Green Fabrication of Bioactive Silver Nanoparticles Using Mentha pulegium Extract under Alkaline: An Enhanced Anticancer Activity.

Fabrication of silver nanoparticles (AgNPs) using Chinese herbal medicine is popular as the bioactive components included in them would generate potential synergistic effect with the metal nanoparticles. The leaf of Mentha pulegium, whose extract contains a range of phytochemicals and exhibits a wide spectrum of bioactivities, is used as Chinese herbal medicine after drying naturally. Thus, the green synthesis of AgNPs using Mentha pulegium has aroused interests from analysts. However, the biosynthesis of AgNPs under alkaline conditions and the biological activities remain elusive, where alkaline conditions may influence the physicochemical properties and the biological activities of biosynthesized AgNPs. In this study, we were stimulated to fabricate bioactive AgNPs using Mentha pulegium extract under alkaline conditions, accompanied by a systematic evaluation on the effect of biosynthesis parameters on the formation, average size, and polydispersity of AgNPs. Our results showed that alkaline conditions could accelerate the formation of AgNPs with a small average size but at a disadvantage to the polydispersity. Additionally, the as-prepared AgNPs had a hexagonal structure and spherical shape with an average size of 15.7 ± 0.1 nm, existing in the monodispersed form and revealing a high degree of stability. The AgNPs exhibited potent antioxidant and significant inhibitory activity for both bacterial and cancer cell lines. The MIC values of AgNPs for Staphylococcus aureus and Escherichia coli were both 50.0 μg·mL-1, and the IC50 values for HCT116, HepG2, and HeLa cells were 9.0, 14.5, and 31.5 μg·mL-1, respectively. The AgNPs biosynthesized using M. pulegium under alkaline conditions, which had a smaller size and more surface loads, are entirely different with those synthesized under acidic conditions, and the anticancer activity increased significantly. The internalization of AgNPs inside these five cells displayed a variant trend with variable AgNPs concentrations, suggesting the different mechanism of cell death. For two pathogens, HCT116 and HepG2 cancer cell lines, both cell wall and intracellular damage may be responsible for the cell death. However, for Hela cell line the cell death may be rooted in oxidative stress or intracellular penetration. These results confirmed that the AgNPs biosynthesized from M. pulegium extract under alkaline conditions would act as better anticancer agents in biomedicine.

Introduction

Due to their high biocompatibility, lower toxicity, and broad spectrum of bioactive activities like antimicrobial and anticancer activities, silver nanoparticles (AgNPs) have been widely applied as the active ingredient delivery vehicles, diagnosis, and therapy.[1−14] Thus, the synthesis of AgNPs is a subject matter with great scientific interest. Although various physicochemical methods such as autoclaving, γ-ray radiation, and the use electrochemical techniques and chemical reduction can produce high yield of AgNPs, they consume lots of energy and may cause adverse biological effects because of the introduction of toxic reducing and capping agents.[15−21] Therefore, bioresources like plant extracts are more popular in the synthesis of AgNPs for biomedical purposes.[22−26] Biological agents are not only biocompatible, degradable, environment friendly, and cost-effective but also provide the stable coating layers to prevent the aggregation of particles and afford a reduction force to metal precursors.[27] Many studies have shown that when these biological agents displayed biological activities, a synergistic effect between the metal nanoparticles and these bioactive molecules would be obtained.[22,27] Thus, traditional Chinese medicines have attracted considerable attention from analysts due to medicinal components in them.[28−34] To date, numerous Chinese herbal medicines such as Sea buckthorn,[30,35]Zizyphus mauritiana,[36]Nelumbo nucifera,[37,38] and Cornus officinalis(31,33,39,40) have been employed for the synthesis of bioactive AgNPs.

