Novel silver-platinum bimetallic nanoalloy synthesized from Vernonia mespilifolia extract: Antioxidant, antimicrobial, and cytotoxic activities

2.1 Materials

Silver nitrate (AgNO₃, ≥ 99%), potassium tetrachloroplatinate (II) (K₂PtCl₄ ≥ 99.9%), Folin–Ciocalteu reagent, anhydrous sodium carbonate, aluminum trichloride (AlCl₃), sodium nitrite (NaNO₂), sodium chloride (NaCl), 2,2 diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), vanillin, TPTZ (tripyridyl triazine), Mueller Hinton agar, Mueller Hinton broth, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin solution, sodium acetate, ferric chloride, ascorbic acid, hydrochloric acid, sodium hydroxide, and phosphate buffer. All chemicals used in this study were of analytical grade and purchased from Sigma-Aldrich (Johannesburg, South Africa). Whole V. mespilifolia plants were collected and identified by Mr. Tony Dold of Selmar Schonland Herbarium, Rhodes University, South Africa. All solutions were prepared with deionized water.

2.2 Preparation of plant material and extract

V. mespilifolia was prepared as previously reported (Unuofin et al., 2018). 1 g of finely ground powder of V. mespilifolia whole plant was mixed with 100 mL ethanol and heated at 60 °C for 20 min and then filtered. The plant extract was kept at 4 °C until further use.

2.3 Synthesis of AgNPs and AgPtNPs and UV–Vis spectroscopy

To synthesize AgPtNPs, 10 mL of ethanolic extract of V. mespilifolia (EVM) was mixed with 50 mL of 1 mM AgNO₃ and 1 mM of K₂PtCl₄ solution in a flask and stirred at 85 °C for 60 min. The reaction was monitored with UV/vis absorption spectroscopy (PerkinElmer Lambda 60 UV–vis spectrophotometer). The nanoparticles that were obtained were centrifuged and washed several times with water to remove any unreacted salts and extracts. The pellets obtained were then oven-dried at 40 °C to obtain the nanoparticles in powder. As a control, the same procedure was followed to synthesize monometallic AgNPs. The nanoparticles of desired weight were dispersed in deionized water, sonicated for 10 min and used for further characterization and biological applications unless otherwise stated.

2.4 Characterization

Dried powdered samples of the purified nanoparticles were used for characterization. The FTIR spectra of extract-reduced AgNPs and AgPtNPs were studied using a PerkinElmer Frontier FT-IR fitted with an ATR detector in the range of 4000–500 cm⁻¹. The morphology and particle size of the nanoparticles were determined by dropping a dispersed solution of the purified nanoparticles onto a carbon coated copper grid and then allowed to dry before analysis using transmission electron microscopy (TEM) on a JEOL JEM 2100 running at 200 kV voltage. Energy dispersive X-ray spectra (EDS) were obtained using a TEM equipped with EDS. Nanoparticle stability was determined using Dynamic Light Scattering (DLS) technique on a Malvern Zetasizer Nano-ZS (Malvern Corp, UK).

2.5 Quantitative phytochemical analysis and antioxidant properties

2.6 Quantification of radical scavenging activity

The radical scavenging capacities of AgNPs and AgPtNPs were measured using DPPH radical scavenging activity, ABTS radical scavenging activity, and ferric reducing antioxidant power (FRAP) assay.

2.7 DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging activity assay

The antioxidant potential of AgNPs and AgPtNPs against DPPH radicals was determined using a previously described method with slight modifications (Khan et al., 2012). The DPPH assays were performed in 96 well plates. The test sample (100 µL) from a stock solution was poured into the respective wells of the plate and then 100 µL of DPPH solution was added to each well to make 200 µL final volume. Ascorbic acid was used as the reference standard. The mixture was incubated at room temperature for 30 min. $$DPPHscavengingactivity\left(\%\right)=\frac{\mathit{Abscontrol}-Abssample}{\mathit{Abscontrol}}X100$$ where Abs control is the absorbance of DPPH + methanol and Abs sample is the absorbance of DPPH radical + sample/or standard.

