Sustainable fabrication of dimorphic plant derived ZnO nanoparticles and exploration of their biomedical and environmental potentialities

Although, different plant species were utilized for the fabrication of polymorphic, hexagonal, spherical, and nanoflower ZnO NPs with various diameters, few studies succeeded in synthesizing small diameter ZnO nanorods from plant extract at ambient temperature. This work sought to pioneer the ZnO NPs fabrication from the aqueous extract of a Mediterranean salt marsh plant species Limoniastrum monopetalum (L.) Boiss. and assess the role of temperature in the fabrication process. Various techniques have been used to evaluate the quality and physicochemical characteristics of ZnO NPs. Ultraviolet–visible spectroscopy (UV–VIS) was used as the primary test for formation confirmation. TEM analysis confirmed the formation of two different shapes of ZnO NPs, nano-rods and near hexagonal NPs at varying reaction temperatures. The nano-rods were about 25.3 and 297.9 nm in diameter and in length, respectively while hexagonal NPs were about 29.3 nm. The UV–VIS absorption spectra of the two forms of ZnO NPs produced were 370 and 365 nm for nano-rods and hexagonal NPs, respectively. FT-IR analysis showed Zn–O stretching at 642 cm−1 and XRD confirmed the crystalline structure of the produced ZnO NPs. Thermogravimetric analysis; TGA was also used to confirm the thermal stability of ZnO NPs. The anti-tumor activities of the two prepared ZnO NPs forms were investigated by the MTT assay, which revealed an effective dose-dependent cytotoxic effect on A-431 cell lines. Both forms displayed considerable antioxidant potential, particularly the rod-shaped ZnO NPs, with an IC50 of 148.43 µg mL−1. The rod-shaped ZnO NPs were superior candidates for destroying skin cancer, with IC50 of 93.88 ± 1 µg mL−1 ZnO NPs. Thus, rod-shaped ZnO NPs are promising, highly biocompatible candidate for biological and biomedical applications. Furthermore, both shapes of phyto-synthesized NPs demonstrated effective antimicrobial activity against various pathogens. The outcomes highlight the potential of phyto-synthesized ZnO NPs as an eco-friendly alternative for water and wastewater disinfection.


Plant material collection and processing
Field surveys were conducted along the salt marshes habitat in El-Alamein region situated at the western Mediterranean coastline of Egypt (Latitude 30° 55′ 338″, Longitude 28° 29′ 365″, Altitude 11) for the collection of L. monopetalum specimens.The relevant national and international guidelines 33 were followed during the plant material collection.The permission for the collection of the species for the study purposes was acquired from the Department of Environmental Sciences at Alexandria University.The identification of the collected plant specimens was conducted by Dr. Marwa Waseem A. Halmy following Täckholm 34 and Boulos 35 .Voucher specimens were prepared and placed in the public herbarium of Tanta University (TANE) under voucher numbers 14210-14220.The plant grinded aboveground parts were prepared as described in our previous work 36 .Briefly, the plant aerial parts were thoroughly washed with running tap water and then deionized water.Eventually, it was dried at 60 °C, ground to a fine powder, and stored in sealed containers.
Sustainable ZnO NPs fabrication from L. monopetalum extract 20 g L −1 of the L. monopetalum powder was added in distilled water (DW) at 70 °C for 30 min, left to cool at ambient temperature, then filtered using filter paper (Whatman), and then stored at 4 °C for further use.
For the phyto-synthesis of ZnO NPs; a simple, low cost, and energy efficient approach was implemented as stated by Naiel et al. 36 .L. monopetalum extract was mixed with 0.5M Zinc acetate dihydrate [Zn (CH 3 COO) 2 ⋅2H 2 O] in a ratio of (1:10).Subsequently, 2 M sodium hydroxide [NaOH] was added drop by drop to maintain the mixture pH to 12, the reaction was maintained at ambient temperature.
The effect of the reaction temperature on the synthesis of the ZnO NPs was assessed by preparing another mixture.This mixture was prepared and stirred at 70 °C for 30 min until a color change from yellow to white was observed and started to precipitate indicating the completion of the reduction process and formation of ZnO NPs [36][37][38][39] .The formed precipitate was then decanted, washed with DW, and dried-up at 70 °C overnight to produce ZnO NPs powder with two different shapes.The produced powder was kept at ambient temperature in sealed bottles for additional characterization and application.The process is illustrated in Fig. 1.

