Biosynthesis of silver nanoparticles using plant extracts and assessment of anthelmintic activity against Haemonchus contortus

Introduction

In India, along with agriculture, livestock plays a significant role in improving the socioeconomic conditions of farmers. Small ruminants, such as sheep are especially valuable as an additional source of daily income. Sheep are susceptible to many diseases of bacterial, viral, fungal and parasitic origin. Globally, parasitic infections in livestock production pose a serious health challenge that severely limits the productivity of livestock by causing a debilitating impact on animals due to morbidity and mortality of the affected animals. The primary challenge facing by the livestock industry is the rising issue of drug resistance or the expensive nature of widely available anthelmintics, along with their adverse effects outweighing their effectiveness in treating the host (Jegede et al., 2021) [13] . H.contortus stands out as the most harmful gastrointestinal nematode in small ruminants, as it feeds on blood from the sheep's abomasum, resulting in severe anemia and potentially fatal consequences if left untreated. The issue regarding use of commercial anthelmintics extends beyond resistance and the indiscriminate use of these drugs also poses a threat to public health by raising concerns about residues of drugs found in milk and meat (Cabardo and Portugalzia, 2017) [3] . The utilization of medicinal plants holds significant importance in meeting fundamental healthcare requirements within developing nations. All parts of the M. oleifera have medicinal properties so, it is also known as "Miracle tree" (Fatima et al., 2014) [9] and bioactive compounds of M. charantia also have many medicinal properties to treat diseases. The process of synthesizing Ag NPs using plants is environmentally friendly and economical compared to conventional physical and chemical approaches (Moodley et al., 2018) [15] . Nanomedicines deal with the organic application of medicines at nanoscale level with promising results of treating diseases and controlling the illness at cellular level.

Nanotechnology tools like nanomaterials, nano sensors, microfluidics have the capacity to address issues concerning animal health, productivity, reproduction, as well as disease prevention and treatment (Patil et al. 2009) [16] . Nanotechnology holds significant promise in enhancing both diagnosis and treatment methods, particularly in drug delivery, offering innovative solutions in the realm of animal production (Ali et al. 2021) [2] . Nanoparticles improve bioavailability of drugs by enhancing solubility, increases half-life for clearance and targeted drug delivery to specific location in the body, which results in reduction in quantity of the drug required, drug dosage toxicity and protection of nontargeted cells from side effects.

Materials and methods

Fresh leaves of M. oleifera and fruits of M. charantia were procured and recognized by the Department of Botany, Sri Venkateswara University, Tirupati.

Preparationof plant extracts

The plant extract (aqueous) was prepared as per the descriptions reported earlier (Abaduet al., 2021) [1] . Extract obtained was refrigerated at 4 ºC for later use.

Fabrication of silver nanoparticles

Ag NPs were prepared by adding 10 mL of M. oleifera leaf extract and 20 mL of M. charantiafruit extract to 90 mL and 80 mL of 2 mM of silver nitrate solution, respectively. After addition, the solution was stirredfor about 1 hr and thereafter incubated for 24 hoursat room temperature, to complete bioreduction and stabilization for formation of Ag NPs.SynthesizedNPs were initially identified by a colour change from light to dark. After incubation, the solution was centrifuged and the sediment was dried, scraped and stored in a container for further characterization studies.

Characterization of silver nanoparticles 2.3.1 UV-Visible spectroscopic analysis

Ag NPs were identified by their surface plasma resonance peak using UV-Visspectrophotometer (Goel et al., 2020) [11] , scanning the hydrosol sample from 200-800 nm.

Fourier Transformed Infrared Analysis (FTIR)

The potential functional groups involved in formation of Ag NPs was identified by FTIR analysis. The dried sample of Ag NPs was placed over ATR (attenuated total reflection) crystal and pressed with anvil of FTIR and measuredbetween 500-3500 cm -1 wavelength.

Dynamic light scattering (DLS) and zeta potential

This method is used to analyse theparticle size distribution and colloidal stability of Ag NPs.

Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray spectroscopy (EDX)

The surface configuration,size and shape of the nanoparticles was monitored by using SEM; and EDX spectroscopy is used for elemental analysis according to the method narrated by Gandhiraj et al., (2018) [10] .

Egg Hatch Assay (EHA)

Worms were obtained from sheep's abomasum slaughtered at abattoir in Tirupati. Eggs were recovered from the adult female worms as per the techniquedescribed by Kakar et al. (2013) [14] with slight modifications. EHAwas conducted based on the method proceeded by Coles et al. (1992) [4] . Egg suspension (nearly 200 eggs/mL) was treated with test compounds, incubated 48 hrs at room temperature. An inhibition percent of egg hatching for different test compounds was calculated using the following formula (Rabel et al., 1994; Davuluri et al., 2019) [18,6] .

Where a-no. of hatched eggs in control group, b-no. of hatched eggs (larva) in different concentrations.

Adult Motility Test (AMT)

The test was performed as per the procedure described by Eguale and Giday (2009) [9] with slight modifications. Ten live active worms were subjected to various concentrations and the cessation of movement was considered a sign of worm mortality. The data was noted attime interval (0, 1, 2, 4, 6, 8, 10 and 12 hr) and the mortality (%) was evaluated through the formula (Egualeet al., 2007; Rabel et al., 1994) [7,18] . Mortality percent = No. of dead worms / Total no. of worms in petri dish × 100

Statistical analysis

Mean percentages of EHA and AMT at different concentrations were performed by one-way AN0VA. Probit analysis of data of AMT-LC 50 , LC 90 and EHA-IC 50 , IC 90 was done by using the software SPSS software.

Results

Characterization of synthesized Ag NPs 3.1.1 UV-Visible spectroscopy

The maximum absorption peak of M. oleiferamediated Ag NPs and M. charantiamediated Ag NPs was recorded at 361.6 nm at an absorbance of 0.2 Au and 357.6 nm at an absorbance of 0.1 Au, respectively (Figure 1).

Dynamic Light Scattering (DLS) and Zeta potential

Hydrodynamic diameter (HDD) of the Ag NPs of M. oleiferaobserved was 88.1 nm with Polydispersity Index (P.I) of 0.224 and the mean zeta potential value was -41.9 mV and the HDD of M. charantia mediated Ag NPs was observed at 76.1 nm with Polydispersity Index of 0.633 and the mean zeta potential value was 52.7 mV (Figure 2).

Fourier Transformed Infrared (FT-IR) spectroscopy

FT-IR spectrum analysis of M. oleifera leaf extract mediated Ag NPs revealed peaks for functional groups at 3849. 3).

Scanning Electron Microscopy

SEM images showing polydispersed Ag NPs. The shape of the nanoparticles was found to be irregular and truncated (Figure 4).

Energy Dispersive X-ray analysis

In EDX analysis, M. oleifera Ag NPs showeda spike at 3 keV and weight % of silver as 54.2;M. charantiaAg NPs had a spike at 3 keV and weight % of silver as 26.42 (Figure 5).

Anthelmintic activity of synthesized compounds 3.2.1 Egg hatch assay (EHA)

The test compounds exhibited concentration dependent anthelmintic activity (Figure 6a).Egg hatch inhibition of H. contortuswithM. oleifera mediated Ag NPs was 100,92.66, 79.0, 65.33, 53.33 and 35.0% at 4, 3, 2, 1, 0.5 and 0.25 mg/mL; M. charantiamediated Ag NPs was 100, 94.33, 79.0, 64.0, 50.33 and 32.66% at 4, 3, 2, 1, 0.5 and 0.25 mg/mL, respectively. Inhibitory concentration (IC) values of test compounds showed in Table 1.

Adult motility test (AMT)

M. oleiferamediated Ag NPs (4, 3, 2, 1, 0.5 and 0.25 mg/mL) were screened against adult H.contortus. At4 mg/mL, 43.33%motility inhibition occurred within 1 hr, with complete inhibition by 6 hrs. M. charantia mediated Ag NPs (4, 3, 2, 1, 0.5 and 0.25 mg/mL) exhibited 40.66% motility inhibition at 4 mg/mL within 1 hr, with complete inhibition by 6 hrs (Figure 6b). Lethal concentrations (LC) of test compounds in Table 1.

