Characterization of silver nanoparticles
The coloration of the reaction medium after mixing the metallic salt and plant extracts (biological reducing agents for the formation of AgNPs by green synthesis routes) is considered one of the first indications of the formation of stable AgNPs and is attributed to the effect of surface plasmon resonance (RPS) that happens due to the collective and spontaneous oscillation of free electrons that collide while the bioreduction reaction occurs, providing the appearance of a band in the spectrum in the visible region (Daniel & Astruc, 2004). In this sense, the dark color observed in the samples (Figure 2) resulted from the reduction of silver ions with consequent formation of AgNPs. Corroborating with Baran et al. (2021) who also observed a change in color from yellow to dark brown after mixing the pineapple peel extract with the AgNO3 solution at 10 mmol L-1.
The UV/Vis spectra of the AgNPs in the analyzed samples showed an absorption peak at 460 nm (Figure 3), confirming the presence of silver nanoparticles in suspension, corroborating Lima et al. (2021) who synthesized AgNPs from leaf extracts of native Brazilian plants. It was observed that the intensity of absorbance is different, but follows a concentration-dependent increase, except for the AgNPs of concentration 3 mmol L-1, synthesized by means of ultrasound. The increase in absorbance intensity may be due to the greater amount of nanoparticles formed, as a result of the reduction of silver ions by the pineapple peel extract.
It can be seen that the shape and peak of the curves coincide, indicating that the average size and shape of the nanoparticles present in the samples, even with different volumes (or concentrations), are similar. For according to Mulfinger et al. (2007) the size and shape of nanoparticles are the main factors that determine the absorption peak. As for energy sources, it was observed that, for all concentrations, ultrasound had the highest absorbance values compared to water bath (Figure 3).
These results were similar to those obtained in other studies involving the synthesis of AgNPs from pineapple extracts, such as that by Baran et al. (2021) with a maximum absorption peak of 463 nm and Acharya, Mohanta and Pandey (2021) at 442-452 nm. However, different from the results obtained by Das et al. (2019) when observing maximum absorption at 485 nm from the readings of AgNPs at a concentration of 1 mmol L-1, and by Agnihotri et al. (2018) who obtained an intense absorption band at 411 and 416 nm when synthesizing AgNPs with 1 and 5 mmol L-1, at the same concentrations used in the present study.
The average diameter values (Z-average), the polydispersity index (PdI) and the Zeta potential (PZ) of AgNPs synthesized by water bath, right after synthesis and after 30 and 60 days of storage, are shown in Table two.
Table 2. Data obtained by DLS and Surface Zeta Potential of AgNPs at different concentrations synthesized by water bath (BM) with the aqueous extract of pineapple peel under storage under two conditions: room temperature (TA) at 22ºC and in a refrigerator (GEL) at 4ºC. Values are represented as the mean ± standard deviation of the mean of measurements obtained from three individual readings.
