain udayagiri.

1 Department of Materials Science and Nanotechnology, Yogi Vemana University, Kadapa, Andhra Pradesh India

Siva Sankar Sana

2 School of Chemical Engineering, Yeungnam University, Gyeongsan, 38541 Republic of Korea

Lakshman Kumar Dogiparthi

3 Department of Pharmacognosy, MB School of Pharmaceutical Sciences, Mohan Babu University, Tirupati, Andhra Pradesh India

Ramakrishna Vadde

4 Department of Biotechnology and Bioinformatics, Yogi Vemana University, Kadapa, Andhra Pradesh 516 005 India

Rajender S. Varma

5 Centre of Excellence for Research in Sustainable Chemistry, Department of Chemistry, Federal University of São Carlos, São Carlos, SP 13565-905 Brazil

Janardhan Reddy Koduru

6 Department of Environmental Engineering, Kwangwoon University, Seoul, 01897 Republic of Korea

Gajanan Sampatrao Ghodake

7 Department of Biological and Environmental Science, Dongguk University-Seoul, Ilsandong-Gu, Goyang-Si, 10326 Gyeonggi-Do South Korea

Adinarayana Reddy Somala

Vijaya kumar naidu boya, seong-cheol kim, rama rao karri.

8 Petroleum and Chemical Engineering, Faculty of Engineering, Universiti Teknologi Brunei, Bandar Seri Begawan, BE1410 Brunei Darussalam

Associated Data

The datasets used and analysed during the current study are available from the first author (Prof. Vijaya Kumar Naidu Boya) upon reasonable request.

The synthesis of metal nanoparticles through bio-reduction is environmentally benign and devoid of impurities, which is very important for biological applications. This method aims to improve ZnO nanoparticle's antibacterial and anti-biofilm activity while reducing the amount of hazardous chemicals used in nanoparticle production. The assembly of zinc oxide nanoparticles (ZnO NPs) is presented via bio-reduction of an aqueous zinc nitrate solution using Echinochloacolona ( E. colona ) plant aqueous leaf extract comprising various phytochemical components such as phenols, flavonoids, proteins, and sugars. The synthesized nano ZnO NPs are characterized by UV–visible spectrophotometer (UV–vis), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (X-RD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and elemental composition by energy-dispersive x-ray spectroscopy (EDX). The formation of biosynthesized ZnO nanoparticles was confirmed by the absorbance at 360–370 nm in the UV–vis spectrum. The average crystal size of the particles was found to be 15.8 nm, as calculated from XRD. SEM and TEM analysis of prepared ZnO NPs confirmed the spherical and hexagonal shaped nanoparticles. ZnO NPs showed antibacterial activity against Escherichia coli and Klebsiella pneumoniae with the largest zone of inhibition (ZOI) of 17 and 18 mm, respectively, from the disc diffusion method. Furthermore, ZnO NPs exhibited significant anti-biofilm activity in a dose-dependent manner against selected bacterial strains, thus suggesting that ZnO NPs can be deployed in the prevention of infectious diseases and also used in food preservation.

Introduction

Nanomaterials (NMs) are made up of nanoscale particles and have dimensions of 1 to 100 nm 1 . Because of their massive surface area to unit volume ratio, nanoparticles (NPs) showed enhanced catalytic reactivity, chemical stability, thermal conductivity, and nonlinear optical performance 2 – 4 . Metal nanoparticles have many optical features that make them interesting for use in biomedical applications. One such property is surface plasmon resonance (SPR), which allows the material to modulate the optical field. Since metal nanoparticles are tiny, they can more easily penetrate biological membranes, which are often impervious to other macromolecules 5 , 6 .

The straight wide gap (3.37 eV, 387 nm), deep violet/borderline ultraviolet (UV), and strong exciton-binding energy (60 meV) of ZnO make it a good n-type semiconductor 7 . Nanoscale ZnO is an important semiconductor that has garnered remarkable attention for its wide range of applications, including electronics, optics, optoelectronics, and biomedicine. They are commercially deployed in diverse industries, namely for producing various materials 8 , leather and rubber industry 9 , and as a protective agent from sunlight 10 . Because of the antimicrobial properties of ZnO, it has been used in the food industry and even in cement production as an additive 11 , 12 . Different types of physical and chemical approaches have been used to generate metal nanoparticles in bulk production, such as chemical precipitation 13 , microwave 14 , sol-gel 15 , pulsed laser deposition (PLD) 16 , and polyol 17 , among others.