2.8 ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid) radical scavenging activity

The ABTS radical scavenging abilities of AgNPs and AgPtNPs from the ethanolic extract of V. mespilifolia followed a method previously described (Unuofin et al., 2017). Briefly, the ABTS radical was formed by reacting solutions of 7 mM ABTS and 2.45 mM potassium persulfate for a period of 12 h at room temperature in the dark. A working solution was developed by mixing 1 mL ABTS radical with 60 mL of methanol, and the absorbance was adjusted to 0.700 ± 0.008 at 734 nm. Thereafter, 100 µL of the ABTS solution was mixed with 100 µL of each extract/standard (25 to 400 µg/mL). The absorbance of the resulting mixture was read at 734 nm after 7 min of incubation in the dark. All samples were run in triplicates. The ABTS radical-scavenging potential of the extracts/standard was determined using the following equation: $$ABTSscavengingactivity\left(\%\right)=\frac{\mathit{Abscontrol}-Abssample}{\mathit{Abscontrol}}X100$$ where Abs control is the absorbance of DPPH + methanol and Abs sample is the absorbance of DPPH radical + sample/or standard.

2.9 Ferric reducing antioxidant power (FRAP) assay

The ability of AgNPs and AgPtNPs obtained from the ethanol extract of Vernonia mespilifolia to reduce ferric ions was carried out using the method described (Ahmed et al., 2015). The working FRAP reagent used for this study was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (tripyridyl triazine) solution, and 20 mM FeCl₃·6H₂O solution at a ratio of 10:1:1 respectively. Briefly, 50 µL of each nanomaterial (1 mg/mL) and 50 µL of the standard solutions of gallic acid (20 to 100 µg/mL) was added to 950 µL of FRAP working reagent. Absorbance was read at 593 nm after 15 min of adding the working reagent. The blank sample consisted of 950 µL of FRAP working reagent and 50 µL of methanol. The reducing power capability of the various extracts was expressed as an equivalent concentration of antioxidants that gave a ferric reducing ability equivalent when compared to the gallic acid standard (GAE). $$\mathit{GAE}=\frac{\mathit{CV}}{M}$$ where GAE = gallic acid equivalent of sample extract (mg GAE/g of sample);

C = concentration of gallic acid extrapolated from the standard curve in mg/mL;

V = volume of the sample extract in mL;

M = weight of the sample extract in g

2.10 Preparation of the microbial suspension

Microorganisms were standardized using the method of CLSI guidelines (Wiegand et al., 2008). All the tested microbes were grown in nutrient broth for 24 h, followed by the matching of bacterial suspension to the turbidity equivalent to 0.5 McFarland solution (1 × 10⁸ CFU/mL) with the addition of sterile saline.

2.11 Antimicrobial activity

The antimicrobial activity of the synthesized AgPtNPs was tested using the serial dilution method (Elisha et al., 2017) against one gram-positive bacterium (Staphylococcus aureus) (ATCC: 25923), a multidrug-resistant gram-negative bacterium (Escherichia coli) (ATCC: 25922), and a fungus (Candida albicans) (ATCC: 10231) (Anatech, Kwik-stick®). A 1 mg/mL AgPtNPs dispersed in deionized water (stock solution) was prepared and serially diluted in a 96 well plate with an overnight culture of microorganisms to a final concentration range of 7.8–1000 µg/mL. The AgNPs was used as a control for the experiment. Plates were incubated at 37 °C for 24 h, and 20 µL of 0.2 mg/mL of the microbial growth indicator Resazurin dye (Sigma-Aldrich®) was added to all wells. All experiments were performed in triplicate.

2.12 Cytotoxicity assay

The MTT reduction assay was used to assess the cytotoxicity of the nanoparticles on human embryonic kidney (HEK 293) and human breast cancer (MCF-7) cell lines. Cells were grown in tissue culture flasks and harvested using 2% trypsin in DMEM. Cells were counted and 100 µL of cells were seeded into a 96 well plate (5 × 10⁴ cells/well) and the plates incubated for 24 h at 37 °C with 5% CO₂. After incubation, the media were aspirated and treated with 100 µL of varying concentrations of dispersions of AgPtNPs and AgNPs in culture media and incubated for 24 h. The plates were treated with 20 µL (5 mg/mL) MTT solution and incubated for another 3 h. Afterwards, the media were carefully removed and then DMSO were added to dissolve the formazan crystals. The plates were read at 570 nm using an ELISA plate reader (ThermoFisher Scientific, Varioskan Flash, Finland) and the mean of the triplicate results calculated.