Sample
|
Storage temperature
|
Time after synthesis (days)
|
Z-average (d.nm)
|
PdI
|
Zeta Potential
|
1 WB
|
|
0
|
138.4 ± 0.814
|
0.167 ± 0.021
|
-12.3 ± 4.97
|
22ºC
|
30
|
148.0 ± 4.521
|
0.226 ± 0.016
|
-17.5 ± 1.14
|
60
|
140.5 ± 1.935
|
0.171 ± 0.057
|
-21.4 ± 1.60
|
4ºC
|
30
|
146.5 ± 5.541
|
0.220 ± 0.051
|
-17.4 ± 2.39
|
60
|
138.1 ± 4.704
|
0.194 ± 0.042
|
-18.6 ± 3.25
|
3 WB
|
|
0
|
127.1 ± 2.066
|
0.235 ± 0.025
|
-13.4 ± 1.11
|
22ºC
|
30
|
136.9 ± 7.300
|
0.322 ± 0.016
|
-18.6 ± 1.29
|
60
|
119.3 ± 2.268
|
0.233 ± 0.016
|
-15.6 ± 1.04
|
4ºC
|
30
|
129.2 ± 1.060
|
0.266 ± 0.024
|
-17.5 ± 3.23
|
60
|
122.5 ± 2.401
|
0.235 ± 0.020
|
-10.1 ± 2.30
|
5 WB
|
|
0
|
141.0 ± 1,152
|
0.241 ± 0.021
|
-17.0 ± 3.69
|
22ºC
|
30
|
136.9 ± 2,579
|
0.204 ± 0.033
|
-19.4 ± 0.44
|
60
|
130.5 ± 2,100
|
0.210 ± 0.012
|
-16.0 ± 1.12
|
4ºC
|
30
|
137.8 ± 6,035
|
0.249 ± 0.055
|
-16.8 ± 2.21
|
60
|
131.9 ± 1,872
|
0.200 ± 0.010
|
-16.5 ± 1.17
|
It is noteworthy that for all analyzed variables (Z-average, PdI and PZ) there was no direct relationship between the increase in concentrations (1 WB, 3 WB and 5 WB) with changes in their values. It was found that the Z-average values, a measurement attribute obtained based on volume and intensity distributions, were similar for all evaluated samples, regardless of storage temperature and measurement time after synthesis. For samples 1 WB, 3 WB and 5 WB the average Z-average values were 142.3 ± 3.5, 127.0 ± 3.30 and 135.6 ± 2.8, respectively.
According to Pardeike et al. (2009), the considered average size of nanoparticles for pharmaceutical or biomedical applications ranges from about 40 to 1000 nm. The formulations closest to the ideal being those with an average diameter of less than 200 nm (Stecanella, 2011). In this way, the average sizes of the particles obtained in the present study are within the ideal range, as the values ranged from 119.3 nm to 148 nm. Furthermore, when stored at room temperature or in a refrigerator, they can be used for up to 60 days without major variations in the size of the synthesized nanoparticles.
The PdI reflects the sample's degree of dispersion, with values closer to 0 corresponding to more uniform particles and, closer to 1.0, more polydisperse (Araújo Neto; Pereira & Silva 2020). The 3 WB formulation, obtained after 30 days of storage at room temperature, showed the highest PdI value (0.322), suggesting a lower uniformity in the sizes of the nanoparticles in relation to the other samples. In addition, PdI variations were observed over time from 0 to 30 days, indicating a possible aggregation of nanoparticles (Stecanella, et al., 2013).
According to Stecanella et al. (2013), formulations with characteristics close to the ideal must present PdI<0.2. In this sense, in the present work, only the 1 WB formulations obtained at 0 and after 60 days, stored at room temperature and at 4ºC, presented characteristics close to the ideal, indicating greater homogeneity in the size of the particles.
Espinoza et al. (2020), characterizing liposomes loaded with silver nanoparticles obtained by green synthesis, obtained PdI values ranging from 0.193 □□0.01 to 0.315 □ 0.02, classifying the degree of dispersivity of the samples as moderate. Thus, except for the 1 WB formulations obtained shortly after preparation and after 60 days of storage at room temperature and at 4ºC, all other formulations in the present work showed moderate polydispersity.
Regarding the Zeta potential, which reflects the measurement of the net electric charge on the surface of the particles, providing information on the colloidal stability of the system (Santos Júnior, 2021), values ranging from -21.4 ± 1.6 to - 10.1 ± 2.3, revealing that the surface of the particles is negatively charged in an aqueous medium, in which all synthesized nanoparticles showed incipient colloidal stability (-10 to -30 mV).
According to Marcato (2009), nanoparticles with Zeta potential greater than ± 30 mV have excellent stability due to greater electrostatic repulsion between particles and, consequently, less possibility of aggregation, flocculation or sedimentation. Thus, the 1 WB formulation, kept at room temperature for 60 days after synthesis, showed a higher Zeta potential (in modulus), compared to the other formulations, demonstrating that in addition to being homogeneous (PdI<0.2), it is more stable in aqueous suspension, making it difficult to agglomerate.
