Antibacterial activity of seed aqueous extract of Citrus limon (L.) mediated synthesis ZnO NPs: An impact on Zebrafish (Danio rerio) caudal fin development.

Among the different metal oxide nanoparticles, zinc oxide nanoparticles have gained significant importance due to their antibacterial properties against clinically pathogenic bacteria during the organal development. In the present study, biogenic zinc oxide nanoparticles were synthesized using seed extract of Citrus limon by a simple, cost-effective, and green chemistry approach. The synthesized ZnO NPs were characterized by UV-Vis spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, Dynamic Light Scattering, and Scanning Electron Microscopy. Next, the antimicrobial activity of ZnO NPs was tested against clinically pathogenic bacteria, i.e., Pseudomonas fluorescens, Escherichia coli, Enterobacter aerogenes, and Bacillus subtilis. Followed by, ZnO NPs were evaluated for the development of caudal fin in Zebrafish. The UV-Vis spectram result showed a band at 380 nm and FTIR results confirmed the ZnO NPs. The average crystallite size of the ZnO NPs was 52.65 ± 0.5 nm by the Debye Scherrer equation and SEM showed spherical-shaped particles. A zone of inhibition around ZnO NPs applied to P. fluorescens indicates sensitive to ZnO NPs followed by B. subtilis. Among the four different bacterial pathogens, E. aerogenes was the most susceptible compared to the other three pathogens. The calculated sub-lethal concentration of ZnO NPs at 96 h was 153.8 mg/L with a 95% confidence limit ranging from 70.62 to 214.18 mg/L, which was used with partially amputated zebrafish caudal fin growth. A significant (p < 0.5) development (95%) in the amputated caudal fin was detected at 12 days post-amputation. Low concentrated ZnO NPs can reduce developmental malformation. Collectively, suggested results strongly proved that lemon seed-mediated synthesized ZnO NPs had a good pathogenic barrier for bacterial infection during the external organal development for the first time.

Introduction

Nanotechnology is the art and science of manipulating new themes for the nanoscale with enormous potential applications to change modern society. The superior properties of nanomaterials significantly impacted various innovative applications such in biomedical, environmental, industrial, and food agriculture sectors [1, 2, 3, 4]. Recently, several types of transition metal, as well as metal oxide nanoparticles, have been employed in various fields, owing to their unique properties [5].

In the past, many methods have engaged for fabricating the nanoparticles. Off them, particularly green synthesis affords simple, low-cost and eco-friendly nanomaterials [6]. It is well known that plant parts like roots, leaves, stems, seeds, fruits and peel of fruits have been in usage for zinc oxide nanoparticles (ZnO NPs) synthesis [7]. Besides, it is wealthy in phytochemicals and acts as a reducing and stabilization agent [8, 9]. Earlier reports suggested the detailed methodology of synthesis of ZnO NPs by Citrus aurantifolia, Jacaranda mimosifolia, Carissa edulis, Trifolium pretense and Nephelium lappaceum flower aqueous extract [10, 11, 12, 13].

Citrus limon (L.) Burm. f. is an evergreen leaves tree with edible yellow colour fruit and is abundantly available in all seasons. An important medicinal plant of Lemon is the Rutaceae family, which contains Vitamin C, phytochemicals, citric acid, and it is non-toxic nature. It has been known in traditional medicine since ancient times, include treatment of high blood pressure, common cold and irregular menstruation. Also, C. limon oil is a good remedy for cough [14]. C. limon is rich in alkaloids, which have been reported to have antibacterial properties in the raw extracts of various parts of the lemon (i.e., leaves, stem, root, seed and flower) against clinically significant bacterial strains [15]. Its flavonoids have a huge range of biological activities such as antibacterial, antifungal, antidiabetic, anticancer and antiviral activities. Also, they play a defensive role against pathogens such as bacteria, fungi and viruses [16, 17]. Furthermore, other phytochemicals such as phenolic acid, mangiferin, phenolic esters, triterpenoids and thiamine are rich in the seed of C. limon. Recent studies are displayed that cell singnaling pathways are promoted by flavonoids [18]. Phytocomponends are well known to have antioxidants [19], anti-aging [20], anticancer, hepatoregenerating and cardioprotective activities [21] and wound healing [22]. Phytocompounds may include the possible response for the reduction of zinc ions to ZnO NPs [23].

