Biogenesis of Zinc Oxide Nanoparticles Using Lawsonia Inermis Leafextract and Their Mosquitocidal, Antimicrobial, Anticancer, Andmoderate Toxic Side Effects on Predatory Copepods and Fish

Pandiyan Amuthavalli Bharathiar University Jiang-Shiou Hwang (  jshwang@mail.ntou.edu.tw ) National Taiwan Ocean University Hans-Uwe Dahms Kaohsiung Medical University Lan Wang Shanxi University Jagannathan Anitha Bharathiar University Murugan Vasanthakumaran Kongunadu Arts and Science College Arumugam Dhanesh Gandhi Thiruvalluvar University Kadarkarai Murugan Bharathiar University Jayapal Subramaniam Bharathiar University Manickam Paulpandi Bharathiar University Balamurugan Chandramohan Bharathiar University Shivangi Singh Kaohsiung Medical University


Introduction
Worldwide, mosquitoes (Diptera: Culicidae) are threatening human individual and public health as vectors of parasites and pathogens 1 .
Mosquitoes provide a substantial threat when compared to other disease-transmitting insects as they spread disease causing pathogens. Anopheles stephensi is a vector which transmits the globally most threatful contagious disease malaria 2 . The most serious health problem caused by malaria affects 214 million cases in 2015 3,4 . The appearance of multi-drug resistance of the disease causing protists belonging to Plasmodium spp. possess a major obstacle to successful chemoprophylaxis and chemotherapy of this disease 5 .
Then exposure to acoustic vibrations within determined frequency bands leads to dorsal tracheal trunk (DDTs) wall rupture in mosquitoes, resulting in the discharge of gases into the body cavity, that block larval development, increase mortality rates or rendering adult mosquitoes ightless. Phyto-constituents that are naturally synthesized by medicinal plants can be utilized for ecofriendly applications in vector control 6 . Nanocomplexes using phyto-and microorganisms will minimize the side effects caused by synthetic drugs and also the toxicity to target organisms 7 in an environmentally friendly manner 8 . Drug resistance provides the main drawback in executing chemotherapy in cancer 9 . The development of e cient versatile drugs against both mosquito-borne diseases and cancer were highlighted 10 .
In spite of increasing evidence for the outstanding mosquitocidal potency of phyto-synthesized nanocompounds and their toxicity against natural predators of mosquitoes, their effects have rarely been studied with respect to sub-lethal doses on predation 11 . Water predators, including juvenile instars of dragon ies, tadpoles, beetles, shes, and crustaceans 12 . The impact of predatory animals on water bodies is important because such predators were introduced throughout many warm regions of the world for mosquito control 13 .
Metabolities such as carbonyl, hydroxyl and functional groups of amines, especially the OH-group of avonoids that react with metal ions leads to reduced size of metal ions used for nanoparticle synthesis. Zinc oxide nanoparticles can show several morphological varieties such as nano owers, nanosheets and even nanorods which were shown to successfully inhibit the growth and development of the bacteria Escherichia coli, Staphylococcus aureus, and Klebsiella pneumonia. Plant derived nanomaterials exhibit various shapes and sizes when compared to nanoparticles produced by other organisms such as algae, fungi, and bacteria. There is no published report that evaluates zincoxide nanocomplex toxicity against the non-target predatory e ciency of Mesocyclops aspericornis. Traditional medicine records the plant Lawsonia inermis L. as a potential natural dye with numerous medicinal applications. Nanoparticles that are synthesized from the plant materials showed several applications in elds such as medicine, agriculture and the food industry. ZnO NPs (zinc oxide nanoparticles which exhibit remarkable properties such as binding energy (large) and band gap (wide). These were the properties which made zinc oxide nanoparticles biocompatible, safe, and non-toxic. Zinc oxide nanoparticles are known to have different applications, such as in medical and biological industries, optoelectronics, as antiplatelet agents, anti-in ammatory, anti-angiogenesis, especially as auspicious anti-cancer agents, providing catalytic and semiconductor properties. Zincoxide nanoparticles are known to show insecticidal properties. ZnO NPs were synthesized from L. inermis in the present study are also used to resolve the following issues: a) the lethal effect against the A. stephensi malaria vector, to nd out the larvicidal and pupicidal effect; b) the predatory e ciency of the Gambussia a nis shes and small crustacean Mesocyclops aspericornis against younger instars of Anopheles larvae in ZnO NPs contaminated water environments; c) antimicrobial potential of nano-formulations against pathogenic microorganism and; d) in-vitro cytotoxicity against the cancer Hep-G2 cells.