However, conventional methods such as physical and chemical approaches typically involve using hazardous reducing agents and organic solvents, most of which are extremely reactive, expensive and toxic to the environment, which can impart various health risks 18 . A new approach known as ‘green synthesis’ uses extracts from plants and microbes to prepare nanoparticles with potential uses in biomedicine 19 . Numerous benefits of this approach include safety, biocompatibility, affordability, and environmental friendliness. Furthermore, many studies have demonstrated the potent antibacterial qualities of metal/metal oxide nanoparticles produced by green synthesis techniques 20 .

The synthesis of NPs via phytochemical route involves various plant parts, including roots, leaves, seeds, stems, and fruits, because their potent extracts have been shown to work as both reducing and stabilizing agents 21 , 22 . Plant-based polyphenols are bioactive and exhibit cytotoxic, antiproliferative, and strong antioxidant activity 23 , 24 . Various water-soluble phytoconstituents, such as proteins, reducing sugars, amino acids, quinones, flavonoids, catechins, and terpenoids, could play key roles in synthesising stable high-surface-area metal nanoparticles 25 – 27 . The factors influencing nanoparticle commercial production entail inexpensively expeditious production with low toxicity and minimal use of hazardous reagents.

Green nanotechnology has such distinctive and appealing attributes regarding material design that has become the most important factor in contemporary scientific studies. Greener synthesis of nanoparticles deploying plant extracts does not require complicated processes and is more appealing than NPs from microbes involving repeated filtration methods and preservation of cell cultures 28 . The Food and Drug Administration of the United States has acknowledged that ZnO is an antimicrobial metal oxide that is safe for the environment and biocompatible with human cells 29 , 30 . Consequently, deploying environmentally friendly methods of producing natural nanoparticles of the desired morphology and size is imperative. For this reason, these are regarded as environmentally friendly protocols for producing desired morphological natural nanoparticles.

The plant Eechinochloa colona is is an annual weed belonging to the family of Poaceae. Except in Greenland and Antarctica, the annual or perennial grass Echinochloa colona is found across the world's warm climates. It is typical in regions with a lot of rainfall and fluctuating temperatures. In India, Echinochloa colona , a terrestrial, tufted, upright grass, is frequently called "jungle rice". This weed is also an alternate host of diseases, insects, and nematodes 31 . It is rich in phytochemicals, like alkaloids, steroids, carbohydrates, glycosides, tannins, phenols, flavonoids, etc., which could be exploited for the bio-reduction process 32 . In this work, ZnO NPs prepared through a green route using an aqueous leaf extract of E. colona highlight the nanoparticle growth at high temperatures and are used as antibacterial and anti-biofilm agents.

Materials and methods

Zinc nitrate hexahydrate (Zn (NO 3 ) 2 ·6H 2 O) was received from Merck, Mumbai. The leaves of the E. colona plant were collected during flowering time. Dr. Madhava Chetty, a taxonomist at Sri Venkateswara University in Tirupathi, India, recognized and verified the plant. The first Author (Hussain Udayagiri) has permission to collect E. colona, and all the methods were carried out according to relevant guidelines and regulations. Double distilled water was used in all steps for the preparation of nanoparticles. The fresh and young healthy leaves of E. colona were cleaned with purified water to eliminate unwanted compounds on the surface of the leaves. 10 g of dry leaf powder was combined with 100 mL of distilled water and then heated the mixture to 80 °C for 30 min. Then, the extract was cooled to room temperature and filtered. Further, it was subjected to a centrifuge at a speed of 3000 rpm for 5 min to remove heavy molecules, and finally, the extract was collected and stored at 4 °C for future use. Because these compounds function as both reducing and protecting agents during the production of metal oxides, the various phytochemicals comprising the leaf extract were identified chemically.

Phenolic content

The quantitative analysis of phenol was performed using the method previously developed with minor modifications 33 . The leaf extract (140 µL) mixed with 600 µL of reagent (Folin-Ciocalteu) was allowed to stand for a few minutes. Afterwards, 460 µL of 7.5 w/v % sodium carbonate (Na 2 CO 3 ) was added, and the entire apparatus was kept at 45 °C for 30 min before being incubated for one hour at room temperature. Results are presented in gallic acid equivalents per gm (GAE/gm) extract (gallic acid equivalents per gram) based on absorbance measured at 764 nm using standard gallic acid.