Zn is a well-defined trace element for all forms of living beings and plays an essential role in regular thyroid function, blood clotting, cognitive functions, fetal growth and sperm production [24]. Zn, combined with metalloenzymes, possibly plays a significant role as metal in the formation of new bones and tissues [25]. Many works demonstrate that ZnO nanostructures successfully promote the growth, multiplication and differentiation of many cell lines combined with the rise of hopeful antibacterial activity [26].

ZnO NPs have high antimicrobial activities rather than macroparticles due to their unique properties such as size (<100 nm) and high surface area [27, 28]. Zebrafish (Danio rerio) have many advantages in disease diagnosis, which makes it a wonderful model animal. These advantages have created the zebrafish a popular and valuable vertebrate for the research model in the past three decades [29, 30]. Therefore, this present study focused on the objective of using the wildly available C. limon fruit waste of seed extract has not been investigated as a reducing agent for synthesising ZnO NPs. Furthermore, the present studies were to assess the antipathogenic effect against Pseudomonas fluorescens, Escherichia coli, Enterobacter aerogenes and Bacillus subtilis and to investigate the possible effect on amputated zebrafish cadul fin development. The investigation findings provide more information about green synthesised ZnO NPs for organal development during bacterial infection.

Materials and methods

Chemicals

Zinc acetate dehydrate [Zn(O2CCH3)2(H2O)2], clove oil, and Hanks solution purchased from Sigma-Aldrich and Sodium hydroxide [NaOH] from Fisher Scientific, India.

Collection and preparation of seed aqueous extracts

Citrus limon (L.) Burm. f. collected from the local outlets of Tirunelveli, Tamil Nadu, India. Seeds were separated from C. limon fruits and washed with the help of Tween 20 for surface sterilization.

Green synthesis of zinc oxide nanoparticles

1 mM Zinc acetate was dissolved in 50 ml of deionized water. The reaction mixture was kept in a magnetic stirrer at 400 rpm for 3 h at room temperature. After 3 h, 25 mL of 2 M NaOH solution was added slowly into the Zinc acetate mixture, followed by 25 mL of seed aqueous extract (Figure 1). The white colour mixture changed into pale yellow after 3h of stirring [31]. The colour change was to confirm the synthesis of ZnO NPs. The precipitate was separated from the reaction solution by centrifugation (8000 rpm; 4 °C for 15 min). The pellet was obtained and dried at 80 °C overnight. Then the samples were calcinated at 400 °C for 5 h and stored in air-tight containers for further analysis [32].

Figure 1

Schematic presentation of C. limon seed aqueous extract mediated synthesizing method of ZnO NPs.

Characterization of C. limon mediated synthesized ZnO NPs

The reduction of Zn ions into ZnO NPs monitored by using a double beam UV-Vis spectrophotometer (UV-Vis spectra) (Lambda 25, PerkinElmer) in a wavelength range from 200 to 800 nm. Functional groups of ZnO NPs determined by Fourier-transform infrared spectroscopy (FTIR). The FTIR results were obtained from a PerkinElmer Spectrum Two FTIR Spectrometer at 4000-400 cm−1 resolution. The quality and quantity of the compounds investigated by X-ray diffraction (XRD) analysis (PAN analytical X-Pert PRO). For this need, powder particles are employed. XRD scanning is done with Cu Kα radiation (k = 0.1540 nm) and a scanning rate of 0.02 °s−1 at a region of (2θ) from 20° to 80°.