Discussion

Small ruminants, notably sheep and goats, act as a key means of sustenance of millions of rural inhabitants in many developing nations, offering significant contributions through meat, wool, and hides. Their presence contributes to the socioeconomic stability of farming systems. Among nematodes, H. contortus poses a significant threat as a highly pathogenic parasite in small ruminants, frequently resulting in high mortality rates. The prevalent use of anthelmintics at incorrect dosages and increased treatment frequency has frequently resulted in the emergence of anthelmintic resistance (Singh and Gupta, 2010) [21] . One of the most valuable weapons in the battle to conserve susceptibility in nematode population is the ability to detect resistance, as anthelmintic resistance is an important management component to take up control measures (Das and Singh, 2005) [5] . Nanotechnology holds immense promise for transforming the agriculture and livestock industries. Nanotechnology applications such as nanomaterials, microfluidics, and nano sensors offer the potential to animal wellness, performance, reproductive efficiency and disease management (Patil et al. 2009) [16] . Nanotechnology has significant promise in enhancing both diagnosis and treatment methods, particularly in drug delivery, while also offering innovative tools for advancements in animal production (Ali et al. 2021) [2] . Prasad and Elumalai (2011) [17] demonstrated that Moringa oleifera leaf extract mediated Ag NPs exhibited absorption peak at 430-440 nm and (Rashid et al. 2016) [19] reported that Momordica charantia fruit extract mediated Ag NPs exhibited absorption peak at 400 nm, supporting the UV-Vis spectroscopy data in present study.

The DLS and zeta potential of Moringa oleifera mediated Ag NPs and Momordica charantia mediated Ag NPs in present study were observed as 88.1 nm, -41.9 mV and 76.1 nm, 52.7 mV, respectively. The results were in contrast with (Ruman and Kia, 2021) [21] reported that Momordicacharantia mediated Ag NPs showed 17.5±2.1 nm of particle size.

The FTIR results in present study were in comparison with (Moodley et al., 2018) [15] reported that Moringa oleifera mediated Ag NPs exhibited peaks in the range of 3000-3300, 2800-3000, 1626, 1400-1550, 1380-1403 and 1000-1100 cm -1 that are associated with hydroxyl groups in alcohols or phenolic compounds. (Rashid et al. 2017) [20] reported that Momordica charantia mediated Ag NPs showed peaks at 3354.21 cm -1 (N-H for primary amine), 2916.01 cm [15] and (Gandhiraj et al. 2018) [10] . In the present investigation, EDX analysis of synthesized Ag NPs showed a spike at 3 keV. The results were totally coherence with Moodley et al. (2018) [15] and (Gandhiraj et al. 2018) [10] . Silver nanoparticles of M. oleifera seeds showed 81% inhibition on eggs of H.contortus at 8 mg/mL was stated by (Ilavarashi et al. 2019) [12] supports the current findings.

Conclusion

In the current experiment, plant extracts mediated Ag NPs showed better anthelmintic activity at low concentrations. The presence of enhanced phytochemicals and the advantages of employing nanoparticles in herbal formulations might explain the effective egg hatching suppression and mortality of H. contortus, even at relatively low doses. The present study may conclude that biocompatibility is greatly valuable for pharmaceutical industries as functioning application of nanoparticles without any adverse side effects. So, the anthelmintics from plant origin may be used safely in combination with the nanoparticles for the desirable results. In vitro techniques offer a quick way to screen for possible anthelmintic effects. However, due to notable differences in factors such as metabolic biotransformation, absorption, and interaction with feed substances, findings from in vitro experiments may not directly apply to in vivo activity.

Acknowledgement

AcknowledgementThe authors express gratitude to Sri Venkateswara Veterinary University, Tirupati. Institute of Frontier Technology, RARS, ANGRAU, Tirupati.

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