Lagenaria siceraria – synthesised ZnO NPs – a valuable green route to control the malaria vector Anopheles stephensi

Abstract

Malaria is a dangerous disease affecting humans and animals in tropical and subtropical areas worldwide. According to recent estimates, 3.2 billion people are at risk of malaria. Many drugs are in practices to control this disease and their vectors. Eco‐friendly control tools are needed to fight vectors of this important disease. Nanotechnology is playing a key role in the fight against many public health emergencies. In the present study, Lagenaria siceraria aqueous peel extract was used to prepare zinc oxide nanoparticles (ZnO NPs), then tested on Anopheles stephensi eggs, larvae and pupae. The L. siceraria ‐synthesised ZnO NPs were characterized additionally by FTIR, AFM, XRD, UV‐Vis spectroscopy, EDX, and SEM spectroscopy The ovicidal, larvicidal, pupicidal and repellent activities of L. siceraria and green‐synthesised ZnO NPs were analysed on A. stephensi. The potential mechanism of action of ZnO NPs was studied investigating the changes in various enzyme activities in A. stephensi IV instar larvae. Furthermore, the smoke toxicity of L. siceraria ‐based cones against A. stephensi evoked higher mortality if compared with the control. Overall, the present study concluded that L. siceraria peel extract and its mediated green synthesised ZnO NPs represent a valuable green option to manage against malaria vectors.

1 Introduction

Mosquitoes (Diptera: Culicidae) are route cause of several prevailing diseases such as yellow fever, Japanese encephalitis, malaria, Zika virus, dengue and filariasis [1, 2, 3]. Additionally, they give rise to allergic reactions in humans, which includes local systemic and cutaneous reactions. From the above‐mentioned diseases, malaria is triggered by the parasites from the Plasmodium genus that was transferred by female Anopheles stays a serious concern regarding the public's health in the countries of subtropics and tropics. In the total population of the world, only one‐third of them are surviving in non‐endemic areas and for every year, >200 million people show the symptoms and signs of malaria [4]. According to the World Health Organization report, there were 212 million new cases of malaria worldwide in 2015. Malaria is still a major killer of children under five, claiming the life of a child every 2 min [5, 6]. Plasmodium falciparum, the most dominant and the most pathogenic among the four species (P. malariae, P. falciparum, P. ovale and P. vivax) of malaria parasites infecting humans, is the main cause of almost all morbidities and all deaths from malaria in tropical countries [7, 8]. Despite the significant efforts to control malaria, the disease still stays as a major health concern for the people. Since there avails no current powerful vaccine against the prevention of malaria, vector control is one among the key strategies utilised to regulate this disease. At present, strategies for controlling the mosquitoes are mainly influenced by the application of synthetic insecticides [9]. The application of insecticides for controlling the mosquitoes has directed to several problems such as poisonous effects on humans, poisonousness to organisms that were not targeted and the resistance improvement in the aimed mosquitoes. All these concerns emphasise the need for urgency to advance new insecticides that must be safe, effective, target‐specific and biodegradable [10]. One possible source for such an affordable treatment lies in the use of traditional herbal remedies [11, 12].

Nanotechnology is an emerging field in which various plant sources have been applied for the synthesis of nanoparticles (NPs) [13]. The use of plants for the green synthesis of NPs is an inexpensive, rapid, safe process and environmentally friendly for using therapeutically in humans [14, 15]. The process of NPs green synthesis by applying environmentally benign methods (i.e. microorganisms, extracts and phytochemicals from whole plants, plant parts and marine algae) is interesting especially if it is intended for cosmetic, medicine and pesticide science [16, 17, 18].

These days, zinc oxide (ZnO) NPs have expanded the attractiveness because of their enthralling and distinguishing properties. In recent years, extraction of plants and several natural sources have turned out as an energy‐efficient, low‐cost and non‐toxic approach for synthesising ZnO NPs [19, 20]. Additionally, ZnO NPs have a higher level of robustness, stability, longer life and biocompatibility than any other antimicrobial agents [21].

Lagenaria siceraria, also known as bottle gourd, is applied in conventional Indian medicine for treating the broad spectrum of diseases. This species has an anti‐oxidant, anti‐diabetic, anti‐ulcer, anti‐inflammatory, anti‐helminthic, analgesic and anti‐septic activities [22]. L. siceraria has also been screened for its repellency effect against Culex pipiens L. mosquitoes by Fouda [23].