Dynamic Light Scattering (DLS) is used for size and size distribution pattern of nanoparticles. The aliquot sample was transferred to the cuvette, which was followed by ten times dilution using Milli-Q water. After that analysis was done using DLS (Malvern Zetasizer, Nano Z500 UK). Maintained the sample holder temperature (25 °C) and scattering intensity (27193 cps). Morphological analyses of synthesized ZnO NPs performed by Scanning Electron Microscopy (SEM) (Carl Zeiss). Thin-film of nanoparticle powder samples prepared with gold-coated tape by adhering a small amount of dried fine powder of samples on the grid, excess samples removed with the help of blotting paper. The film on the SEM grid was allowed to dry under a mercury lamp for 5 min.

Antibacterial assay

Antibacterial assay of ZnO NPs were evaluated against four clinical bacterial species strains i.e., Pseudomonas fluorescens ATCC4163, Escherichia coli ATCC25922, Enterobacter aerogenes ATCC35028 (presently known as Klebsiella aerogenes) and Bacillus subtilis ATC-C6749 by the agar well diffusion method. The wells are made on Mueller–Hinton Agar (MHA, bioMerieux) having the test microorganism and filled with various concentrations ZnO NPs (100, 120, 140 and 160 μl).

Zebrafish maintenance

Zebrafish (SL, 2.5 ± 0.5 cm; weight, 0.18 ± 0.1 g) were purchased from an ornamental fish supplier (AM fish farm, Madurai, Tamil Nadu, Indian) and maintained in recirculating tanks containing oxygenated reverse osmosis water. The water was filtered through nylon mesh, denitrified by dust filtration, and finally disinfected by ultraviolet light exposure. Fish was fed twice per day with commercial food pellets. The room temperature is maintained at 26 ± 5 °C, and the conditions of lighting are 14:10 h (light: dark) [33].

Acute toxicity test

LC50 concentrations of ZnO NPs exposed with diverse concentrations (100, 200, 300, 400 and 500 mg/L) for 96 h against sexually mature male adult zebrafish (n = 8). The exposure was performed in a 5 L plastic tank according to OECD 203 [34]. According to Litchfield and Wilcoxon, LC50 values of 96 h exposure were calculated by test animals [35]. Similarly, the same procedure was followed for control with the Hanks solution [36].

Fin amputation and ZnO NPs exposure

Treated fish samples were anesthetized with clove oil. Caudal fins were amputated with the help of surgical scalpel blade No.10 at the beginning point of lepidotrichia. 96 hr LC50 concentrations were diluted at different dilution factors such as 1/5, 1/50, and 1/100 (30.76, 3.07, and 1.53 mg/L). Eight fishes (preferably male) were used for each test concentration, as well as positive and negative controls employed with zinc acetate and aqueous seed extract of C. limon. The experiments were performed for three replicative with the same concentration.

Measurement of fin development

Digital Vernier Calliper was used for measuring the amputated caudal fin development, which was performed with anesthetized Zebrafish from 0th to 12th day post-amputation (dpa). The development of the caudal fin was observed with the help of a light microscope (Olympus, SZ61) to examine the morphological differentiation of the Zebrafish caudal fin. The results are expressed as mean ± standard error.

Statistical analysis

All the data were analysed using the SPSS windows tool (version 22.0).

Results and discussions

Visual observation

Colour change is one of the primary indicators, which confirms the formation of ZnO NPs from zinc acetate by the reduction process. The reduced mixture turned into pale yellow from white colour (Figure 2). Hence, seed extract of C. limon mediated synthesized ZnO NPs gained significant attention due to their superior optical properties [31, 37]. Presence of phytochemicals in seed extract induced the formation of ZnO NPs.

Figure 2

UV-vis spectrum of ZnO NPs synthesized using aqueous seed extract of C.limon. (A) 1 mM Zinc acetate solution; (B) C. limon seed water extract and (C) final solution.

