Green nanocolloids play an important role in cancer diagnosis, antibacterial activity, antimicrobial, anticancer, antifungal and antiviral etc., (Iravani et al. 2014) The green nanotechnology paved the way for the development of eco-friendly nano-based solution to plants, animals and human beings. Herein we developed green silver nanocolloid using citronella extract. Citronella contains various phytocompounds such as steroids, phenols, tannins, alkaloids, flavonoids, phytosteroids, glycosides, which could be harnessed for its antibacterial, antifungal and anticancer activity. These phytochemicals of Citronella paved the way to explore more on drug development and its action on treating several diseases. The phytochemical tests are also consistent with previous literatures through confirming the presence of bioactive compounds such as alkaloids, anthocyanins, carotenoids, flavonoids, phenols, tannins, terpenoids, saponins and steroids. Plant phenols possess antioxidant activity which helps in reducing free radicals and it interferes with all stages and types of cancer due to its antioxidant activity. Flavonoids can inhibit the formation of peroxides. Phenols, Tannins, flavones, flavonoids, alkaloids, terpenoids and saponins possess significant antibacterial, antiviral, anti-inflammatory and anti-tumor properties (Shashank Kumar et al. 2013).
Biogenesis of CANCs was physio-chemically characterized by UV Vis spectrum, FTIR, PSA, DLS, FESM, EDAX. By observing the UV Vis spectrum SPR peak confirmed the synthesis of CANCs. It was reported that the peak absorbance of silver nanoparticles produced through microwave irradiation is higher than that of room temperature reaction (Abdalla et al. 2014). It was reported that thermal conditions modify the size and shape of synthesised nanoparticles (Raut Rajesh et al. 2009) and temperature increases the biogenesis of silver nanoparticles (Kanchana et al. 2010).
Phytocompound enriched citronella extract acted as an excellent reducing agent for the synthesis of CANCs. This triggered the conversion of Ag+ to AgO which occurs during the formation of enol/keto form of those phytocompounds (Siemieniec 2013). Citronella is rich in terpenoids, which was reported to play a key role in reduction of silver ions (Mittal et al. 2015). Flavonoids, proteins, polysaccharides are reported to bind to the surface of nanoparticles (Sathishkumar et al. 2009). Water soluble phytocompounds flavonoids and terpenoids played crucial role in the reduction of silver ions upon microwave irradiation. The FTIR spectra was used to find the potential bioactive compounds which are responsible for the capping and reduction during the synthesis of CANCs (Ranjani et al. 2019a). Thus, the FTIR results revealed the mixture of phytocompounds present in the extract assist the reduction and stabilization of CANCS synthesis simultaneously. The size and stability of colloidal nanoparticles was measured using the Brownian movement of the nanoparticles shows CANCs were in nanometer in range and stable without aggregation. FESEM EDAX showed the presence of other elements C, O, Si, Cl, S could be derived from the bioactive compounds which were in the citronella extract, which may be bound to the topical surface of silver nanocolloids, which may played the role in bioreduction (Ndikau et al. 2017). Apart from Ag, elements such as C, Cl, Si, S has potent antibacterial activity. These elements work synergistically in imparting antibacterial, antifungal and anticancer activity. Toxicity study of CANCs on Buffalo oocyte maturation confirmed its non-toxicity even at the reproductive and embryonic developmental stages. Upon treatment it indicates the occurrence of complete maturation and readiness of the oocytes to undergo fertilization.
In this present study CANCs were found to be very efficient antibacterial activity, which was confirmed through bacteriostatic (MIC) and bactericidal (MBC) assays. The phyto constituents present in the citronella extract possess good antibacterial activity along with the Ag, Si, S, Cl. It was reported that Silver nanoparticles exert their antibacterial effects by attaching to the membrane and modulating cellular signalling pathways by means of penetrating inside the cell. The main mechanism by which silver nanoparticles exhibit antibacterial properties is by means of dephosphorylating the trypsin residues. After penetration into the bacteria, silver nanoparticles inactivate the enzymes, which stop many of the signalling pathways and metabolic pathways, eventually lead to bacterial cell death (Fatem et al. 2011). Previous studies reported that Citronella nanoparticles possess antibacterial activity against multiple drug resistant hospital isolates of E. coli, S. typhi, P. mirabilis K .pnuemoniae, S.aureus.3 There was also report on the green silver nanoparticles, which effectively control the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus (Saliem et al. 2016). CANCs showed effective biofilm inhibition activity, which could be due to its better penetrating ability of nanocolloids into the slimy layer of biofilm and successfully reduced the formation of biofilm by controlling the bacterial growth. The shape, size, and surface charge of CANCs play critical role in penetrating into the biofilm layer and kill the organism by anyone of the mechanism such as ROS production, cell wall damage, DNA damage etc. In previous studies it was reported that polyphenolic compounds, tannins, terpenoids, flavonoids, which are present in the plant extracts, acted as a siderophores, which chelates the metal from the growth medium and degrades the biofilm (Sharma et al. 2016).Similar results were supporting in our study, in such a way that citronella extracts were rich in polyphenols, tannins, terpenoids, flavonoids which acted as a reducing agent during nanocolloid synthesis, which reacted as quorum quenchers by disturbing the biofilm formation by preventing the cell to cell communication (Qing et al. 2018).
