Green Synthesized ZnO Nanoparticles (ZnO-NPs) and Its Acaricidal Action against Rhipicephalus (B). microplus

Parasitic diseases in particular ectoparasitic infestation is a global problem and considered as major obstacle in the health and production performance of animal industries. Among the ectoparasites, ticks and tick-borne diseases (TTBDs) continue to be the major constraint globally on profitable livestock production (Mondal et al., 2013). TTBDs were ranked high globally in terms of their impact on the livelihood of resource-poor farming communities in developing countries including India (Minjauw and McLeod, 2000). Calculated costs for control of TTBDs affecting Indian livestock was 498.7 million US $ per annum (Minjauw and McLeod, 2003). Rhipicephalus (Boophilus) microplus, is the most prevalent cattle tick in various agro climatic zones of India and infesting all age groups of cattle, horse, sheep, goat, deer (Ghosh et al., 2007), mithun (Rajkhowa et al., 2005), yak (Mondal, 2006) and wild herbivores (Singh et al., 1978).

At present, tick control is predominantly based on large-scale repeated use of synthetic acaricides, viz. cypermethrin, deltamethrin, fenvalerate, diazinon, amitraz, flumethrin and ivermectin. Application of chemical acaricides has had limited efficacy in reducing tick infestations and is often accompanied by serious drawbacks, including the development of acaricide resistant ticks, environmental contamination and residual contamination of acaricides in milk and meat (Kumar et al., 2020).  As per the report published by FAO, the tick population in India has developed resistance against all commonly available acaricides and there have been only limited research activities to develop newer classes of acaricides due to the enormous costs involved (FAO, 2004). Nanotechnology has enormous potential in livestock sector various nanoparticles have been prepared for diagnostic and treatment purpose. Nowadays plant mediated nanoparticles synthesis has attracted attention of researchers globally.

Annona squamosa is a multipurpose tree with edible fruits and is of an enormous source of medicinal and industrial products (Zahid et al., 2018). Aqueous extract of A. squamosa seed extract with 8% concentration revealed lethal to 70.8% tick population by 24 h of treatment (Magadum  et al., 2009). Metal nanoparticles such as Zinc Oxide, Copper, Silver, Nickel have been tested for their acaricidal efficacy against R. (B). microplus (Kirthi et al., 2011; Ramyadevi et al., 2011; Santoshkumar et al., 2012; Rajkumar et al., 2013). ZnO nanoparticles showed potent acaricidal activity against Hyalomma spp (Norouzi et al., 2019). ZnO NPS could be used for pest management for stored product pest (Hamdy et al., 2023). There are limited studies available on the use of A. squamosa as an acaricide to control R (B). microplus. In addition to this, synthesis of ZnO-NPs using the seed extract of A. squamosa has not been reported till date. In the present study, an attempt was made for synthesis and characterization of A. squamosa seed based ZnO-NPs to develop potent acaricide against R. (B) microplus without hampering the normal body system of the host animal.

Green synthesis of A. squmosa based ZnO-NPs

The work was conducted at medicine division of ICAR-IVRI, UP during 2018-2019. A. squamosa seed aqueous extract based ZnO-NPs were prepared by the method described earlier with some modifications (Banumathi et al., 2016). Briefly, zinc acetate dehydrate was added to 50 ml of distilled water under constant stirring using a magnetic stirrer. Eight per cent (8%) of aqueous extract of A. squamosa seed was introduced into the above solution after 10 minutes of stirring. The solution was kept under vigorous stirring for 6 hours thereafter; the synthesized nanoparticles were collected by centrifugation at 12000 rpm for 45 minutes. The pellet was subjected to lyophilization and the product was preserved in air tight container for further use.

Characterization of green synthesized ZnO-NPs   

Scanning electron microscopy (SEM) Analysis

Scanning electron microscope was used to study the morphology of nanoparticles. Drop of sample was gold coated with JFC-1600 auto fine sputter gold coater. The coated sample was viewed under SEM (JEOL JSM-6610LV) and imaging was performed.

Zeta analysis

Zeta potential of the ZnO-NPs was obtained with the aid of zetasizer.