Materials And Methods
Several aspects of below M&M are similar to and detailed by our earlier paper Jaganathana et al. (2016) 14 . All methods were performed in accordance with the relevant guidelines and regulations of international law and the IAECC of Bharathiar University (see below statement).

Anopheles stephensi cultivation
Details are provided in our earlier paper Jaganathana et al. (2016) 14 . Anopheles stephensi eggs were collected from a local breeding habitat, a fresh water tank in Kalveerampalaiyam, Coimbatore (Tamil Nadu, India) and laboratory reared for egg hatching (80% relative humidity and 27 o C and a photoperiod of 14:10 h (L/D). Emerging larvae and pupae were used for toxicological testing as outlined below.

Leaves collection and processing
Plant sample (Lawsonia inermis) was collected in Maruthamalai hill, Coimbatore, Tamil Nadu, India). It was authenticated at the Botanical Survey of India, whose voucher specimens number was BSI/SRC/5/23/2019/Tech and deposited at the Department Zoology, Bharathiar University. The leaves were rinsed by tap water and dried at room temperature (28 ± 2 °C), and nely powdered. Powdered leave material (10 g) were boiled with 100 mL of double distilled water for further nanocomposite preparation.

Synthesis of ZnO NPs
The leaf broth was combined with 1-mM ZnNO 3 (Sigma-Aldrich, India) solution and was stirred at room temperature (35 ± 2 °C) for 1 h. A brown-yellowish precipitate was heated under stirring at 60 °C for 4 h and further, the solution was continuously stirred at room temperature for 24 h. The precipitate was dried at 100 °C. The obtained sample was ground gently using a pestle and mortar and nally, the sample was calcined at 400 °C for 3 h.

Characterization
The synthesized ZnO NPs samples were analyzed by a UV-vis diffuse re ectance spectroscopy (UV-vis DRS) at a wavelength range of 200-700 nm, using a UV-vis spectrophotometer (Shimadzu -UV 2600, Tokyo, Japan). Fourier transform infrared spectroscopy (FT-IR) analysis was carried out using a spectrum 65 FT-IR spectrometer (PerkinElmer Co., Ltd., Massachusetts, USA). ZnO NPs were used for scanning electron microscopy (FEI QUANTA-200; SEM), energy-dispersive X-ray spectroscopy (EDX) 15 . XRD pattern were recorded using Cu Kα radiation at a wavelength of 1.54060 Å, with a nickel monochromator in the 2θ range from 10 to 80° using an analytical X-PERT PRO, diffractometer.

Acute toxicity assessment against A. stephensi
In the laboratory the larvae and pupae of A. stephensi (I, II, III, or IV instars) were exposed for 24 h at concentrations of 20, 40, 60, 80 and 100 ppm of L. inermis broth and 2, 4, 6, 8 and 10 ppm of ZnO NPs in triplicates. Dechlorinated water without acetone served as a control. Predation assays under standard laboratory conditions Details are provided in our earlier paper Jaganathana et al. (2016) 14 . Here, predation e ciency of G. a nis adults was assessed against A. stephensi (I -IV) instar larvae. In each trial mosquitoes, n = 200 larvae were introduced with one G. a nis adult in a 2-L glass arena lled with dechlorinated water and ve replicates were conducted. Control arenas contained dechlorinated water only. All arenas were checked every 24 h for 5 days and the number of prey missing that were assumed to be eaten by mosquito sh was recorded. After each check, the missing mosquito larvae were replaced with new ones. Predation e ciency was calculated by (Eq. 2). Predation e ciency = (Number of consumed mosquitoes/ total number of mosquitoes) × 100 (Eq. 2). We con rm that the experimental protocol was approved by the here named institutional committee: Institutional Animal Ethical Clearance Certi cate (IAEC) of the Bharathiar University, Coimbatore -641046 (see appended original document signed by IAEC Chairman Prof. V. Vijaya Padma).
Predatory e ciency of G. a nis species after treatment with synthesized ZnO NPs Details are provided in our earlier paper Jaganathana et al. (2016) 14 . Predation assays in contaminated aquatic environments: the predation e ciency of G. a nis adults was assessed against I-IV instar larvae of A. stephensi, after a mosquitocidal treatment with standard and green-synthesized ZnO NPs. For both mosquitoe species, n = 200 I-IV instar larvae were introduced with one G. a nis adult in a 2 L glass tank lled with dechlorinated water plus 1 mL of the desired concentration of NPs (i.e. 5 ppm of ZnO NPs, 1/3 of the LC 50 calculated against I instar mosquito larvae) 16 . For each mosquito species, three replicates were used. Control was dechlorinated water only. All experimental tanks were checked every 24 h at day and night time and the number of prey eaten by mosquito shes was recorded. After each checking, the predated mosquito larvae were replaced by new ones. Predation e ciency was calculated using the above-mentioned formula (Eq. 2).