UV-vis spectrum analysis

The synthesized ZnO NPs was confirmed by UV-vis spectrum analysis. As seen in Figure 2, it denoted that UV-vis spectra of synthesized ZnO NPs by aqueous extracts of C. limon seed. The absorption peak (λ-max) was found at 380 nm with a broad spectrum and monodispersed in nature, which indicated the reduction of Zinc. The broad surface plasmon resonance bands are founded with an absorption end in the longer wavelengths, which may occur due to the size distribution of the particles [38]. Accordingly, the aqueous extract of Passiflora caerulea and Limonia acidissima leafs derived ZnO NPs obtained at 350 nm [39, 40]. Furthermore, the attained results correlated with Eichhornia crassipes leaf extract mediated synthesized ZnO NPs, in which the UV-Vis spectrum peak was found at 378 nm [41].

FTIR analysis

Substance-specific vibrations of the molecules lead to the particular signals acquired by FTIR spectroscopy, which helps to understand the purity and nature of the nanoparticles. FTIR spectra expressed the peaks at the range between 4000-400 cm−1 (Figure 3). Seeds aqueous extract of C. limon mediated synthesized ZnO NPs expressed broad and narrow peaks. The broad peaks found at 3467, 2958, and 2498 cm−1, which is corresponding to OH stretching, H–C–H asymmetric and symmetric stretching, and hydrogen-bonded O–H stretching vibrations.

Figure 3

FTIR spectrum of green synthesized ZnO NPs.

Whereas narrow peaks were displayed at 1441, 887, and 701 cm−1, which corresponded to C–N stretching, C–O stretching and C–H bending, respectively. The small peak was found at 596 cm−1 due to the presence of a metal oxide group (Table 1) [42]. These results are in line with the previously reported result on P. caerulea leaf aqueous extract mediated synthesized ZnO NPs [40]. Kumar and Rani [43] reported similar peaks obtained from Borassus flabellifer fruit extracted mediated synthesized ZnO NPs in their investigation.

Table 1

Presence of functional groups in synthesized ZnO NPs by FT-IR.

S. No.	Absorption peak (cm−1) in ZnO NPs	Bond/functional groups
1	3467	OH stretching vibrations
2	2958	H–C–H asymmetric stretch
3	2498	H–C–H symmetric stretch
4	1441	C–N stretch
5	887	C–O stretch
6	701	C–H bend
7	596	Zn–O stretch

XRD analysis

XRD analysis employed phase confirmations of green synthesized ZnO NPs. The XRD patterns of green synthesized ZnO NPs showed in Figure 4. 2θ values were found in ZnO NPs at 31.8°, 34.44°, 36.29°, 47.57°, 56.61°, 67.96° and 69.07°, which was corresponding to (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 1 2) and (2 0 1). Lattice plane values of face-centered cubic (fcc) are mentioned as crystal structured ZnO NPs. All the XRD peaks are correlated very well with the hexagonal phase (wurtzite structure) [44]. JCPDS card No.89-7102 was used as a reference to access the lattice plane value compared with obtained peaks. Santhoshkumar et al. [40] reported similar results from P. caerulea leaf extract mediated synthesized ZnO NPs in their investigation.

Figure 4

XRD Spectrum of C. limon seed aqueous extract mediated synthesized ZnO NPs.

Furthermore, the results of 2θ values at 31.8°, 34.44°, 36.29°, 47.57°, 56.61°, 67.96° and 69.07° from P. caerulea leaf extract mediated synthesized ZnO NPs were closely similar with C. limon mediated synthesized ZnO NPs [40]. The strong and narrow diffraction peaks indicated that well crystalline nature of ZnO NPs. The average crystallite size of the nanoparticles was calculated by the Debye Scherrer equation stated in Eq. (1). The average size of the synthesized nanoparticles was found at 52.65 ± 0.5 nm.where Dp is the average crystallite size, β is the line broadening in radians, θ is the Bragg angle, and λ is the X-ray wavelength.

DLS analysis

The size and size distribution of synthesized ZnO NPs were analyzed by DLS, which showed that narrow and brand spectrums represented small and larger particles, respectively [31]. DLS results found that different sized particles like 2 nm–444 nm. The average size of synthesized ZnO NPs is 150 nm (Figure 5) and the Polydispersity Index (P.I.) is 0.753.

