The reaction of bio-reduction between the Ag+ cations and the phytochemical compounds of the aqueous extract of L. coccinea leaves occurred instantaneously (before of the five minutes), evidenced by the presence of a dark brown colored complex (Fig. 3A), indicative of the formation of nanoparticles (Vadlapudi and Amanchy 2017; Sarkar and Goutam 2017; Khan et al. 2018). UV-Visible spectroscopic absorbance (Fig. 3B) evidenced a signal with λmax around of 470 nm due to strong surface Plasmon resonance, which reaffirms the formation of SNP (Travieso et al. 2018). The release of H+ ions since the first minutes of the reaction was verified by the gradual decrease of the pH in the aqueous media (Fig. 3C). Authors had explained the oxidation process of Ag+ by phytochemical compounds from plants, which are accompanied by the release of H+ ions (Zuorro et al. 2019). Also, it has been proposed that tautomer transformations of other compounds from plants (Ex. flavonoids) from the enol form into the keto form can release reactive hydrogen atoms that reduce metal ions to coordination complex of Ag forming nanoparticles. Nevertheless, due to the complex composition of naturals extracts actually there is no enough proof about which specific phytochemicals are responsible for the formation of nanoparticles (Jeevanandam et al. 2016).
Phytochemical screening showed the presence of secondary metabolites such as phenols, triterpenes and steroids, flavonoids, free aminoacids and leucoantocianidine in the aqueous extract of leaves of Leea coccinea (Table 1). Some of these compound podrían reaccionar with Ag+ to form the coordination complex so kinetic and chemical studies should be carrying out to the enlightenment of the reaction of bio-reduction due to the presence of several functional groups with reducing properties.
Currently, there are very few reports available on the phytochemistry of L. coccinea. Previous studies on chemical compositions of others species of Leea genera agreement with ours previous results. Flavonoids and flavonoid glycosides are found to be the major constituents of the genus (Lakornwong et al. 2014). Chemical studies of Leea indica reported six phenolic compounds, flavan-3-ols, flavonoids/flavonoid glycosides, dihydrochalcones and dimeric catechins (Singh et al. 2019). Similar studies based on NMR, MS, and ECD spectra reported one new lignan, one new lactam, five known lignans, four favonoid glycosides between other compounds were isolated from the ethanol extracts of the aerial parts of Leea aequata (Tun et al. 2019).
Table 1 Phytochemical screening of the aqueous extracts of Leea coccinea leaves
Table Key: - Absent + Presence
FTIR spectrum confirmed the phytochemicals screening (Fig. 4). Measurements were carried out to identify the functional groups responsible for the reduction of the Ag+ ions and capping of the bio-reduced silver nanoparticles synthesized by L. coccinea leaf extract. FTIR spectrum of SNP shows the presence of signals such as strong peaks between 3083 and 3076 cm-1 (peak 8) with a signal at 1538 cm-1 (peak 16) like aromatic with multiple substitutions(C=C stretching); other peak around 1616 cm-1 (peak 15) (C=O stretching) of carboxylic group. The absorption at about 1334 cm-1 (peak 18) is indicating residual amount of (NO3)-1 ion in the solution. Others signals appear at 1207 cm-1 (peak 19), between 3881 and 3831 cm-1 (peak 1), 3752 cm-1 (peak 2), 3565 cm-1 (peak 3), 3529 cm-1 (peak 4), between 3414 and 3335 cm-1 (peak 6) suggest polyphenolic OH group along with the peak around 880 cm-1 which represents the aromatic ring C-H vibrations. Others peaks around of 3270 cm-1(peak 7), 3025 cm-1 (peak 9), between 2932 and 2925 cm-1 (peak 10), 2235 cm-1 (peak 11), 2171 cm-1(peak 12), 1747 cm-1 (peak 13), 1688 cm-1 (peak 14), 1451 cm-1 (peak 17), 1156 cm-1 (peak 20) and between 1107 and 1035 cm-1 (peak 21) indicate the complexity of samples containing nanoparticles from natural sources and suggest the necessity of continuous studies about chemicals structures of compounds implicated in the bio-reduction.
The SEM images (Fig. 5 A y B) of SNP showed the presence of abundant nanoparticles with high grade of aggregation which was confirmed with confocal microscopy images where the emitted fluorescence was detected at 479 nm, demonstrating the occurrence of overlapping of smaller particles (Fig. 5 C). Numerous authors have reported agglomeration due to strong electrostatic interactions of metal ions. The fluorescent properties suggest to the presence of phytoconstituents or antioxidants with fluorophores groups. Others metal nanoparticles synthesized from plants have been reported with fluorescent properties (Talamond et al. 2015; Donaldson and Williams 2018).
The results of the qualitative analysis by EDX (Fig. 6) showed a high percentage of Ag evidenced in strong signals in the range of 2-4 keV, as well as the presence of carbon and oxygen indicative of hybrid NPs, with an organic component (Ex flavonoid) covalently bound to the metallic element. Likewise, the high compaction demonstrates its crystalline nature due to the reduction of Ag + cations by the reducing compounds present in the aqueous extract of L. coccinea leaves. (Singh et al. 2019; Kasithevar et al. 2017; Moodley et al. 2018 and Oluwasogo et al. 2019).
SNP showed promising activity against tested Gram negative bacterial strain of X. phaseoli pv phaseoli (Fig. 7). In previous studies in which the activity of AgNPs synthesized from the residual extract of the hydrodistillation of the essential oil of Thymus vulgaris was evaluated, the similar activity against this bacterium (Travieso et al. 2018) was demonstrated. However, in the present study in which the precipitated NPs were evaluated, the little diffusion of these nanostructures was found, so biological evaluation studies must be completed with the addition of stabilizing agents. On the other hand, the antimicrobial activity of AgNPs depends on parameters such as Ag concentration, as well as the size and shape of the nanostructures (Duval et al. 2019). However, in recent years other parameters have been demonstrated that influence antibacterial activity such as stabilization in the aqueous medium and the accessibility of the surface of NPs (Duval et al. 2019).
The formation of aggregates and agglomerates are the main destabilizing factor and reduce the accessible surface area in contact with the bacterial cell, and also inhibit the diffusion capacity in the in vitro tests (diffusion in agar), interfering with the results. Silver nanoparticles’ diffusion coefficients are generally related to size and physicochemical characteristics (Xiaoxue et al. 2020). For this reason, diffusion methods are used in initial or screening studies, however they are not valid for the determination of Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (CMB), suggesting tests such as serial dilutions in those that, in addition, the liquid medium facilitates the interaction of the substances to be evaluated with the microorganism through agitation. Authors have addressed the limitations of the antimicrobial activity studies of AgNPs suggesting methods with a liquid medium (macrodilution and microdilution), the Kirby-Bauer method, prior knowledge of the diffusion coefficient of the sample, among other standardized ones.