Antioxidant activity
The 2,2-diphenyl-1-picrylhydrazyl; DPPH assay was implemented to evaluate the antioxidant capability of the phyto-synthesized ZnO NPs.To summarize, 0.1 Mm of 1 mL DPPH was mixed with 3 mL of the dissolved ZnO NPs in ethanol at 1.95-1000 µg mL −1 concentrations.The mixture was maintained at pH range of 5-6.5.After that, stirring vigorously and kept at ambient temperature for 30 min.The method was done in triplicate and the absorbance was estimated at 517 nm using UV-VIS spectrophotometer (Milton Roy, USA).DPPH Scavenging Activity (%) was calculated and expressed as IC 50 42,43 : where A 0 is the control absorbance, and A 1 is the test absorbance.

Antimicrobial activity
The effect of the plant-mediated ZnO NPs using L. monopetalum as potential antimicrobial agents was investigated against pathogenic species.The study focused on pathogenic microorganisms responsible for medical and environmental implications, particularly: Gentamycin was utilized as a positive control for bacterial strains, whereas Fluconazole was used as control for fungal species.A total of 100 µl of phyto-synthesized ZnO NPs and positive controls were used in the assessment.Antimicrobial activity was detected via the estimation of the inhibition zone diameter (in mm) by means of the 'agar well diffusion' method [44][45][46] .

Surface properties and phytochemical profile
The spectra of the two ZnO NPs forms attained different intensity peaks; the form prepared at ambient temperature appeared at 370 nm, while that prepared by heating to 70 °C emerged at 365 nm (Fig. 2a,b).The two peaks are characteristic and distinct for ZnO NPs formation.These results are consistent with earlier studies 7,47 .
The functional groups of FT-IR revealed the detection of Zn-O stretching at 642 cm −1 indicating the successful synthesis of ZnO NPs as reported by Khalaf et al. 23 .The plant extract spectrum had a broad peak at 3410 cm −1 corresponding to-OH group.This peak was shifted in the ZnO NPs FT-IR spectrum of both ZnO NPs to 3443 cm −1 and 3434 cm −1 for rod-shaped and hexagonal NPs, respectively, similar to Rani et al. 48.These peaks (1) may be correlated with the phenolic, alcoholic, and terpenoid content of the plant extract.As depicted in Fig. 2c, 1624 cm −1 peak in the plant extract spectrum corresponded to H-O-H bending, which shifted to 1560 cm −1 , 1567 cm −1 , and 1640 cm −1 in the spectrum of ZnO NPs.The peaks of plant extract shifted to another wavelength or disappeared in the FT-IR spectrum of the gained ZnO NPs, demonstrating the importance of these phytochemicals for the stability and reduction of ZnO NPs 49 .The analysis of the phytochemical profile of the plant extract using Gas Chromatography-Mass Spectrophotometry (GC-MS) helped identify the phytochemicals that might be accountable for reducing ZnO NPs and stabilizing its formation (Fig. 3 and Table 1).Four categories of bioactive compounds were identified and classified into monoterpenoid alcohols, oxanes, and esters.The main compounds were identified in Table 1.These phytochemicals may have acted as reducing, stabilizing, and capping agents for ZnO NPs, as suggested by previous studies [50][51][52] .These phytochemicals contain hydroxyl groups that could assist in the reduction of the precursor zinc acetate dihydrate to ZnO NPs.They are also responsible for the development of the stable form of ZnO NPs via capping process 53 .Moreover, the aforementioned FT-IR analysis was consistent with the occurrence of the bioactive compounds detected by the GC-MS.

Morphological structure
The data acquired from Transmission Electron Microscope (TEM) and Energy Dispersive X-ray (EDX) revealed that two different morphologies of ZnO NPs were obtained with varying reaction temperatures (see TEM images Fig. 4).Rod-shaped ZnO NPs were formed at ambient temperature, and the nearly hexagonal shape was formed at 70 °C.The average size of rod-shaped ZnO NPs was ~ 25.3 nm and 297.9 nm in diameter and in length, respectively whereas that of the hexagonal NPs was ~ 29.3 nm as demonstrated in Fig. 4a,b respectively.These results followed with Kotresh et al. 54 who revealed that reaction temperature is a key factor affecting the morphological features of nanoparticles.In contrast to our results; Thi et al. 22 reported large coagulated ZnO nano-rods with lengths and widths of ∼ 370 nm and ∼ 160 nm, respectively using orange peel extract.A summary Table 2. Illustrating the previously prepared ZnO NPs using various reaction temperatures and plant extracts.The EDX spectra affirmed the occurrence of two robust peaks with high intensities of Zinc and Oxygen at ~ 1keV and ~ 0.5keV respectively (Fig. 4c,d).The atomic percentage of Zn was 42.33% and Oxygen was 57.67% for rod-shaped ZnO NPs.On the other hand, the atomic percentage of Zn was 37.52% and oxygen was 62.48% for the near-hexagonal NPs (Fig. 4c,d).No impurities were detected, confirming the purity of the phyto-synthesized NPs 58 .

