We investigated the effects of phytochemical, ascorbic acid and total phenolic content on reduction of silver ions from plant materials and the toxicity of these silver ions on Drosophila melanogaster as an in vivo model system. In the presence of ascorbic acid concentration 38.88µg/gm and 35.5088µg/gm and the total phenolic contents concentration 97.89µg/gm and 26.99µg/gm in Chenopodium and Marigold extracts respectively, the multiple plasmon peaks were observed at the range of 360-426nm with absorbance of 0.780- 0.925cm-1 in Chenopodium extract with absorbance 0.392-0.569 cm-1 in marigold extract. The appearance of multiple surface  plasmon peaks indicated the spherical shape of silver nanoparticles (primary characterization). Obtained multiple plasmon resonances at 360- 426nm and 310-450nm in Chenopodium and Marigold extracts, respectively may be indication of maximum reduction of Ag+ and this enhancement of maximum reduction of Ag+ due to the presence of ascorbic acid and total phenolic content higher concentrations. On the other hand, the quantitative measurement of AgNPs toxicity on Drosophila melanogaster a significant concentration-dependent decrease in survival rate, duration of development and melanization defects were observed for flies fed with AgNPs as compared to  control Drosophila flies.

Drosophila melanogaster, Nanotoxicity, Silver nanoparticles, Ascorbic acid, Total phenolic contents,  Pigmentation

Biosynthesis of nanoparticles using plant extract is a cost-effective approach because the preparation of plant extract normally utilizes biomass wastes such as leaves, flowers, roots, fruit peels, etc. These parts of plant can   be used either fresh or dried. But the dry form is more preferable due to differences in water content within different plant tissues (Tiwari et al. 2011). Nanotechnology and nanofabrication has opened its doors to a world of metal nanoparticles synthesis with easy preparation protocols, less toxicity and a wide range of applications according to their size and shape (Tyagi et al. 2012). Controllability in biological methods is far easier to achieve than with other methods (Tyagi 2016).In recent years, noble metal nanoparticles have been the subject of focused research due to their unique optical, electronic, mechanical, magnetic and chemical properties that  are  significantly different from those of bulk materials. Silver nanoparticles play a profound role in the field of biology and medicines due to their attractive physiochemical properties. There has been intense interest recently among  the public and the media in the possibility that increased intake of dietary antioxidants may protect against chronic diseases, which include cancers, cardiovascular, and cerebrovascular  diseases.  Antioxidants  are  substances  that, when present at low concentrations, compared with those of an oxidizable substrate, significantly prevent or delay a pro-oxidant–initiated oxidation of the substrate (Prior & Cao 1999). A pro-oxidant is a toxic substance that can cause oxidative damage to lipids, proteins, and nucleic acids, resulting in various pathological events or diseases. The synthesis of metallic nanoparticles can be done by reducing metal ion using some chemical molecules. Plants contain an ample of free radical scavenging molecules such as phenolic compounds, nitrogen compounds,vitamins, reducing sugar, terpenoids and some other metabolites that are rich in antioxidant activity. The plants used to synthesize nanoparticles are known to be rich in polyols and antioxidant. The hydroxyl and carboxylic group present in plants may act as reducing agent and stabilizing agents in the synthesis of nanoparticles (Vilchis-Nestor et al. 2008). Song and his co-workers reported that for the M. Kobus extract, the proteins and terpenoids are believed to act as reducing agent (Song et al. 2009). According to Amin et al. functional groups such as phenolics and alkaloids are responsible for capping and stabilizing of nanoparticles reduced (Amin et al. 2012).The reduction mechanism is also capable to controlling the size and stability of the nanostructured produced. The stability of nanoparticles can attributed to the formation of stable bonding between metallic nanoparticles and phytochemicals present in the leaf extract (Kanchana et al. 2010).

