2.1 Materials

Silver nitrate was purchased from Merck Millipore. Polyvinylpyrrolidone 4000 (PVP), standards of plumbagin, myricetin, quercetin, hyperoside, ellagic acid, isorhamnetin, were purchased from Sigma Aldrich. Ramentaceone was obtained from the University of Pretoria, Republic of South Africa. Droserone was synthesized by dr E. Paluszkiewicz from the Technical University of Gdansk, according to the method described by Razzakova et al. (1973) and Behrman et al. (1995) with several modifications. Plants: Drosera indica, Drosera binata, Drosera spatulata, and Dionaea muscipula were grown on solid (0.75% agar), half strength Murashige and Skoog (½ MS) medium (Murashige and Skoog, 1962) with 2% of sucrose and 0.15% activated charcoal at 20 °C, light 30–35 µmol/m² × s, photoperiod 16/8h, pH 5.5 (Fig. 1). During the whole experiment deionized water was used.

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Fig. 1

Growth of carnivorous plants on ½ MS medium + 2% sucrose + 0.15% activated charcoal: A. Drosera binata, B. Drosera indica, C. Drosera spatulata, D. Dionaea muscipula.

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2.2 Preparation of plant extract

Five grams of fresh plant tissue were placed in a 250 mL borosilicate flask. Then 100 mL of water was added. The flask was microwaved for 2 min (Sharp microwave model R-242(IN)E, 800 W) with a 30-s break after the first minute. After the extract had cooled to room temperature, it was filtered through Whatman No. 1 filter paper. Prior to use, the extract was filtered through 0.22 µm mixed cellulose ester syringe filter to remove residual impurities.

2.3 Preparation of AgNPs – Water bath

Firstly, 400 mg of PVP was added to a 250 mL borosilicate flask. Then 10 mL of plant extract and 90 ml of water was added and everything was thoroughly mixed until complete dissolution of PVP. Subsequently, 100 mL of 8 mM AgNO₃ was added to obtain the final 4 mM silver nitrate concentration. Flasks were incubated in a water bath for 2 h at 70 °C. After the nanoparticle solution had cooled down to room temperature, it was centrifuged for 15 min at 15,000 RCF. The obtained nanoparticle pellet was resuspended in 4 mL of water. The sample was stored at room temperature in darkness for the duration of the tests.

2.4 Preparation of AgNPs – Microwave

Firstly, 200 mg of PVP was added to a 250 mL borosilicate flask. Then, 5 mL of plant extract and 45 mL of water was added and everything was thoroughly mixed until complete dissolution of PVP. Subsequently, 50 mL of 8 mM AgNO₃ was added to obtain the final 4 mM silver nitrate concentration. Flasks were microwaved for 2 min with a 30-s break after the first minute. After the nanoparticle solution had cooled down to room temperature it was centrifuged for 15 min at 15,000 RCF. The obtained nanoparticle pellet was resuspended in 4 mL of water. The sample was stored at room temperature in darkness for the duration of the tests.

For the determination of power consumption, GreenBlue GB202 Energy Meter was used each time a synthesis was performed. For the microwave synthesis, the whole energy usage was registered for the sample. For the water bath synthesis, the energy usage was divided between the samples in the water bath. The energy used was measured in Watt-hours.

2.5 Characterization of nanoparticles

AgNPs synthesized with the use of carnivorous plants extracts were characterized by using transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS). For TEM analysis samples were placed on grids (Sigma-Aldrich, St Louis, MO, USA) coated with a 2% collodion solution (Sigma-Aldrich). Samples absorbed to the surface were examined using a Philips CM100 electron microscope at 80 kV (FEI Company, Eindhoven, the Netherlands).

The nanoparticles morphology was investigated using scanning electron microscopy (Nova Nano SEM 200, FEI). The observations have been performed taking into account the chemical analysis of specimens in microareas with energy dispersive X-ray spectroscopy (EDS, EDAX). The observations were carried out in low vacuum conditions in the secondary electron mode, using Helix detector. Results are presented in the form of spectra and tables demonstrating the qualitative and quantitative chemical composition of elements occurring in the specimens. The EDS analysis confirmed chemical composition of examined silver nanoparticles.

