Comparative antileishmanial efficacy of the biosynthesised ZnO NPs from genus Verbena

Abstract

This study describes ZnO NPs biosynthesis using leaf extracts of Verbena officinalis and Verbena tenuisecta. The extracts serve as natural reducing, capping and stabilization facilitators. Plant extracts phytochemical analysis, revealed that V. officinalis showed higher total phenolic and flavonoid content (22.12 and 6.38 mg g ⁻¹ DW) as compared to V. tennuisecta (12.18 and 2.7 mg g ⁻¹ DW). ZnO NPs were characterised by ultraviolet–visible spectroscopy, Fourier transform infrared, X‐ray diffraction, scanning electron microscope, transmission electron microscopy (TEM) and energy dispersive X‐ray. TEM analysis of ZnO NPs reveals rod and flower shapes and were in the range of 65–75 and 14–31 nm, for V. tenuisecta and V. officinalis, respectively. Bio‐potential of ZnO NPs was examined through their leishmanicidal potential against Leishmania tropica. ZnO NPs showed potent leishmanicidal activity with 250 µg ml⁻¹ being the most potent concentration. V. officinalis mediated ZnO NPs showed more potent leishmanicidal activity compared to V. tenuisecta mediated ZnO NPs due to their smaller size and increased phenolics doped onto its surface. These results can be a step forward towards the development of novel compounds that can efficiently replace the current medication schemes for leishmaniasis treatment.

1 Introduction

Leishmaniasis, caused by a vector borne protozoan parasite Leishmania, is a serious public health issue among various infectious diseases endemic in almost 98 countries across the globe [1]. These figures are expected to soar even further owing to an increase in the disease carrying vectors because of global warming [2]. The drugs in use currently for the treatment of Leishmaniasis poses striking disadvantages like high prices, toxicity to human cells and development of antibiotic resistivity. Regular outbreaks and development of antibiotic‐resistant strains incited the thrust for discovery of novel antimicrobial agents among the scientific community [3]. The current study aims at analysing the potential role of biosynthesised zinc oxide nanoparticles (ZnO NPs) as novel leishmanicidal agents owing to their enhanced volume‐to‐surface area ratio, improved optical properties and capacity of reactive oxygen species (ROS) production within the microbial cells [4].

ZnO NPs have exhibited tremendous application in drug delivery, semiconductors, solar cells, agriculture, biosensors, bio‐imaging probes, cosmetics and diagnostics [5, 6, 7]. Additionally, ZnO NPs are also biocompatible and bio‐safe with unusual structural, thermal and electrical properties, which could be changed with respect to particle orientation and morphology (size and shape) [8]. Moreover, the fabricated ZnO NPs were utilised for silymarin (collective name for flavonolignans such as silychristin, silydianin, silybin or silibinin) molecule sensing, extracted from Silybum marianum [9]. Owing to noteworthy applications, which are yet to be scrutinised, a substantial research area is focusing on ZnO NPs synthesis to achieve the desirable functionality [10, 11]. The synthesis of NPs having controlled size and shape is a challenging task which is highly reliant on the design of the protocols. Previously, various physio‐chemical approaches for ZnO NPs synthesis have been reported [12, 13, 14]. Majority of these approaches are noxious and non‐ecofriendly. The need for high vacuum, sophisticated equipment, high‐priced chemicals, high‐energy input, laborious experimental procedures and production hazardous wastes raises further concerns over the use of these approaches [14]. There is an urgent demand of mitigating the aforementioned problems via adoption of environmentally benign and simple green method for NPs synthesis. The alternative to chemical method is biological approach being greener, cost effective and energy saving. Relating to stability, the NPs fabricated via biological method are more biocompatible and innocuous than the NPs prepared by chemical methods due to coating of bio‐molecules [15]. Use of phyto‐extracts has gained superiority among various green synthesis methods due to ensued eco‐friendly synthesis protocols, easy availability and extensive distribution of plants [8, 16]. Furthermore, the plants extract‐mediated NPs are more stable and showed more morphological (size and shape) variations compared to those synthesised by other organisms [8]. Recently, leaves extract of various plants such as Pongamia pinnata, apple pectin, Aloe vera, Nerium oleander, Solanum nigrum and Vitex negundo L. was used for the eco‐friendly fabrication of ZnO NPs [17]. Plant extracts act as natural reservoirs of various phytochemicals that are involved in capping and bioreduction of fabricated NPs [18]. Plant extracts differ in their bioreduction capacity because of variable phytochemicals that directly affect the physio‐chemical and morphological properties of the fabricated NPs [19].

