2.1. Synthesis and Characterization of Gold and Silver Nanoparticles
Several reaction conditions were tested for the process of synthesis of the nanoparticles by modifying the extract concentrations, the metal salt concentration, temperature, and time of the reaction. The optimal conditions were established after the study of the UV-Vis spectra obtained for the different reactions performed. In all cases, during the synthesis, a change in color was perceived after the reduction of the metal salt. The color changed to red/purple in the case of gold nanoparticles and to yellow/orange in the case of silver nanoparticles.
a shows the UV-Vis spectra of gold nanoparticles for a fixed concentration of DA extract and different concentrations of HAuCl4
. In all the cases, the appearance of the surface plasmon resonance (SPR) band of gold at around 500 nm can be observed, while in the spectra of the extract, there is no band. It can be observed how the SPR band varies depending on the gold concentration. It can be noted that with the lowest concentration of gold tested, the bands that appeared were broad and not really intense. When a higher concentration was added, the band became narrower and more intense. In addition, a variation in the λmax
of the SPR band was observed, which shifted to lower values when the concentration of gold was increased. With the highest concentrations of gold, the particles obtained were not stable and tended to aggregate and precipitate.
Regarding silver nanoparticles, Figure 1
b–e show the UV-Vis spectra of their synthesis led by DA extract. The reactions were performed at 100 °C in order to synthesize more homogeneous nanoparticles in a faster reaction, since it was observed that at lower temperatures, the reactions did not take place or heterogeneous nanoparticles were obtained. In the UV-Vis spectra, we observed the difference on the characteristic SPR band of silver at around 400 nm when the extract/water ratio is changed and the concentration of silver (Ag) is modified. Figure 1
b shows that the characteristic SRP band of silver nanoparticles does not appear when employing the concentrated extract with any of the Ag tested. When diluting the extract, the SPR band appeared for all the silver concentrations.
With the red seaweed I. cordata
, in order to attain a narrow size distribution, it was necessary to further decrease the concentration of the extract, which, as previously mentioned, already had a lower concentration than that of the other macroalgae studied. After several trials, a narrow SPR band with a maximum wavelength at 539 nm was obtained for [email protected]
, while [email protected]
showed the characteristic SPR band with λmax
at 418 nm, as shown in Figure 2
To accurately determine when the gold nanoparticles synthesis was completed, measurements of the absorbance in the maximum wavelength were acquired. Figure 2
c shows the spectrum obtained for [email protected]
, and it can be noted that the reaction started when HAuCl4
was added. A quick increase in absorbance, which corresponds to the change in color, was observed between 0 and 5 h. After that, the reaction slowed down, and a slight increase in absorbance was observed without color change progression. The measurements were stopped after 15 h.
In the case of [email protected]
d), it can be observed that the use of a lower concentration of the extract could have affected negatively the kinetics of the reaction. The reaction might be divided into three stages. The first one, between 0 and 5 h, corresponds to the activation process. Next, an increase in absorbance appeared between 5 and 25 h, which corresponds to the change in color observed and the time when the nucleation process might take place. After that, a stabilizing process occurred. The reaction was stopped after 60 h.
pH was measured at room temperature prior to and after the synthesis of nanoparticles, as shown in Table 2
. It must be noted that a decrease in pH was observed in all cases, although it is worth mentioning that during the synthesis of gold nanoparticles, the decrease in pH was more significant than in the case of silver nanoparticles. These results are in accordance with previous reports that suggest the involvement of hydroxyl or amino in the reduction of gold(III) and silver(I) to gold(0) and silver(0). The reaction proposed involved the oxidation of hydroxyl or amino groups, which will lead to the liberation of protons and therefore the decrease of pH [6
The Z potential values obtained for the samples are collected in Table 2
, where it can be seen that the particles carry a negative electrostatic surface charge. This result suggests the involvement of the anionic polysaccharides present in the extract in the stabilization process of the nanoparticles, since the negative values obtained are in accordance with other studies where marine polysaccharides were employed for the synthesis of nanoparticles [25
]. It is interesting to highlight the values obtained for the nanoparticles synthesized since, according to Z potential guidelines, nanoparticle dispersions with Z potential values ˃±30 mV are highly stable [27
]. When samples are preserved at 4 °C, their stability in the long term (>6 months) has proven to be high.