Predation of Mesocyclops aspericornis against malaria mosquitoes
Details are provided in our earlier paper Jaganathana et al. (2016) 14 . In this experiment, the predation e ciency of Mesocyclops aspericornis adults was assessed against A. stephensi larvae. For each instar, n = 100 mosquitoes were introduced, with 10 copepods, in a 500-mL glass beaker containing 250 mL of dechlorinated water. Mosquito larvae were replaced daily by new ones. For each mosquito instar, ve replicates were conducted. Control was 250 mL of dechlorinated water without copepods. All beakers were checked after 1, 2, 3, 4 and 5 days and the number of prey consumed by copepods was recorded. Predatory e ciency was calculated using the following formula (Eq. Here, we evaluated the predation e ciency of M. aspericornis adults against A. stephensi larvae, after a mosquitocidal treatment with synthesized ZnO NPs. For each instar, n = 100 mosquitoes were introduced with 10 copepods in a 500-mL glass beaker lled with dechlorinated water treated with synthesized ZnO NPs (i.e. for both species, 1/3 of the LC 50 calculated against rst instar larvae).
Mosquito larvae were replaced daily with new ones. For each mosquito instar, ve replicates were conducted. Consumed by copepods was recorded. Predatory e ciency was calculated using the above-mentioned formula (Eq. 2). Control was dechlorinated water without copepods. All beakers were checked after 1,2,3,4 and 5 days and the number of prey consumed by copepods was recorded.

Antimicrobial inhibitory assay
All were provided by the Microbial Type Culture Collection and Gene Bank Institute of Microbial Technology Sector 39-A, Chandigarh-160,036 (India). Antimicrobial activity of Li-ZnO NPs was tested against the selected bacteria (Escherichia coli and Bacillus subtilus) and the fungal strains (Alternaria alternate and Aspergillus avus) using the disk diffusion method 17,18 . The standard inoculum suspension (10 6 CFU/ml) was streaked over the surface of the media using a sterile cotton swab to ensure con uent growth of the organisms. 10 µL of synthesized ZnO NPs was diluted with two volumes of 5% dimethyl sulfoxide (DMSO), impregnated on lter paper disks that were placed on the surface of the agar plates. Petri plates were kept for incubation at room temperature (27 °C ± 2) for 24 h. Inhibition was measured in millimeters using a photomicroscope (Leica ES2, Leipzig, Germany) and compared with standard positive controls, i.e. tetracycline (for bacteria) and uconazole (for fungi).
Cytotoxicity on liver cancer cell lines Human liver cancer cell line (Hep-G2) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cell lines were cultured and incubated according to the procedure given and used for further toxicity studies.

MTT assay
After 48 h of incubation, 15 µL of MTT (5 mg/ mL) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4 h. The medium with MTT was then icked off and the formed formazan crystals were solubilized in 100 µL of DMSO and then measured at 570 nm using a microplate reader. Following the below mentioned formula the cell viability will be calculated (Eq. 3) Percentage Cell Viability (%) = (Mean experimental call absorbance (A620) / (Mean control call absorbance (A620)) × 100 (Eq. 3) Data analysis SPSS 16.0 version was used for all analyses. The average larval and pupal mortality data were subjected to probit analysis for calculating LC 50 , LC 90 , and other statistics at 95% con dence limits, and chi-square values were calculated using the SPSS Statistical software package 13.0 version. Results with P < 0.05 were considered as statistically signi cant.