Based on the report by Chang et al . 34 , the quantitative analysis of flavonoids was measured by the aluminium chloride method. The plant extract (25 µL) and alcohol (75 µL) were mixed well, and to this, AlCl 3 (5 µL) and potassium acetate (CH 3 COOK) (5 µL) were added and made up the volume up to 260 µL using distilled water. The absorbance at 415 nm was measured after the solution was incubated for 40 min at room temperature. The standard used was Rutin, and results were reported as Rutin equivalents per gram (RE/gm) of extract.

Total sugars

Using the Anthrone method, the quantitative analysis of sugar content was measured 35 . In brief, 3 mL of anthrone reagent and 1 mL of leaf extract were mixed well and incubated for 10 min in the water bath. The absorbance was measured at 630 nm against a reagent blank in a spectrophotometer using glucose as a standard.

Protein content

Using the Biuret method, the quantitative analysis of proteins in plant extract was performed 35 . In brief, 3 mL of biuret reagent and 1 mL of extract were mixed perfectly and subjected to 30 min of incubation. Bovine serum albumin (BSA) was used as a standard for the absorbance measurement at 540 nm.

Preparation of ZnO NPs

For the preparation of ZnO NPs, an aqueous extract of E. colona (30 mL) was mixed with 3 g of zinc salt, and the solution was stirred at 70–80 °C for four hours to achieve a deep, yellow-colored paste. This product was dried at 70 °C for 6 h and subjected to calcination for 3 h at 300 °C. Finally, the ensuing nanoparticles were collected in powdered form.

Characterization

UV–visible spectrophotometer (Model 3092, Lab India, Mumbai, India) was used to characterize the optical features of biosynthesized ZnO NPs. The resultant nanopowder was suspended in sterile milli-Q water and observed at a wavelength range of 300–600 nm. The functional groups, including phytochemical components that are associated with the reduction and stabilization of ZnO NPs, were analyzed by Fourier transform infrared spectroscopy (FTIR), recorded with Perkin Elmer Frontier Spectrophotometer with a resolution of 4 cm −1 in 400–4000 cm −1 range by KBr pellet method. RIGAKU smart lab X-ray diffractometer with a 1.5406 Å (CuKα) irradiation wavelengths was used for X-ray diffraction spectroscopic (XRD) analysis of dried ZnO NPs to access the purity and the crystalline size of the synthesized nanopowder; scanning angle was analyzed at 0.02 ͦ step in 10◦ to 80◦ range. The morphology of the particles was examined by scanning electron microscope (SEM), JEOL JSM-6310LV, operating at 200 kV to study the surface structure of the synthesized ZnO NPs. Energy-dispersive X-ray (EDX) spectroscopy was employed to analyze the elemental composition of the sample and determine its purity. The morphology, size, and crystallinity of NPs were studied using transmission electron microscopy (TEM). After coating onto a copper grid followed by drying, the ZnO nanoparticle suspension was analyzed via TEM JEOL 3010 with an acceleration voltage of 200 kV.

Antimicrobial activity

The antimicrobial activity of green and biosynthesized ZnO NPs was measured against two different bacterial strains such as Escherichia coli (E. coli) and Klebsiella pneumoniae ( K. pneumoniae )strains acquired from Chandigarh's Institute of Microbial Technology. The disc diffusion method was used to examine ZnO NP's antibacterial activity. Using the disc diffusion method, the antibacterial activity of ZnO NPs was assessed at various doses, including 20, 40, 60, and 80 µg of NPs, in conjunction with a standard antibiotic drug (ampicillin) 36 . The MHA plates were prepared with respective bacterial strains at 10 8  CFU. ZnO NPs filled discs in varying concentrations are positioned at the corners of the plates. After 36 h of incubation, the ZOI was determined. Five separate experiments were conducted, and the clear zone that developed around the discs was measured and reported in millimeters (mm).

SEM investigations were performed to verify the morphological alterations in microorganisms brought about by biosynthesized ZnO NPs. After 8 h of growth in standard broth at 37 °C, microbial suspensions were treated with 80 µg/mL ZnO NPs and incubated for 12 h at 37 °C. Assorted microbes were centrifuged at 5000 rpm for 5 min at 4 °C, washed with PBS (0.1 M, pH 7.4) three times for 10 min each, then fixed in 2.5% (v/v) glutaraldehyde in PBS (0.1 M, pH 7.4) for 2 h (4 °C). The microbial specimen was dehydrated with 50, 70, 80, 90, and 100% ethanol for 10 min each, followed by tertiary butyl alcohol treatment. Following CO 2 drying, the microbial specimens were coated in an ion coater (2 min) with gold. Using a scanning electron microscope (JEOL, JSM-6310LV operating at 200 kV), changes in the morphology of the bacterial cells treated with biogenic ZnO NPs were observed.

Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Anti-biofilm activity determination

Minimum inhibitory concentration ( MIC) estimation was performed as per the ASM (American Society for Microbiology) manual according to CLSI guidelines as described by Bauer et al . 36 and Eloff et al . 37 . MIC and MBC of nanoparticles were determined as follows. To 1 mL of nutrient broth, different concentrations of nanoparticles were added, and various concentration-containing tubes were used for treatment. Bacterial culture in the log phase was taken and checked for absorbance in a spectrophotometer to get 0.6 to 0.8 OD values equivalent to 1.5 × 10 8  CFU/mL. The culture was diluted 20 times and added this diluted culture (100 μL) was placed directly into the tubes and incubated for 16 h. The tubes were then filled with 40 μL of iodonitrotetrazolium (INT) at a concentration of 0.2 mg/mL, and they were incubated for 30 min at 37 °C. Tubes were observed to change color from yellow to pink or white. A color change signifies the existence of bacteria, whereas a colorless area denotes the absence of microorganisms. The MIC of the nanoparticle was determined by serially arranging tubes containing varying concentrations and watching for color changes.

Nevertheless, after a 24-h incubation period, a portion (10 μL) of the aforementioned MIC experimental solution from the tube where no color change was detected was collected, and spread over the nutrient agar plate to measure the MBC observed for bacterial colonies. The MBC concentration was found using a plate to examine for no bacterial colonies. Nanoparticles (µg) containing MIC and MBC were administered. These attributes are significant as several such characteristics are important requirements in various industries, including paint, sunscreen, pharmaceuticals, and cosmetics.

The anti-biofilm activity of ZnO NP was evaluated through the previously described method with some modifications 36 , 37 . Starting with 10 mL nutrient broth, it was inoculated with a loopful of tested microorganisms from overnight culture on nutrient agar. Diluted the culture until it reached the OD value of 1.0 at 600 nm. Every individual well in the flat bottom 96 well microtiter plate was filled with 180 μL of diluted culture with inoculated sterile broth served as blank. Afterwards, 10 µL of ZnO NPs or positive control ampicillin was added and mixed thoroughly. The culture plates were incubated at 37 °C for 24 h. After incubation, all the contents were gently removed and floated, including non-adherent cells in wells. The wells were washed thrice with 200 μL of normal saline and air-dried for 30 min. Biofilms of bacteria that remained adherent to the walls were fixed with 2% sodium acetate and stained with 0.1% crystal violet for 10 min. The excess stain was incubated for 30 min, washed with saline, and air-dried. These plates were then de-stained with 200 μL of 95% ethanol for 10 min and measured absorbance at 620 nm using a Microplate Reader (Biorad 680, USA). The percentage of inhibition of biofilm formation was calculated using the following formula:

Statistical analysis

The data presented in tables and figures represents mean ± SE of five individual determinations. Data was evaluated using one-way ANOVA post-hoc multiple comparisons from the Duncan Multiple Range (DMR) Test at a significance level of p  ≤ 0.05.

Results and discussion

The water leaf extract of E. colona was identified for polyphenols, flavonoids, and proteins, and the extract was found to contain 17.4 ± 1.6 GAE/gm, 5.8 ± 0.05 RE/gm, 18.53 ± 0.3 and 210.33 ± 3.3 mg/gm of polyphenols, flavonoids, proteins, and sugars, respectively. The UV–vis spectroscopy approach was used to describe the optical property and determine the generation of ZnO NPs. The distinctive ZnO absorption peak is located at 370 nm in ZnO NPs dispersion in the UV–vis absorption spectrum, as illustrated in Fig.  1 a. This peak is indicative of a pure sample since it occurs at the same time as the ZnO undergoes an electrical band gap between its valence as well as conduction, states 38 . The functional groups in the E. colona extract that might serve as reducing agents during the production of ZnO NPs were found using FT-IR spectroscopy. The current study discovered that zinc nitrate was reduced into ZnO NPs by the secondary metabolites of E. colona . FT-IR spectrum is depicted in Fig.  1 b. The broad peak at 3245 cm −1 could be attributed to hydroxyl groups (O–H) originating from the phenolic chemicals present in the plant extract, indicating the existence of hydrogen-bonded groups 38 . A peak at 1034.79 cm -1 observed can be ascribed to the presence of phenolic groups and alcohols. It was identified that the Zn–O bond possessed a significant absorption band at 450 cm −1 . The absorption peak at 1626 cm −1 indicates the stretching bands of C=O of the amide group 39 . Narrow peaks at 2922 cm −1 and 2852 cm −1 are attributed to C–H stretching vibration, which depicts the presence of alkanes group 39 . Flavonoids and polyphenols consist of many –OH groups of Zn-flavonoid complex, which resulted in the production of ZnO NPs by calcination. Previous studies have suggested that C–O, C–O–C, and C = C groups of heterocyclic compounds could have stabilizing properties. The aqueous plant extract could bind the zinc surface with phenols and flavonoids, causing zinc nitrate to synthesize and regulate ZnO NPs. It can be mentioned that functional groups of phenols and flavonoids in the extract could donate electrons that could reduce zinc ions (Zn 2 + ) to ZnO.