Verbena officinalis Linn., commonly known as verbena and vervain, belongs to family verbenaceae. It is a perennial weed found across North Africa, Europe, China and Japan. For thousands of years, this herb has been utilised as folk medicine and approved as dietary supplement and herbal medicine by several regulatory statuses in many countries. Verbena is mostly situated in pleasant areas and is well known in folk medicine due to its expectorant, anti‐inflammatory and diuretic properties. Different types of extracts from V. officinales have shown antioxidant, antifungal, analgesic, antibacterial, nerve growth factor‐potentiating and anti‐rheumatic activities [20, 21, 22]. Consequently, V. officinalis can be utilised to treat enteritis, acute dysentery, depression and amenorrhea [23, 24]. The V. officinalis extracts have also been reported for its anti‐inflammatory [25, 26] properties.

Verbena tenuisecta Briq. also belongs to family Verbenaceae. It has originated from South America [27]. Many Verbena species are utilised in folk medicines of South and Central American against fever, diarrhoea, sexually transmitted disease, gastrointestinal disorders [28], and anti‐inflammatory topical purposes [29]. The main chemical constituents of Verbena species include iridoid glycosides such as verbascosides [30], flavonoid compounds e.g. kaempferol [31], luteolin, volatile oils [32], ursolic acid [33], dihydrochalcone [34], sterols [35], phenylethanoids [36], anthocyanidin [21] and verbenalin and its derivatives [37].

2 Materials and methods

2.1 Plants collection and identification

The fresh leaves of V. officinalis and V. tennuisecta were collected from Quaid‐i‐Azam University Islamabad, Pakistan (latitude: 33°44′48.62′, longitude: 73°3′27.18′) during the month of June 2016. The leaves were identified and authenticated by the Department of Plant Sciences, Quaid‐i‐Azam University Islamabad, Pakistan.

2.2 Fabrication of ZnO NPs using V. officinalis and V. tennuisecta leaf extracts

The freshly plucked leaves of V. officinalis and V. tennuisecta were cleaned thoroughly under running tap water to remove dust and debris followed by three times washing with distilled water. The leaves were dried in open air before weighing. Extracts of V. officinalis and V. tennuisecta were prepared by boiling 20 g leaves separately in the microwave oven for 10 min in Erlenmeyer flask (500 ml) containing 100 ml distilled water. These ensued mixtures were placed in an incubator at 40°C for 24 h and re‐boiled for 10 min. During boiling, a clear light brown and dark greenish coloured extract was formed from V. officinalis and V. tennuisecta, respectively. The extract was brought down to room temperature before filtration using Whatman No. 1 filter paper and stored at 4°C for further usage.

For ZnO NPs biosynthesis, zinc acetate dihydrate was utilised as precursor of zinc ion (Zn⁺²). The protocol reported by Gnanasangeetha and Saralathambavani [19] was used for the synthesis of ZnO NPs. Briefly, 1 ml of both plant extracts was separately mixed with 50 ml of 0.02 M zinc acetate solution (ZAS) and the pH was adjusted at 12 by simply adding 2 M NaOH drop wise under constant stirring using magnetic stirrer. The solutions were kept for 3 h at 80°C under vigorous shaking. The white precipitates formed in the reaction mixtures were settled down by keeping the solutions overnight at room temperature. The crude precipitates were subjected to centrifugation for 15 min at 6000 rpm. The resulting pellets were re‐suspended once in ethanol and thrice in distilled water accompanied by 15 min centrifugation at 6000 rpm. The resulting pellets were kept overnight in the oven at 60°C for drying. For further analyses, dried pellets were ground into fine powder.