In view of the above, it should be noted that the size of the AgNPs (<200 nm), together with the high value of the Zeta potential (in modulus) and the low or moderate polydispersity, constitute a low tendency for these particles to aggregate in aqueous solution and high probability of use in several areas. Table 3 shows the results of mean diameter (Z-average), polydispersity index (PdI) and Zeta potential (PZ) of AgNPs synthesized by ultrasound and analyzed right after synthesis and after 30 and 60 days of storage.
Table 3. Data obtained by DLS and Surface Zeta Potential of AgNPs at different concentrations synthesized by ultrasound (U) with the aqueous extract of pineapple peel under storage under two conditions: room temperature (TA) at 22ºC and in a refrigerator (GEL) at 4ºC. Values are represented as the mean ± standard deviation of the mean of measurements obtained from three individual readings.
Sample
|
Storage temperature
|
Time after synthesis (days)
|
Z-average (d.nm)
|
PdI
|
Zeta Potential
|
1 U
|
|
0
|
172.8 ± 5.478
|
0.198 ± 0.029
|
-5.24 ± 1.35
|
22ºC
|
30
|
155.1 ± 4.339
|
0.266 ± 0.015
|
-13.1 ± 3.35
|
60
|
146.6 ± 1.650
|
0.230 ± 0.027
|
-15.4 ± 7.21
|
4ºC
|
30
|
149.9 ± 2.103
|
0.233 ± 0.010
|
-20.9 ± 1.25
|
60
|
144.7 ± 2.818
|
0.235 ± 0.030
|
-17.5 ± 2.33
|
3 U
|
|
0
|
116.4 ± 2.639
|
0.228 ± 0.021
|
-21.7 ± 0.40
|
22ºC
|
30
|
120.2 ± 1.266
|
0.275 ± 0.011
|
-12.6 ± 3.61
|
60
|
114.2 ± 1.758
|
0.242 ± 0.013
|
-14.9 ± 2.55
|
4ºC
|
30
|
119.0 ± 1.097
|
0.257 ± 0.004
|
-17.9 ± 0.95
|
60
|
115.3 ± 2.150
|
0.281 ± 0.025
|
-13.4 ± 5.81
|
5 U
|
|
0
|
136.0 ± 3.109
|
0.235 ± 0.027
|
-18.8 ± 2.42
|
22ºC
|
30
|
137.8 ± 3.496
|
0.253 ± 0.034
|
-15.2 ± 6.63
|
60
|
124.7 ± 2.219
|
0.217 ± 0.017
|
-15.1 ± 2.81
|
4ºC
|
30
|
132.0 ± 3.940
|
0.24 ± 0.044
|
-9.07 ± 5.16
|
60
|
128.4 ± 1.818
|
0.218 ± 0.014
|
-11.2 ± 4.11
|
As a result, for all formulations synthesized by ultrasound, particles with an average size smaller than 200 nm, low (<0.2) to moderate polydispersity PdI and negative Zeta potential, ranging from -5.24±1.35 to -21.7±0.41 mV. In general, all nanoparticles synthesized in the present study, regardless of how they were obtained and concentration: 1, 2 or 3 mmol L-1, showed good colloidal stability and, consequently, advantages in their use .
The images obtained by TEM for the AgNPs prepared both by the water bath method and by the ultrasound method show particles with predominantly spherical morphologies, smooth and irregular edges, in addition to populations of particles with a homogeneous diameter in the majority (Figure 4a, 4b, 4c , 4d, 4e, 4f), corroborating the polydispersity and hydrodynamic size data obtained by DLS and confirming the results achieved by UV/Vis. Another important aspect to be highlighted is the “core-shell” structure (nucleus-shell) covering the surface of AgNPs, a characteristic phenomenon to be observed in micrographs of green nanomaterials prepared via plants, due to the immobilization of biomolecules from plant extracts that act as stabilizers of AgNPs, increasing their stability (Ahmad & Sharma, 2012; Yassin et al., 2022).
Das et al. (2019) synthesized AgNPs through the aqueous extract of pineapple peel and in morphological analyzes using scanning electron microscopy (SEM) the authors identified an almost spherical morphology of AgNPs, with the presence of some clusters. Similarly, Baran et al. (2021) and Poadang, Yongvanich and Phongtongpasuk (2017) obtained spherical and polydisperse populations of AgNPs from pineapple peel with a mean diameter between 12 and 22 nm by TEM.