( a ) UV–visible absorption spectrum of biosynthesized ZnO NPs and ( b ) FT-IR spectrum of biosynthesized ZnO NPs using E. colona extract.

Moreover, the negative functional groups present in the extract could have a stabilizing effect 29 . Sanaz et al . reported that negatively charged biomolecules (proteins and carboxylic acids) are possibly involved in stabilizing nanoparticles 40 . Therefore, our investigation explained the importance of plant metabolites such as proteins, terpenoids, and phenolic compounds in the production and stability of ZnO NPs. Proteins have the ability to cover NPs in a protective shell that increases their dispersibility and keeps them from clumping in water. Furthermore, ZnO NPs can form and be more stable due to the interaction of free amino and carboxylic groups with the zinc surface 40 .

The XRD pattern of ZnO NPs that were biosynthesized using E. colona extract is shown in Fig.  2 . In XRD pattern, at two theta degrees 31.85 (100), 34.47 (002), 36.37 (101), 47.57 (102), 56.64 (110), 63.03 (103), 66.53 (200), 68.05 (112), and 77.21° (202) confirms that ZnO NPs have a hexagonal wurtzite structure which match those of (JCPDS card No. 36–1451). The biosynthesized ZnO NPs of 14 nm size at the peak associated with the (101) planes employing Deby-Scherrer's formula 40 . The XRD pattern only displayed the expected ZnO peaks, further demonstrating the purity of the synthesized ZnO NPs, and the results matched the previous results 40 . The morphology of biosynthesized ZnO NPs was examined using the SEM (Fig.  3 a–b), as well as their chemical composition was determined using EDX. The SEM images finalized the ZnO NPs, which had a regular distribution of both spherical and hexagonal shapes morphologically. The EDX spectrum of the nanoparticles of zinc oxide produced is depicted in Fig.  3 c. EDX spectrum shows that zinc oxide nanoparticles have 50.21 Zn % and 22.08% by weight. This confirmed the existence of O and Zn in the prepared material. The formation of the ZnO NPs has been verified by employing a resolution transmission electron microscopy examination (Fig.  3 d and e), and it is observed that neat ZnO NPs are spherical and hexagonal shaped. The high degree of crystallinity of the ZnO NPs is further demonstrated by the fact that the lattice fringes are presented clearly and without distortion. The selected area electron diffraction (SAED) pattern (Fig.  3 f) demonstrates that ZnO NPs are made up of a sequence of rings interspersed with bright spots, which may be interpreted as evidence of crystallinity. Diffraction patterns in the SAED picture, as well as XRD spectral peaks, provide additional evidence that ZnO NPs have a hexagonal wurtzite crystalline structure 40 .

XRD pattern of biosynthesized ZnO NPs using E.colona plant extract.

Scanning electron microscopic images of ZnO NPs ( a ) and ( b ), ( c ) energy-dispersive X-ray spectroscopy spectrum of ZnO NPs, ( d ) and ( e ) transmission electron microscopic images of ZnO NPs, and (f) SAED pattern of ZnO NPs.

The antibacterial activity of ZnO NP was measured qualitatively and quantitatively using zone of inhibition (ZOI), minimum inhibitory concentration (MIC), and minimum bacteriocidal concentration (MBC). The results are presented in Table ​ Table1. 1 . The synthesized ZnO NPs are subjected to antibacterial activity through the disc diffusion method. ZnO NPs exhibited significant antibacterial activity by inhibiting growth in terms of ZOI and multiplication of bacteria E. coli, and K. pneumonia at lower concentrations, and inhibition concentration increased with a higher concentration of nanoparticles. These results are compared with a standard positive control drug (ampicillin) (Fig.  4 and Table ​ Table1). 1 ). Teichoic acid and lipoteichoic acid are typical components of the cell walls of Gram-positive bacteria, which contain more layers of peptidoglycan polymer and are thicker (20–80 nm). These two acids act as chelating agents, allowing zinc ions to be transported into the cell from ZnO NPs. The outer layer of gram-negative bacteria cells has a thin coating of peptidoglycan (7–8 nm) and porins, thus allowing ZnO NPs to passively enter the cells 41 , 42 .