2.3 Phytochemical analysis

Using Folin–Ciocalteu's (FC) reagent and following Singleton and Rossi [38] procedure, total phenolic content (TPC) was determined. Briefly, 20 µl of the sample was mixed with 90 µl FC reagent (ten times diluted with distilled water) and incubated at 25 ± 2°C for 5 min. Ninety microliters of 6% sodium carbonate were added to the above mixture and absorbance at 630 nm was recorded on a microplate reader. Gallic acid of various concentration (25, 20, 15, 10 and 5 μg ml⁻¹) were employed as standard for calibration curve plotting (R ²  = 0.986) and TPC was denoted as gallic acid equivalent (GAE)/g of dry weight (DW) equivalents of gallic acid.

Similarly, total flavonoid content (TFC) was calculated by using the Zia‐Ul‐Haq et al. [39] method. Briefly, 20 µl of extracted sample was mixed with AlCl₃ (10 µl) (10%, w/v) and potassium acetate (1 M, 10 µl) followed by distilled water (160 µl) addition. Under room temperature, this mixture was incubated for 30 min and the absorbance was recorded on a microplate reader at 415 nm. Different concentrations of Quercetin (40, 20, 10, 5 and 2.5 μg ml⁻¹) and 20 μl of methanol were used as a positive and negative control, respectively, and the TFC was denoted as quercetin equivalents/g of DW.

2.4 Characterisation

Ultraviolet–visible (UV–vis) spectroscopy was used to determine the optical properties of V. officinales mediated ZnO NPs (VOZNPs) and V. tennuisecta mediated ZnO NPs (VTZNPs). Biosynthesis of ZnO NPs was recorded during the reaction as a function of time with 1 h interval. HALO DB‐20 spectrophotometer was exploited for monitoring the UV–vis spectra of the reaction mixtures in the 180–800 nm range. Solution prepared by re‐suspending both type of biofabricated ZnO NPs (1 mg/ml, w/v) separately in distilled water was used for determining the ZnO NPs stability. At room temperature, these aqueous suspensions were sonicated for 30 min followed by vortexing for 5 min in order to uniformly distribute the ZnO NPs. For stability determination, UV–vis spectra were recorded with 1 h interval for a total of 24 h time period, for each of the respective samples.

X‐ray diffraction (XRD) analysis was done to determine the biosynthesised ZnO NPs crystalline nature by X‐ray diffractometer (Shimadzu‐Model, XRD‐6000) supplied with 30 mA current at 40 kV, and radiation of Cu Kα (λ  = 1.5406 Å) with 2θ ranging between 20° and 80°. The average size of biosynthesised ZnO NPs was determined by Debye–Scherrer equation

$$D=k\lambda /\beta \cos \theta$$

where θ is the Bragg's angle; β is the full width at half maximum (FWHM) in radians; λ is the X‐ray wavelength (λ   =  1.5418 Å); k is the shape factor (0.94).

Fourier transform infrared (FTIR) spectrophotometer (Bruker V70) was used to record the reflectance, for determining the possible functional groups responsible for ZnO NPs reduction and capping, which works beneath a dehydrated air flow in reflectivity mode, connected with an attenuated total reflectance accessory consisting of gold crystal. On gold crystal, powder samples were placed in the median of the infrared region (600–4500 cm⁻¹) and wave number was measured. FTIR spectra were recorded in FTIR spectroscopy at a resolution of 4 cm⁻¹ and 64 scan evaluation was average.

Scanning electron microscope (SEM) was used for determining the morphology of biosynthesised ZnO NPs via exploiting the SIGMA model (MIRA3 TESCAN) supplied with a high voltage (10 kV). Finely dispersed ZnO NPs were placed on a carbon covered copper grid simply by placing very minute amount of NPs on grid. Prior to SEM images collection at various magnifications, the thin layer was subjected to drying for 10 min under the mercury lamp.

The morphological features of both types of ZnO NPs were analysed by transmission electron microscopy (TEM) using JEOL model operated at 120 kV accelerating voltage. The TEM slides were made simply by placing very minute quantity of samples on a carbon‐coated copper grid. Under a table lamp, the film was dried for 5 min, and at various magnifications, TEM images were captured. For analysing the elemental composition and purity on ZnO NPs, the dried ZnO NPs were drop coated onto carbon film. Using the energy dispersive X‐ray (EDX) detector connected with TEM, EDX analysis was performed.

