Kinetics of the Cationic bio-reduction involved in the biosynthesis of silver nanoparticles from Leea Coccinea Leaves

The resistance of microorganisms to conventional antimicrobials is one of the most serious health problems that affect not only the human, but also animals and plants, making the search for antimicrobial active ingredients a priority of global research. Green synthesis of antimicrobial silver nanoparticles (AgNPs) is a simple, cost-effective, rapid, reproducible, and environment friendly alternative for which numerous plant species have been reported for this purpose. Previous studies have shown the potential of Leea coccinea leaves in to the biosynthesis of antimicrobial silver nanoparticles. The current research aimed to study the kinetics of the reaction of synthesis of AgNPs by cationic bio-reduction from this botanical bioresource. A technology for the synthesis of AgNPs was established and the inuence of operational parameters such as the bio-reduction conditions and the kinetics of the reaction were studied. AgNPs were characterized at different times by UV-VIS spectroscopic method, scanning electron microscopy, determination of particle size, and Z potential through Dynamic Light Scattering technique (DLS). Addition of tensoactive substances was evaluated for the stabilization of the suspension of nanoparticles. The results showed that spherical AgNPs smaller than 100 nm were obtained, which were visually identied by the formation of a dark brown complex with maximum absorption at 470 nm. Kinetic studies demonstrated the inuence of the initial plant material on speed and performance, making evident a complex phenomenology with the possible occurrence of parallel reactions, which points to the possible reaction of different reducing compounds contained in this natural source. Addition of surface agents, such as SDS (0,5 %) or maltose (0,5 %), improved the stabilization in the aqueous medium, suggesting the continuation of studies to develop pharmaceuticals formulations based on AgNPs. The present research reports the kinetic of the bio-reduction to biosynthesize uorescent silver nanoparticles from aqueous extract of L. coccinea fresh and dry leaves. Drying process of the plant material showed to be a critical point that determined the kinetics of bio-reduction, which were complex and involved possible parallel reactions. The biosynthesized AgNPs had a spherical shape and a size less than 100 nm, and they showed a λ max of 440 nm in the UV-Vis spectrum, due to the presence of potentially reducing compounds such as polyphenols (avonoids). The addition of surfactants to the medium, with the exception of Tween 80 (-19.9), improved the stabilization of the nanostructures with respect to the aqueous medium (-21.3), being SDS the best (-41.8). Leea coccinea leaf was conrmed as a valuable bioresource to the synthesis of antibacterial silver nanoparticles through a simple, fast and environmentally safe technology.


Introduction
Antimicrobial Resistance (AMR) has been identi ed by the World Health Organization (WHO) as one of the greatest threats to global health (Quiñones 2017;WHO 2016). AMR is a complex, genetic and environmental phenomenon that occurs when microorganisms (bacteria, fungi, viruses and parasites) undergo changes when exposed to conventional antimicrobials (antibacterials, antifungals, antivirals, antimalarials or anthelmintics) and modify their behavior and sensitivity (WHO 2017; UN 2017). It is a global problem that not only affects humans, but also animals and plants (WHO 2017;OIE 2013;FAO 2016;Serra 2017). The search for new antimicrobial active ingredients is a global research priority. With the emergence of nanotechnologies, the scienti c community has accessed tools that are revolutionizing approaches to the discovery of new active principles, due to modi cations in a property as important as particle size with the use of these technologies, sizes in the order of the nanoscale are achieved, with the consequent impact on the bioavailability of these molecules at the site of action (Travieso et al. 2017). All this has allowed nanotechnology to attract great interest today and to be projected as a promising solution to problems in the scienti c and technological eld. However, many of the technologies applied to obtain nanoparticles (NPs), fundamentally based on physical and chemical processes, show disadvantages not only from the economic (high costs) or technological (need for specialized equipment) points of view, but also from the environmental aspects because of the required use of highly toxic organic solvents and other aggressive chemical compounds, reasons among others, that make current efforts focus on more feasible and environmentally friendly technologies. Such is the case of the biological methods (Singh et al. 2019) that use natural sources (microorganisms, plants, algae, or derived substances). Although dissimilar methods have been used to obtain NPs from plants (Travieso et al. 2017), the bio-reduction of metal cations is the most used and promising method for the formation of biogenic nanoparticles with antimicrobial activity (Mohammadlou et al. 2016;Roy 2017), being the metal salts of the (groups 10, 11 and 12) the most used as sources of the metallic cations of silver (Ag + ), gold (Au 3+ ), copper (Cu 2+ ) and zinc (Zn 2+ ) ( sources in the biosynthesis of AgNPs. However, sustainability is a critical point to take into account in the design of a technology based on natural sources, with a view to guaranteeing the required production levels. Leea coccinea which belongs to a genus known for its great richness in phytochemical compounds such as avonoids , and it was recently identi ed as a promising source of reducing compounds (Travieso et al. 2021). The objective of this work was to study the kinetics of cationic bio-reduction by the reducing compounds in the aqueous extract of Leea coccinea leaves, as well as to evaluate surfactants for the stabilization of the biosynthesized silver nanoparticles in the aqueous medium. to the quality and performances of the nanostructures were identi ed. Operational parameters were studied which included the initial quality of the plant raw material, and speci cally the use of fresh (F) or dry (D) plant material; the bio-reduction conditions (kinetics). Also, surfactant-stabilizing agents for AgNPs in the aqueous medium were evaluated to the future design of active nuclei for different types of formulations.