MIC and MBC of ZnO nanoparticles against E. coli and Klebsiella pneumoniae bacteria.

Antibacterial activities of ZnO NPs in the zone of inhibition at different concentrations against E. coli and K. pneumoniae bacterial strains along with positive control (Ampicillin).

ZnO NPs and the intracellular components they release, as well as the chelated zinc they contain, can trigger cell death by compromising the phospholipid bilayer due to the presence of ZnO NPs. Alkaline phosphatase, polymerases, and carboxy peptidases are only a few of the enzymes that might be inhibited by Zn ions. Strong interactions may be formed between zinc ions and the cysteine, histidine, and aspartate side chains of proteins at nanomolar concentrations, as demonstrated by Chulhun and Herbert 43 . The breakdown of bacterial cell walls causes a decrease in osmotic pressure and ionic strength in pathogenic species. Cellular functions, including growth, metabolism, and reproduction, are stifled by this drop in osmotic pressure. Smaller ZnO NPs may have an easier time breaking through bacterial membranes because of their larger interfacial area. As a result, their antimicrobial properties would become much stronger. The most potent bactericidal and fungicidal response was significantly influenced by NP size; smaller ZnO NPs displayed the highest levels of antibacterial activity 44 . SEM results (\* MERGEFORMAT Fig.  5 a–d) clearly show that ZnO NPs treatment reduced the colony counts of the studied microbes and distorted cell wall shape and size, respectively ( \* MERGEFORMAT Fig.  5 c, d) in comparison to untreated microbes (\* MERGEFORMAT Fig.  5 a, b). Despite the thickness of bacterial cell walls, intracellular leakage is observed in these results, which corroborates previous reports that ZnO NPs can be used to prevent pathogenic microbial infections in living cells. The possible mechanism of the antibacterial activity of ZnO NPs is presented in \* MERGEFORMAT Fig.  6 .

Scanning electron microscopic images of ( a ) E. coli, ( b ) E. coli treated with ZnO NPs, ( c ) Klebsiella pneumonia, and ( d ) Klebsiella pneumonia treated with ZnO NPs.

Possible mechanism of antibacterial activity of Echinocloa colona extract mediated synthesized ZnO NPs.

Biosynthesized NPs are bactericidal because their antibacterial effect is directed at the respiratory chain and cell division, both of which ultimately result in cell death. One of the most crucial roles that bacterial cell walls and membranes play is protecting against medicinal chemicals like NPs 45 .There is a great deal of diversity in the classification of bacteria based on the components of their cell walls. The cell wall of a Gram-negative bacterium, also known as the cell envelope, is composed of two layers of lipopolysaccharides. On the other hand, the thicker cell walls of gram-positive bacteria are almost entirely made up of peptidoglycans, a single family of chemicals. The presence of ZnO NPs suspensions significantly increases the generation of reactive oxygen species (ROS) 46 , 47 . Ahmed M. et al . investigated the enhanced antibacterial activity of bio-ZnO NPs, and the results were expressed in ZOI, particularly with a 22 mm ZOI against S. aureus and a 17 mm ZOI against E. coli . The study results demonstrate that ZnO nanoparticles are more effective against gram-positive bacteria due to structural variations in their cell walls 48 . Thana et al . studied the antibacterial activity against S. typhi , E. coli , S. pneumoniae , and S. aureus, which were found to be 21.4 mm, 15.8 mm, and 15 mm, respectively, in antibacterial activity. The amount of zinc oxide affected the antibacterial activity of the ZnO NPs in their prepared state 49 .