2.5 Antileishmanial efficacy of biosynthesised ZnO NPs

In vitro leishmanicidal efficacy of biosynthesised ZnO NPs, MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H ‐tetrazolium bromide] (purchased from Sigma Chemical Co., St. Louis, MO) assay was performed against the Leishmania tropica promastigotes. RPMI 1640 medium containing 10% FBS, 4.5 mg ml⁻¹ glucose (Sigma) and 292 µg ml⁻¹ L‐glutamine was used for culturing the L. tropica promastigotes at 25°C. The cultures were refreshed in vitro by subculturing them on every fifth day. L. tropica promastigotes of logarithmic phase (1 × 10⁵ parasites ml⁻¹) were seeded in 96‐well microplates. Fifty microlitres of ZnO NPs solution (prepared in PBS) of each concentration (25–250 µg ml⁻¹) were added in three wells and plates were incubated at 25°C for 24 h. Wells with RPMI 1640 (100 ml) media only were used as a control. Ten millilitres of MTT were added to each well and plates were again incubated at 25°C for 3 h. Enzymatic activities were halted by adding 100 ml dimethyl sulfoxide. Under continuous agitation, plates were re‐incubated for 30 min at room temperature. Due to formazan formation, purple colour appeared. Relative optical density was then measured at 540 nm. Linear regression curve analysis was performed for determining the 50% inhibitory concentrations (IC₅₀).

3 Results and discussion

3.1 Phytochemical analysis

Phytochemical constituents, e.g. flavonoids, aromatic compounds, tannins or secondary metabolites, serve as defence mechanism against various microbes. The therapeutic nature of medicinal plants is possibly due to the existence of diverse secondary metabolites, namely flavonoids, phenolic compounds, tannins, alkaloids, phytosterols and saponins [40].

Verbena species are well known for their bioactive secondary metabolites, e.g. iridoid glycosides such as verbascosides, verbenalin and its by‐products, flavonoid compounds, namely luteolin, kaempferol, ursolic acid, volatile oils, sterols, dihydrochalcone, anthocyanidin and phenylethanoid. These bioactive compounds have shown strong activities against fever, diarrhoea, gastrointestinal disorders and some sexually transmitted disease, and in anti‐inflammatory topical applications as well. Thus because of these antioxidant phytoconstituents, Verbena species can act as a potent reducing and stabilising agent in ZnO NPs biosynthesis. Verbena species showed significant variations in the TPC and TFC accumulation (Table 1). V. officinales and V. tennuisecta extracts showed maximum TPC (22.12 and 12.18 mg g⁻¹) and TFC (6.38 and 2.7 mg g⁻¹) as compared to their respective biosynthesised Zno NPs (TPC: 4.7 and 0.7 mg g⁻¹; TFC: 0.01 and 0.005 mg g⁻¹). Our results are in harmony with that of Rehecho et al. [41] and confirm that both phenolics and flavonoids actively participated in stabilisation of ZnO NPs. The higher values of TPC in biosynthesised ZnO NPs suggest that phenolics from verbena species are more dominant in reduction and capping of ZnO NPs as compared to flavonoids.

Table 1.

Phytochemical analysis of V. officinales and V. tenuisecta extracts and their respective biosynthesised ZnO NPs

Samples	TPC (mg g−1 DW)	TFC (mg g−1 DW)
V. officinales extract	22.12	6.38
V. tenuisecta extract	12.18	2.7
VOZNPs	4.7	0.005
VTZNPs	0.7	0.01

3.2 UV–vis absorption studies of biofabricated VOZNPs and VTZNPs

The ZnO NPs biosynthesis can be monitored visually by observing the colour changes in reaction mixture. Both reaction mixtures (V. officinales extract + ZAS and V. tenuissecta extract + ZAS) changed their colour from darker brown to white. UV–vis spectra ranging from 180 to 800 nm wavelength is used to monitor biosynthesis of ZnO NPs. In case of VOZNPs, broad absorbance spectra with narrow peak were observed at 410 nm. This is attributed to the reduced size and maximum yield of VOZNPs [42]. Several reports demonstrated that the morphology (size and shape) of NPs in liquid medium could be studied via UV–vis spectra [43]. Generally, the optical absorption peaks of NPs shifting to lower wavelength are a sign of reduction in particles size [44, 45].