Materials
The study of the kinetics of the reaction was carried out by monitoring the reduction of Ag cations and presence of the colored complex indicative of the formation of AgNPs at different times (t 0 , 5, 30, 60, 120, 180, 240, 360, 720, 1080, 1320, 1440, 2160 and 4320 minutes) by UV-Vis spectrophotometry. At each time, an aliquot of the AgNPs suspension was taken and diluted with distilled water (1:8) (V/V) to characterize of the colloidal suspension by UV-Vis spectrophotometry (Vis-723 G spectrophotometer, Rayleigh) in a l range between 380 nm and 800 nm. The variation of the H + ion concentration (product of the bio-reduction reaction) over time was determined in the entire reaction pool by means of a pH meter (Mettler Toledo Instrument).
Curves of Maximum absorbance (A) vs time (minutes), as well as pH variation versus time (minutes) were designed. The type of reaction was determined by the differential method.
The reaction was stopped at different points (6, 12, 18 and 24 hours) by centrifuging at 10000 rpm for 15 minutes (Eppendorf ™ Centrifuge 5424R) and removing excess cation donor solution. The precipitated AgNPs (pellets) were stored at room temperature (25 ± 2 °C) in the dark for the physicochemical and morphological characterization studies as a function of time.
In uence of the initial plant material Taking into account the importance of the quality of the plant raw material, the in uence of drying on the quality and performance of the synthesized nanostructures was studied. Synthesis from fresh and dry plant material was comparatively evaluated. The leaves were dried at room temperature (27-30 °C) in the shade. The dried plant material was kept in nylon bags for 9 months. The yield of the synthesis of AgNPs was determined, both from fresh and dry plant material, by measuring of the dry weight of 1 mL of the suspension for triplicate on an infrared balance (Sartorious MA35) and calculated with relation to the initial mass of the plant material.
In uence of surfactant-stabilizing agents Surface agents, such as tween 80 at 0,5 % (TW 0,5 %), maltose at 0,5% (Mt 0,5 %) and SDS at 0,5 % (SDS 0,5 %), were evaluated. Their in uence on the particle size and the Z potential were determined as a measure of the stabilization of the nanostructures in the aqueous medium through Dynamic Light Scattering technique Characterization of the AgNPs Determination of particle size and Z potential The puri ed NPs contained in each vial were reconstituted in 2 mL of distilled water (Lichrosolv water for chromatography, Merck) using the mechanical shaker (IKA-VF2), and subsequently sonicated in an ultrasonic bath (WiseClean) for 10 minutes. An aliquot of 1 mL was taken and placed in polyethylene cuvettes for DLS particle size determinations using a particle size analyzer (Nano ZS90 instrument, Malvern, UK).
Likewise, the effects of the surfactants were evaluated at 12 hours. A selection of four samples (12h WT, 12h TW 80, 12h Mt, y 12h SDS) was made from those previously analyzed to determine the zeta potential by using the same Particle Size Analyzer (Malvernzetasizer). Each vial was shaken at high speed with the mechanical shaker and subsequently sonicated for 10 minutes. An aliquot of 2 mL was taken for each measurement.
The structural properties of the nanostructures formed at 6, 12, 18 and 24 hours were determined by SEM (MIRA 3 LMU TESCAN). The suspended sample was placed as a thin layer on carbon adhesive tape and once dry it was coated with gold-vanadium (5 nm) using the Desk Sputter Coater DSR1. The quantitative analysis was carried out using EDX (Oxford Instrument) to determine the main elements present and con rm the formation of NPs.