Results
As shown in Fig. 1, the absorption spectra obtained for biosynthesized ZnONP were at 364 nm when investigated under UV-spectroscopy.
This absorption was con rmed by the formation ZnO NPs. When L. inermis extract was analyzed by FTIR, its spectrum showed several vibration peaks at 3575, 3272, 2073, 1636 and 489 cm −1 . Thereafter, synthesized ZnO NPs spectrum showed vibration peaks at 1473, 878, 668, and 575 cm −1 (Fig. 2). The nanostructure of synthesized ZnO NPs was seen in SEM which acquired 5 nm given in Fig. 3. EDX analysis of synthesized ZnO NPs showed dual peaks which were situated between 1.2 and 8.6 keV, where it is for zinc characteristic lines K and L shell, as shown in Fig. 4 in that order as shown in Fig. 5. Table 1 Table 2 shows the toxicity against larva and pupa of synthesized ZnO NPs. The toxicity was found to be higher at doses of 2, 4, 6, 8 and 10 ppm whose LC 50 were found to be 5.494 (I), 6.801 (II), 9.336 (III), 10.736 (IV), and 12.710% (pupae).
Food feeding competence of G. a nis shes were calculated, against I to IV instar larvae of A. stephensi. Very small doses of synthesized ZnO NPs were treated with water under standard laboratory conditions where the shes were introduced, their predation rate subsequent after 24 h was 45% (I) to 25.83 (IV). The food utilization of G. a nis was 71.33 (I) to 34.25% (IV), respectively (Table 3). M. aspericornis adults predate on A. stephensi young larval instars. The predatory e ciency per copepod per day was 4.06, 2.87, 1.79 and 1.08 larvae (I, II, III, and IV, respectively). During post-treatment with sub-lethal doses of synthesized ZnO NPs, the predation e ciency was boosted to 4.06, 2.87, 1.79 and 1.67 larvae (I, II, III, and IV, respectively) ( Table 4).

Antimicrobial effects of synthesized ZnO NPs against selected pathogens like E. coli and B. subtilis (bacteria) and fungal species like
Alternaria alternate, and Aspergillus avus were evaluated in the present investigation. Synthesized ZnO NPs were highly effective in inhibiting the growth of E. coli (13.3 mm) which were then followed by B. subtilus (8.4 mm) respectively given in Table 5 and Fig. 6. Similarly, a maximum zone of inhibition was achieved for the fungus Alternaria alternate (11.5 mm), followed by Aspergillus avus (7.8 mm). Synthesized ZnO NPs treated with Hep-G2 cell lines were tested to ensure its cell viability after 24 h. The cytotoxicity on Hep-G2 cell lines mediated by ZnO NPs exhibited a dose-dependent relationship as shown in Fig. 7. Here, IC 50 values were found to be 21.63 µg/mL (R 2 =0.942; P<0.001), respectively, and its morphology and cell inhibition was shown in Fig. 8.