To determine the ZnO NPs biosynthesis reaction time completion, we scrutinised the wavelength peaks and absorbance intensities of both reaction mixtures at various time intervals (1–24 h). In case of VTZNPs biosynthesis, an expansion of reaction time was accompanied with an apparent enhancement in the absorbance intensity before 6 h, but in absorption no significant increase was noticed after 6 h (Fig. 1 a). While, in case of VOZNPs, the reaction was terminated within 5 h and in absorbance intensity, no significant change was noticed till 24 h (Fig. 1 b).

Fig. 1.

VOZNPs and VTZNPs, UV–vis spectra

(a) Absorption bands of VTZNPs after 6, 12 and 24 h, (b) Absorption peaks of VOZNPs after 6, 12 and 24 h, (c) Reaction completion time of VOZNPs and VTZNPs after 6 h

These results demonstrated that the VOZNPs biosynthesis was completed in shorter time compared to VTZNPs. This might be because of enhanced phytochemicals (TPC and TFC) in V. officinales extract than V. tenuissecta extract, which accelerates the Zn²⁺ ions reduction to Zn⁰. Both of the biosynthesised ZnO NPs under investigation in current study depicted high absorption peaks at 390 nm, because of surface plasmon resonance features (Fig. 1 c). The naturally available phytochemicals of the phyto extracts play crucial role in NPs reduction and stabilisation [44] as suggested by several other reports in case of silver NPs [42, 46].

3.3 XRD pattern of VOZNPs and VTZNPs

The XRD analysis was done to confirm the crystallinity and phase purity of biosynthesised ZnO NPs. VTZNPs diffractogram (Fig. 2 a) displayed characteristic peaks at 15.44, 26.11, 27.05, 29.73, 31.67, 38.4 and 49.28, which corresponds to 100, 002, 101, 102, 110, 103 and 200 reflection lines of ZnO NPs hexangular wurtzite configuration (JCPDS 36‐1451) [47]. Likewise, VOZNPs diffractogram (Fig. 2 b) displayed diffraction peaks at 31.81, 34.61, 36.44, 53.47, 56.61, 58.59, 63.13 and 68.15, which are in close correspondence with 100, 002, 101, 102, 110, 103 and 200 reflection lines of ZnO NPs hexagular wurtzite configuration as well (JCPDS 36‐1451).

Fig. 2.

XRD pattern

(a) VTZNPs, (b) VOZNPs

Previously, the hexangular wurtzite configuration of biosynthesised ZnO NPs has been reported over and over again [47, 48]. Both biosynthesised ZnO NPs showed diffraction peaks of narrow width and strong intensity which disclose the highly crystalline nature of biosynthesised NPs. Moreover, peaks associated with secondary phase or any impurity were not found, indicating the superiority of biosynthesis proposed mechanism over others which could be applied for highly pure ZnO NPs synthesis.

Debye–Scherrer equation was used to calculate the average size of biofabricated ZnO NPs. The average size of VOZNPs was found to be 18.49 nm, whereas VTZNPs were 63.51 nm in size. These findings clearly demonstrate that both of the biosynthesised ZnO NPs exhibit the similar crystalline structure but their sizes were different. V. officinales was involved in the production of small‐sized ZnO NPs and thus hypothesised to be more potent in their microbicidal and leishmanicidal activities compared to VTZNPs owing to the facile penetration and movement of smaller‐sized NPs inside the microbial cells [49].