In these aforementioned studies, by complementary analyzes of the elemental composition of AgNPs using EDX, the presence of silver in the samples was confirmed as predominant, however the occurrence of other elements such as carbon, oxygen, sulfur, nitrogen, chlorine, originating from the biomolecules of the plant extract , for example proteins, flavonoids and phenolic acids, was also detected.
Biochemical analyzes
The results of the biochemical analyzes of total phenolic compounds and antioxidant activity by the FRAP and ABTS methods are presented in Table 4.
Table 4. Results of the biochemical analyses, mean values of total phenols, ABTS and FRAP of the extract and AgNPs of the pineapple peel.
Variable
|
Extrato
|
Banho Maria
|
Ultrasound
|
1 mmol L-1
|
3 mmol L-1
|
5 mmol L-1
|
1 mmol L-1
|
3 mmol L-1
|
5 mmol L-1
|
Total phenols (mg GA 100 g-1)
|
132.00±6.00
|
159.92±7.98
|
231.85±0.40
|
316.27±11.05
|
171.39±4.59
|
245.20±2.78
|
326.90±10.58
|
ABTS (%)
|
88.16±2.25
|
19.11±3.09
|
56.26±1.79
|
77.44±0.86
|
23.15±8.78
|
59.88±3.82
|
77.90±3.00
|
FRAP (mg FeSO4 g-1)
|
3.72±0.04
|
4.49±0.72
|
4.81±0.45
|
6.83±1.00
|
5.53±0.70
|
5.33±0.89
|
4.52±0.12
|
Phenolic compounds act as antioxidant agents by donating hydrogen ions to stabilize free radicals. thus preventing oxidation reactions. This type of reaction is beneficial to health. since it can help in the prevention of diseases. The average content of total phenols obtained for the pineapple peel extract was 132.00 ± 6.0 mgGA 100 g-1. value close to those detected by Barbosa Júnior et al. (2022) for fresh fruit pulp ranging from 108.7 mg to 133.3 mgGA 100 g-1.
Rasheed et al. (2012) found that the pineapple peel had a higher content of total phenols when compared to the pulp: 130.23 mgGA 100 g-1 against 65.78 mgGA 100 g-1. respectively. According to the same authors. the bark is more susceptible to attack by pests and diseases. therefore. more phenolic compounds will be present in this part of the fruit to protect it.
Studying the nutritional and technological properties of fruit peels. Ortiz (2016) observed that the aqueous extract of pineapple peel had 76.91 ± 0.42 mgGA 100 g-1. lower value than the one obtained in the present work. This fact can be explained by the different climatic conditions. growth stage. Storage conditions. geographic origin. pesticide application. maturation stage. processing type. extraction methods. among others (Silva et al.. 2014; Rasheed et al.. 2012).
The synthesized AgNPs showed higher concentrations of total phenolics (159.92 ± 7.98 to 326.90 ± 10.58 mg AG 100 g-1) when compared to the aqueous extract of pineapple peel (132.00 ± 6.00). Similar results were reported by Salari et al. (2019). Sultana et al. (2015) and Abdel-Aziz et al. (2014) found a higher total phenol content in synthesized AgNPs compared to Prosopis farcta fruit extract and Chenopodium murale and Houttuynia cordata leaf extracts. respectively.
There was an increase in total phenol contents with the increase in AgNO3 nitrate concentration. In a general way. all the synthesized formulations showed relevant concentrations of phenols. whose values ranged from 159.92 ± 7.98 to 326.90 ± 10.58 mgGA 100 g-1. Numerically. nanoparticles synthesized by ultrasound showed the highest values of total phenols in all concentrations evaluated when compared to those obtained by water bath. corroborating with the maximum absorption peaks observed in the UV/Vis analyzes that directly reflect the formation of the provided AgNPs. among other aspects. to the presence of phenolic compounds that through chelation with silver ions reduce and promote the stabilization of nanostructures.