Mentha pulegium, which belongs to the family Lamiaceae and is native to humid regions like China and Iran, is a traditional medicine to treat colds, sinusitis, cholera, food poisoning, bronchitis, and tuberculosis.[41] Studies have shown that Mentha pulegium contains a range of bioactive chemicals like essential oils, isomenthone, menthoacetate, peppermint isoflavourin, and rosmarinic acid, which exhibit a wide spectrum of antioxidant, antimicrobial, and anticancer activities.[41−44] It is possible to fabricate AgNPs with superior bioactivities using Mentha pulegium. On this basis, Abdol-Khalegh Bordbar[45] and Hadi Faranak[46] have successfully synthesized AgNPs using Mentha pulegium leaf extract, which revealed significant inhibition of bacterial strains as well as the cancer cell lines but no cytotoxicity for human peripheral blood mononuclear cells (PBMCs). We noticed that both Abdol-Khalegh Bordbar and Hadi Faranak used the primary extract of the Mentha pulegium leaf, which is an acidic solution. It has been indicated that an alkaline solution may accelerate the formation of AgNPs with a small size and mediate the surface charge, which are directly related to the biological activities.[22,31] The AgNPs with different sizes, shapes, and ζ-potentials would exhibit different antimicrobial and anticancer activities due to displaying various degrees of membrane permeability and electrostatic adhesion between AgNPs and the cell membrane.[22] What the physicochemical properties and biological activities of biosynthesized AgNPs under alkaline conditions are is a fascinating subject. Additionally, it is clear that evaluating the effects of biosynthesis parameters on the physicochemical properties of nanoparticles will play an important role in regulating their biological activities. However, these studies remain elusive to a large extent in past investigations. Obviously, further study on the green synthesis of AgNPs using Mentha pulegium should be proceeded.

Herein, we present the biosynthesis of AgNPs using Mentha pulegium extract under alkaline conditions at ambient temperature (Scheme ), where a systematically investigation about the effects of biosynthesis parameters on the reducing reaction and physicochemical properties of AgNPs was performed. Additionally, the crystalline nature, size, shape, surface properties, stability, and biological activities, including antioxidant, antimicrobial, and anticancer activities, of biosynthesized AgNPs were assessed thoroughly and were entirely different from those of AgNPs synthesized under acidic conditions. The mechanism of cell death was also preliminarily speculated by investigating the internalization of as-prepared AgNPs. These results reveal that the reducing reaction and physicochemical properties of as-prepared AgNPs are sensitive to the biosynthesis parameters, and the AgNPs biosynthesized using Mentha pulegium at alkaline conditions could also act as a promising candidates for antioxidant, antimicrobial, and anticancer drugs.

Scheme 1

Schematic of the Green Fabrication of AgNPs using Mentha pulegium Leaf Extract As the Bioreductant

Results and Discussion

According to previous studies,[47−49] biosynthesis parameters like the pH of the extract, the material proportion, and the incubation time could influence the formation and physicochemical properties of nanoparticles, playing pivotal roles in controlling the chemical, physical, electronic, and optical properties of these nanoscopic materials. Thus, the effects of biosynthesis parameters on the formation, average size, and polydispersity of AgNPs were evaluated systematically.

Effect of Biosynthesis Parameters on the Formation of AgNPs

The primary extract of Mentha pulegium leaf (pH 5.0) was initially used to prepare AgNPs by mixing it (5.0 mL) with 10 mM AgNO3 (5.0 mL) under ultrasonic radiation at ambient temperature. After 1.0 h of incubation, however, there were not any obvious changes in the color of the mixed solution (Figure S1). As shown in previous studies,[50,51] the formation of AgNPs would turn the color of mixed solution to dark brown. This result indicates that the primary extract of Mentha pulegium is helpless for reducing Ag+ to Ag0 within 1.0 h. This may originate from the slow formation rate, which results in very little AgNP generation. To further confirm this conclusion, UV–vis spectroscopy measurements were performed where AgNPs had a distinct absorption peak in 400–500 nm as the surface plasmon resonance (SPR). Figure A shows the UV–vis spectrum of the mixture after 1.0 h of incubation, where the absorbance of the Mentha pulegium leaf extract and AgNO3 were negligible. The spectrum of mixture in the 400–500 nm range is almost similar to that of Mentha pulegium leaf extract alone, which essentially agrees with the observation in color by the naked eye.

Figure 1

UV–vis spectra of the mixture after the reaction between the Mentha pulegium leaf extract and AgNO3 at different (A) pH levels, (B) material ratios, and (C) incubation times.