According to our results, ZnO NPs have a high degree of biofilm destruction capability against both bacteria (Fig.  7 ). The schematic representation of the anti-biofilm activity of ZnO NPs is presented in Fig.  8 . This is comparable to ZnO NPs that have been shown in the past to stop the synthesis of exo-polysaccharides and prevent the formation of biofilms, which in turn stopped the growth of bacteria. According to another study, biologically generated metal oxides inhibited the biofilm growth before it reached the irreversible adhesion stage. It's interesting to note that first-stage biofilm activity was shown to be suppressed at the MIC values 50 . The investigation by Kaweeteerawat et al . suggests that metal oxide nanoparticles may interact with bacterial cell membranes to induce oxidative stress, which is the mechanism through which NPs reduce the biofilms 50 . Microscopy images that showed bacterial cell deformation, external cell roughness, and cell wall shrinkage have been deployed in an earlier investigation to interpret the anti-biofilm action.

Antibiofilm activity of synthesized ZnO NPs using Echinocloacolona plant extract.

Schematic mechanism of anti-biofilm activity of ZnO NPs.

Furthermore, less biofilm was developed, and fewer live cells were seen. Reactive oxygen species (ROS) generated by ZnO NPs and surface ions inhibited biofilm formation. Many variables, such as the size and dispersibility of the nanoparticles, influence the extent of growth inhibition. Similar to our results, Zahra et al . reported that a significant inhibition of biofilm formation was found at 85% and 97% against S. aureus  ATCC 25,923 and P. aeruginosa ATCC 27,853 biofilm, respectively 51 . This finding demonstrated that the synthesised ZnO NPs could quickly and efficiently separate biofilm and suggest their usage as agents to disrupt the biofilms 52 . According to a prior explanation, the extracellular ROS production caused by biogenic synthesized ZnO NPs may be the origin of the bio mechanism of anti-biofilm activity, which destroys microorganisms' biofilm exo-polysaccharides 52 .

The MIC data suggest the significantly improved antibacterial activity of ZnO NPs is due to, in part, the greater impact on the surface along with the surface area of these particles. One study that examined the antibacterial properties of ZnO NPs found that smaller particles showed more antibacterial activity than larger ones. These nanoparticles may have enhanced antibacterial activity due to their high liposolubility index. Overtone postulated that the lipid barrier surrounding cells would only allow chemicals that are also soluble in lipids to enter through. Therefore, lipophilicity could be a significant feature of antibiotics 53 . Diminutive, synthesized nanoparticles can penetrate and rupture the cell membrane, leading to the death of the cell as a secondary mechanism of antibacterial activity. ZnO NPs cause the release of H 2 O 2 molecules from the cell surface, which aids in the killing of bacteria.

Hsueh et al . have reported that ZnO NPs weakened the structural properties of the (epoxy polysaccharides) EPS that omprised the biofilm, leading to its dissolution. A. faecalis and P. gingivalis biofilm production decreased by 92.27 1.22% and 95.27 1.28%, respectively, when biosynthesized ZnO NPs were utilized 54 . The outcomes of this research were consistent with those of others that looked at S. aureus, E. coli, and Pseudomonas aeruginosa biofilms. Ankush et al . synthesized ZnO NPs using the leaf extract of the Saracaasoca  plant, and the antibacterial and anti-biofilm activity of NPs was measured against the biofilm-producing bacteria Bacillus subtilis. The results showed that biofilm growth was suppressed by approximately 45%, 64%, and 83% at 0.5 × MIC, 0.75 × MIC, and 1 × MIC value, respectively. The concentration-dependent biofilm biomass produced or matured biofilms by ZnO NPs was assessed to be 68%, 50%, and 33% at concentrations of 0.5 Η MIC, 0.75 × MIC, and 1 × MIC, respectively  55 .

Conclusions

This reports the synthesis of ZnO NPs using the bio-reduction method. The Eechinochloacolona plant leaf extract comprises a significant number of polyphenols, flavonoids, sugars, and proteins, which act as reducing as well as stabilizing agents in the production of ZnO NPs. The UV absorption peak confirmed the formation of ZnO NPs at 370 nm, and the hexagonal wurtzite structure was affirmed by XRD and TEM investigations, with a size range of  17 nm. Anti-biofilm assay results suggested that the synthesized ZnO NPs demonstrated a high degree of biofilm detachment property over the range of concentrations examined. Both Gram-positive and Gram-negative bacteria were unable to proliferate in the presence of nanoparticles, confirming the strong antibacterial activity of the synthesized ZnO NPs. The nanoparticle's ability to efficiently destroy particular diseases or preservation of food products can be enhanced by functionalizing them with specific chemicals or ligands. The impact of these nanoparticles on the environment and untargeted creatures should be the main focus of research. Additional research could be looked into how well they treat various medical conditions, control patient infections, and healing of wounds.