In the present investigation, we proposed an economic and rapid technique for the green synthesis of ZnO NPs using the aqueous peel extract of L. siceraria. Bio‐reduced ZnO NPs were characterised by Fourier‐transform infrared spectroscopy (FTIR), ultraviolet (UV), energy‐dispersive X‐ray spectroscopy (EDX), atomic force microscopy (AFM) and scanning electron microscope (SEM). The critical toxicity level of L. siceraria aqueous peel extract and the ZnO NPs were measured in comparison with the pupae, larvae and eggs of Anopheles Stephensi (Fig. 1). Also, we have measured the toxicity level of the smoke from the herbal cones prepared from the peel extract of L. siceraria on the A. stephensi adults. From our gathered information, this is the first report on the mosquitocidal activity of L. siceraria peel extract and green‐fabricated ZnO NPs prepared using phytochemicals from its peel. The mechanism of action of ZnO NPs was studied investigating the changes in various enzyme activities in A. stephensi larvae.

Fig. 1.

Green route to control malaria vector A. stephensi

2 Experimental section

2.1 Collection of plant material

The fresh L. siceraria fruits were collected from Vellore, India. The species identification was done by Prof. Jayaraman of the Plant Anatomical research Centre, Chennai, India. The voucher specimen (PARC/2015/3172) was numbered and deposited in our research laboratory for the reference in the near future.

2.2 Preparation of aqueous extract

L. siceraria fruits which are healthy and fresh were washed completely with distilled water and exposed to dry in a cloth. The fruit was carefully peeled to separate the epicarp and immediately dried under shade. The dried peels were made into fine powder. About 10 g of L. siceraria peel powder was weighed and transferred into a beaker containing 100 ml of distilled water, mixed thoroughly and the mixture is heated for the next 15 min. The extract acquired through the process was filtered with a Whatman No. 1 filter paper, and the filtrate was collected in a separate flask and stored in a refrigerator for further use [24].

2.3 Synthesis of ZnO NPs

About 100 ml zinc nitrate [Zn (NO₃)₂] (5 mM) was prepared mainly for the synthesising process of ZnO NPs. Then, with 25 ml of L. siceraria peel extract, Zn (NO₃)₂ was mixed in a quantity of 75 ml, and then continuously stirred for ten complete hours [24, 25].

2.4 Characterisation of ZnO NPs

ZnO NPs green synthesis was established by samples of the reaction mixture collected at fixed intervals and by keeping the wavelength of 200–800 nm for UV–visible (UV–vis) spectra, the absorption maxima were scanned. Furthermore, the reaction mixture was subjected to centrifugation at 15,000 rpm for 20 min; the pellet obtained was dissolved in distilled water and filtered through a Millipore filter. An aliquot of this filtrate comprising ZnO NPs was used for FTIR, X‐ray diffraction (XRD), SEM, EDX, AFM, dynamic light scattering (DLS) and zeta potential. Regarding the studies of XRD, on the XRD grid the dried NPs were used for coating purpose, and with the help of PAN analytical X‐Pert PRO the spectra were filled and operated at a 30 kV voltage and a 40 mA current through the CuKα radiation. By the application of SEM, the size and shape were examined. Through the AFM, ZnO NPs topography was defined. At 4 cm⁻¹ resolution, the ZnO NPs’ surface groups were validated qualitatively by FTIR spectroscopy and the elemental ingredients of the synthesised NPs were categorised with the help of energy dispersive X‐ray analysis (EDAX) [26, 27].

2.5 A. stephensi rearing

A. stephensi larvae were directly collected from Cooum river, Chennai and species were identified by Dr. Elumalai Kuppusamy, Entomologist, Government Arts College, Nandanam, Chennai. The larvae which are collected were kept in enamel and plastic trays holding tap water and nurtured in the laboratory at 75–85% R.H. and 27 ± 2°C under the ratio of 10:14 dark and light cycles. Larvae were nourished under a diet of dog biscuits, algae and brewer's yeast in 3:1:1 proportion, respectively. From the trays, all the pupae were transmitted to a cup having tap water and were kept under insectary, where they emerge as adults. In glass cages, all the adults were preserved and with a cotton wick constantly delivered with a 10% sucrose solution in a jar. Nourishment for adults is provided through the blood meal with a young chick in cages immediately on the account of the fifth day. With the intention for oviposition, the Petri dishes containing tap water of 50 ml lined by means of filter paper were placed inside the cage [28].