According to a previous study,[52] adjusting the pH of a plant extract to alkalinity may accelerate AgNP formation. Thus, we then tried to performed the reaction at pH 6.0. The color of the mixed solution changed from yellow to brown after 1.0 h of incubation (Figure S1), suggesting the generation of AgNPs. The UV–vis spectrum for the Mentha pulegium leaf extract and AgNO3 at pH 6.0 obtained after the 1.0 h reaction reveals a strong absorption band that peaks at 412 nm (Figure A). The spectrum characteristics essentially resembled those from previous studies about the SPR peak of AgNPs,[30,52] further confirming that AgNPs were successfully prepared. Subsequently, the pH of the Mentha pulegium leaf extract was successively increased. When the pH was increased to 10.0, the absorption band around 412 nm sharply increased, accompanyied with the slight shift of SPR peak to 407 nm (Figure A). The enhanced SPR band intensity should reflect the increased formation amount of AgNPs. The slight blue-shift of the SPR peak from 412 to 407 nm suggests that the formed AgNPs have different sizes (see below). This may originate from the increased bioavailabity of functional groups at high pH that favors the AgNP product, leading to a faster nucleation rate. This result is in accord with the perspective obtained from previous studies that biosynthesis of AgNPs is a pH-dependent reaction.[52]

Then, to assess the effect of the material proportion on the formation of AgNPs, the experiment was performed at pH 10.0, where the volumes of AgNO3 (5.0 mL) and the mixed solution (10.0 mL) were kept constant. Figure B displays the UV–vis spectra of mixed solutions at different ratios after 4.0 h of incubation at ambient temperature. When the reaction was performed on the ratio 1:20 (Mentha pulegium leaf extract/AgNO3), a weak band with a maximum absorbance around 422 nm was observed. Additionally, as the material proportion of Mentha pulegium leaf extract and AgNO3 gradually increased to 1:1, the peak intensity also increased, accompanyed with the band shift to 403 nm. The increased peak intensity and band shift to blue indicate the increased production yield and different sizes of AgNPs, respectively. It seems that more Mentha pulegium leaf extract would be benefit the generation of AgNPs. However, it is difficult for us to obtain an extract with larger concentrations, since the extract is thick enough.

Figure C shows the absorption spectra of AgNPs after incubating 5.0 mL of Mentha pulegium leaf extract (pH 10.0) and 5.0 mL of AgNO3 with different times. The featured SPR band of AgNPs was clearly observed after 2.0 h of ultrasonic radiation at ambient temperature. Additionally, the absorption intensity enhanced steadily with the passage of time and reached a maximum after 6.0 h, indicating an increased amount of AgNPs and the fully completed reduction reaction within 6.0 h. The UV–vis spectra display a negligible band shift within 6.0 h, but with an obvious band shift after 6.0 h of incubation. The band shift may also result from different sizes of biosynthesized AgNPs. It is clear that biosynthesis of AgNPs with 5.0 mL of 10 mM AgNO3 and 5.0 mL of Mentha pulegium leaf extract at pH 10.0 would have the maximum efficiency.

Effect of Biosynthesis Parameters on the Average Size of AgNPs

Then, the effects of biosynthesis parameters on the average size of biosynthesized AgNPs were systematically assessed, which could further confirm the origin of spectral evolution. The average size of biosynthesized AgNPs was measured by the dynamic light scattering (DLS) method, and the results are shown in Figure and Table S1. It is clear that when the material ratio of Mentha pulegium leaf extract and AgNO3 is 1:1 the average size of biosynthesized AgNPs at an alkaline pH is smaller than that at an acidic pH (Figure A), which is basically in accordance with Sathishkumar[53] and Heydari’s[54] view that an alkaline solution could accelerate the formation of AgNPs with a small size. This distribution of average size could also illustrate the evolution of our UV–vis spectrum shown previously. However, there are still some difference between our results and those from previous studies. It was shown that at pH 7.0 the biosynthesized AgNPs have a larger average size at almost all the incubation times. The biggest (66.3 ± 1.7 nm) and smallest (15.7 ± 0.1 nm) average sizes of AgNPs could be obtained at pH 8.0 after 1.0 h of incubation and pH 10.0 after 5.0 h of incubation, respectively. The influence of the material ratio on the average size of AgNPs was also evaluated at pH 10.0 after incubation for 4.0 h. As shown in Figure B and Table S2, with the addition of Mentha pulegium leaf extract into the mixture, the average size of AgNPs gradually decreased from 122.2 ± 2.7 to 22.6 ± 0.9 nm. This may originate from the faster nucleation rate, since more reducing substances are included in the reaction mixture.