Acknowledgements

The authors would like to express their gratitude partly to the National Research Foundation of Korea (NRF), which was funded and supported by the Ministry of Education (2020R1I1A3052258). Hussain Udayagiri thank Dr. Madhava Chetty, a taxonomist at the Department of Botany, Sri Venkateswara University in Tirupathi, India, for providing the leaves.

Author contributions

Hussain Udayagiri, Siva Sankar Sana: Conceptualization, Investigation, Writing -Original Draft; Lakshman Kumar Dogiparthi, Ramakrishna Vadde, Rajender S. Varma: Review-editing, Data Analysis and Validation; Janardhan Reddy Koduru, Gajanan Sampatrao Ghodake, Adinarayana Reddy Somala, Vijaya Kumar Naidu Boya, Seong-Cheol Kim, Rama Rao Karri: Writing review-editing.

Data availability

Competing interests.

The authors declare no competing interests.

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These authors contributed equally: Hussain Udayagiri and Siva Sankar Sana.

Contributor Information

Vijaya Kumar Naidu Boya, Email: moc.liamg@ayobyajivrd .

Seong-Cheol Kim, Email: rk.ca.uny@70mikcs .

Rama Rao Karri, Email: [email protected] .

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Green synthesis and characterization of zno nanoparticles using pelargonium odoratissimum (l.) aqueous leaf extract and their antioxidant, antibacterial and anti-inflammatory activities.

1. Introduction

2. materials and methods, 2.1. chemicals, 2.2. plant collection and processing, 2.3. preparation of p. odoratissimum leaf extract, 2.4. qualitative phytochemical screening, 2.5. hplc-analysis, 2.6. estimation of total phenolic and flavonoid contents (tpc and tfc), 2.7. green synthesis of zno nanoparticles, 2.8. characterization methods of zno nps, 2.8.1. uv-vis spectroscopy, 2.8.2. dynamic light scattering (dls), 2.8.3. fourier transform infra-red spectroscopy (ftir), 2.8.4. x-ray diffraction (xrd), 2.8.5. field emission-scanning electron microscopy (fe-sem), 2.8.6. high-resolution transmission electron microscopy (hrtem), 2.9. estimation of antioxidant activity—dpph radical scavenging activity, 2.10. estimation of antibacterial activity, 2.10.1. bacteria strains, 2.10.2. antibacterial assay, 2.11. estimation of anti-inflammatory activity, 2.12. statistical analysis, 3. results and discussion, 3.1. qualitative phytochemical screening (qps), 3.2. hplc-analysis, 3.3. characterization of zno nps, 3.3.1. visual observation, 3.3.2. uv-vis spectroscopy, 3.3.3. dynamic light scattering (dls), 3.3.4. ftir analysis of biosynthesized zno nps and p. odoratissimum ale, 3.3.5. x-ray diffraction (xrd) analysis of zno nps, 3.3.6. fe-sem of zno nps, 3.3.7. energy dispersive x-ray analysis (edx) spectrum of zno nps, 3.3.8. hr-tem of zno nps, 3.4. antioxidant activity, 3.5. antibacterial activity, 3.6. anti-inflammatory activity, 4. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, conflicts of interest.

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Abdelbaky, A.S.; Abd El-Mageed, T.A.; Babalghith, A.O.; Selim, S.; Mohamed, A.M.H.A. Green Synthesis and Characterization of ZnO Nanoparticles Using Pelargonium odoratissimum (L.) Aqueous Leaf Extract and Their Antioxidant, Antibacterial and Anti-inflammatory Activities. Antioxidants 2022 , 11 , 1444. https://doi.org/10.3390/antiox11081444

Abdelbaky AS, Abd El-Mageed TA, Babalghith AO, Selim S, Mohamed AMHA. Green Synthesis and Characterization of ZnO Nanoparticles Using Pelargonium odoratissimum (L.) Aqueous Leaf Extract and Their Antioxidant, Antibacterial and Anti-inflammatory Activities. Antioxidants . 2022; 11(8):1444. https://doi.org/10.3390/antiox11081444

Abdelbaky, Ahmed S., Taia A. Abd El-Mageed, Ahmad O. Babalghith, Samy Selim, and Abir M. H. A. Mohamed. 2022. "Green Synthesis and Characterization of ZnO Nanoparticles Using Pelargonium odoratissimum (L.) Aqueous Leaf Extract and Their Antioxidant, Antibacterial and Anti-inflammatory Activities" Antioxidants 11, no. 8: 1444. https://doi.org/10.3390/antiox11081444

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