Biogenic Synthesis of Silver Nanoparticles Using Guava (Psidium guajava) Leaf Extract and Its Larvicidal Action against Anopheles gambiae

The progress in the field of nanotechnology has contributed to the development of tools for combating the most critical problems in developing countries. The requirements that such tools should meet are low-cost and resource settings, environmental protection, ease of use, and availability. The use of plant properties for the generation of nanoparticles (NPs), which serve as bioinsecticides to combat the plasticity and resistance of mosquitoes and parasites, is considered possible. Here, we report for the first time the larvicidal activity of silver (Ag) NPs (AgNPs) synthesized from Psidium guajava (P. guajava) extract, which targets the 4 instar larvae of Anopheles gambiae. Concentrations of AgNPs between 0 and 200 ppm were used and their LC50 at 24 h and 48 h were determined as 19.55 ppm and 8.737 ppm, respectively. The AgNPs were stable and highly effective against the larvae of A. gambiae and thereby we anticipate that they can be used to combat vector-borne diseases in developing countries.


Introduction
Mosquitoes are the principal vector of vector-borne diseases, which affect human beings and animals [1]. Diseases transmitted by mosquitoes lead to commercial and labor output losses, particularly in countries with tropical and subtropical climates [2]. Mosquitoes represent a huge threat for millions of people worldwide, since they spread various tropical diseases, especially malaria, which is transmitted by female A. gambiae mosquitoes [3].
Infected female Anopheles mosquitoes transmit malaria parasites to people and animals via their bites during their blood meal. Marked progress has been achieved in malaria control, including the discovery of artemisinin (for which the Nobel Prize was awarded to Y. Tu), development of the first vaccine against Plasmodium falciparum malaria, and decrease in the rate of malaria infections worldwide and particularly in sub-Saharan Africa, which contributes to the bulk of malaria burden [4] [5] [6]. However, resistance to existing antimalarial drugs, such as the gold-standard medication artemisinin, particularly in the Greater Mekong sub-region in Southeast Asia, is a growing problem [7], which has hampered the global progress in malaria control. In 2016, the malaria cases were estimated to be 216 million, with an increase of about 5 million cases compared to 2015; the death rates in both years reached approximately 445,000. The majority of the malaria cases (92%) and related deaths (93%) occurred in Africa [5], where the principal vectors of malaria are A. gambiae sensu stricto (s.s.) and Anopheles arabiensis [8].
Synthetic insecticides are widely used to control insect spread as indoor insecticides and residual spraying in treated nets [9]. Their abuse leads to both human and environmental toxicity, thereby potentially eliminating non-target organisms [10]. The adaptation of mosquitoes to new environmental conditions is a result of the development of physiological resistance, and alternative selective measures to prevent such resistance are urgently needed. Vector control is a crucial necessity in epidemic situations. The new methods for mosquito control must be both economical and efficient, while being safe for non-target organisms and the environment. They must be adapted to the conditions prevailing in endemic countries [11]. The use of impregnated mosquito nets or indoor spays are measures to slow the transmission of the disease by killing or preventing infected mosquitoes from biting humans [12]. Secondary metabolites of various plants including Azadirachta indica (neem), Clerodendron infortunatumis (glorybower), Schoenocaulon officinale (neotropical lily), and Chrysanthemum pyrethrum (African daisy) [13] [14] have been used for controlling the spread of mosquitoes [1] [15]. Since most malaria-affected countries are poor, the main challenges are to reduce the costs of the toxicological tests and to make the biopesticides available despite the low incomes and economic weakness of these markets, as well as to limit intellectual property. Other factors include the quality control and lack of stability of these metabolites depending on the environmental conditions. In addition, there is competition with other biopesticides and A. A. Ntoumba et al. Journal of Biomaterials and Nanobiotechnology biocontrol agents which reduce their efficiency [14]. Moreover, movements in the global distribution and burden of infectious diseases with climate change are observed [16]. By generating NPs obtained from plant metabolites with therapeutic potential, the scientific community is aiming to overcome these challenges and to develop biocontrol agents against mosquitoes and microbes. Plant extracts are considered eco-friendly bioreactors due to the simple process of Ag + reduction. Studies have shown that when present in the reaction mixture surface-active molecules or stabilizers such as ionic liquids create electrostatic interactions, thereby increasing the stability of the NPs [17]. Controlling the NP/secondary metabolite interface would make it possible to modulate the nanostructure and to adapt the properties of the materials for specific applications. The number of studies focusing on the cost-effective use of nanomaterials for human health is increasing rapidly [12]. Nanotechnologies have the potential to revolutionize pest control and larval management. The production of plant-based NPs is advantageous over chemical and physical methods, since it is cheap, single-step, and does not require high pressure, energy, temperature, or the use of highly toxic chemicals [18]. In the present study, we report for the first time the larvicidal action of green Ag NPs synthesized from P. guajava L. leaf extract against 4 th instar larvae of A. gambiae (s.l.). The efficacy of NPs was compared to that of their precursors, namely, plant extract and Ag + . In the bioassay, the P. guajava leaf extract was the dispersion medium, capping, and reducing agent.
P. guajava and their AgNPs P. guajava (Myrtaceae) is a native bush species from South America known as "goiaba", which is commonly used in traditional medicine. Among the conditions treated with goiaba are gastrointestinal infections; malaria, respiratory infections, oral and dental infections, skin infections, diabetes, cardiovascular disease and hypertension, cancer, malnutrition, gynecological issues, pain, fever, and liver and kidney conditions [19]. The following two varieties of P. guajava are commonly cultivated: P. guajava var. pomifera and P. guajava var. pyrifera. The fruit of P. guajava is highly appreciated in the tropical and subtropical cuisine and used widely in traditional medicine [20]. The P. guajava is a small-branched tree with smooth, mottled bark that can peel off in flakes. Its leaves (6 inches long and 3 inches wide) are aromatic and oppositely arranged along the stems with prominent lateral veins on the dorsal side [21]. Moreover, QG5 helps against acute non-infectious diarrhea and menstrual colic.
Previous studies have characterized the synthesis process of AgNPs from P. guajava leaf extracts (Table 1 and references therein [27]- [50]). Potent antimicrobial action [46] [47] [49] [50], cytotoxicity [34], and dye fabric degradation [44] of AgNPs have been described. This has resulted in the formulation of the following guidelines: 1) plant extracts can be obtained by aging, sohxlet extraction, microwave, or ultrasound methods; 2) 1 mM Ag nitrate (AgNO 3 ) is a favorable concentration for the reaction; 3) the reaction condition and state of agglomeration have plasmon resonance bands between 380 and 490 nm, as obtained using UV-Vis spectroscopy; 4) the stability in water of the AgNPs obtained from P. guajava extract is up to 30 weeks; 5) rapid synthesis as the use of microwave heating tends to produce pure AgNPs; 6) TEM shows nanometer range spherical NPs while SEM shows aggregates; and 7) IR spectroscopy is an appropriate method to validate biomolecule presence at metallic interface.