X-ray diffraction (XRD) analysis

XRD was used to analyze the crystalline pattern of ZnO-NPs. ZnO-NPs were coated on XRD grid and the measurements were recorded at an acceleration voltage of 40 kV and current of 30 mA.

Fourier transform infrared spectroscopy (FT-IR) analysis

The FTIR spectrum was recorded in the wave numbers ranging from 400 to 4000 cm^-1 using FTIR spectroscopy.

U.V. visible spectroscopic analysis

The optical properties of ZnO-NPs were measured by UV-spectrophotometer. The study was carried out for 6 hours at room temperature and the absorption was measured at different wavelength starting from 300 nm to 800 nm using A. squamosa seed extract as reference.

Acaricidal activity of green synthesized ZnO-NPsby adult tick immersion test

Fully engorged adult female R. (B). microplus ticks were collected from infested cattle, washed with distilled water and dried using clean soft tissue paper. Adult tick immersion test (ATI) was performed as per the protocol described earlier with minor modification (Drummond, 1983). Different concentrations of ZnO -NPs were soaked on Whatman filter paper No. 1 in a 90 mm petridish. The control papers were soaked with distilled water. Petridishes were placed at a temperature of 28-30°C and 80% relative humidity (RH) in an incubator. Petridishes were opened at distinct time intervals (24, 48, 72 hours) and the number of live and dead ticks was recorded. Adult mortality was determined by using Abbott’s formula.

                                                      

In vitro population limiting properties of green synthesized ZnO-NPs

The surviving ticks from the ATI test were weighed individually and kept in a tick rearing tube for oviposition. When oviposition ceased, the eggs laid in each tube were weighed separately and recorded to calculate the reproductive index. The reproductive index (RI), alteration in the egg mass production (DO %), alteration in the weight of adult tick (DR %) and the percent efficacy of the A. squamosa extract and ZnO NPs (E %) of ticks were calculated as follows:

 
                                                               
 
                                                              

PMTV = Mean weight of adult female of treated group
PMTC = Mean weight of adult female of control group
 
 
 
PATV = Mean weight of eggs of treated group
PATC = Mean weight of eggs of control group
E % (Efficacy of drug) = 100 ×[1-(CRT-CRO)]
CRT = Reduction in weight of adult female (PMTV/PMTC)
CRO = Reduction in egg laying capacity (PATV/PATC)
 
Hemolytic activity of green synthesized ZnO NPs

Spectrophotometric method
 
Hemolytic potency of ZnO NPs was assessed by spectrophotometer method (Raghava et al., 1994).  Drug samples were dissolved in 1 ml of PBS and after sonication, two fold serial dilutions were made. Twenty micro liter (μl) of RBC suspension was added to 96 well plates to which 100 μl of each concentration of drug was added. For complete haemolysis, cells were suspended in 100 μl of distilled water (Positive control). For negative control cells were suspended in PBS with pH 7.4. Plate was incubated at 37°C for 90 minutes after that content was transferred to eppendorf  tubes and centrifuged at 3000 rpm for 5 min. Supernatants were directly transferred to new 96 well plates by using multipipette. Optical density of each well was taken at 543 nm by using UV spectrophotometer.
 
                                                                    
 
The per cent hemolysis caused by ZnO NPs at a given concentration was calculated by the following formula
Where H, OD_S, OD₀, OD₁₀₀ are per cent haemolysis, optical density in the presence of ZnO NPs, the presence of drug solubilising buffer and the presence of water, respectively.

Microscopic method
 
Haemolytic potency of ZnO NPs was assessed as described earlier with minor modifications (Simundic et al., 2013). Two fold serial dilutions of the ZnO NPs were prepared in 1 ml of 1X PBS. Ten μl of ZnO NPs were mixed with 50 μl of final RBC suspension and kept at 37°C in incubator. After 24 hr of incubation, blood smears were prepared out of the sample and examined under microscope along with control.

Green synthesis of ZnO-NPs

In our study, A. squamosa seed extract based ZnO-NPs were prepared and subjected to characterization and toxicity studies.

