The extraction procedure from dried R. hymenosepalus
roots yielded a dark liquid which we examined spectroscopically. The UV-Vis and NMR experiments confirm the presence of polyphenols in the Rh extract. The absorbance of the sample presented a peak in the range of 277 to 280 nm as characteristic of polyphenol molecules. On the other hand, the 1
H NMR proton spectra display a wealth of peaks characteristic of plant extracts (Additional file
: Figure S2). We have identified some of these signals as corresponding to polyphenol molecules
] (Additional file
: Figures S3 and S4). In particular, some peaks correspond to catechines and stilbene molecules. For instance, at least five chemical shifts of our spectra match those of epicatechin, as reported in the SDBS spectral database of organic compounds (no. 22007HSP-44-526). The coincidences are shown in Additional file
: Table S1. The chemical shifts also match those reported for epicatechin gallate and epigallocatechin gallate (Additional file
: Table S1). In the Additional file
: Figure S5, we display the chemical structure of these molecules. On the other hand, ten of the peaks match those reported for a stilbene compound extracted from roots of the Terminalia sericeae
] (Additional file
: Table S1). These signals correspond to a stilbene molecule known as stilbene glycoside (Additional file
: Figure S6). The NMR results obtained so far allow us to assess a significant presence of polyphenolic compounds in the plant extract of R. hymenosepalus
. These compounds are potential reductor agents in the synthesis mechanism of silver nanoparticles. From UV-Vis calibration curves (using pure compounds), we estimate the concentration of two of the reducing molecules: epicatechin (241 μM) and epicatechin gallate (91.1 μM). Additional NMR experiments are under way in order to further characterize this plant extract. The results will be published elsewhere.
Since the R. hymenosepalus
extract contains polyphenols, we can anticipate that it will serve as reducing agent for the nanoparticle synthesis. In fact, the same molecular mechanisms that give antioxidant properties to these molecules must promote the reduction of Ag+
ions to Ag atoms. The main mechanism is hydrogen abstraction
] due to the OH groups in the polyphenol molecules.
We have thus prepared silver nanoparticles using the R. hymenosepalus
extracts as reducing agent. For all the AgNO3
concentrations, the samples changed their visual appearance shortly after addition of the plant extract, indicating that a reduction reaction took place. Initially, the reacting mixture was a slightly yellowish liquid; as the reaction proceeded, the solutions became orange, red, and brown. This is a strong indication of the formation of silver nanoparticles: the change in color is due to the strong absorption of visible light due to excitation of the nanoparticle surface plasmons
]. In Figure
, we show vials with reacting samples for different AgNO3
concentrations (0, 2.5, 5, 7.5, 10, and 15 mM), and different times after the reaction started (24, 48, 72, and 96 h); the clear time evolution is a signal of the growth of silver nanoparticles. The time scale of the visual evolution depends on the AgNO3
concentration. For instance, for CAgNO3
= 5 mM, nanoparticle formation is visually appreciable after 4 min of the beginning of the reaction.
Figure 1 Visual appearance of vials containing the Rumex hymenosepalus extract and AgNO3 solution after different reaction times. The vials correspond to different AgNO3 concentrations: (a) pure extract, (b) 2.5 mM, (c) 5 mM, (d) 7.5 mM, (more ...)
The change in color, and thus the formation of silver nanoparticles, was confirmed by the UV-Vis experiments. In Figure
, we show the spectra for a reaction time of 96 h. The curves display a pronounced peak around 425 nm, as expected from the plasmon resonance of silver nanoparticles. The UV-Vis peak is more pronounced for higher AgNO3 concentrations, indicating that more nanoparticles per unit volume are formed when this concentration increases. Note that in all the spectra displayed in Figure
, the polyphenol peak (observed in the Rh extract) is also clearly visible around 278 nm. In the inset of Figure
, we also display the UV-Vis spectra of the AgNO3 solution; it has a peak around 217 nm, as expected for Ag+ ions.
Figure 2 UV–vis absorbance for samples with different values of AgNO3 concentrations. (a) Pure extract, (b) 2.5 mM, (c) 5 mM, (d) 7.5 mM, (e) 10 mM, and (f) 15 mM. The peak around 425 nm corresponds to the (more ...)
Note that we have performed control experiments in order to discard the action of ethanol and microorganisms as reducing agents. In the case of ethanol, the UV-Vis experiments show no significant Ag+
ions reduction when AgNO3
was dissolved, without Rh extract, in pure ethanol and in an ethanol/water mixture (see Additional file
: Table S2 and Figure S7). On the other hand, we have verified the absence of microorganisms in the samples. We have performed aerobic plate count experiments for mold, yeast, and aerobic mesophilic bacteria
], for the reacting sample where the silver nitrate concentration was 15 mM. In the case of the aerobic mesophilic bacteria test, we used plate count agar as culture medium; the sample was incubated at 35°C for 48 h. The results show that no mesophilic bacteria grow in the plate (see Additional file
: Figure S8). In fact, the colony forming unit (CFU) is <1 CFU/ml. For the mold and yeast count test, we used potato dextrose agar; the sample was incubated at 25°C for 5 days. No mold or yeast was detected in the plate (the resulting CFU is <1 CFU/ml) (see Additional file
: Figure S8). Thus, we can be sure that the synthesis of nanoparticles was performed under fully abiotic conditions.