2.5.1 Larvicidal and pupicidal bioassays against A. stephensi

Totally, 25 A. stephensi IV instars pupae and larvae were left in a glass beaker for 24 h containing 250 ml of dechlorinated water along with ZnO NPs (2, 4, 6, 8 and 10 ppm) or the required concentration level of aqueous peel extract of L. siceraria (100, 200, 300, 400 and 500 ppm). About 0.5 mg of larval food was delivered for every verified level of concentration. Each concentration was duplicated five times against the IV instar pupae and larvae. As a control, dechlorinated water was functioned [10, 29, 30, 31]. The percentage calculation of mortality is as follows:

$$\begin{array}{rl}\mathrm{mortality}(\text{\%})& =(\mathrm{number}\mathrm{of}\mathrm{dead}\mathrm{individuals}/\\ & \mathrm{number}\mathrm{of}\mathrm{treated}\mathrm{individuals})\times 100\end{array}$$

2.5.2 Ovicidal bioassay

Eggs were collected from the Department of Zoology, Government Arts College, Nandanam, Chennai. Eggs were kept open to the diverse concentration of the ZnO NPs and peel extract of aqueous L. siceraria ranging from 100–500 and 2–10 ppm, respectively. Eggs exposed to distilled water serve as a control. For every treatment, five replicates were retained with 20 eggs for every one replicate. After completing the treatment, from each concentration, the eggs were shifted to one tray having distilled water for the purpose of hatching. The rate of hatching is calculated as 48 h post‐treatment, and the percent hatch was calculated in each instance [32]

$$\text{\%}\mathrm{hatch}=\frac{\mathrm{number}\mathrm{of}\mathrm{hatched}\mathrm{larvae}}{\mathrm{total}\mathrm{number}\mathrm{of}\mathrm{eggs}\mathrm{exposed}}\times 100$$

2.5.3 Repellent activity

In evaluating the repellent, a control and treated cotton pad were kept soaking in the chick blood and positioned exactly in the opposite direction inside a glass container. Then, the treated pads were drenched in with diverse concentration levels of L. siceraria peel extract (100–500 ppm) and ZnO NPs (2–10 ppm). In each container, 20 A. Stephensi females were released and the record was maintained for calculating the amount of female landing on every single pad [33]. The percentage of repellency of cotton pads, after treatment, can be evaluated with the help of the upcoming formula

$$\mathrm{repellency}(\text{\%})=C-T/C\times 100$$

where C and T are the number of mosquitoes on the control and treated pad.

2.6 Smoke toxicity assays against A. stephensi

L. siceraria aqueous peel extracts were utilised for preparing the cones for toxicity of smoke evaluates against A. stephensi. To prepare the L. siceraria‐ based cones and related bioassays, the method by Murugan et al. was followed. Each trial and for each treatment (L. siceraria peel‐centred coil; negative and positive controls) was repeated on five individual days with five times. The overall mosquitoes were kept open for 1 h toward the smoke from the burning coils. Later on, completing the experiment, the number of unfed (dead) and feds (alive) mosquitoes was calculated [29, 34, 35, 36]. The provided protection through the plant samples’ smoke against A. stephensi can be evaluated in the sort of percentage of unfed mosquitoes due to treatment

$$\frac{\mathrm{no}.\mathrm{of}\mathrm{unfed}\mathrm{mosquitoes}\mathrm{in}\mathrm{treatment}-\mathrm{no}.\mathrm{of}\mathrm{unfed}\mathrm{mosquitoes}\mathrm{in}\mathrm{negative}\mathrm{control}}{\mathrm{number}\mathrm{of}\mathrm{treated}\mathrm{mosquitoes}}\times 100$$

2.7 Mechanism of ZnO NPs and plant extract on mosquito larvae

The plant extract and the action of ZnO NPs on the activities regarding enzyme involved in the mosquito larvae metabolism were analysed. The changes in terms of quantity of overall phosphatases, esterases and proteins in entire body homogenates of fourth‐instar larvae of A. stephensi kept open to ZnO NPs, and plant extract was examined.