2.13 Data analysis

All assays were conducted in triplicate, and the obtained results were expressed as (mean ± SD). Statistical analysis was done using MINITAB 17 and a one-way ANOVA by Fischer’s least significant difference (LSD) to determine significant differences in all parameters. Values were considered statistically significant at *p < 0.05.

3.1 Preparation and characterization

Preliminary phytochemical screening of the EVM plant extract confirmed the presence of bioactive compounds such as polyphenols, proanthocyanidins, tannins, and flavonoids, thereby confirming its use as an eco-friendly means of synthesizing metal nanoparticles (Unuofin et al., 2018). The bioreduction of mixed Ag and Pt ions to form AgPtNPs using EVM extract was monitored using UV–vis spectroscopy with the reaction completed in 1 h. A final brownish-green color was observed indicating the formation of AgPtNPs (Fig. 1). The UV–vis spectra of the EVM capped AgPtNPs showed surface plasmon resonance (SPR) at 415 nm after 60 min (Fig. 2A). Unlike core–shell bimetallic nanoparticles, which display two bands, the single band formation in the region of Ag likely indicated that more of the Ag than Pt was reduced. Hence, we suspected the possible formation of a nanoalloy (Elemike et al., 2019; Xia et al., 2013). The monometallic AgNPs control showed a characteristic peak appearing at approximately 413 nm (Fig. S1). The peaks at 538, 610, and 672 nm could be ascribed to some intensely absorbed bioorganic oxidation products originating from the extracts (Elemike et al., 2019).

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Fig. 1

Photographs showing the color of whole plant ethanolic extract (A), Ag and Pt mixed solution (B), and colloidal AgPtNPs nanoparticles (C).

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Fig. 2

UV–vis absorption spectra (A), TEM image (B) EDS spectra (C), and SAED (D) of the extract synthesized AuPtNPs.

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The TEM image of the AgPtNPs synthesized from the dried EVM extract showed predominantly spherical shaped nanoparticles (Fig. 2B) with a mean diameter of 35.5 ± 0.8 nm (Fig. S2) and polydispersity index (PDI) of 0.2. The average particle diameter calculated for AgNPs was 33.4 ± 1.0 nm with a spherical morphology (Fig. S3). Both AgPtNPs and AgNPs were of mean diameter lower than 40 nm, which have been reported to easily taken up by cells and can enter the nucleus (Wu et al., 2019). The energy-dispersive X-ray (EDS) spectra (Fig. 2C) showed peak signals from Ag and Pt, indicating that both metals were present in the extract synthesized nanoparticles. Similarly, the EDS spectra of the control AgNPs contained Ag elemental presence (Fig. S4). The Cu signals were attributed to the carbon coated copper grid upon which the nanoparticle samples were drop-dried for the analysis. The bright spots in the selected area electron diffraction pattern (SAED) showed that the formed AgPtNPs are crystalline (Fig. 2D). The results corresponded with the absorption spectra data and demonstrated the efficiency of the EVM to form metallic nanostructures.

3.1.1

3.1.1 FTIR analysis

The FTIR spectrum of the dried EVM extract (Fig. 3) showed peaks at 3331, 2918, 1736, 1602, 1377, 1263, and a strong and intense peak at 1028 cm⁻¹. The peak at 3331 cm⁻¹ represents a strong hydroxyl group found mostly in phenolics, carbohydrates, and flavonoids. The peaks at 2918, 1736, 1602, 1377, 1263, and 1028 cm⁻¹ are associated with C-H (stretching), C = O (carboxylic stretching), N-H (bending), O-H (carboxylic bending), C-N (primary aromatic amine stretching), and C-C (stretching) respectively. The presence of almost all these characteristic peaks of EVM in the biosynthesized AgNPs and AgPtNPs strongly indicated effective capping and stabilization of the nanoparticles. The intensity of the peaks of AgNPs and AgPtNPs was reduced, as shown in their spectra, thus indicating a reduced concentration of EVM extract in the stabilization and possible conjugation with the nanoparticles. These results support the key role that EVM extract played in the reduction of the metal ions to their corresponding metal nanoparticles.