Materials and Methods
Plant collection and preparation of the extract Leaves of Psidium guajava L. (Figure 1) were collected at Massoumbou 2885/SRFK). The plant extract was obtained according to a previously published method [29]. The plant reactor was used for not more than 1 week to avoid the gradual loss of viability due to prolonged storage [51]. The extract concentration was determined as per previously reported procedures [52].

Biosynthesis of AgNPs
The AgNPs were synthesized as previously described with slight modifications [27]. The bioreduction process was performed by adding 10 mL of freshly prepared aqueous extract to a 50 mL aqueous solution of AgNO 3 (1 mM). The mixture was incubated 5 h at 25˚C -28˚C in dark to minimize the photo activation of AgNO 3 . The incubation was performed under static conditions until the color changed to brown (Figure 2). The mixture was then centrifuged (D-7200; Hettich, Tuttlingen, Germany) at 7000 rpm for 1 h and washed twice with distilled water and once with 95% ethanol. Reaction was verified by treating the obtained filtrate with sodium chloride. Purified pellets were placed in a petri dish, dried in an oven at 60˚C for 24 h, and used for NP characterization. The characterization of the AgNPs is in the supplement material: see Figure A1 (UV-Vis), Figure A2 (IR), Figure A3 (PXRD), Figure A4 (DLS) and Figure A5 (SEM and EDX).