3.4 FTIR spectra of VOZNPs and VTZNPs

The potent interfacial phytochemicals responsible for the reduction and capping of VTZNPs and VOZNPs was characterised and identified via FTIR analysis. FTIR spectra of V. tenuissecta extract and V. officinales extract displayed a lot of peaks which reflects that these extracts contain phytochemicals of complex nature (Fig. 3). V. officinales extract displayed broad absorption peaks at 732.91, 1012.58, 1022.2, 1226.67, 1433.04, 1539.12, 1639.41, 2042.52, 23.04.82, 2856.44 and 3234.47 cm⁻¹ which correlates with the bond stretching of –N–H (amine), –C–N (aliphatic amine), –OH (carboxylic acid), –C–C (aromatic ring), –C–O (carbonyl group), –C–H (alkanes) and –O–H (phenol), respectively. While the VOZNPs displayed absorption peaks at 1022.2, 1218.9, 1334.67, 1589.2 and 2823.65 cm⁻¹ which represent the involvement of amino group, aliphatic amine, carboxylic acid, carbonyls, phenols and alkanes (Fig. 3 a) [8, 50]. V. officinales extract and VOZNPs FTIR spectra exhibit similar absorption peaks in 750–1250 cm⁻¹ region, but the peaks intensity was maximum in V. officinales extract in the region of 1400–2350cm⁻¹ than VOZNPs, proposing the potential involvement of carboxylic acid, carbonyl group, aromatic rings, phenolics and alkanes in the capping and stabilisation of VOZNPs.

Fig. 3.

FTIR analysis

(a) V. officinales extract and VOZNPs, (b) V. tenuissecta extract and VTZNPs

FTIR spectrum of V. tenuissecta extract displayed intense absorption peaks at 756.0, 1016.43, 1217.02, 1332.75, 1400.25, 1500.54, 1593.12, 2044.45, 2854.51 and 3240.26 cm⁻¹ which correlates the vibrational bond stretching of –N–H (amine), –C–N– (aliphatic amine), –C–Cl (alkyl halide), –C–C– (alkenes), –C–H (aromatic group), –C–H (alkanes) and –O–H (phenol) [51]. VTZNPs illustrated peaks at 893.0, 1078.15, 1245.95, 1498.62 and 2972.16 cm⁻¹ which also represent the same functional groups as mentioned in V. tenuissecta extract (Fig. 3 b). However, low‐intensity peaks were observed in VTZNPs spectrum as compared to V. tenuissecta extract. Interestingly, peaks present in the range 1598–3250 in V. tenuissecta extract were diminished in VTZNPs spectrum. Thus, we assume that disappearance of these characteristics peaks may be assigned to the concept that the amino group, aliphatic amine, carboxylic acid, carbonyl groups, alkanes and phenols are involved in the reduction of Zn²⁺ to Zn⁰. Many reports are available in support of our results which suggest that in the ZnO NPs bioreduction and capping, the above‐mentioned groups are possibly involved [8, 48].

3.5 SEM, TEM and EDX analysis of VOZNPs and VTZNPs

The shape and size of biosynthesised ZnO NPs was determined via SEM analysis. VTZNPs seemed to be rod shaped with weak NPs agglomeration (Fig. 4 a). While the VOZNPs were found to be flower shaped with uniform NPs distribution (Fig. 4 b). Like our results, various shapes (rod, spherical, flower‐like and hexagonal) of ZnO NPs have been recorded previously [16, 52]. By exploiting the Sigma Scan Pro software connected with SEM, mean values of ZnO NPs size were calculated. VTZNPs observed in 65–75 nm range while VOZNPs in 14–31 nm. Average ZnO NPs size determined by SEM was in harmony with the XRD results. Hence, SEM results also supported the findings of XRD. The SEM studied crystallites size was supported and observed in detail via TEM analysis (Figs. 4 c and d). To confirm the purity and elemental zinc and oxygen composition of biosynthesised ZnO NPs, EDX analysis was performed. EDX spectra (Figs. 5 a and b) in the range of 2.0–3.0 keV exhibited sharp peaks of zinc and oxygen. Besides highly intense peaks, small weak peaks of P, Cu and Si were also observed because of the bounded biomolecules entangled onto the biosynthesised ZnO NPs. These results are in harmony with the previous reports [52].

Fig. 4.

SEM and TEM analysis of NPs where

(a) SEM image of VTZNPs, (b) SEM image of VOZNPs at 2 and 5 µm magnification bar, (c) TEM image of VTZNPs, (d) TEM image of VOZNPs at 0.5 and 0.2 µm magnification bar

Fig. 5.