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Fig. 3

Fourier transform infrared spectra of dried V. mespilifolia (EVM) leaf extract before and after biosynthesis of AgNPs and AgPtNPs.

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3.1.2

3.1.2 Nanoparticles stability

The stability of the purified nanoparticles dispersed in water was measured for 7 days using a dynamic light scattering (DLS) technique. The AgNPs and AgPtNPs showed a negligible zeta-potential value increase during this period (Fig. 4). The negative charges may be attributed to the strong adsorption of plant phytochemicals on the formed nanoparticles, thereby improving their stability and preventing aggregation (Elemike et al., 2019). Similarly, a polydispersity index (PDI) of 0.2 was calculated for AgPtNPs. A low degree of dispersity indicated the good quality and monodispersity of the AgPtNPs with respect to the particle size distribution which is beneficial for biological activity. The PDI and size distribution are highly essential properties as it affect the stability, cellular uptake, accumulation, and ultimately the biological performance induced by nanoparticles (Danaei et al., 2018).

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Fig. 4

Zeta-potentials of the extract synthesized AgNPs and AgPtNPs measured for 7 days. Data are presented as mean ± standard deviation (n = 3).

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3.2 Phytochemical constituents

We investigated the role of polyphenolic compounds in the biosynthesis of AgNPs and AgPtNPs from an ethanol extract of V. mespilifolia by quantifying the total phenolic content (TPC), total flavonoids content (TFC), and proanthocyanidins (PA) content at baseline (0 min) and after 60 min of reaction time. The information from the above is essential in highlighting the possible mechanism of the reaction. Phenols, flavonoids, proanthocyanins, polyamines, and polyamides belong to a family known as polyphenolic compounds. In recent times, phenols, flavonoids, and proanthocyanins have been shown to possess the ability to donate an electron, act as metal chelators, and as singlet and triplet oxygen quenchers (Bakhtiar et al., 2015; Unuofin et al., 2018). In our previous study, ethanol extract of V. mespilifolia showed the presence of 106.9 ± 0.0 mg GAE/g dry weight TPC, 464.7 ± 0.0 mg QE/g dry weight TFC, and 83.9 ± 0.0 mg CE/g dry weight PA (Unuofin et al., 2018). In the synthesized nanoparticles (AgNPs and AgPtNPs), a reduction in the amount of TPC (28.0 ± 0.9 and 13.6 ± 0.1 mg GAE/g dry weight) and TFC (366.2 ± 17.0 and 126.6 ± 0.3 mg QE/g dry weight) was observed, while the PA content of AgNPs increased as shown in Table 1. The reduction observed mainly in the TPC and TFC of AgNPs and AgPtNPs and PA of AgPtNPs after 60 min of synthesis could be attributed to the nucleophilic nature of the aforementioned phytochemicals, which play a major role in the reduction and chelation of transitional metals and also aid in stabilizing them (Raghunandan et al., 2010; Sivaraman et al., 2009).

3.3 Antioxidant activity

The antioxidant activities of synthesized AgNPs and AgPtNPs were evaluated by DPPH, ABTS, and FRAP assays, with ascorbic acid being used as a standard. The DPPH and ABTS radical scavenging abilities of AgNPs and AgPtNPs are shown in Fig. 5. These biosynthesized nanoparticles exhibited great potential in scavenging free radicals in a dose-dependent manner. The AgPtNPs showed a greater ability to scavenge free radicals, especially DPPH and ABTS (IC₅₀ 19.5 and 21.6 µg/mL) respectively, comparable to AgNPs and ascorbic acid, as shown in Table 1. The AgPtNPs showed a markedly higher inhibition of DPPH radicals compared to ABTS. The FRAP assay estimates the reducing potential of an antioxidant substance reacting with a ferric tripyridyltriazine complex, thus yielding a colored ferrous tripyridyltriazine. In this assay, free radical chains are broken via the donation of a hydrogen atom. FRAP values of AgNPs and AgPtNPs were 18.5 and 44.1 mg GAE/g of the nanomaterials respectively, as shown in Table 2.