We have characterized the silver nanoparticles with transmission electron microscopy. The size and abundance of the resulting particles depend on the AgNO3 concentration. Their diameter is in the range of 2 to 40 nm. In Figures
, we present micrographs of the obtained silver nanoparticles after 24 and 96 h of the beginning of the reaction, for the different AgNO3 concentrations. For a reacting time of 24 h (Figure
), we can appreciate that for CAgNO3 = 2.5 mM (micrograph A), the population is composed mainly of scattered, small nanoparticles. As the CAgNO3 increases, bigger nanoparticles are observed, while the proportion of small nanoparticles decreases. This trend is somehow maintained for a reacting time of 96 h (Figure
). From the micrographs, we can observe that a population of big nanoparticles, in coexistence with a small proportion of small particles, is clearly appreciated. Furthermore, the size of the bigger particles increases as CAgNO3 is increased, while at the same time, the proportion of small nanoparticle decreases. Note that we do not observe particle coalescence, probably due to a stabilizing effect produced by the antioxidant molecules.
TEM micrographs of the silver nanoparticles obtained for different AgNO3 concentrations. (A) 2.5 mM, (B) 5 mM, (C) 7.5 mM, and (D) 15 mM, after a reaction time of 24 h.
TEM micrographs of the silver nanoparticles obtained for different AgNO3 concentrations. (A) 2.5 mM, (B) 5 mM, (C) 7.5 mM, and (D) 15 mM, after a reaction time of 96 h.
We have quantified these tendencies by statistically analyzing a population of more than 500 nanoparticles for each reaction time. The results are shown in Figure
, where for matters of clarity, we present the full histograms for 96 h of reaction time, and only a representative curve for 24 h. For the shorter reaction time (24 h, black curves in Figure
), most of the particles are small, with an average diameter around 3 to 5 nm. For 96 h after the beginning of the reaction, two populations are clearly distinguishable in the histograms. The first one is a subpopulation of small nanoparticles of average diameter around 4 to 5 nm. However, there exists also a considerable fraction of nanoparticles with larger average diameters, of the order of 10 to 20 nm. The average diameter of these larger particles grows with an increase in the AgNO3 concentration.
Figure 5 Size distribution of the obtained silver nanoparticles for different values of the AgNO3 concentration. (A) 2.5 mM, (B) 5 mM, (C) 7.5 mM, and (D) 15 mM, and two reaction times (24 and 96 h). For clarity, we display (more ...)
The chemical composition of the obtained particles was assessed by EDS spectroscopy. From the EDS spectra (see Additional file
: Figure S9), we have confirmed that the nanoparticles are mainly composed of silver (subtracting the Cu, Si, and C contributions from the TEM grid and the detector window). Some amount of oxygen is also displayed in the EDS results (see Additional file
: Table S3), probably meaning that some trace amount of the extract is still present in the TEM grid.
The crystallographic analysis confirms that the nanoparticles are indeed silver crystals. For instance, in Figures
, we show HR-TEM images of two representative nanoparticles, with the corresponding FFT plot. Very interestingly, these results show that the nanoparticle population has a combination of two kinds of crystal symmetries: face centered cubic (fcc) and hexagonal (4H). The prevalence rates of these geometries are 79% (fcc) and 21% (4H). We have computed the interplanar distances from the micrographs and the FFT plots. In the case of the fcc nanoparticles, the interplanar distances are d1
= 2.316 Å, d2
= 1.517 Å, and d3
= 1.159 Å. They are, respectively, associated with the planes (111), (220), and (222) corresponding to the fcc structure of a silver crystal. On the other hand, the interplanar distances for the 4H structure are d1
= 2.405 Å, d2
= 2.275 Å, d3
= 1.407 Å, d4
= 1.249 Å, and d5
= 1.149 Å, corresponding to the planes (101), (1-12), (110), (008), and (203) of a hexagonal 4H structure
]. We have characterized the nanoparticle population for both the fcc and 4H structures, analyzing 100 particles. The results are shown in Figure
. We observe that the fcc nanoparticles display two size populations: one with a small average diameter (around 10 nm) and a second one with a larger diameter (around 28 nm). On the other hand, the hexagonal nanoparticles have only one size population and larger diameters (around 38 nm). Note that the results shown in Figure
correspond to samples where the reaction time is of 30 days.
Figure 6 HR-TEM images of a representative nanoparticl, with fcc structure. HR-TEM image of a silver nanoparticle, the crystal planes correspond to a fcc structure (A) with its corresponding FFT plot (B). The other figure (C) is an integrated image from the FFT (more ...)
Figure 7 HR-TEM images of a representative nanoparticle, with hexagonal (4H) structure. HR-TEM image of a silver nanoparticle, the crystal planes correspond to a hexagonal (4H) structure (A) with its corresponding FFT plot (B). The other figure (C) is an integrated (more ...)
TEM micrograph displaying both fcc and 4H nanoparticles. The population histogram for each crystal structure is also displayed. The statistical analysis has been performed with 100 nanoparticles. The reaction time was 30 days.
The observed features in the TEM, UV-Vis,, and visual observation experiments can be summarized and understood as follows. The polyphenols contained in the R. hymenosepalus extracts act effectively as reducing agents for the Ag+ ions due to their antioxidant activity. The reduction reaction promotes the nucleation and growth of nearly spherical Ag nanoparticles. As expected, the kinetics of nanoparticle formation, as well as the resulting nanoparticle populations, depends on the AgNO3 concentration. Higher silver nitrate concentrations yield more nanoparticles for reacting times of 24 h, because more material is available for the nanoparticle growth. However, when the reaction time is 96 h, two populations of nanoparticles are present. In this case, most of the silver atoms are accommodated in large nanoparticles.