Figure 5

The size and size distribution profile of C. limon seed aqueous extract mediated synthesizing ZnO NPs.

SEM analysis

SEM analysis was engaged in identifying the morphological pattern of C. limon seed extract mediated synthesized nanoparticles (Figure 6). Synthesized ZnO NPs attained in spherical shape along with the number of aggregates. Similar SEM results were reported on P. caerulea and C. gigantea leaf extract mediated synthesized ZnO NPs [31, 40].

Figure 6

SEM analysis of green synthesized ZnO NPs (2 μm magnification).

Green synthesized ZnO NPs were employed for the well diffusion method to execute the antibacterial assay. Zone of inhibition was observed in a dose-dependent manner (Figure 7). The green synthesized ZnO NPs expressed the highest zone of inhibition against E. aerogenes (12.13 ± 0.6 mm), E. coli (11.66 ± 0.58 mm) and P. fluorescens (11.3 ± 0.56 mm) and minimum zone of inhibition of B. subtilis (10.74 ± 0.58 mm) for 160 μg/ml (Figure 8). Zone of inhibition was compared with control as well as standard antibacterial components (not shown). It has been reported that ZnO NPs can impact the survival of microorganisms by agglomeration on the bacterial surface and also change the properties of lipids, peptidoglycan, proteins and DNA due to their high surface area and size [45].

Figure 7

Antibacterial activity of C. limon seed aqueous extract mediated synthesized ZnO NPs. (A) Escherichia coli; (B) Enterobacteraerogenes; (C) Bacillus subtilis and (D) Pseudomonas fluorescens.

Figure 8

Zone of inhibition of ZnO NPs against bacterial pathogens.

Gram-negative bacteria are more sensitive compared to gram-positive bacteria due to the surface peptidoglycan. The gram-positive bacterial cell wall contains thick peptidoglycan, while the gram-negative bacterial cell wall contains thin peptidoglycan, which favors the penetration of nano-sized particles into bacteria [45]. Diverse mechanisms of action are involved in the biocidal activity of nanoparticles against gram-positive and gram-negative bacteria. This may be attributed to the different structural compositions of nanoparticles [28]. Green synthesized ZnO NPs from P. caerulea leaf aqueous extract showed maximum zone of inhibition against urinary tract infecting pathogens Klebsiella sp. and Streptococcus sp. [40]. In addition, a maximum zone of inhibition was reported in P. aeruginosa and E. coli by C. neilgherrensis leaf aqueous extract mediated synthesized ZnO NPs. It indicates the biocidal capability of ZnO NPs. Moreover, green synthesized ZnO NPs were performed as an excellent antimicrobial agent against Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger [46]. Hence, the ZnO NPs was formulated via green way using C. limon seed aqueous extract may possibly use as an antimicrobial agent.

Acute toxicity of ZnO NPs to zebrafish with increased concentrations of ZnO NPs was displayed in Figure 9, which was demonstrated by dose dependency. As displayed in Figure 9, no mortality was produced for 100 mg/L concentration of ZnO NPs at 24 h exposure and as in the control group (24–96 h). 100% mortality was found at 500 mg/L concentration of ZnO NPs and calculated 96 h LC50 of 153.8 mg/L with a 95% confidence limit ranging from 70.62 to 214.18 mg/L. The median LC50values are calculated at diverse times of exposure to ZnO NPs presented in Table 2. A higher concentration of ZnO NPs toxic action was relatively signs of stress, which caused irregular movement and increased swimming.

Figure 9

Mortality percentage in zebrafish exposed to different concentrations of ZnO NPs at various time periods.

Table 2

Median LC50 of ZnO NPs on Zebrafish with a diverse time of exposure.