Figure 2

Average size of AgNPs biosynthesized using Mentha pulegium leaf extract at different (A) pH levels and incubation times and (B) material ratios.

Effect of Biosynthesis Parameters on the Polydispersity of AgNPs

The polydispersity index (PDI), which is an important parameter for nanoparticles and could affect the characteristics of nanoparticles, was also assessed thoroughly. Studies have reported that if the sample has a PDI value lower than 0.3, it would exist in the monodisperse form.[55]Figure and Table S3 display the PDIs of biosynthesized AgNPs under different biosynthesis parameters. As shown in Figure A, when the material ratio of Mentha pulegium leaf extract and AgNO3 is 1:1, the biosynthesized AgNPs at an acidic pH have smaller PDI values compared with those synthesized at an alkaline solution on the whole. The smallest and largest PDI values were obtained when the experiments were performed at pH 5.0 after 2.0 h of incubation and at pH 10.0 after 6.0 h of incubation, respectively. The smallest PDI value is 0.23 ± 0.01, indicating a superior monodispersity. The material ratio also has significant influence on the PDI of biosynthesized AgNPs (Figure B and Table S4). At pH 10.0 after incubation for 4.0 h, the best monodispersed AgNPs were obtained with the material ratio of 1:3, where the PDI value was 0.21 ± 0.01.

Figure 3

Polydispersity index (PDI) of AgNPs biosynthesized using Mentha pulegium leaf extract at different (A) pH levels and incubation times and (B) material ratios.

To obtain more products with a small size for further characterization and biological activity studies, we prepared AgNPs using 5.0 mL of 10 mM AgNO3 and 5.0 mL of Mentha pulegium leaf extract (pH 10.0) after 5.0 h of incubation.

Characterization of Biosynthesized AgNPs

The experiments of energy dispersive X-ray spectroscopy (EDX), TEM, HRTEM, selected area electron diffraction (SAED), and DLS were performed to characterize the proper synthesis, crystalline nature, size, shape, and surface properties of biosynthesized AgNPs. The EDX spectrum (Figure A) displays a strong absorption peak around 3 keV, which relates to the presence of elemental silver in the nanoparticles. Another two strong signals in the EDX spectroscum are for carbon and copper, which should partially originate from the carbon-coated copper grid used in the TEM and EDX analysis. The EDX result further confirms the generation of AgNPs after a period of incubation of Mentha pulegium leaf extract and Ag+.

Figure 4

(A) EDX spectrum, (B) TEM image, (C) SAED image, and (D) HRTEM image.

Figure B shows the TEM image for biosynthesized AgNPs, which are mainly spherical in shape and have good dispersion and scattering in nature for a large proportion of the particles. It is obviously that the biosynthesized AgNPs have diameters roughly in the range of 10–20 nm basically in accordance with DLS results (15.7 ± 0.1 nm), which are smaller than those synthesized using Mentha pulegium extract under acidic condition.[45,46]

The SAED pattern and HRTEM spectrum as shown in Figure C and D, respectively, were used to prove the crystalline nature of as-prepared AgNPs. Three circular rings with d-values of 2.255, 1.420, and 1.159 Å in the SAED pattern (Figure C) were observed, which corresponded to the characteristic reflections of hexagonal crystalline silver for (1 1̅ 2), (1 1 0), and (2 0 3) facets. This indicates the formation of hexagonal crystalline AgNPs. The lattice fringe was also measured to illustrate the crystalline nature of biosynthesized AgNPs. The measured d-value was 2.510 Å (Figure D), which was assigned to (0 0 4) facets of hexagonal crystalline AgNPs, further confirming the result about the crystalline nature obtained from the SAED pattern. The crystalline nature of biosynthesized AgNPs herein is different from that biosynthesized by Abdol-Khalegh Bordbar[45] and Hadi Faranak,[46] which may originate from the different nanocomposite.[56]