Regarding the antioxidant activity by the ABTS radical capture method, it was observed that the pineapple peel extract had the highest value (88.16% ± 2.25) in relation to the synthesized AgNPs (19.11% ± 3.1 to 77.90% ± 3.00). By the iron reduction method (FRAP). it was found that the capacity of AgNPs to reduce Fe3+ to Fe2+ was greater (4.49 ± 0.72 to 6.83 ± 1.00 mg of FeSO4 g-1) than that of aqueous extract of pineapple peel (3.72 ±0.04 mg of FeSO4 g-1). Result similar to that obtained by Salari et al. (2019) when verifying the greater ability of AgNPs. in different concentrations (0.2-1 mg mL-1). in reducing Fe3+ to Fe2+ in relation to Prosopis farcta extract.
In vitro biological tests
Antibacterial activity of AgNPs
The antibacterial activity of AgNPs was tested in pathogenic bacteria by the broth microdilution method in a wide range of concentrations to identify values capable of inhibiting bacterial growth. The antibacterial assay showed that AgNPs synthesized from the aqueous ex-tract of pineapple peel were effective against Gram-negative and Gram-positive bacteria (Table 5). The inhibitory effects of synthesized AgNPs were confirmed by MIC and MBC values obtained in this study. highlighting the 5 mmol L-1 formulation. obtained by water bath. for presenting better inhibitory and bactericidal activity for the three types of microorganisms studied.
E. coli showed less sensitivity to the 3 mmol L-1 formulation obtained by water bath. with MIC and MBC values of 5.31 μg mL-1. Pseudomonas aeruginosa, on the other hand, showed lower sensitivity for the vast majority of AgNPs samples. independent of concentration and route of synthesis. presenting MIC and MBC between 10.62 and 21.25 μg mL-1. These results are. in general. more efficient than those obtained by Lima et al. (2019) who obtained MIC in the range of 8 to 31 μg mL-1 for AgNPs synthesized with Euterpe oleraceae pulp, Theobroma grandiflorum, Byrsonima crassifolia and Spondias mombin.
Table 5. MIC and MBC values of plant extract and biogenic AgNPs synthesized under different concentrations of silver nitrate and under different energy sources.
MIC / MBC (µg mL-1)
|
Bacterium
|
Extract
|
Banho Maria
|
Ultrasound
|
1 mmol L-1
|
3 mmol L-1
|
5 mmol L-1
|
1 mmol L-1
|
3 mmol L-1
|
5 mmol L-1
|
|
CIM
|
CIM
|
CBM
|
CIM
|
CBM
|
CIM
|
CBM
|
CIM
|
CBM
|
CIM
|
CBM
|
CIM
|
CBM
|
E. coli ATCC 25922
|
5450
|
2.6
|
2.6
|
5.31
|
5.31
|
2.6
|
2.6
|
2.6
|
2.6
|
2.6
|
2.6
|
2.6
|
2.6
|
Pseudomonas aeruginosa ATCC 9027
|
5450
|
10.62
|
21.25
|
21.25
|
21.25
|
10.62
|
10.62
|
10.62
|
21.25
|
21.25
|
21.25
|
21.25
|
21.25
|
S. aureus ATCC 25923
|
5450
|
21.25
|
21.25
|
21.25
|
42.5
|
21.25
|
21.25
|
21.25
|
21.25
|
21.25
|
42.5
|
42.5
|
21.25
|
MIC ranged from 2.6 to 21.25 µg mL-1 for Gram-negative bacteria and from 21.25 to 42.25 µg mL-1 for Gram-positive bacteria. pointing out Gram-negative bacteria as the most susceptible to the inhibitory and bactericidal actions of the evaluated AgNPs. However. no direct relationship was observed between the concentrations and/or source of energy used to obtain AgNPs in the MIC and MBC results.
It was found that the aqueous extract of pineapple peel showed high MIC values. suggesting that it does not perform an effective inhibitory action. This way. based on these results. highlights the importance of using AgNPs synthesized from biological sources in the elimination of pathogenic microorganisms. allowing the reuse of waste that would normally be discarded.