Characterization of green synthesized ZnO-NPs

Scanning electron microscopy (SEM) analysis

SEM (JOEL JSM-6610LV) was used to determine the size and shape of nanoparticles. SEM picture depicting the ovoid structure of nanoparticles is shown in Fig 1.



Minimum size of  ZnO NPs was 250 nm with a maximum of 303 nm and the average of 292.75 nm. The functional performance of nanoparticles depends on size, morphology and physical state (Galindo-Rodriguez et al., 2004). Plectranthus amboinicus leaf extract-assisted biosynthesis of ZnO-NPs revealed rod shaped nanoparticles with a diameter range of 50-180 nm (Fu and Fu, 2015).  Green synthesized ZnO-NPs using leaf extract of Ixora coccinea had spherical shaped nanoparticles with a diameter range of 80-130 nm (Yedurkar et al., 2016).  These variations in the size and shape of nanoparticles are due to the nature of plant, amount of plant extract used, kind of parent zinc salt and application of particular centrifugal speed and time upon nano-structured component.

Zeta analysis

ZnO-NPs were dissolved in distilled water and after sonication (40 volts, 15 min), samples were subjected to zeta analysis (Malvern zeta sizer). Polydispersity index (PDI) and zeta potential of ZnO-NPs were 0.5 and -38.9, respectively (Fig 2).



Zeta potential states the potential stability of synthesized nanoparticles. Zeta potential of ZnO NPs prepared using Myristica fragrans was (-) 22.1 mV (Faisal et al., 2021). If the sample has large negative or positive zeta potential values, particles will repel each other and there will be no aggregation (Hughes et al., 2015). On the other hand, if the sample has small zeta potential values there is more chance for aggregation of particles. Optimum zeta potential value is greater than (+) 30 mV or smaller than (-) 30 mV ((Yedurkar et al., 2016). Obtained zeta potential (-38.9 mV) in the present study indicated the stability of particles in colloids.

X-ray diffraction (XRD) analysis

ZnO-NPs showed the characteristic intense peaks at 2θ of 31.89° and 45.64° due to its crystalline nature. In addition to this, moderately intense peaks at 2q of 34.6° and 56.4° were also observed (Fig 3).



These findings are consistent with previous studies (Fakhari et al., 2019; Unni et al., 2022). Almost similar values were reported (Thirunavukkarasu et al., 2016) for ZnO-NPs prepared by sol gel method with the characteristic intense peaks at 2θ of 31.85°, 34.32°, 36.47°, 47.61°, 56.62°. Likewise, another study also reported 2θ values at 31.84°, 56.7° for green synthesized ZnO-NPs (Yedurkar et al., 2016). This indicated crystalline nature of ZnO-NPs in the present study.
       
Fourier transform infrared spectroscopy (FT-IR) analysis

Possible functional groups present in the synthesized ZnO-NPs were confirmed by FTIR analysis. Peak observed at 3754.73 cm^-1 may be due to-OH stretching and deformation. Similarly, peaks at 1643.05 cm^-1 and 732.81 cm^-1 correspond to zinc oxide stretching and vibration respectively (Fig 4).



Similar results were reported by authors who reported absorption peak at 3398 cm^-1 and OH stretching  at (3517 cm^-1) for ZnO-NPs prepared by sol gel method, respectively (Yedurkar et al., 2016; Thirunavukkarasu et al., 2016). A previous study by Unni et al., (2022) reported FTIR peaks at 3640 cm^-1 (hydroxyl group), 2100 cm^-1 (C𝄘C terminal alkyne), 1739 cm-1 (C-O stretching) and 1490 cm^-1 (C-H bending).  Therefore, this inference confirmed the presence of ZnO-NPs of the present study.

U.V. visible spectroscopic analysis

Optical absorption property of ZnO-NPs was recorded by UV-spectroscopic analysis. ZnO-NPs showed a strong absorbance peak at 300 nm in the entire time span (1-6 hour) which confirmed the stability of synthesized nanoparticles (Fig 5). 