Lastly, the ζ-potential was used to assess the surface state and the long-term stability of biosynthesized AgNPs. Previous studies reported that the nanoparticles with ζ-potentials between −25.0 and +25.0 mV would have interparticle attractions and eventually lead to the aggregation of nanoparticles.[54] In parallel, the nanoparticles with ζ-potentials outside of the interval would have superior stability. As shown in Table S5, the ζ-potential of AgNPs biosynthesized by Mentha pulegium leaf extract at pH 10.0 (−24.0 mV) was close to boundary of −25.0 mV, indicating a high degree of stability. Practically, the average size of the biosynthesized AgNPs after 45 days (21.7 ± 0.5 nm) reveals only a slightly change. Here, the ζ-potential obtained at pH 10.0 is smaller than that obtained by Abdol-Khalegh Bordbar (−15.9 mV) using Mentha pulegium(45) and us at lower pH (Table S5). The negative ζ-potential also suggests the electronegative capping agents in the surface of AgNPs. These molecules may be flavonoids, terpenoids, polyphenols, and proteins contained in Mentha pulegium extract, which have been confirmed by previous FTIR studies from Abdol-Khalegh Bordbar[45] and Hadi Faranak.[46]

Antioxidant Activities of Biosynthesized AgNPs

Based on previous studies,[41,42]Mentha pulegium contained more than 25 species, such as a high content of essential oils. Several authors have documented the antioxidant activity of Mentha pulegium extracts and the essential oils originating from Mentha pulegium.[42] As AgNPs would be coating by the bioactive constituent involved in the Mentha pulegium leaf extract, the AgNPs biosynthesized using Mentha pulegium leaf extract are anticipated to exhibit powerful antioxidant activity. Thus, we performed the DPPH experiment to evaluate the antioxidant activity of biosynthesized AgNPs, where the DPPH method is considered as one of the quickest methods. DPPH is a stable free radical, and its color would change from purple to yellow when it accepted hydrogen and electrons from the donors. Different concentrations of biosynthesized AgNPs were added into DPPH solution for the purpose of scavenging free radicals. As shown in Figure , the as-prepared AgNPs reveal significant DPPH scavenging activity, which increased in a dose-dependent manner and had scavenging activities of 67.2% in 200.0 μg·mL–1 of AgNPs. However, when increasing the amount of AgNPs to 400.0 μg·mL–1, the scavenging activity slightly changed to 70.2%. The DPPH scavenging activity of AgNPs is comparable with those from previous reports by KÜP[57] and Kivcak,[58] indicating that the AgNPs synthesized by using Mentha pulegium leaf extract could be used in treatment of many diseases caused by oxidative stress.

Figure 5

Free radical scavenging rate of biosynthesized AgNPs against DPPH.

Antibacterial Activities of Biosynthesized AgNPs

The antibacterial activities of as-prepared AgNPs against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were evaluated by measuring the absorbance of bacteria at 600 nm. Figure displays the growth kinetics for these two pathogens after treatment with different doses of AgNPs, exhibiting a potential dose-dependent manner. It clearly reveals that even after treatment with 3.1 μg·mL–1 AgNPs, the growth of both pathogens would be inhibited. When incubating these two pathogens with 50.0 μg·mL–1 AgNPs, the growth of both E. coli and S. aureus was effectively inhibited within 9.0 h, indicating that the minimum inhibition concentrations (MIC) of as-prepared AgNPs were 50.0 μg·mL–1. The tests of zone inhibition, which were performed using the oxford cup method, also show significant inhibition for both bacterial strains against the nanomaterials (Figure S2). The MIC of AgNPs obtained herein is slightly different from those obtained by Abdol-Khalegh Bordbar (E. coli and S. aureus, 25 μg·mL–1)[45] and Hadi Faranak (S. aureus, 61.5 μg·mL–1; E. coli, 125 μg·mL–1)[46] using Mentha pulegium leaves. We speculated that might arise from different chemical constituents of the Mentha pulegium extract used in the biosynthesis of AgNPs, which have a significant effect on the affinity of AgNPs for phosphorus- and sulfur-containing compounds on the cell.[59]

Figure 6

Antibacterial activity of biosynthesized AgNPs for (A) E. coli and (B) S. aureus.