2.7.1 Preparation of whole body homogenates

The fourth‐instar larvae were exposed to ZnO NPs and plant extract. Then, the exposed larvae were completely rinsed with twice over distilled water, and by using tissue paper its body surface was blotted for the purpose of removing the adhering water. In eppendorf tubes, the larvae (every ten individuals) were distinctly homogenised through a teflon hand homogeniser in ice‐cold sodium phosphate buffer of 500 µl (20 mM, pH 7.0) for estimating the activities of phosphatases, esterases and proteins. For 20 min at 4°C and at 8000 rpm, the entire body homogenates were centrifuged, and at 4°C the pure supernatants were kept till it is utilised for the biochemical analyses.

2.7.2 Determination of protein concentrations

The available protein content in the larval homogenates (50 µl each) was precipitated first by collaborating with 80% ethanol for 20 volumes, and the concentration of proteins was purely estimated by the Lowry method [37].

2.7.3 Acetylcholinesterase (AchE) assays

AchE activity in the larval homogenates was spectrophotometrically measured according to Ellman [38] with slight modification using acetylthiocholine iodide as substrate [39]. About 200 µl of homogenate was mixed with 200 µl of sodium phosphate buffer and incubated at 30°C for 10 min. To this mixture, 50 µl of 10 mM 5,5‐dithio‐bis [2‐nitrobenzoic acid] and 50 µl of 12.5 mM acetylthiocholine iodide were added for a total volume of 1 ml. After incubation for 5 min at room temperature, the reaction was measured at absorbance 400 nm using UV spectrophotometer [40, 41].

2.7.4 Carboxylesterase assays

The beta (β)‐ and alpha (α)‐carboxylesterase activities in the larvae were evaluated by the Van Asperen method [42] along with few modifications in it. The larval homogenate (test and control) for 200 µl was incubated using 2 ml of β and α ‐naphthyl acetate solution at room temperature correspondingly for 30 min. To each reaction mixture, fast blue sodium dodecyl sulphate (SDS) reagent of 500 µl was added [22.5 mg fast blue salt is mixed in distilled water of 22.5 ml and in 0.2 M phosphate buffer of pH 7.2, 5% w/v SDS is added] to terminate the enzymatic reaction, and at 28°C the colour was permitted to progress for 15 continuous minutes. The sample optical density was evaluated in the spectrophotometer at 588 nm (β ‐carboxylesterase) and 430 nm (α ‐carboxylesterase) against the particular reagent blank [40, 41].

2.7.5 Acid and alkaline phosphatase assays

The contents of these two phosphatases in the larvae homogenates (A. stephensi) were evaluated against the subsequent Asakura procedures [43] with minor or small modifications to it. The activity of acid phosphatase was evaluated through 450 µl of 50 mM sodium acetate buffer at 4.0 optimal level of pH by assorting 50 µl of larva in it. For estimating the activity of alkaline phosphatase, larval homogenate for 20 µl was prepared up to 500 µl by Tris‐hydrochloric acid buffer for 50 nM at 8.0 optimal level of pH of 50 mM and blended with the same volume of the corresponding buffer having only p‐nitrophenyl phosphate for 12.5 mM. It was placed in water bath for incubation at 37°C for 15 min, after which 100 µl of 0.5N sodium hydroxide solution was added to stop the enzymatic reaction and centrifuged at 4000 rpm for 5 min. At 440 nm, the resulting transparent supernatants’ absorbance was studied [41].

2.8 Statistical analysis

The 16.0 version of SPSS software package was used. Information was collected from larvicidal, pupicidal and ovicidal experiments for the purpose of examining through the probit analysis calculating LC₉₀ and LC₅₀. In the experiment of herbal toxicity, certain analyses are made on several unfed, dead and fed mosquitoes. The difference in the levels of repellence against mosquito bites, as well as differences in numerous biochemical parameters among the treated and control larvae, was analysed by analysis of variance and Tukey's honestly significant difference (HSD) test.

3 Results and discussion

3.1 Characterisation of ZnO NPs

The NPs sizes play a significant role in modifying the whole properties of materials. Usually, UV–vis spectroscopy is conducted to validate the ZnO NPs synthesis. Fig. 2 signifies the UV–vis range of newly produced ZnO NPs. The highest level is obtained at 385 nm and it revealed that in the reaction mixture the existence of ZnO NPs was proved. In agreement with these findings, the ZnO NPs fabricated using Eclipta prostrata showed an absorption peak at 372 nm [25]. Recently, Santhosh kumar showed that the P. caerulea‐ mediated ZnO NPs evoked at 380 nm, an extreme peak level of absorbance [26]. According to the study, Talam et al. [44] showed the UV–vis band at 355 nm because of ZnO NPs. Similarly, Jin et al. [45] also show the UV–vis band at 355 nm due to ZnO NPs. However, Khorsand Zak [46] examined the UV–vis absorbance spectrum of ZnO NPs from 200 to 1000 nm. Overall these findings show the different sizes of ZnO NPs.