It was in accordance with previous study wherein author synthesized ZnO NPs from leaves of L. leschenaultiana and reported a strong absorbance peak at 383 nm (Banumathi et al., 2016).  So, the present study reconfirmed the stability of nanoparticles. ZnO NPs prepared from green alage showed absorbance peak at 370 nm confirmed successful formation of nanoparticles using L.pruinosum (Naiel et al., 2022).

Acaricidal activity of green synthesized ZnO-NPs by adult tick immersion test

Adult tick immersion test (ATI) (Fig 6) depicted no consistent tick mortality from 2 to 10% of aqueous extract (AE) at 24 hr, 48 hr and 72 hr.



However, significantly (p<0.05) increased tick mortality was observed in both 48 hr and 72 hr as compared to 24 hr study in AE treated group. In contrast, significant reduction in tick mortality with 1% AE of A. squamosa was observed as compared to higher concentrations of 2 to 10% A.E (Table 1).

Contrary to AE of A. squamosa, green synthesized ZnO-NPs depicted inverse concentration dependent tick mortality at 24 hr, 48 hr and 72 hr with 2 to 10% concentrations. However, similar trend was not observed with 1% concentration of ZnO-NPs at any time intervals. Critical analysis of the study revealed consistent tick mortality in different time intervals with 2% concentration of ZnO-NPs (Table 1).



Similarly, green synthesized ZnO NPs exhibited excellent acaricidal activity against all developmental stages of Hyalomma dromedary (Abdel-Ghany, 2022).
 
Evaluation of population limiting property of green synthesized ZnO-NPs

There was a significant reduction in DR% (reduction in adult tick weight) with higher concentration AE as compared to ZnO-NPs. Highest DO% (reduction in egg weight) was observed with 10% AE as compared to ZnO-NPs. There was consistently significant variation in the efficacy (E%) of AE as well as ZnO-NPs from 10% to 2% concentrations. Regarding reproductive index (RI), ticks exposed with ZnO-NPs depicted consistent values at all concentrations as compared to AE. Critical analysis of reproductive index revealed consistent efficacy with the potent acaricidal property of 2% ZnO-NPs even in the next generation (Table 2) and may be a key component to reduce tick load from diverse pasture in the future.


 
Hemolytic assessment by spectrophotometer method

Suspension of RBC treated with two fold serial dilutions of ZnO NPs and % hemolysis estimated by spectrophotometric evaluation revealed positively correlated concentration dependent cytotoxicity at higher concentrations (4% and 8%) which continues to be toxic at further higher concentration (10%). Both 2% and 1% concentrations showed negligible per cent hemolysis. Increased concentration of nanoparticles depicted a positive correlation with cytotoxicity as was evident in Fig 7a.



The study revealed concentration dependent cytotoxicity of ZnO-NPs but not affecting the host beyond the target species at lower concentrations but highly toxic at higher concentrations.
 
Hemolytic assessment by microscopic method

After 24 hr incubation of RBC suspension with two fold serial dilutions of ZnO-NPs revealed, an absence of aggregation as well as other morphological changes of RBC. Morphological changes in RBC of nanoparticles, positive control and negative control and treated groups are shown in Fig 7b. Interaction of nanoparticles with RBC can result in transformation of erythrocyte shape (Suwalsky et al., 2005). Aggregation of RBC was observed at higher concentrations of ZnO-NPs which were synthesized chemically (Simundic et al., 2013; Raguvaran et al., 2015). Absence of aggregation as well as morphological changes in RBC was evident in the present study. Toxicity study of ZnO NPs on mice revealed non-significant changes in hematobiochemical parameters which was also eviden in histopathology of liver and kidney (Abdel-Ghany et al., 2022).

In the present study, potent acaricidal property with consistent efficacy was recorded with 2% ZnO-NPs without any cytotoxicity to the host beyond the target species i.e. tick, revealed present green synthesized ZnO NPs as a promising and potent acaricidal agent for further clinical use on livestock for tick control program.

Authors sincerely thank Director, ICAR-IVRI Bareilly, UP for extending facilities.

There is no conflict of interest among the authors.