Nanoparticles can modify the physicochemical properties of the material as well as create the opportunity for increased uptake and interaction with biological tissues. This combination of effects can generate adverse biological effects in living cells that would not otherwise be possible with the same material in larger form. Nanoparticles have the ability to cross biological membranes and access the cells, tissues and organs through inhalation or ingestion. The toxicological studies indicates that toxicity percentage inhabitation of chemically synthesized AgNPs was much greater than the biologically synthesized AgNPs synthesized from apple onion, garlic and followed by papaya and observed PI value indicated that the gut microbial community probiotic B. subtilis and E. coli was killed in higher percentage of chemically synthesized AgNPs as compare to biologically synthesized AgNPs synthesized from apple, onion, garlic and papaya(Tyagi et al. 2013 a, 2016a). Bio-AgNPs is the most suitable metallic coating material coat to drugs instead of CH-AgNPs in pharmaceutical industries (Tyagi et al. 2013b).The Drosophila has been chosen as a model organism to study the toxic effects of nanoparticles. Today’s the Drosophila has quickly attracted the attention of many research groups, establishing  it  as  the standard model organisms in the field of nanotoxicology research. In this context, a particular example is represented by gold nanoparticles (AuNPs), which displayed significant toxicity, despite the well-known biocompatibility of gold in bulk form (Sabella et al. 2011). In fact, it has been observed that AuNPs administered by ingestion to Drosophila were equally distributed along various organs and tissues, causing a strong reduction   in lifespan and fertility of flies (Pompa et al. 2011), and disorder in gene expression (Vecchio et al. 2012), concentration-dependent (Tyagi et. al. 2016) and metabolism (Wang et al. 2012). However, the most striking  result obtained was during the analysis of the effects induced by AuNPs in Drosophila was the discovery of aberrant phenotypes in the untreated progeny derived from flies fed with nanoparticles.

10gm of Chenopodium leaves and Marigold leaves were taken and thoroughly washed in distilled water. Washed leaves were crushed with motor pestle and then mixed into sterile deionize water after that it was boiled for around 10min. Extract of samples were filtered through whatman filter paper (Pore size 45 µm) and centrifuged for 10min at 4000rpm. The aforementioned extracts were used immediately for biosynthesis of silver nanoparticles.

In this study AgNPs were synthesized from Chenopodium and Marigold which are easily available and exhibit medicinal properties. Bio reduction is main phenomenon which is responsible for the NPs synthesis. Extracts prepared from these plants have good amount of various kind of sugar, proteins, vitamins, ascorbic acid and phenolic substances. Previous studies indicated that various kind of reducing agents are present in the plant  extract which provide as chance for ecofriendly synthesis of nanoparticles. During the synthesis of AgNPs from chili researchers believed that polyphenols and ascorbic acid played a significant role in synthesis of AgNPs (Jha& Prasad 2011).

Ascorbic acid concentration was determined after biosynthesis of AgNPs from samples. Results indicated that ascorbic acid concentration 38.88µg/gm and 35.5088µg/gm were observed in Chenopodium and Marigold  extracts respectively. On the other hand, the total phenolic contents concentration 97.89µg/gm and 26.99µg/gm were found in Chenopodium and Marigold extracts respectively (Table 1). The multiple plasmon peaks were observed at the range of 360-426nm with absorbance range of 0.780-0.925cm-1 in Chenopodium extract and 310-450nm with absorbance range 0.392-0.569 cm-1 in marigold extract. As shown in Figure 1a and 1b, the appearance of the wide range in absorptions and UV-visible nanometer scale indicated to enhancement of Ag+ reduction (found multiple plasmon peaks) in the reaction mixture of both Chenopodium and Marigold extracts. Obtained results, indicated that the phytochemical ascorbic acid and total phenolic  contents  concentrations present in the plant extracts enhance the reduction of Ag+ in reaction mixture. We observed at the time of measuring  the  concentration  of  some  samples  at  UV-VIS  spectrophotometer  the  variation  in  concentration(µg/gm) of phytochemicals (ascorbic acid and total phenolic contents) were found. Those samples that contained minimum concentration of ascorbic acid and total phenolic contents no Plasmon resonance was observed and a curved single peak was observed. Samples which have maximum concentration of ascorbic acid  and  total phenolic contents multiple plasmon resonance was observed. Some samples were not following this observation so it may need further investigation. On the other hand, the multiple surface plasmon peaks representing the spherical shape of AgNPs (primary characterization).As shown in figure 2a &2b, multiple plasmon resonances were found to enhance the absorption in the presence of ascorbic acid and total phenolic content concentration over a broad wavelength range. In previous data some researchers indicated that ascorbic acid is  main factor which responsible for biosynthesis of silver nanoparticles (Caroling et al. 2013). It is proven in various studies that biochemicals in plant leaves play significant role in reduction of ionic salts. Bioreduction is understood as main cause by which nanoparticles are synthesized. There are various kind of reductants used for chemical synthesis   of nanoparticles (Tyagi et al. 2013b). Chemical reductants are costly and toxic some of them may produce toxic byproducts. 