Fig. 2.

UV‐visualisation of the absorption spectrum of ZnO NPs synthesised using the L. siceraria extract

FTIR spectroscopy was completed to recognise the potential biomolecules in the peel extract of L. siceraria that may take the responsibility for the stabilisation and synthesis of ZnO NPs. Fig. 3 reveals the FTIR spectrum of ZnO NPs is made by using the peel extract of L. siceraria. The transmittance peaks at the spectrum are displayed at 3404.36, 2929.87, 2852.72, 1612.49, 1514.12, 1435.04, 1190.08, 1112.93, 1083.99, 887.26, 711.73, 582.50 and 459.06. The peaks at 3404.36 correspond to OH stretch. The 2929.87 and 2852.72 peaks correspond to C–H stretch in the alkyl group, and the peak 1612.49 shows strong aromatic C = C stretch (Table 1). The peak at 1514.1 refers to the amide C = O vibration stretch. Strong C = C aromatic stretch is shown by the peak value of 1435.04. The peak at 1190.08 corresponds to strong C–F stretch, whereas 1112.93 show C–O–C stretch and 1083.99 correspond to C–OH stretch. C–Cl stretch was shown by the peak values of 887.26 and 711.73, C–Br was found in 582.50 peak and C–I stretch as found in 459.06 peaks (Table 1). Recently, Plectranthus amboinicus extract was studied by FTIR spectroscopy to recognise the functional groups are responsible for the process of capping and synthesis of ZnO NPs [47]. Kalpana et al. [48] examined the FTIR bands of ZnO NPs by using L. siceraria and showed the 2920.23 (bond: C–H stretch) and 3313.71 (bond: O–H stretch); this gives the evidence of the appearance of strong aromatic rings and carboxylic acid that may be accountable for the ZnO NPs synthesis using L. siceraria. This same finding was supported by Prajapati et al. [49] on phytochemical constitutions. The FTIR outcomes have specified that secondary structures of proteins were not influenced in consequence of binding with ZnO NPs or reaction with zinc ions. This proof recommended that the discharge of extracellular protein molecules might achieve the responsibility in an aqueous medium for the stabilisation and formation of ZnO NPs in it.

Fig. 3.

FTIR spectrum of ZnO NPs synthesised using the L. siceraria extract

Table 1.

FTIR spectral peaks characterising the L. siceraria – synthesised ZnO NPs

No.	Absorption peak, cm−1	Bond and related functional groups
1.	3404.36	OH stretching vibrations
2.	2929.87	C–H alkyl stretch
3.	2852.72	OH carboxylic acid stretch
4.	1612.49	C = C aromatic stretch
5.	1514.12	C = O amide stretch
6.	1435.04	C = C aromatic stretch
7.	1190.08	C–F stretch
8.	1112.93	C–O–C stretch
9.	1083.99	C–OH stretch
10.	887.26	C–Cl stretch
11.	711.73	C–Cl stretch
12.	582.50	C–Br stretch
13.	459.06	C–I stretch

The crystalline nature of ZnO NPs was confirmed from the XRD analysis. The XRD pattern of freeze‐dried NPs exhibited peaks at 31.6°, 34.2°, 43.5° and 59.8° which correspond to the (100), (101), (102) and (103) reflection of fullerene‐containing carbon, respectively, that was in an important contract using the JCPDS file no. 36121 and it is indexed in the shape of hexagonal wurtzite of ZnO NPs. The XRD results revealed that the formed ZnO NPs were naturally crystalline. A high‐intensity peak indicated the anisotropic growth and crystallites orientation of the ZnO NPs at (101) (Fig. 4). These results were in line with the study of (100), (002), (101), (200), (112) and (202) [48]. Similar to this, the study of Sangeetha et al. [47] also highlight the same finding. The structure of the ZnO NPs was confirmed as a hexagonal wurtzite structure, and stiff and narrow diffraction peaks confirmed the crystalline nature of the NPs. Scherrer's formula was used to calculate crystalline size which resulted in 45–57 nm. According to the report of the average particle size in the range of 43–56 nm, the average crystalline size is 48.6 nm [50]. The synthesised NP’ crystalline nature was matching in the previous reports through Miller leaf extract of Aloe barbadensis for the process of synthesising the ZnO NPs’ [47] SEM of the ZnO NPs synthesised by the reduction of Zn (NO₃)₂ revealed the spherical and hexagonal NPs ranging from 35 to 112 nm with 49 ± 0.2 nm as average size (Fig. 5). EDAX spectroscopy of ZnO NPs has clearly explained the presence of ZnO. The chemical profile of ZnO NPs was analysed using EDAX (Fig. 6). SEM of ZnO also revealed the presence of NP agglomerates. [51]. AFM analysis detailed the size and shape of NPs. Fig. 7 reveals the smooth NPs by phytochemical capping for the surface of NPs.