From the result of CTX-M-15 gene expression studies, it can be hypothesized that CANCs suppress CTX-M-15 gene expression which in turn reduce the transcription and translation of enzyme which is responsible for antibiotic degradation. There were previous report supporting that silver ions directly interact with DNA, RNA and protein, which disturb the central dogma of the cell (Qing et al. 2018). Thus, reproductive mechanism of the organism will be prevented, which ultimately reduces the pathogenicity of microorganism through various means such biofilm inhibitor, suppressing the antibiotic resistant genes etc.,
The antifungal activity of CANCs was validated against three different plant pathogenic fungi A. niger MTCC (281), Fusarium udum MTCC (2204), Fusarium graminearum MTCC (2089). This is the first report to study the antifungal activity of CANCs. All these plant pathogenic fungi cause severe damages to crop, cereals, fruits and vegetables in course of pre and post harvesting and greater economic loss to the country. This study confirmed that CANCs is a potent broad-spectrum antifungal agent. The difference in the inhibition rate was observed because each fungus differs in their growth rate by the physiological changes induced by the CANCs. There are few literatures supporting that the lemon grass oil has potent antifungal activity, CANCs with the synergistic effect of both silver and phytocompounds played a crucial role in inhibiting the fungal growth. There are few literatures supporting that silver nanoparticles affect the morphology of mycelium and affect the normal growth of hyphae. Green nanoparticles attach to the cell membrane, which in turn damages the cell membrane and changes the permeability of the cell wall. By altering the membrane permeability, the CANCs can easily enters the cell, disturbs the normal metabolism, and damages the cell organelles. Previous studies showed that silver nanoparticles damage the cell membrane, reduce the enzyme activity and affect the replication, transcription and translation process (Shunyu et al. 2019).
CANCs induced apoptosis via enhancing the release of lactate dehydrogenase enzyme in MCF-7 cells. MTT assay is an important process to recognize the activity of NAD (P) H dependent oxido-reductase enzyme that are capable to convert MTT to formazan in viable cells, on the contrary, LDH measures the released LDH enzyme from damaged plasma membrane of the cells. Similarly, SRB assay measures the amount of protein present in viable cells. Therefore, the therapeutic interventions of CANCs encapsulated molecules might be challenging chemoprevention candidate, with its ameliorative efficacy in cancers, including breast cancer (Shariq et al. 2019).
In this study, we adopted new strategy of green nanocolloid synthesis using Citronella extract by means of microwave irradiation. The microwave irradiation method was found to be rapid and efficient approach involving environmentally friendly and using low cost reductant flora, besides using toxic chemicals, for synthesizing CANCs. CANCs showed effective antimicrobial, biofilm quenching agents on biofilm forming E. coli. Further CTX-M-15 gene expression studies showed effective down regulation of CTX-M-15 gene in clinical isolate upon treatment with CANCs. These CANCs may be formulated as different types of nanoformulations such as nanogel, nanospary to fight against these deadly pathogens. Further, CANCs was validated against three phytopathogenic fungi A. niger MTCC (281), F. udum MTCC (2204), F. graminearum MTCC (2089) and confirmed it as effective antifungal agent. It was found that CANCs significantly reduces the growth rate of fungi through affecting morphology of hyphae. Hence, CANCs could be an effective alternative to chemical fungicides. This CANCs can be formulated as nano spray in effective way of controlling fungal infection in agricultural farming of cereals, fruits, vegetables and in long term storage of food commodities. CANCs can be used to eliminate the contamination of food grains with mycotoxins and protect the living beings from associated health effects. In addition, Cytotoxic activities of CANCs in MCF-7 cells revealed the efficacy of CANCs in decreasing the cell viability and can be used in formulating anticancer drugs at sub cellular, molecular and translational studies, however further in vivo studies are warranted. Further studies on optimisation of CANCs in different nanoformulations will help us in mass production and commercialization for various applications.