X-ray diffraction (XRD)
The sharp peaks exhibited by the prepared ZnO NPs confirmed their crystalline nature (Fig. 5).Diffraction peaks of rod-shaped ZnO NPs were at ~ 31.8°,34.1°, 36.4°,47.8°, 56.8°, 62.9° and 68.2°.The peaks of the hexagonal ZnO NPs were located at ~ 32.01°, 34.3°, 36.6°,47.9°, 57.01°, 63.07°, and 68.4°.The peaks were compatible with JCPDS Card (2300112) for rod-shaped ZnO NPs and (1011258) for hexagonal NPs.The ZnO NPs attained a space group P 63 mc (no.186), which was also established by other previous studies 29,59 .The detected slight XRD peaks could be attributed to the crystallization of the detected phytochemicals (capping and stabilization of ZnO NPs) as was cited in the literature 59 .Using the Debye-Scherrer's equation 60 , the average crystalline sizes of rod-shaped and hexagonal ZnO NPs were ~ 12 nm and ~ 11 nm, respectively.The size of NPs differs by using XRD and TEM as XRD provided data about the grain size which might be related to the polycrystalline of large particles resulting from the fusion of smaller grains 61,62 .

Thermogravimetric analysis (TGA)
As shown in the thermogravimetric analysis (Fig. 6), it demonstrates the weight loss (%) of ZnO NPs samples in response to temperature increases.The produced ZnO NPs proved remarkable thermal stability.The mass loss profile was steady up to 28.3 and 13.9% of the actual mass for nano-rods and the hexagonal NPs, respectively.This loss might be due to the elimination of moisture and carbonaceous phytochemicals in the prepared samples of ZnO NPs 22,63 .
The TGA analysis revealed two distinctive phases of weight loss.The TGA pattern of Rod ZnO NPs-RT (Fig. 6a) revealed a weight loss of 19.3% up to 296°C at the first phase and a slight weight loss of 9% from 296 to 947 °C at the second phase.A slightly higher thermal stability pattern was obtained by hexagonal ZnO NPs-70 °C (Fig. 6b) as it showed minor weight loss of 10.1% up to 286 °C at the first phase and 3.8% from 286 to 947 °C at the second phase.The initial weight loss might be due to moisture elimination and the second weight loss might be related to the decomposition of Zn (OH) 2 to ZnO NPs [64][65][66] .

Anticancer activity and cytotoxicity
The results showed that the rod-shaped ZnO NPs at RT had superior cytotoxicity toward cancer cells than hexagonal ZnO NPs at 70 °C.The IC50 of the phyto-synthesized rod-shaped ZnO NPs and hexagonal NPs was 93.88 ± 1 μg mL −1 and 187.15 ± 0.86 μg mL −1 respectively, against A-431 cancerous cell lines.This could be attributed to the small diameter and surface morphology of the obtained NPs 67 .Similar studies confirmed the cytotoxicity of ZnO NPs against A-431 cell lines (Fig. 7a).For instance, Chelladurai et al. 68 proved that the IC 50 of ZnO NPs was 125.3 μg mL −1 using Alpinia calcarata rhizome extract.In addition, Naiel et al. 36  that the IC 50 for Limonium pruinosum aqueous extract was 409.7 μg mL −1 ZnO NPs.This finding confirms that ZnO NPs derived from L. monopetalum aqueous extract had higher cytotoxicity than previously reported.
The MTT assay results that were obtained after the cell lines were exposed to the produced nanoparticles, revealed that the ZnO NPs had a significant impact on the viability of the tumor cells.Using a concentration of 125 g mL −1 of rod-shaped ZnO NPs, the viability of the malignant cells significantly decreased to 24.3%.At a concentration of 250 g mL −1 , the hexagonal ZnO NPs significantly decreased the viability of tumorous cells to 20.08%.Ali et al. 29 indicated that the synthesized ZnO nano-rods from Leea asiatica leaf extract could be used as a substitute anticancer medication.Sadhukhan et al. 69 revealed the functionality of ZnO nano-rods for bioimaging and drug delivery usages.The prospective anticancer effect of ZnO NPs may be connected to the release of zinc ions, high oxidative stress, and finally cell apoptosis through DNA impairment and protein denaturation 70 .
Both obtained ZnO NPs showed higher cytotoxicity for cancer cells compared to normal cells.The IC 50 of the phyto-synthesized rod-shaped ZnO NPs and near hexagonal NPs was 179.61 ± 3.63 μg mL −1 and 684.42 ± 19.1 μg mL −1 respectively against normal HFB4 cell lines (Fig. 7b).These findings confirm the reliability, and selectivity of phyto-synthesized ZnO NPs using L. monopetalum aqueous extract, which is in accordance with the findings by Babayevska et al. 13 , which proved that ZnO NPs and nano-rods were more destructive to cancerous cells than to the normal ones at the similar concentration.
The enhanced permeability and retention (EPR) effect may be related to the selectivity of ZnO NPs.They may increase the amount of zinc in cells, which may lead to protein disequilibrium and a rise in oxidative stress 11,71 .
Morphological changes in cancerous cells (A-431) and normal cells (HFB4) were also investigated as shown in Fig. 8a,b.These figures shows that at high concentrations of ZnO NPs, the cells became rounded and shrunk indicating the cytotoxic effect of those concentrations.It was detected that high concentrations can raise ROS levels, leading to DNA fragmentation, cell shrinkage and apoptosis 67,72 .The produced ZnO NPs appear to be more toxic to A-431 cell lines, confirming the biocompatibility of ZnO NPs.