Fig. 4.

XRD pattern of ZnO NPs synthesised using L. siceraria extract

Fig. 5.

SEM of ZnO NPs synthesised using the L. siceraria extract

Fig. 6.

EDAX spectrum of ZnO NPs synthesised using the L. siceraria extract

Fig. 7.

AFM of ZnO NPs synthesised using the L. siceraria extract

Dynamic light scattering result gives the information about the size distribution of NPs. Fig. 8 indicates the size of ZnO NPs situated in between 53 and 144 nm range with an average particle size 81.8 nm of ZnO NPs. The average hydrodynamic diameter as calculated by DLS is 81.8 nm, which is quite larger than the sizes reported by XRD. Synthesised ZnO NPs are highly crystalline, hexagonal in shape with an average size 81.8 nm and stable for 2 months at 4 C due to high zeta potential (26.6 mV) under optimum conditions (Fig. 9). Polydispersity is used to describe the degree of ‘non‐uniformity’ of a distribution and in the case of NP suspensions could be related to the occurrence of NPs as aggregates or agglomerates, in turn causing variability in calculated particle size in comparison with the actual size of the particles.

Fig. 8.

DLS of ZnO NPs synthesised using the L. siceraria extract

Fig. 9.

Zeta potential of ZnO NPs synthesised using the L. siceraria extract

3.2 Larvicidal, pupicidal and ovicidal activities

In laboratory conditions, L. siceraria aqueous peel extract and ZnO NPs was discovered as poisonous against the fourth‐instar larvae of A. stephensi. For L. siceraria, peel extract the LC₅₀ values were found to be 185.78 ppm, LC₉₀ were found to be 389.05 ppm (Table 2) and LC₅₀ for synthesised ZnO NPs were found to be 3.873 ppm, and LC₉₀ were found to be 8.83 ppm (Table 2). The comparative LC₉₀ and LC₅₀ values of Plectranthus amboinicus‐ synthesised ZnO NPs with the fourth‐instar larvae of A. stephensi were 4.6 and 3.11 mg/l correspondingly [52] Sargassum wightii ‐synthesised ZnO NP was extremely functional in the laboratory experiments that were executed against the pupae and larvae of A. stephensi. LC₅₀ was found to be 4.330 ppm (I), 5.057 ppm (II), 5.696 ppm (III), 6.434 ppm (IV) and 7.430 ppm (pupae) [53].

Table 2.

Larvicidal activities of L. siceraria extract and L. siceraria ‐synthesised ZnO NPs against A. stephensi

Treatment	LC50, ppm	LC90, ppm	Regression equation	Slope	r 2	P value
L. siceraria plant extract	190.54	1096.4	y  = 1.945x  + 0.5591	1.945	0.962	0.031
ZnO NPs	3.491	7.834	y  = 3.653x  + 3.013	3.653	0.935	0.0072

ZnO was the most commonly used NPs as they have usages in a large diversity of sectors starting from the personal care products to catalysts and coatings in environmental remediation [54, 55]. The research paper of Banumathi et al. [56] recommend that the Lobelia leschenaultiana plant is a dependable agent source to create nanomaterials containing high toxic level against Aedes aegypti larvae, which is green‐capped ZnO NPs shows 100% mortality at 10 mg/l, despite the fact that at 60 mg/l the zinc acetate shows only 65.33% mortality. The researchers of Elumalai et al. [57] has examined the activities of larvicide with silver NPs of green synthesised by the means of Achyranthes aspera leaf extract for the fourth‐instar larvae of A. aegypti, A. stephensi and Culex quinquefasciatus attaining the LC₅₀ values of 3.68 and 2.48 mg/ml correspondingly.