TEM images obtained for the characterization of the silver and gold nanoparticles synthesized by the Antarctic macroalgae are shown in Figure 3
. Regarding silver nanoparticles, all the samples analyzed showed the presence of spherical nanoparticles, as seen in Figure 3
a,b. [email protected]
had a mean diameter of 13.7 ± 3.1 nm (Figure 3
a). The nanoparticles obtained with DM using the same reaction conditions were bigger, with a mean size of 17.8 ± 2.6 nm [9
]. The mean diameter of silver nanoparticles obtained with the red algae Iridaea cordata
b) is similar to that obtained with the brown algae DM, with sizes of 17.5 ± 3.7 nm. The smallest nanoparticles were obtained with PD extract, being the only silver nanoparticles synthesized in the context of the present study with a diameter lower than 10 nm [9
]. Previously, the stability of these nanoparticles had been confirmed with the determination of the Z potential (Table 3
). This stability can be due to the fact that the nanoparticles are embedded in the extract matrix, as Figure 3
a,b clearly illustrate, even after the centrifugation and purification step in the TEM sample preparation.
In the case of the gold nanoparticles synthesized, the extract cannot be clearly observed in the images acquired (Figure 3
c,d). In the case of [email protected]
, this could be due to the lower concentration of extract employed for the synthesis and the centrifugation step for TEM sample preparation. A predominance of spherical nanoparticles was observed in both samples, and the mean diameters obtained were 12.6 ± 1.9 and 12.3 ± 1.6 nm for [email protected]
and [email protected]
, respectively. These sizes are very similar to those obtained with the brown algae D. menziesii
, with mean diameters of 11.5 ± 3.3 nm, while in the case of [email protected]
, the nanoparticles obtained were the biggest ones, with mean diameters of 36.8 ± 5.3 nm [9
In this study, the FTIR technique was employed for the evaluation of the changes observed in the functional groups of the molecules in the extracts before and after the nanoparticles were synthesized. Figure 4
a,b show the spectra and assignation of bands for DA and IC samples, respectively. For the assignation of the bands, previous studies were used as reference, mostly from the same species, but also from the same genus or other Antarctic species [9
In general, O-H stretching vibrations of the hydroxyl group in alcohols and N-H stretching vibrations in amides and amines are assigned to the broad band that appeared in all spectra between 3437 and 3391 cm−1
, while the weaker signal at 2961–2927 cm−1
could be related to C-H stretching vibrations of the hydrocarbon chains. Carboxylate groups are typically assigned to two bands; one more intense at around 1653–1648 cm−1
, corresponding to an asymmetrical stretching, and a weaker band at 1412–1416 cm−1
, which is assigned to symmetrical stretching from amide I and II of proteins. The sugar ring and glycosidic bond C-O stretching vibrations might account for the signals at 1070 cm−1
. The C-O-S bending vibration observed at 800 cm−1
and S-O stretching vibration at 1260 cm−1
ascribed to sulfated esters point to the presence of sulfate groups in the polysaccharide structure. Some studies on brown seaweed suggest that the band at 800 cm−1
is characteristic of mannuronic acid residues [28
It must be noted that in the case of IC extract, the band at 1200 cm−1
is more intense than in the case of DA extract. According to some studies, the intensity of this band is an indicator of the degree of sulfurization of the polysaccharides [31
When comparing DA extract with [email protected]
and [email protected]
, a different behavior related to the shifts in bands could be observed. Firstly, in the case of [email protected]
, the peak of 3400 cm−1
shifted to lower wavelengths, while in [email protected]
, it shifted to higher wavelengths. These shifts might indicate an involvement of either the hydroxyl functional groups from polyphenols and polysaccharides or the amino groups of proteins in the bioreduction of the salts employed. This could be related to other studies that have synthesized nanoparticles using alginate, which is a polysaccharide isolated from brown seaweed [32
The peak at 1600 cm−1
, which corresponds to carbonyl stretching, also shifted to lower wavelengths in [email protected]
, but there was no change in [email protected]
This is in consonance with other studies, which have suggested that the carbonyl group from proteins can effectively bind metals, so it is likely that proteins could cap silver nanoparticles to impede agglomeration [33
Lastly, significant differences were observed in both [email protected]
and [email protected]
in the bands between 1200 and 1000 cm−1
when compared with DA extract. The shifts to lower wavelengths and differences in intensity observed could indicate a role of the sulfonic groups from polysaccharides in metal binding.
Regarding IC extract, when compared with [email protected]
and [email protected]
, there were minor shifts in the position, shape, and intensity of the bands at 3429 cm−1
and in the region between 1400 and 1200 cm−1
in the case of [email protected]
On the other hand, no major changes were observed in [email protected]
, with just some differences in the intensity and broadness of the bands.