Antioxidant activity
The antioxidant effect estimated for the two phyto-synthesized forms of ZnO NPs using DPPH assay revealed that both showed a concentration dependent free radical scavenging activity as was indicated in previous studies 73 .The rod-shaped ZnO NPs exhibited higher DPPH scavenging activity than hexagonal NPs (Fig. 9a).IC 50 was 148.43 and 475.7 µg mL −1 for the phyto-synthesized rod-shaped ZnO NPs at RT and hexagonal ZnO NPs at 70 °C respectively.The small diameter of rod-shaped ZnO NPs relative to that of the hexagonal NPs might be the cause of their higher antioxidant capability 74 .In contrast to the current study, Saleemi et al. 75 found low antioxidant activity for ZnO NPs with an IC 50 > 500 µg mL −1 .The phytochemicals that were related to reducing and stabilizing ZnO NPs might be responsible for their scavenging capabilities as stated by Sivasankarapillai et al. 43 .
The resultant formation of the hydroxyl and hydrogen radicals, may be responsible for the potential antioxidant effect of the synthesized ZnO NPs (Fig. 9b) as reported in other literature 43,[76][77][78] .The spectrophotometric assessments of antioxidants' ability to scavenge DPPH radicals serve as the basis for this assay.Taking a hydrogen atom from the antioxidants reduces the solitary electron of the nitrogen atom in DPPH to hydrazine 79 .

Antimicrobial activity
The merit of sustainable nanofabrication is to promote the use of nanomaterials as disinfectants against pathogenic microorganisms in water and wastewater.Water conventional methods for disinfection applying chlorination and ozonation create toxic and harmful byproducts 80 .While the sustainable nanomaterials could minimize the generated byproducts during the disinfection and corrosive suppression 81 .Food-borne diseases have also exerted pressure on health and subsequent economic sectors.These pathogens can be transmitted via contaminated irrigation water, soil, and manure to various crops and eventually to humans 82 .Fungal pathogens, for instance, Candida is a significant cause of invasive infections, linked to high morbidity, mortality rates and prolonged hospitalization with high costs.Some fungal pathogens can also produce secondary byproducts that contribute to microbial corrosion in water distribution systems.Candida can exist in aquatic ecosystems contaminated with human or animal feces and also in potable water in hospitals [83][84][85] .Aspergillus flavus can grow in different kinds of food and produce toxic aflatoxins 86 .One of the main complications of fungal and bacterial species is the deleterious impacts on public health (see Table S1).
The results (Table 3) demonstrated that the ZnO NPs produced through phytosynthesis means exhibited a wide range of antimicrobial efficacy.When utilizing hexagonal ZnO NPs at 70 °C in comparison to the common antibiotic Gentamycin, the broadest inhibition zone was seen against Gram-negative bacteria Pseudomonas aeruginosa (ATCC 90274) at 30 mm and Gram-positive Bacteria Bacillus Subtilis (ATCC 6633) at 29 mm.These findings are in line with those of earlier research investigations [87][88][89] .
Hexagonal ZnO NPs revealed slightly higher antibacterial and antifungal efficacy against all tested microorganisms except Aspergillus flavus.The efficacy of the antibacterial activity of ZnO NPs may be ascribed to their shape, morphology, and surface-to-volume ratio 67,90 .According to previous literature, the current findings proved higher inhibition zones applying the phyto-synthesized ZnO NPs as concluded in (Table 4).
According to these findings, the antimicrobial effect of the produced ZnO NPs might be attributed to the capability to bind to the microbes' cell membrane and enhancing the formation of ROS leading to oxidative stress, DNA damage, and finally cell death 26,42,73,92 .Sportelli et al. 10 also demonstrated that zinc oxide has outstanding antimicrobial action, particularly at the nanoscale.Additionally, the FT-IR and GC-MS results recognized the occurrence of capping and stabilizing phytochemicals such as monoterpenoid alcohols, esters, and fatty acid methyl esters.These bioactive constituents demonstrated good antibacterial and antifungal activity, as mentioned by Zhumakanova et al. 93