Discussion
As shown in Fig. 1, a UV-vis DRS analysis of synthesized ZnO NPs showed an absorption peak at 364 nm, revealing a blue shift at a band gap value of 3.40 eV. The direct band gap of the synthesized ZnO NRs was evaluated using Eq. (4); Eg = 1240/λ max Eq. (4), where Eg provides the band gap (eV) and λ max is the wavelength (nm) of the absorption edge within the spectrum. As a red shift indicates an increase in particle size and a blue shift indicates a decrease in particle size. Generally, green synthesized ZnO NPs show an absorption peak at 375 nm and a band gap at 3.30 eV. The higher band gap ZnO NPs was suitable for biological application, which highly promoted the generation of ROS in NPs. In this study, synthesized ZnO NPs exhibited a blue shift of an absorption peak at 364 nm higher ban gap value at 3.31 eV. The same trend was observed with previous reports 19,20,21 .
FT-IR spectrum of L. inermis extract exhibited various stretching and transmittance peaks corresponding to various functional groups including alkyl halides, amine, alkynes, and alcohols. Here, peptide bonds of proteins correspond to N-H and C = O stretching frequencies exhibiting a peak at 3575 cm − 1 . A similar banding pattern at 3402 cm − 1 was reported by Natarajan et al. 22 and or alcohols and/or phenols, as well as aliphatic amines support the presence of polyphenols at 1027 ~ 1092 cm − 1 for C-N vibration 23 . The peaks at 802 cm − 1 , stay for the C-H stretching frequencies within the free catechins in the aromatic ring 24  NPs was related to plant species and bioactive compounds 26,27 . EDX spectra indicates O and Zn elements having energy levels of 0.5, 1.2, and 8.6 keV, respectively. Moreover, two additional peaks of Cl and Ag were found as well. The major peak of the sample represented Ag, which was due to the sputter coating process with silver (Ag). XRD analysis of synthesized ZnO NPs demonstrated a well-crystallized structure. The three distinctly high diffraction peaks at 2θ = 31.75, 34.40, and 36.25° corresponded to the planes of (100), (002), and (101) 28,29,30 . This was con rmed by the hexagonal structure of ZnO. This was also matching well with the JCPDS card no. 36-1451.
The toxicity on larval and pupal stages of A. stephensi caused by ZnO NPs might be due to the reduction of nanoparticles caused by the plant extract. The tiny NP spheres penetrate cells and interfere with physiological processes such as molting 31 . The present study corroborates with the ndings of Gandhi and Madhusudhan 32 , who postulated the e ciency of Momordica charantia leafes reducing ZnO NPs against C. quinquefasciatus and A. stephensi. Murugan et al. 33 found that Sargassum wightii-synthesized ZnO NPs were highly effective in killing the larvae and pupae of the malaria vector A. stephensi. In contrast, silver nanoparticles were found to be e cient at lower doses against the malarial vector A. stephensi -however, only against its young instars. Equally high toxic effects against larvae and pupae of A. stephensi. were provided by leaf extracts from both S. occidentalis and Ocimum basilicum. A dose dependent effect was in agreement with previous evidences from other plant extracts 34 . Our results clearly indicate that ZnO NPs affect pathogen growth by cell wall disruption. ZnO NPs may reduce surface hydrophobicity of bacterial cells and its oxidative stress-resistance genes were downregulated, causing nally degradation and the death of cells 35 . ZnO NPs has freshly achieved individual notices concerning possible electronic applications due to its unique optical, electrical, and chemical properties 36 . Its heterogeneous catalytic property might be the cause for bacterial growth inhibition through different mechanisms as known from conventional antibiotics 37,38 . Previous reports of 70-74 postulates higher MIC of ZnO NPs against E. coli, Listeria monocytogenes, Salmonella typhi, and S. aureus provide supportive evidence for the present microbial evaluation. Most of the fungal strains like F. solani, A. alternate, and A. avus had shown antifungal drug resistance 39 . Hence it was in need to formulate nanoparticles with potent chemical and structural nature to overcome such drug resistance 40 .
Similarly, Chobu et al. 41 demonstrated that Anopheles gambiae was less e ciently predated upon by the mosquito sh G. a nis.
Murugan et al. 42 noted that the teleost guppy sh, P. reticulata actively predates on the larvae of A. stephensi. Subramaniam et al. 43 mentioned another study showing that green synthesized Ag NPs with Mimusops elengi did not affect predation rates of the mosquito sh G. a nis on the mosquitoes A. albopictus and A. stephensi. In a paper by Murugan et al. 44 the predatory e ciency of a single copepod species belonging to M. aspericornis was 8.0, 6.3, 0.8, and 0.2 larvae (instar I, II, III, and IV, respectively) per day after a post-treatment with seaweed-synthesized silver nanoparticles. Mahesh Kumar et al. 45 studied the predatory e ciency of a single adult copepod of M. thermocyclopoides being 6.5, 4.6, 0.76, and 0.14 C. quinquefasciatus larvae per day (instar I, II, III, and IV, respectively). The predatory e ciency was enhanced to 8.7, 5.9, 1.2, and 0.36 larvae day (instar I, II, III, and IV, respectively) after treatment with Solanum xanthocarpum fruit extract.
ZnO NPs acted as anticancer agents with minimum dosage as con rmed by the IC 50 values. Silver nanoparticles and earthworm combinations mediated anti-proliferative activity at increasing concentration as revealed by DNA analysis of Hep-G2 cells 18 . Similarly 46 , Amorphophallus paeoniifolius peels mediated ZnO NPs induced cancer cell apoptosis at increasing concentration also in accordance with our present ndings.

Conclusion
The emergence of multi-drug resistance of vectors and microbes provide a major obstacle for the successful control of mosquitoes, as well as chemoprophylaxis and chemotherapy of diseases. Hence, there is an urgent need to develop a novel, rapid synthesis, and ecofriendly tool in mosquito control. Physicochemically characterized ZnO NPs reduced by leaves of Lawsonia inermis were shown to have multipotency against of A. stephensi larvae and pupae, Hep-G2 cancer cell lines, and selected pathogens including bacteria and fungi. Green synthesis of ZnO NPs provides a potential candidate in controlling young instars of blood feeding malaria vectors, inducing apoptosis in a dose-dependent manner, and inhibiting microbial growth. Therefore, this was an effective regularized and eco-friendly approach that can be used as one of the ways to decrease pathogenic microbial and mosquito populations.

DECLARATION OF COMPETING INTEREST
The Authors declare no con ict of interest.  Predation rates are means ± SD of 3 replicates (1 fish vs. 400 mosquitoes per replicate).

Tables
Control was clean water without G. a nis fishes.
Within the column, means followed by the same letter are not significantly different (weighted generalized linear model, P < 0.05) Table 4. Predation e ciency of Mesocyclops aspericornis on An. stephensi larvae in standard conditions and post-treatment of ZnO NPs. Predation rates are means ± SD of four replicates (1 G. a nis sh vs. 100 mosquitoes per replication) No predation in control (i.e. clean water without G. a nis sh) Within each column, means followed by the same letter are not signi cantly different (P < 0.05)