Other studies that also used pineapple peel for the green synthesis of AgNPs investigated MIC and MBC against different bacterial strains. Das et al. (2019) found values above 50 µg mL-1, higher than the present study. On the other hand, the results presented by Baran et al. (2021) prove that AgNPs of 5 mmol L-1 have MIC from 0.5 to 2.0 µg mL-1 for the same bacteria of the present study. Poadang. Yongvanich and Phongtongpasuk (2017) tested AgNPs at 10 µg mL-1 under S. aureus and P. aeruginosa and obtained a zone of growth inhibition of 10 and 7 mm, respectively.
Although the antibacterial action of AgNPs is extensively known, the mechanisms that involve such action are still poorly understood. Some important aspects of nanoparticles that can cause toxicity in microorganisms are: method of obtaining, size, state of aggregation, stability in a biological environment, chemical nature of the coating, surface charge, among others (Durán et al.. 2019). According to Hashemi et al. (2022) and Durán et al. (2019), the size and morphology of nanomaterials play a significant role in antibacterial activity. Smaller materials have higher surface areas, showing greater ability to interact and penetrate the bacterial cell. Within the bacterial cell, during the oxidation process, AgNPs can release Ag+ ions leading to a greater production of oxidative reactive species and, consequently, damage to the cell structure. may result in death. However. Durán et al. (2019) point out that the exact mechanism of toxicity of silver nanoparticles is still the subject of studies.
Cell viability assays by the MTT method
The anticancer activity of AgNPs and the aqueous plant extract of pineapple peel. with different concentrations (0.625; 1.25; 2.5; and 5%). synthesized by water bath and ultrasound. were tested for the MCF-7 cancer cell line (breast cancer) (Figure 5).
The cytotoxic evaluation of AgNPs was performed using the in vitro MTT method, which is a test that evaluates cell metabolism by colorimetry. It is a quantitative test. very sensitive because there is a linear relationship between cellular activity and absorption. resulting in the formation of a purple colored compound. formazan. which can be solubilized and quantified by spectrophotometry. The concentration of formed formazan crystals is directly proportional to the concentration of viable cells (Galdino et al.. 2014).
Cell viability estimates indicated that cell death occurred in a dose-dependent manner. This way. it was verified that the viability of MCF-7 cells decreases with the increase in concentrations of AgNPs (0.625%, 1.25%, 2.5% and 5.0%), with the anticancer action being more intense for nanoparticles with higher concentration, such as, 5 mmol L-1 and 3 mmol L-1. An expected result due to the fact that silver ions present greater toxicity to cancer cells.
It was found that the aqueous extract of pineapple peel. at all evaluated concentrations did not significantly affect the in vitro cell death profile. showing an effect similar to the negative control. AgNPs at 3 mmol L-1 showed significant cytotoxicity to MCF-7 cells with 77.5%. and 90.6% cell death at 2.5% and 5.0% concentrations. respectively. AgNPs at 5 mmol L-1 showed significant differences for cellular cytotoxicity in all concentrations evaluated. having 37.7%, 69.9%, 92.8% and 96.5% of cell death caused at concentrations of 0.625%, 1.25%, 2.5% and 5.0%. respectively. Like this. it was found that the antiproliferative action of AgNPs at 3 mmol L-1 and 5 mmol L-1 was more intense than at the lowest test concentration.
The results of the anticancer activity of AgNPs synthesized by ultrasound at different concentrations (0.625; 1.25; 2.5; and 5%) and, of the aqueous extract of pineapple peel, for MCF-7 cells (Figure 6).
The AgNPs at 1 mmol L-1 showed significant differences, in relation to the control, only at the 5% concentration, where there was a 55.4% reduction in cell viability. MCF-7 cells when exposed to AgNPs at 3 mmol L-1, at 2.5% and 5% concentrations, for 24 h, showed a significant reduction in viability of 75.3% and 95.1%, respectively. AgNPs at 5 mmol L-1 at concentrations 1.25%, 2.5% and 5% showed cytotoxicity to MCF-7 cells of 65.0%, 93.6% and 96.4% respectively.