Evaluation of larvicidal activities
Eggs of the susceptible Anopheles gambiae (Kisumu strain) were obtained from the Organisation de Coordination pour la lutte contre les Endémies en Afrique central, Yaounde, Cameroon. They were maintained and reared in the Insectarium of the University of Douala, Faculty of Medicine and Pharmaceutical Sciences to obtain 4 th instar larvae. The larvicidal activity of the AgNPs produced from Psidium guajava extract was determined following the standard test procedures of the WHO [53] with some modifications. For the bioassay, 20 4 th instar larvae were placed in plastic bowls (6 cm diameter, 120 mL capacity) with distilled water in 4 replicates. The controls were set up with distilled water, Psidium guajava plant extract, and AgNO 3 at ambient temperature, or AgNO 3 in the dark. Different concentrations of AgNO 3 in the range of 0 -200 ppm were prepared through serial dilutions of 100 mL each. The experiments were carried out at 27˚C ± 2˚C, relative humidity of 75% ± 5%, and a photoperiod of 14 h/10h (light/dark). Larvae were considered dead if they did not respond to contact. The number of dead larvae was counted 24 h and 48 h after treatment and the percentage of mortality was computed as follows: Journal of Biomaterials and Nanobiotechnology

Results and Discussion
Larvicidal activity of synthesized AgNPs P. guajava plant was selected for this study because of its accessibility and word wide distribution, thereby allowing easy translation of the results from lab scale to industrial scale. Different AgNP synthetic schemes have been previously developed in India, China, and Thailand ( Table 1). The synthetic schema, which we selected, is oriented toward environment preservation; in the current study, water was used as solvent and the NP production method used was aging. We obtained 2.42 g/L concentration of P. guajava plant extract, which was used for the synthesis of AgNPs and AgClNPs (supplement 1). Possible reaction schemes leading to the mixtures of Ag and AgCl were described by Awwad and coworkers [54] and by our group [55]. Early 4 th instar larvae of Anopheles gambiae were treated with biosynthesized AgNPs in various concentrations ranging between 0 and 200 ppm and the mortality percentage was assessed. The LC 50 values of AgNPs were determined as 19.55 ppm and 8.737 ppm at 24 h and 48 h, respectively ( Figure 3). The analysis of the larvicidal activity is shown in Table 2 and the mortality percentage is depicted in Figure 4. P. guajava plant extract did not cause larval mortality at all tested concentrations. When used at different concentrations, both photo-activated AgNO 3 and AgNO 3 in the dark killed all larvae of A. gambiae. Mondal and colleagues have previously described the mortality of Culex quiquefasciatus in response to a 10 ppm AgNO 3 solution. At 24 h the mortality rate was 12.5%, at 48 h was 13.04%, Journal of Biomaterials and Nanobiotechnology and at 72 h was 21.74% [56]. The Ag + are accumulated in various organisms (plants, herbivorous organisms, or fishes) isn't environment fiendly [57]. Since the AgNPs aggregate and agglomerate quickly, their isolation and resuspension in water appeared unsuccessful. The plant extract, which we used here, served as a green dispersant and played a capping role, as proved by infrared or energy-dispersive X-ray spectroscopy experiments. Nowadays, environmental safety is crucial when developing novel strategies for combating vector-borne diseases.
An insecticide should be ecofriendly in nature and acceptable by the community to cause the desired mortality against target organisms [2].
The advantages of using the developed here AgNPs as larvicidal substances are that small active quantities are required and that the resistance due to the excessive use of pesticides can be overcome [56]. Ponraj

Conclusion
Vector control is one of the most serious concerns in developing countries and

Funding
CSC provided help for PXRD and DAAD provide support for SEM, EDX, and DLS.

Availability of Data and Materials
All data generated or analyzed during this study are included in this published article and its additional files. nanoparticles and the presence of nano-silver elements was confirmed by EDX at 20 keV. EDX qualitative spectrum shows a strong silver peak (3 kev) along with chloride, oxygen, carbon as main elements. Figure A1. Ultraviolet-visible spectra 1 hour analysis of synthesized nanoparticles. Figure A2. Fourier transform infrared spectrum for synthesized silver nanoparticles using dry plant power (up) and silver nanoparticles (down). Figure A3. X-ray diffraction pattern of the nanoparticles from Psidium guajava; • represents silver nanocrystallites and □ represents silver chloride nanocrystallites. Journal of Biomaterials and Nanobiotechnology  Graphical abstract.