Statistical analysis
All determinations were carried out in triplicate and the results were expressed as the average value in the range of the standard deviation. For comparative studies, data were statistically processed using a simple analysis of variance and the means were compared using the Duncan's multiple range test or T test, with a signi cance level of 5%, using InfoStat but also by the differences in speed and yields of the bio-reduction reaction. In this sense, the reaction with the extract of fresh leaves started instantly (before 5 minutes), and the color drastically changed to dark brown (Figure 1 D), while the reaction with the dry leaves was after one hour ( Figure 1H). Regarding the yields of the crude suspension, they were statistically superior in the synthesis with the fresh plant material p 0.05 ( Figure 2).
Monitoring of the UV-Vis spectra con rmed that the reaction with the fresh material started instantaneously (Figure 3 and 4 A), given by the occurrence of a sudden color change from light green to dark brown, and by the presence of a well-de ned peak at λ max 440 nm. Likewise, the increase in absorbance over time was The results of the study of the kinetics of the bio-reduction reaction when the differential method was applied revealed a complex phenomenology where different kinetic mechanisms were expected to occur due to the presence of dissimilar families of secondary metabolites. These metabolites could have reducing properties and the possible occurrence of concurrent or non-concurrent parallel reactions, self-catalysis, etc., were likely to occur (Figure 6 A and B). Further studies should clarify the possible reactions and end products.
The results obtained point to the technical feasibility of using fresh plant material for the synthesis of AgNPs with L. coccinea because of the higher yields and a faster bioreduction reaction. For this reason, subsequent studies were performed on AgNPs biosynthesized with fresh leaves.

Characterization of the AgNPs
The morphology of the AgNPs synthesized at different times was examined by SEM, and abundant NPs were observed to have spherical shapes and diameters less than 100 nm at all times (6, 12, and 24 hours) of the synthesis ( Figure 7A-D). The magni cation of the images showed average diameters of 70-90 nm. Many authors agree on the strong agglomeration due to the electrostatic interactions between metal ions (Chen et al. 2016).
Determination of particle size The particle size determinations by the DLS method for AgNPs synthesized with fresh plant material showed diameters less than 100 nm (Figure 8), and the high agglomeration that the nanostructures showed in the aqueous medium (-21.30 mV ) (Figure 8 B) ), which corroborated the results obtained by SEM.

Analysis by EDX
The results of the qualitative analysis using EDX (Oxford Instrument) at different times (

Conclusion
The present research reports the kinetic of the bio-reduction to biosynthesize uorescent silver nanoparticles from aqueous extract of L. coccinea fresh and dry leaves. Drying process of the plant material showed to be a critical point that determined the kinetics of bio-reduction, which were complex and involved possible parallel reactions. The biosynthesized AgNPs had a spherical shape and a size less than 100 nm, and they showed a

Con ict of Interest
There are no con icts of interest to be declared.

Funding
These results were supported by National Program of Nanoscience and Nanotechnology of Ministry of Science and Environmental from Cuba.  Bio-reduction reaction during 24 hours of the synthesis of AgNPs with L. coccinea. A: UV-Vis absorption spectra at different times (fresh plant material); B: UV-Vis absorption spectra at different times (dry plant material) Figure 5 Variation of pH during the bio-reduction reaction with L. coccinea (fresh and dry plant material) Figure 6 Curve of the study of kinetics of the bio-reduction reaction from L. coccinea by the differential method. A: Fresh plant material; B: Dry plant material  In uence of surfactants on particle size of the AgNPs synthesized with fresh leaves of L. coccinea Figure 11 In uence of surfactants on the Z Potential. A: Water, B: Maltose (0.5%), C: SDS (0.5%), and D: Tw 80 (0.5%)

Supplementary Files
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