2.2. In Vitro Antioxidant Activity
In the present study, the antioxidant activity of DA and IC extracts was determined and compared with the values obtained for the two seaweed previously studied, D. menziesii
and P. decipiens
]. Furthermore, the antioxidant activity of the four macroalgae extracts after the synthesis of nanoparticles was also analyzed. The values of the reducing activity, total phenolic content, and radical scavenging activity of the aqueous extracts of D. antarctica
, D. menziesii, I. cordata,
and P. decipiens
are represented in Figure 5
It can be observed that DM possesses the highest reducing power, with a value that doubles that of the other Desmarestia.
When compared with the red seaweed, DM presents a reducing power that triples that of P. decipiens
and is four times that of I. cordata.
The reducing power of the two red seaweed is lower than that of the brown seaweed, which is in line with other studies where this relation was maintained [35
Regarding the total phenolic content (TPC) of the samples, it can be observed that the direct relationship that is usually noticed between antioxidant activity and TPC is not maintained. It can be noted that DM extract possesses the highest TPC, with 0.84 ± 0.02 mg gallic acid equivalents (GAE)/g alga. However, D. antarctica and PD, which show quite different values of reducing power, do not show significant differences as regards phenolic content, with values of 0.37 ± 0.01 and 0.34 ± 0.03 mg GAE/g alga, respectively. Surprisingly, the value of TPC obtained for I. cordata is extremely low, to the extent of being negligible.
In the case of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, the lowest IC50 value was obtained with I. cordata, which, in contrast, has the lowest TPC value of the seaweed studied. Both Desmarestia show similar IC50 values, 63.3 ± 0.2 mg/mL for DA and 68.7 ± 1.3 mg/mL for DM. PD has the highest IC50 value, almost double those of both Desmarestia, indicating a lower scavenging activity.
In the literature, information about these species of seaweed is scarce. The studies found focus on the content of lipids and fatty acid composition, as well as on polysaccharides [37
], and they provide data related to the content of hydrocarbons in these species [38
]. For instance, the study conducted by Dhargalkar and Bhosle shows that D. menziessi
has a higher content of hydrocarbons than P. decipiens
but no significant difference in lipids [40
]. The higher content of polysaccharides would account for the more significant reducing power of D. menziesii
in comparison with P. decipiens
. D. menziesii
also shows higher reducing power than D. antarctica
, which could be related to the higher content of phlorotannins described by Iken et al. [42
After the synthesis of the nanoparticles, the reducing power, TPC, and DPPH scavenging activity of the extracts were also determined. In Figure 5
, the results obtained for DA extract, [email protected]
, and [email protected]
are shown. As regards reducing power, gold and silver nanoparticles behave differently. [email protected]
possesses almost half the reducing power after the synthesis, while the value obtained for [email protected]
does not show significant differences compared to that of the extract before the synthesis. Interestingly, in both cases, a significant decrease in the TPC can be observed, which suggests an active role of the phenolic compounds present in DA extract in the reduction process for the synthesis of the nanoparticles. In the case of the DPPH, the same behavior as in the case of the reducing power is observed. The IC50
value of [email protected]
is higher after the synthesis, while it is lower in the case of [email protected]
When the values obtained for DM extract are compared to those obtained after the synthesis of [email protected]
and [email protected]
, the results obtained showed a similar pattern to that of the results obtained with the other Desmarestia
. A decrease in the reducing power was observed in [email protected]
, while a significant increase was observed in [email protected]
Regarding TPC, it is noteworthy that it decreased by half in both samples. The IC50
values calculated for the DPPH scavenging activity showed a significant decrease for [email protected]
, while there was no significant difference for [email protected]
Regarding the comparison of IC extract before and after the synthesis, the results obtained showed a slight increase in the reducing power as well as a diminution in the IC50
value for both [email protected]
and [email protected]
In both cases, the difference in the total phenolic content was insignificant. Due to the low TPC obtained for IC, it could be argued that that they do not intervene in the synthesis of nanoparticles.
Finally, the results obtained for PD extract did not reveal any significant variations in the reducing power and scavenging activity of [email protected]
On the other hand, a notable increase in the reducing power was observed, and consequently, a decrease in the IC50
value was also obtained for [email protected]
In both cases, there was a significant lowering in the TPC obtained, indicating their contribution during the synthesis of the nanoparticles.