EDX spectra

(a) VTZNPs, (b) VOZNPs

3.6 Antileishmanial efficacy of VTZNPs and VOZNPs

Due to emergence of antibiotic resistant strains of pathogen, researchers and the pharmaceutical industries are in constant search for novel antimicrobial [4]. In this report, we tested the leishmanicidal potency of VTZNPs and VOZNPs against L. tropica promastigotes individually at concentrations ranging from 25 to 250 µg ml⁻¹ for their leishmanicidal potency. Mortality of L. tropica promastigotes altered when exposed to ZnO NPs as compared to control. Controls showed strong purple colour showing that L. tropica remained alive. However, upon exposure to ZnO NPs, light purple coloured formazan crystals appeared, showing that the tested ZnO NPs suppressed metabolic activities of L. tropica. Quantitatively, in both types of ZnO NPs with various concentrations, dose‐dependent effect was detected. Highest percentage of promastigotes mortality was shown by ZnO NPs tested at the highest concentration (250 µg ml⁻¹) resulting in 55.2% (VOZNP) and 27.9% (VTZNP) mortality (Fig. 6). Previous studies have also shown dose‐dependent leishmanicidal action of TiO and AgNPs [1, 3]. The IC₅₀ value of VOZNPs (243.42 µg ml⁻¹) was 1.7‐fold less than VTZNPs (414.03 µg ml⁻¹). Both per cent mortality and IC₅₀ values showed that efficacy of VOZNPs was stronger than the VTZNPs. This could be due to variations in their morphology and stabilising agents. In support of our findings, many reports have shown that usually the smaller‐sized NPs have the highest potency of suppressing the Leishmania parasites [53]. Various kinds of metal and metallic oxide NPs are reported for their enhanced induction of ROS, which favours apoptosis and hence mortality process [3, 51]. It is a well‐known fact that any agent/drug that could produce ROS would be a promising leishmanicidal candidate as the Leishmania parasite is oversensitive to ROS [54].

Fig. 6.

Leishmanicidal effect of various concentration of VTZNPs and VOZNPs calculated as percentage promastigotes mortality

3.7 Probable mechanism of antileishmanial efficacy of ZnO NPs

Recently, no satisfactory report is available on the possible mechanism behind the antileishmanial efficacy of ZnO NPs and yet is under debate. In this work, we hypothesised the probable mechanism behind the leishmanicidal efficacy of biofabricated ZnO NPs (Fig. 7). Owing to small size and electrostatic interactions, ZnO NPs get entrance into leishmanial cells. The exposure to ZnO NPs declines the parasite's metabolic activity and proliferation values, which induced intracellular depletion via ROS production (Figs. 7 a –c). Increased production of ROS (O₂, H₂ O₂, OH, etc.) and Zn⁺² causes cell membrane disruption which leads to cytoplasmic leakage and finally protozoa death (Figs. 7 d and e). Owing to ROS high reactivity and oxidising properties, they induced toxicity in protozoa as evident from previous reports [55].

Fig. 7.

Probable mechanism of both types of biofabricated ZnO NPs involved in the leishmanicidal action

4 Conclusion

Exploitation of Verbena species could act as a new and better source of phytochemicals (bioreducing agents) for enhanced ZnO NPs biofabrication. UV–vis spectroscopy, FTIR, XRD, SEM, TEM and EDX analysis disclosed that VOZNPs were more competent than VTZNPs in terms of their physical features such as size, shape, crystallinity and distribution and biomedical potentials like antileishmanial activity. The variation in both types of biofabricated ZnO NPs physical properties and bio‐potent assay is assigned to the differential accumulation of bioactive phytochemicals within the same genera involved in the NPs capping and stabilisation. The TPC of VOZNPs was also much greater than VTZNPs (4.7 and 0.7 mg g⁻¹ DW) which might as well be another possible reason apart from particle size behind improved leishmanicidal activity of VOZNPs as compared to VTZNPs since plant phenolics in itself possess antimicrobial properties. These biosynthesised ZnO NPs are doped with natural phytochemicals and devoid of noxious chemicals that are commonly tangled on the chemi‐synthesised NPs surfaces and can thus prove unhazardous and safe for utilisation in different pharmaceutical industries and biomedical applications.