Anticancer Activities of Biosynthesized AgNPs

The anticancer activities of the biosynthesized AgNPs against HCT116, HepG2, and HeLa were preliminarily assessed by exposing these three cancer cell lines to AgNPs at concentrations in the range of 0–100 μg·mL–1 for 24.0 h. MTT assays were performed to determine the cytotoxicity of AgNPs for these cancer cell lines. As shown in Figure , after treating these cancer cell lines with AgNPs, the cell viability decreased in a dose-dependent fashion. With increased doses of AgNPs, the cell viability of AgNP-treated cell lines significantly decreased. In the cases of HCT116 and HepG2 cell lines, cell viabilities were significantly decreased to 19.9% and 16.7% at 20.0 μg·mL–1 AgNPs and to 6.1% and 11.9% at 80.0 μg·mL–1 AgNPs, respectively. On the other hand, for the Hela cell line, the cell viability was 10.9% at 50.0 μg·mL–1 AgNPs and 6.5% at 100.0 μg·mL–1 AgNPs. The IC50 values of AgNPs against HCT116, HepG2, and HeLa cancer cell lines were determined by fitting the curve of the cell viability using the GraphPad Prism 8.0 software package, and the values were 9.0, 14.5, and 31.5 μg·mL–1, respectively. The AgNPs synthesized by the Mentha pulegium extract herein displayed a higher anticancer activity than the AgNPs synthesized by Hadi Faranak using Iranian Mentha pulegium (∼100 μg·mL–1 for Hela and MCF-7),[46] and other traditional medicines with an anticancer effect, such as Iresine herbstii(60) (51 μg·mL–1 for Hela) and Cornus officinalis(31) (20.68 and 69.72 μg·mL–1 for HCT116 and HepG2, respectively). Additionally, these values were also comparable to those of Sea buckthorn(30) (8.77, 14.59, and 27.98 μg·mL–1 for HCT116, HepG2, and HeLa, respectively), indicating that the AgNPs biosynthesized using Mentha pulegium extract could be acted as candidate for anticancer drugs.

Figure 7

Inhibition effects of biosynthesized AgNPs on HCT116, HepG2, and Hela cell lines.

Internalization of Biosynthesized AgNPs

To clearly elucidate the mode of action between the cell and the nanoparticles, three well-defined mechanisms have been proposed: (i) cell wall and membrane damage, (ii) intracellular penetration and damage, and (iii) oxidative stress.[27,61] Obviously, assessing the internalization of AgNPs into cells would benefit the understanding of the cell death mechanism. Previous studies are available that show granules in cytoplasm could scatter more light in the perpendicular direction (90°) of an incident laser (488 nm), defined as the side scattering channel (SSC).[62,63] It has been hypothesized that the internalized nanoparticles in cells could act as granules and scatter more lights, which could be collected using flow cytometry. Therefore, we performed flow cytometric analysis to assess the internalization of AgNPs in both bacteria strains and cancer cell lines.

Figures and 9 display the density plots of flow cytometry light scattering before and after treating both bacteria and cancer cell lines with 5.0 and 20.0 μg·mL–1 AgNPs for the indicated times. The SSC intensity for both pathogens after treatment with 5.0 and 20.0 μg·mL–1 AgNPs slightly increased in compared with that of the control cells, indicating the internalization of synthesized AgNPs. It is interesting that E. coli displays a higher scattering in comparison to S. aureus, meaning more uptake of the biosynthesized AgNPs. The difference may be due to the distinct adherence to the charged bacterial cell wall or the affinity for phosphorus- and sulfur-containing compounds on the cell within a certain processing time. Moreover, the SSC values obtained after treatment with 5.0 μg·mL–1 AgNPs for both pathogens are slightly higher than those obtained after treatment with 20.0 μg·mL–1 AgNPs. We speculated that after treatment with a high concentration of AgNPs, the cell wall and membrane were damaged and the granules could run off from the cytoplasm.[59] Combining these results with the MICs of AgNPs against both E. coli and S. aureus, it is possible that the cell wall and membrane of S. aureus are susceptible to the AgNPs biosynthesized by the Mentha pulegium leaf extract.

Figure 8

Uptake analysis of AgNPs with concentrations of (A) 0, (B) 5, and (C) 20 μg·ml–1 into E. coli and (D) 0, (E) 5, and (F) 20 μg·ml–1 into S. aureus as assessed by flow cytometry.