Conclusion and recommendations
Our results represent an added value for one of the halophytic species that always has been over looked.Successfully, the aqueous extract of Limoniastrum monopetalum (L.) Boiss mediated the synthesis of two different shapes of ZnO NP for the first time.
The results also demonstrated that reaction temperature played a major role in influencing the morphology, size, and physio-chemical characteristics of the obtained NPs.These outcomes were affirmed through the TEM images that proved the materialization of rod-shaped ZnO NPs at ambient temperature whereas hexagonal ZnO NPs were formed at 70 °C.The potential phytochemicals accountable for the reduction of ZnO NPs include monoterpenoid alcohols, esters and fatty acid methyl esters were identified using GC-MS and FT-IR.The anticancer, antimicrobial, and antioxidant investigations revealed the superior efficacy of the rod-shaped ZnO NPs relative to the hexagonal ones.The IC 50 of rod-ZnO NPs was 93.88 ± 1 µg mL −1 against A-431 cancerous cell lines compared to 187.15 ± 0.86 μg mL −1 for hexagonal NPs.According to the results of the cytotoxicity, the IC 50 values for rod-shaped ZnO NPs and hexagonal NPs against normal HFB4 cell lines were 179.61 ± 3.63 g mL −1 and 684.42 ± 19. g mL −1 , respectively.This underscores the high cytotoxicity of the phyto-synthesized ZnO NPs toward skin cancer cells relevant to the normal cells and their biocompatibility.They also exhibited dose-dependent antioxidant activities, particularly rod-shaped ones.This study also emphasizes the significant effect of ZnO nanoparticle shape on pharmacological activity.Moreover, the two produced ZnO NPs forms can provide a potentially efficient substitute as an effective antimicrobial agent to eradicate resistant bacterial and fungal pathogens which is a challenge in water and wastewater disinfection.In conclusion, these results highlight the prospect of using L. monopetalum for ecofriendly synthesis of ZnO NPs with diverse morphologies in numerous prospective biomedical and environmental applications.Accordingly, this study adds more support for the transformation to green chemistry approach, thereby reducing the harmful effects of high energy consumption, pressure and hazardous chemicals during production.It also calls for the conservation of a highly vulnerable habitat, salt marshes, for pronounced role of halophytes in CO 2 sequestration and contribution for climate change mitigation and adaptation measures.

Figure 2 .
Figure 2. (a, b) UV-Vis spectra of the phyto-synthesized ZnO NPs and (c) FT-IR spectra of L. monopetalum extract and the phyto-synthesized ZnO NPs.

Figure 8 .
Figure 8. Morphological changes of (a) A-431 cancer cell lines and (b) HFB4 normal cell lines at different ZnO NPs concentrations. .

Figure 9 .
Figure 9. (a) The antioxidant activity of phytosynthesized ZnO NPs at RT and 70 °C.(b) The possible antioxidant mechanism of phyto-synthesized ZnO NPs.Data are expressed as mean ± SD (n = 3).

Table 1 .
Phytochemical compounds identified in the extract of L. monopetalum.

Table 2 .
Comparison between previously prepared ZnO NPs in literature using various plant extracts and reaction temperatures.

Table 3 .
Zones of inhibition in mm of different phytosynthesized ZnO-NPs against pathogenic bacteria and fungi.*Control: Gentamycin for bacteria and Fluconazole for fungi.**NA: No Activity.

Table 4 .
Antimicrobial activity of phyto-synthesized ZnO NPs produced in this work compared to the previously reported literature.