Exposure (hr)	LC50 (mg/L)	95% confidence limit (mg/L)		Calculated chi square (X2)
		Lower	Upper
24	387.432	230.806	479.231	0.514a
48	318.399	192.481	399.412	0.589a
72	225.340	128.133	358.719	2.715a
96	153.865	70.629	214.186	2.503a

level of significance is greater than 0.5.

This finding was consistent with a previous report that suggested that Zebrafish is more sensitive to ZnO NPs than zinc sulfate based on their concentration [47]. Boran and Ulutas [48] reported that the larval Zebrafish exhibited at 21.37 ± 1.81 and 4.66 ± 0.11 mg/L for 96 h LC50 of nZnO and Zn(II), respectively. ZnO NPs toxicity is generally recognized to its size and high surface area [49]. Moreover, nanoparticles are considerably more toxic than their bulk counterparts owing to their accumulation in tissues [50]. As compared to other studies, ZnO NPs found to be less toxic [51, 52].

Zebrafish caudal fins development

Zebrafishes are treated with various concentrations of ZnO NPs like 30.76, 3.07 and 1.53 mg/L. The caudal fins were partially amputated and allowed to develop for 12 dpa, and the measurement was taken at every 2 dpa interval (Figure 10B, C, D, E, F, G, H, I, J, and K). Displayed results are compared with control (Figure 10A). Simultaneously, the developmental activity of zinc acetate and C. limon seed aqueous extract were also individually determined as described above. The results showed that the treatment group attained 95% caudal fin development without mortality for 12 dpa. The average fin length rate (%) was calculated by the formula stated in Eq. (2), followed by ZnO NPs, Zinc acetate and C. limon seed aqueous extract exposure [53].where i is the length of the amputated fin, ii is the length of the injured fin at days post-amputation subtracted from the length of uninjured fin and n is the number of fish.

Figure 10

Amputated caudal fins display position-dependent rates of development. (A) Adult zebrafish before amputated, (B to F) images denoted control (without treated) fish caudal fin development on 0th to 12 dpa and (G–K) images denoted ZnO NPs treated fish caudal fin development on 0th to 12 dpa. The magnification is 15×.

Development of caudal fin attained 68.66% at 12 dpa, which increased while compared to controls (Figure 11A, B, C, and D). The cumulative individual development (Figure 11B and C) of control fish in zinc acetate treatment is 56.66% (12 dpa), and aqueous seed extract of C. limon expressed at 55.33% (12 dpa). The average fin length rate was attained at 92% (12 dpa) with 30.76 mg/L (1/5 concentration of LC50) of ZnO NPs exposure, which showed an increase with other treatment groups (Figure 11D). Hence, caudal fin development significantly increased by seed aqueous extract of C. limon mediated synthesized ZnO NPs compare to control (P < 0.05). Interestingly, ZnO NPs induced developmental malformation during the treatment.

Figure 11

Assessment of caudal fins development through dosage depending on ZnO NPs exposure for 12 dpa. (A) 1/5 (30.76 mg/L), (B) 1/50 (3.07 mg/L), (C) 1/100 (1.53 mg/L) concentrations of LC50 for 96 h and (D) cumulative development of fin for diverse concentration of ZnO NPs. Error bars represent the standard deviation of the mean values from triplicate analysis. Asterisk denoted significant differences (p < 0.05) between test concentrations.

Zebrafish caudal fins are used for regeneration because it contains numerous focal points such as easily accessible, fin amputation does not affect the survival, rapid growth and simple structure [54]. Development of zebrafish caudal fin consisted of several specific stages, including epithelial cap formation, dedifferentiation of mesenchymal cells, and differentiation of cells forming a blastemal, which leads to the formation of epithelium, nerves and vessels [55, 56, 57]. The development of the caudal fin in Zebrafish was projected more in different studies among vertebrates. Development of amputated caudal fin in adult zebrafish required 14 days [58]. Initially, more than two dpa is required for epidermal cover development. From 2–4 dpa, the mesenchymal cells were grown in the direction of the epidermis. Originations of a blastema by a proliferation of mesenchymal cells happened during the period of 4–8 dpa. Blastemal tissue separation was made at 8–12 dpa, which required for reaching the full shape of the caudal fin [57]. Several genes, including wnt, hox, fgf, fgfr, and msx have a significant role in regulating developmental mechanisms [59]. Majorly, Fgf signaling contributed to the blastema formation in Zebrafish caudal fin region [57]. The capability of regeneration is associated with amputation through the msx signal pathway. Additionally, ontogenetic factors were evidence for epimorphic regeneration in teleosts and amphibians [60]. Furthermore, the regeneration of the dermal skeleton and re-expression of sonic hedgehog (shh) signaling pathway genes stimulated the zebrafish caudal fin development [61]. Thus, the fin development needed for fin growth may be regulated by the developmental checkpoint.