Figure 9

Uptake analysis of AgNPs with concentrations of (A) 0, (B) 5, and (C) 20 μg·ml–1 into HCT116; (D) 0, (E) 5, and (F) 20 μg·ml–1 into HepG2; and (G) 0, (H) 5, and (I) 20 μg·ml–1 into Hela cell lines as assessed by flow cytometry

The uptake analysis of AgNPs into HCT116, HepG2, and HeLa cancer cell lines was also performed. The SSC values for HCT116 and HeLa cancer cell lines show a significance increase at both concentrations, indicating AgNP internalization. However, for the HepG2 cancer cell line the SSC value decreases from 3.45% to 1.95% after treatment with 5.0 μg·mL–1 AgNPs, which may result from the leakage of granules into the cytoplasm. This result indicates that AgNPs biosynthesized by Mentha pulegium leaf extract may cause necrosis by disintegrating the cell wall and membrane, which is assumed to be primary mechanism of cell death. Briefly, nanoparticle adhesion to the cell membrane would trigger cell morphological changes and thus result in the disruption of membrane permeability and respiratory functions via membrane depolarization. The increased membrane permeability and disruption of the cell wall would lead to the leak of cellular content, including proteins, enzymes, DNA, ions, metabolites, and the energy reservoir, into environment. The disintegrated cell wall and membrane would facilitate the uptake of AgNPs into the cell, which has been confirmed by the flow cytometry analysis at 20.0 μg·mL–1 AgNPs (SSC = 8.43%). Otherwise, we found that after treatment with a high concentration of AgNPs, the changes of the SSC intensity revealed different trends, where the SSC values for HCT116 decreased to 4.95% and those for Hela increased to 56.7%. The slightly decreased SSC value for HCT116 may due to the leak of cellular content, and the increased value indicates the more uptake of AgNPs. It seems that the mechanism of cell death originating from AgNPs in different cell lines is variant. For HCT116 and HepG2 cell lines, both cell wall damage and intracellular damage may be responsible for the cell death. However, for Hela cell line the cell death may be rooted in the formation of ROS or intracellular penetration. To disclose the precise mechanism of cell death, more sophisticated experiments should be performed.

Conclusion

In this study, the bioactive silver nanoparticles (AgNPs) were successfully fabricated using Mentha pulegium leaf extract under alkaline conditions with ultrasonic radiation at ambient temperature. The effects of biosynthesis parameters on the formation, average size, and polydispersity of AgNPs were also assessed. Characterization of as-prepared AgNPs revealed that the crystalline structure, average size, and surface loadings were entirely different from those synthesized by Mentha pulegium under acidic conditions. Furthermore, as-prepared materials were applied for biological activities like antioxidant, antibacterial, and anticancer activities. The results displayed that the anticancer activities were enhanced significantly. The flow cytometric analysis for both pathogens and cancer cell strains displayed a significant change in the SSC value with a variant trend, indicating the different mechanisms of cell death. These results confirmed that the as-prepared AgNPs could be also useful for the development of new alternative biological agents in biomedicine, especially for anticancer efficacy.

Experimental Section

Chemicals

Mentha pulegium was purchased from a local pharmaceutical factory (Shaanxi Sciendan Pharmaceutical Co., Ltd., Xi’an, China). Other reagents, such as silver nitrate (AgNO3, 99.8%), sodium chloride, yeast powder, 1,1-diphenyl-2-picrylhydrazulin (DPPH, > 99.5%), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromine (MTT), dimethyl sulfoxide (DMSO), and so on, were bought as described in our previous studies.[30] The purity of all chemicals was analytical grade, which was not enhanced further. Ultrapure Milli-Q water was used to prepare all solutions.

Synthesis of Silver Nanoparticles

In this study, an easy and eco-friendly method for the green fabrication of bioactive AgNPs was developed using the leaf extract of Mentha pulegium under ultrasonic radiation at ambient temperature. The specific processes are as follows. The Mentha pulegium leaves (5.0 g) were ground to powder and then added into 100 mL of deionized water with ultrasonic radiation for 4.0 h. After filtration, the extract of the Mentha pulegium leaf was obtained, which was stored in 4 °C for further studies. Then, 5.0 mL of the aqueous extract was combined with 5.0 mL of 10 mM AgNO3 to biosynthesize AgNPs under ultrasonic radiation. After 30 min of centrifugation at 10 000 rpm, the pure AgNPs could be obtained from the mixture. The resulting precipitates were washed several times using deionized water and then lyophilized for 12.0 h. The pH of the Mentha pulegium extract was adjusted with a certain concentration of NaOH to desired values. After adding AgNO3 for the reduction reaction, the pH value of the mixture did not have any obvious change, which was monitored and determined by a pH meter.