Table 3 illustrates the significant pupal mortality after treatment with aqueous peel extract of L. siceraria and ZnO NPs. The LC₅₀ and LC₉₀ values of the extracts and ZnO NPs against the pupae of A. stephensi at 48 h are provided in Table 3. Valentina et al. [58] reported that the aqueous extract of selected seaweeds led to high mortality on treated larvae and pupae.

Table 3.

Pupicidal activities of L. siceraria extract and L. siceraria ‐synthesised ZnO NPs against A. stephensi

Treatment	LC50, ppm	LC90, ppm	Regression equation	Slope	r 2	P value
L. siceraria plant extract	202.30	426.57	y  = 3.924x  − 4.051	3.924	0.974	0.0018
ZnO NPs	2.328	6.501	y  = 2.868x  + 3.946	2.868	0.7903	0.0436

Furthermore, the aqueous peel extract of L. siceraria exerted 95% ovicidal activity at 500 ppm against A. stephensi and higher toxicity was found for ZnO NPs (Table 4). Similarly, the ovicidal consequences of different extracts from the Annona reticulata against C. Quinquefasciatus, A. stephensi and A. Aegypti were successfully investigated [59]. Additionally, stated that the Terminalia chebula leaf extract would possess remarkable activities of larvicide and ovicide against C. Quinquefasciatus, A. stephensi and A. aegypti [60]. Saraf and Dixit examined that ethanolic extract of flower heads of Spilanthes acmella is having potent pupicidal, larvicidal and ovicidal activities [27].

Table 4.

Ovicidal activities of L. siceraria extract and L. siceraria ‐synthesised ZnO NPs ZnO NPs against A. stephensi

Treatment	LC50, ppm	LC90, ppm	Regression equation	Slope	r 2	P
L. siceraria plant extract	185.78	389.05	y  = 4.04x  + (−4.17)	4.04	0.85	0.0254
ZnO NPs	3.873	8.83	y  = 3.571x  + (2.9)	3.571	0.821	0.0342

3.3 Repellent activity

The aqueous components of L. siceraria peel extract and ZnO NPs showed significant repellency against A. stephensi (Table 5). The results imply that L. siceraria peel extract and ZnO NPs gave protection against mosquito bites, and additionally the repelling activities mainly rely on the ZnO NPs and plant extract. Reported that the crude extracts of Andrographis paniculata and Eclipta alba were actually operative for controlling A. stephensi.

Table 5.

Repellent activities of L. siceraria extract and L. siceraria ‐synthesised ZnO NPs against A. stephensi

Treatment	Concentration, ppm	Repellency, %
		30 min	60 min	90 min	120 min	150 min	180 min
L. siceraria plant extract	100	79 ± 0.57 d	74 ± 2.6 d	71 ± 1.86 d	68 ± 1.67 e	62 ± 1.91 c	57 ± 1.14 c
	200	83 ± 1.76 d	78 ± 0.57 cd	73 ± 1.46 d	69 ± 1.87 d	64 ± 1.94 bc	61 ± 1.40 c
	300	89 ± 0.88 c	82 ± 1.92 c	77 ± 1.64 c	73 ± 1.70 c	67 ± 1.92 b	69 ± 2.58 b
	400	95 ± 0.33 b	89 ± 1.84 b	84 ± 1.80 b	79 ± 1.76 b	74 ± 1.48 a	71 ± 2.12 ab
	500	100 ± 0.00 a	100 ± 0.00 a	92 ± 1.64 a	81 ± 1.69 a	79 ± 1.69 a	75 ± 1.42 a
ZnO NPs	2	96 ± 1.73 b	91 ± 1.00 c	84 ± 2.82 d	71 ± 2.00 d	62 ± 0.70 c	60 ± 1.41 d
	4	99 ± 2.00 a	93 ± 1.73 bc	89 ± 1.41 c	77 ± 1.84 c	66 ± 1.69 c	64 ± 0.88 d
	6	100 ± 0.00 a	98 ± 0.82 b	93 ± 1.69 c	85 ± 1.58 b	71 ± 2.00 b	70 ± 2.82 c
	8	100 ± 0.00 a	100 ± 0.00 a	97 ± 0.57 b	94 ± 2.82 a	91 ± 1.41 a	87 ± 0.57 b
	10	100 ± 0.00 a	100 ± 0.00 a	100 ± 0.00 a	98 ± 0.70 a	94 ± 1.84 a	92 ± 0.88 a

Within each treatment and column, different letters indicate significant differences (Tukey's HSD, P  < 0.05).