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Fig. 5

Inhibition of DPPH radicals (A) and ABTS radicals (B) by AgNPs and AgPtNPs synthesized from the ethanol extract of Vernonia mespilifolia.

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In the present study, the bimetallic AgPtNPs system was found to possess superior antioxidant activity compared with AgNPs and ascorbic acid as determined by DPPH, ABTS, and FRAP radical scavenging methods, as shown in Table 2. The antioxidant activity exhibited by nanoparticles may be due to the phytochemicals present in the ethanol extract of V. mespilifolia, already known for their antioxidant potential (Unuofin et al., 2018), which form the part of synthesized nanoparticles as capping agents (Meena Kumari et al., 2015; Reddy et al., 2014; Xia et al., 2013).

3.4 Antimicrobial activity

The antimicrobial activity of the synthesized AgPtNPs was evaluated against Staphylococcus aureus, Escherichia coli, and Candida albicans at a concentration range of 7.8 – 1000 µg/mL. For control, AgNPs was used under similar conditions in the experiment. As shown in Table 3, the synthesized AgPtNPs showed significant antimicrobial activity against the tested pathogens. The minimum inhibitory concentration (MIC) of AgPtNPs had the strongest inhibitory activity against the gram-positive strain (S. aureus) with an MIC of 32.5 µg/mL, followed by 250 and 125 µg/mL against E. coli and C. albicans, respectively. These MIC values represented a two-fold inhibitory activity demonstrated by the AgPtNPs on the pathogens compared to AgNPs. On the other hand, a lower inhibition was observed in the multi-drug resistant bacteria, E. coli. A recent study by Ruiz et al., investigated the antimicrobial effect of chemically synthesized AgPtNPs and observed enhanced inhibitory activity against medically important pathogens (Ruiz et al., 2020).

3.5 Cytotoxic activity

The possible cytotoxic effects of the extract-synthesized AgNPs and AgPtNPs against normal human embryonic kidney cells (HEK 293) and human breast cancer cells (MCF-7) were performed. As shown in Fig. 6A, HEK 293 cells responded to the cytotoxic effects of AgNPs and AgPtNPs in a dose-dependent manner. Both AgNPs and AgPtNPs were relatively non-toxic to normal cells (HEK 293) with more than 60% cell viability at a concentration of 12.5 µg/mL. A lethal dose (LD₅₀) value of 54.5 µg/mL and 60.0 µg/mL were calculated for AgNPs and AgPtNPs respectively. A similar dose–response effect was noticed for AgNPs and AgPtNPs in MCF-7 cells. However, AgPtNPs exhibited significant cytotoxic activity compared to AgNPs (Fig. 6B). For example, AgPtNPs demonstrated a remarkable ability to inhibit the proliferation of cancer cells at a concentration of 6.25–100 µg/mL. As shown in Table 4, an LD₅₀ of 10.2 μg/mL was calculated for AgPtNPs, while AgNPs showed an LD₅₀ value of 27.1 µg/mL. The results obtained revealed the variability in LD₅₀ values with the AgPtNPs showing a higher cytotoxic effect in cancerous cells compared to normal cells. We could attribute the enhanced cytotoxicity to the synergistic effect of Ag and Pt as reported (Ghosh et al., 2015).

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Fig. 6

Cell viability assay of AgNPs and AgPtNPs against (A) HEK 293 and (B) MCF-7 cells. Data are expressed as a percentage of the control (always 100%) and are represented as mean ± standard deviation (SD).

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Our results suggested a selective cytotoxic mechanism of action, which may be attributed to several factors. These factors include the physicochemical properties of the nanoparticles, possible tightly packed alloyed systems in which the diffusion of either Ag or Pt ions was restricted, induction of cellular DNA damage, metabolic expressions in the cells, and synergistic effects of the extract and the nanoparticles.