Characterization of Silver Nanoparticles

To monitor the formation of AgNPs, the spectrum of the mixture ranging from 200 to 800 nm was periodically collected by using a UV–vis spectrophotometer (UV-2600, Japan). Transmission electron microscopy (TEM) was performed with a JEM-2100 Plus microscope equipped with an energy dispersive X-ray spectroscopy (EDX) attachment. Both were used to characterize the morphology of the AgNPs, whose crystalline nature was confirmed by HRTEM and selected area electron diffraction (SAED). The polydispersity and surface loads of the AgNPs were evaluated by measuring the polydispersity index and ζ-potential using a Malvern Zetasizer Nanoseries (ZEN 3600, Malvern, UK) instrument.

Antioxidant Activity of Silver Nanoparticles

To assess the antioxidant activity, serial concentrations of AgNPs (1.0 mL) were added to 1.0 mL of a 1,1-diphenyl-2-picrylhydrazulin solution (DPPH, 2.5 μg·mL–1), which was vigorously shaken and incubated for 30 min in the dark at room temperature. The absorbance of DPPH at 517 nm in ethanol would be quenched to some extent due to the addition of antioxidative AgNPs. The absorption of the negative control group (without AgNPs) was subtracted from each group of reaction mixture. The DPPH scavenging rate could be obtained by the following equation:where A0 is the absorbance of the DPPH and ultrapure water at 517 nm, A1 is the absorbance of DPPH and AgNPs at 517 nm, and A2 is the absorbance of ethanol and AgNPs at 517 nm.

Antibacterial Activity of Silver Nanoparticles

The growth inhibition studies of AgNPs against both S. aureus and E. coli were conducted in Luria–Bertani (LB) broth media. The experiments were carried out as per our previous illustration.[30] Briefly, 50 μL of the freshly prepared bacterial suspensions was added into 96-well plates, which contain desired amount of AgNPs and 50 μL of LB broth media in each hole. The plates were then incubated in a rotary shaker at 160 rpm at 37 °C. The increase of bacterial absorption at 600 nm was monitored every hour for 24.0 h. The control experiments, which contained only media and bacteria devoid of AgNPs, were also performed for both pathogens.

Anticancer Activity of Silver Nanoparticles

The well-cultured human colorectal cancer HCT116, hepatoma cancer HepG2, and cervical cancer (HeLa) cell lines were plated out in flat-bottom 96-well plates at a density of 1 × 104 cells per well and allowed to attach for 24.0 h. After removing the supernatant, the desired amount of AgNPs (0.8–100.0 μg·mL–1) was added into 96-well plates, which were cultured at 37 °C in an atmosphere of 5% CO2 for 24.0 h. Then, 20 mL of thiazolyl blue tetrazolium bromide (MTT, 5.0 μg·mL–1) and 150 mL of DMEM medium were injected into each well. After 4.0 h of incubation, the media were gently removed to collect the formed formazan crystals, which were dissolved in 150 μL of DMSO before their absorbance was measured at 490 nm. The cell viability could be obtained by complying with the following formula:where A is the absorbance of the sample, Ab is the bleaching absorbance without the sample, and A0 is the absorbance value of the control.

Internalization of Silver Nanoparticles

According to a previous study,[63] light scattering principles could be utilized to assess the internalization of nanoparticles in both bacteria and cancer cell lines. Thus, flow cytometric analysis was performed. All the cells referred to in the study were seeded in 6-well culture plates and combined with 5.0 and 20.0 μg·mL–1 AgNPs for 12.0 h of incubation. After this, these cells were harvested and resuspended for the flow cytometric experiments, where light scattering signals were collected and detected with corresponding detection channels after excitation with 488 nm lasers. The FlowJo 7.6.1 software was utilized to analyze the output data.