3.4 Smoke toxicity assays against A. stephensi

Table 6 tells about the experiment results on the toxic level of smoking by examining the effectiveness of L. siceraria‐ based cones against A. stephensi. After treatment, the average percentage of unfed mosquitoes was found to be 51.6%. About 93.6% mortality was found in commercial coils which serve as a positive control. Conversely, cones on the basis of green may be possibly valuable substitutes to those based on permethrin. Suresh conducted the smoke toxicity test with Phyllanthus niruri against A. aegypti [35].

Table 6.

Smoke toxicity testing L. siceraria – based cone assays on A. stephensi

Treatment	Fed mosquitoes	Unfed mosquitoes		Total unfed
		Alive	Dead
L. siceraria‐ based cone	23.6 ± 0.33 b	26.3 ± 0.33 b	51.6 ± 0.66 b	77.3 ± 0.33 b
positive control	7.00 ± 1.0 a	38.3 ± 1.20 c	55.6 ± 0.33 c	93.6 ± 0.33 c
negative control	89.3 ± 1.20 c	11.8 ± 2.12 a	0.00 ± 0.00 a	11.1 ± 0.32 a

Within each column, different letters indicate significant differences (Tukey's HSD, P  < 0.05).

3.5 Mechanism of ZnO NPs and plant extract on mosquito larvae

Considering the larvicidal activity of green‐fabricated ZnO NPs, it is essential to recognise their working mechanism. The impact of the ZnO NPs and plant extract on the biochemical components characterising fourth‐instar larvae of A. stephensi was investigated. Similarly, Fouad et al. reported the effect of Ag NPs and the plant extract on the biochemical constituents of fourth‐instar larvae of Aedes albopictus and C. pipiens pallens. The authors found that the tested samples caused variation in normal biochemical constituents with either an increase or decrease in their activity as compared with control. Xie et al. [61] revealed that the mechanism of action of ZnO NPs against C. jejuni.

3.5.1 Quantitative analyses of biochemical constituents

The exposure of the larvae of A. Stephensi to ZnO NPs and L. siceraria peel extract for 24 h significantly reduced (P  < 0.05) the level of total proteins when compared with control. The values are represented in Fig. 10 a. Therefore, the metabolism of protein in the A. Stephensi’ larvae is kept open to ZnO NPs, and plant extract was found to be disturbed. These results propose a direct toxic effect of ZnO NPs in the synthetic protein machinery of mosquito larvae. The exposure of the larvae of A. stephensi to ZnO NPs and plant extract for 24 h significantly decreased (P  < 0.05) the level of acetylcholinesterase activity (Fig. 10 b). The exposure of the larvae of A. stephensi to ZnO NPs and plant extract for 24 h inhibited (P  < 0.05) the activity of α ‐ and β ‐carboxylesterase. The inhibition level is decreased eventually and almost remained at the same level throughout the development period of larvae (Fig. 10 c). The level of acid and alkaline phosphatase also decreased (P  < 0.05) during the development of fourth‐instar larvae (Fig. 10 d).

Fig. 10.

Impact of the aqueous L. siceraria extract and green‐fabricated ZnO NPs

(a) Quantitative analysis – protein estimation, (b) Quantitative analysis – acetylcholineesterase assay, (c) Quantitative analysis – carboxylestersae assays (μg/ml), (d) Quantitative analysis – acid and alkaline phosphatase assays

Impact of the aqueous L. siceraria extract and green‐fabricated ZnO NPs treated fourth‐instar larvae of A. stephensi showing differential responses in total protein, acetylcholine esterase, carboxylesterase and acid and alkaline phosphatase assays (Fig. 10).

4 Conclusion

Herein, it was evaluated the possible role of L. siceraria aqueous peel extract and synthesised ZnO NPs in controlling malaria mosquitoes. In this paper, ZnO NPs were green synthesised using an aqueous peel extract of L. siceraria as reducing and capping agent. This study showed that L. siceraria‐ synthesised ZnO NPs production is easy and could be used at very low level of doses to handle the populaces of vectors of malaria. Moreover, coils based on L. siceraria make smoke repellents that could be safer and cheaper while comparing with the current commercial available coils. Their mechanisms of action were investigated on mosquito larvae, adding knowledge to how NP affects mosquito cellular physiology.