Metallothionein protein (MT) is an essential factor for controlling the diverse concentration of Zn ions in cells by releasing or binding Zn [48]. Moreover, MT protein is expressed by toxic metal ions such as Ag and Cd as well as Cu and Zn. Noteworthy, Zn is an essential trace element for helping the organal development when needed [47]. Finding results showed the expression of MT protein in Zebrafish indirectly. The induction of MT gene is partially supported for the wnt and fgf-signaling pathways. Nano-sized (52.65 ± 0.5 nm) spherical-shaped ZnO NPs have induced the development of the caudal fin. Therefore, the result estimated that minor contrasts in LC50 related to species (larva and adult) [48]. Therefore, these micronutrient particles, such as metal oxide-containing nanoparticles can induce the organal development.

Conclusion

The current study reported the green synthesis of ZnO NPs from aqueous seed extract of Citrus limon. An advantage of the current study is eco-friendly, cost modest synthetic way and large-scale production of ZnO NPs. The UV-Vis spectrum, FTIR, XRD, DLS and SEM analysis denoted the redaction of zinc ions, quantifying functional groups, crystallinity nature, size, and shape of the nanoparticles. From the results, it could be concluded that biogenic synthesized zinc oxide nanoparticles have excellent antibacterial activity against targeted bacterial species. When increasing the concentration of ZnO NPs showing clear growth inhibition of all bacteria tested, but this inhibition is different due to the concentrations of ZnO NPs and the nature of the tested bacteria. Metal nanoparticles induced the expression of MT proteins, and overexpression of protein may be causing the development of Zebrafish caudal fin. Furthermore, a trace amount of Zn helps Zebrafish caudal fin development. The Zebrafish model provides unique advantages for further understanding of ZnO NPs in tissue development. Hence, the current study suggested that phytogenic ZnO NPs act as an excellent barrier for bacterial pathogens during the zebrafish caudal fin development. Also, the fish consuming the C. limon seed extract mediated synthesized ZnO NPs, or its derivatives help tissue development. It was established that the green synthesized metal oxide nanoparticles were perhaps used for therapeutic purposes.

Declarations

Author contribution statement

Selvakumar Sakthivel: Conceived and designed the experiments; Performed the experiments; Wrote the paper.

Anand Raj Dhanapal: Analyzed and interpreted the data; Contributed reagents; Wrote and validated the results throughout the manuscript.

Lilly Pushpa Paulraj: Analysis tools or data; Wrote the paper.

Annadurai Gurusamy: Conceived and designed the experiments; Wrote the paper.

Baskar Venkidasamy: Analyzed and interpreted the data; Contributed reagents; Wrote the paper.

Muthu Thiruvengadam: Materials, analysis tools or data; Wrote the paper.

Rajakumar Govindasamy: Materials, analysis tools or data; Wrote the paper.

Mohammad Ali Shariati: Analysis tools or data; Wrote the paper.

Abdelhakim Bouyahya: Materials, analysis tools or data; Wrote the paper.

Gokhan Zengin: Analyzed and interpreted the data; Wrote the paper.

Mohammad Mehedi Hasan: Analyzed and interpreted the data; Wrote the paper.

Pavel Burkov: Analysis tools or data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.