The most common method to evaluate nanoparticle toxicity in the Drosophila model is through the determination        of survivorship after nanoparticle exposure. As nanoparticle can  gain  entry into the human body by several  ways  such as oral, dermal and inhalation routes, it is essential that such entries are modulated in the Drosophila model.

One possible route of AgNPs exposure to Drosophila is through ingestion. For instance, AgNPs can be added directly to the standard food medium of Drosophila at various concentrations (20%, 50%, 70% and 100%). Newly emerged healthy adult flies were transferred directly to the food containing different concentrations of AgNPs and survivorship and duration of development were accounted every 12 h for a period of 20 days. A significant concentration-dependent decrease in survivorship and duration of development were observed for flies fed with AgNPs as compared to control flies (fed normal maize medium). On the other hand, food without AgNPS did not affect the survivorship and duration of development of flies, except in some replicates having with higher concentration but no significant variation observed, suggesting that the toxicity was caused specifically by AgNPs. The studies of dose-dependent silver nanoparticle toxicity on Drosophila complement our results (Tian et al. 2013). For the study of survivorship and duration of development the 10 Drosophila eggs were exposed to different concentration of AgNPs contain food a significant effect on survivorship was observed during the hatchability and viability stages. Many of the resulting eggs failed to hatch, larvae failed to pupate, and time to pupation was delayed so the duration of development increases so many days. In addition, there was a decrease    in the number of flies leaving the pupa stage and emerging as adult flies compared to control flies. Higher concentrations of AgNPs (100%) was more toxic for eggs as well as larval stages, only 50% hatchability were recorded (5 larvae emerges out of 10egg) and 20% viability (2 flies emerges out of 5 larvae) in flies. Results of 70% and 50% concentrations are very closer with 70% hatchability and 60% viability and 70% both hatchability and viability were recorded respectively. On the other hand, the 20% concentration was showing less toxic effect of eggs or larval stages with 90% hatchability and 80% viability (Figure 3&4). Times to duration of development were slowed by nano-silver ingestion in an increasing concentration of AgNPs. The time taken by flies for

emergence at 20% is 13 days followed by 50% for 16 days , 70% for 17 days, 100% for 20days at 230C. On the other hand, the control flies emerges within 11 days for the same temperature (Table 2).

The adult Drosophila flies fed with different concentration of AgNP containing food during larval stage exhibits  defects in cuticle development and melanization. Drosophila that  ingested  AgNPs  has non-pigmented soft cuticle     as compared to controls (Armstrong et al. 2013; Silver Key et al. 2011; Panacek et al. 2011; Philbrook et al. 2011; Posgai et al. 2011). The pigmentation defect is due to the influence of AgNPs on copper transporters. Excess Ag   results in competitive inhibition of copper uptake at the copper  transporters, causing a depletion of  copper  in cells.  As the copper-dependent tyrosinase is required for melanin synthesis, its decline in activity may be the reason for AgNP-induced pigmentation defect (Armstrong et  al. 2013). Indeed,  the  phenotypic modification was first reported  by Rapoport and the reduction in body pigmentation was thought to be related to treatment with silver salts  (Di  Stefano 1943). Flies that survived higher concentration (100%) of AgNPs ingestion had a score of zero pigmented cuticles. No such effect was observed in normal-fed flies as  control.  On  the  other  hands,  the  control  flies  has higher pigmented in all abdominal segments  as  well  as  thoracic  segments  as  compare to  AgNPs  feds  flies.  On  the other hands, zero pigmented flies was observed in 100% AgNPs  treated  feds,  light  pigmented  flies  was  observed in 70% AgNPs treated feds slightly  significant  differences  observed  in  70%  and  50%  AgNPs  treated feds, more pigmented flies was observed in 20% AgNPs treated,  its pigmentation score  is near  to control  flies  (Figure 5). As epidermal pigments are secreted by the cuticle, the cuticle defect is likely the root cause of these phenotypes.

This work was supported in part by a grant from the Department of Science and Technology under the Women Scientist  Scheme-A (WOS-A)project no  SR/WOS-A/LS-1171/2014 government of  India.