The anticancer activity of AgNPs at 1, 3 and 5 mmol L-1, synthesized by water bath, with different concentrations (0.625 - 5%) and aqueous extract of pineapple peel, for B16F10 murine melanoma tumor cells is found up in Figure 7.
There were statistically significant differences for AgNPs 5 mmol L-1 in all concentrations evaluated. in relation to control. showing cytotoxic activity for B16F10 cells of 32.5%, 70.3%, 96.6% and 96.6% at concentrations 0.625%, 1.25%, 2.5% and 5%. respectively. As the concentration increases (%) there is an increase in the efficiency of AgNPs 5 mmol L-1 in decreasing the viability of cancer cells. except for the 2.5% and 5.0% concentrations that showed the same percentage of cytotoxic activity.
AgNPs 1 mmol L-1 showed a significant difference in relation to the control at concentrations of 2.5% and 5.0%. The reduction in cell viability caused by 1 mmol L-1 AgNPs at the mentioned concentrations was 20% and 62%, respectively. At concentrations of 1.25%, 2.5% and 5.0%, AgNPs 3 mmol L-1 showed a cytotoxic effect on B16F10 cancer cells of 41%, 76% and 96%, respectively, where at the highest concentration the effect was more significant.
The results of the cell viability test of the B16F10 lineage after exposure for 24 hours to AgNPs, synthesized by ultrasound (U) at different concentrations, and to the aqueous extract of pineapple peel, are shown in Figure 8. It was verified that the AgNPs 5 mmol L-1 showed significant differences, in relation to the control, in all the evaluated concentrations, showing cytotoxic activity to B16F10 cells of 44.0%, 67.4%, 96.8% and 97.4% in the concentrations 0.625%, 1.25%, 2.5% and 5.0%, respectively. This way. cell viability estimates indicated that cell death occurred in a dose-dependent manner.
At concentrations of 1.25%, 2.5% and 5.0%, AgNPs at 3 mmol L-1 showed a significant reduction in cell viability, compared to the control, of 48%, 73% and 96%, respectively. AgNPs at 1 mmol L-1 differed from the control group only at 2.5% and 5.0% concentrations. in which it showed a reduction in cell viability of 31% and 67%, respectively.
The results of the present study demonstrate that the AgNPs obtained by water bath or ultrasound in the three concentrations showed significant cytotoxic activity, in at least one of the different concentrations, for cancer cells of the MCF-7 and B16F10 strains. The aqueous extract of pineapple peel did not significantly affect the viability of MCF-7 and B16F10 cells compared to the control group. in none of the evaluated concentrations.
From the data obtained through cell viability assays, the IC50 (effective concentration to inhibit 50% of cell growth) of in vitro cell viability after 24 hours of exposure of MCF-7 and B16F10 cells to Ag-NPs was calculated. synthesized by water bath or ultrasound, with different concentrations of AgNO3. Similar IC50 values were observed for AgNPs obtained from different energy sources (water bath or ultrasound), but with the same concentration of metallic salt (Table 6), indicating that there is no difference in the use of AgNPs synthesized by the routes proposed in the present study. It was also found that the IC50 values were similar for the two cell lines. MCF-7 and B16F10, after being exposed to AgNPs of the same concentration, regardless of energy sources.
Table 6. IC50 values of AgNPs and plant extract obtained for biological assays of cell viability in MCF-7 and B16F10 cells.
Samples
|
MCF-7
|
B16F10
|
|
IC50 (µg mL-1)
|
IC50 (µg mL-1)
|
|
Extract
|
10015
|
2350
|
1 mmol L-1 – WB
|
4.25
|
4.8
|
1 mmol L-1 – U
|
4.2
|
4.35
|
3 mmol L-1 – WB
|
7.74
|
7.54
|
3 mmol L-1 – U
|
7.36
|
7.76
|
5 mmol L-1 – WB
|
10.35
|
10.5
|
5 mmol L-1 – U
|
11.1
|
11
|