The particle size distribution in the samples was assessed by dynamic light scattering (DLS) analysis using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK).

The UV–Vis spectra of all samples were recorded with the use of Evolution 300 double beam spectrophotometer equipped with the thermostatic cells. The 200–800 nm range for the measurements was used. All spectra were registered at room temperature. The optical path for all experiments was 1 cm. Spectra were registered for both types of synthesis at intervals ranging from 15 min to 24 h.

Infrared spectra (FT-IR) of AgNPs and carnivorous plant extracts were recorded with the use of potassium bromide (KBr) pellets and Brüker Infrared Spectrometer in the range of 399–4599 cm^–1. AgNPs samples, as well as extracts, were lyophilised before the forming of KBr pellets.

X-ray Photoelectron Spectroscopy analysis (XPS) was carried out with the use of X-ray photoelectron spectrometer (Omicron NanoTechnology) with a 128-channel collector. XPS measurement has been done at room temperature in ultra-high vacuum conditions below 1.1 × 10⁻⁸ mBar. The photoelectrons were excited by a Mg-Kα X-ray source. The X-ray anode was operated at 15 keV and 300 W. Omicron Argus hemispherical electron analyser with round aperture of 4 mm was used for analysis of emitted photoelectrons. The binding energies were corrected using the background C1s line (285.0 eV) as a reference. XPS spectra were analysed with Casa-XPS software using a Shirley background subtraction and Gaussian–Lorentzian curve as a fitting algorithm.

2.6 HPLC analysis

The chromatographic separation was carried out using Beckmann Gold System equipped with a Thermo Separations Spectra 100 variable wavelength detector and a Rheodyne 6-way injection valve. For the stationary phase, an Agilent Zorbax SB-Phenyl (4.6 × 150 mm, 3.5 µm) was used. The flow rate used was 1 mL min⁻¹. The sample injection volume was 10 µL. The mobile phase for the analysis consisted of 0.1% (v/v) trifluoroacetic acid in acetonitrile as eluent A; 0.1% (v/v) trifluoroacetic acid in water as eluent B. The separation gradient was 0 min (10% A)-> 5 min (10% A)-> 12 min (90% A)-> 20 min (90% A) followed by a 10-min column regeneration. Chromatographic separations were carried out at room temperature. Typical compounds present in carnivorous plant tissues (droserone, hyperoside, ellagic acid, myricetin, ramentaceone, plumbagin, quercetin, isorhamnetin) were used as standards to determine the extracts composition. Three level standard curve was used for determining the concentration of the compounds 4-point. Monitoring was performed at 254 nm. All analyses were performed in triplicate.

2.7 DPPH radical scavenging assay

The antioxidative properties of water extracts from Drosera and Dionaea species were evaluated with the use of the DPPH (2,2-diphenyl-1-picrylhydrazyl free radical) assay, which measures the ability of chemicals to reduce DPPH radical. Free radical scavenging activity of extracts used in silver nanoparticles synthesis was determined according to the method described by Abdelwahab et al. (2014) with minor modifications. The final concentration of DPPH radical in the assay was 0.1 mM. Working solutions of extracts and DPPH radical were prepared in pure methanol. Each extract was tested at the concentration ranging from 1.25 to 25 mg of fresh weight (FW) mL⁻¹. Conversion of 2,2-diphenyl-1-picrylhydrazyl free radical into diphenyl-1-picrylhydrazyl (DPPH) was measured spectrophotometrically at 517 nm (EnVision Multilabel Reader, Perkin Elmer) after 30 min incubation at room temperature in the dark. The inhibition of DPPH radical absorbance was calculated according to the formula:

2.8 Study of antimicrobial and antifungal susceptibility

The antimicrobial and antifungal activity of AgNPs were determined using the Broth Microdilutions Method according to guidelines provided by the Clinical and Laboratory Standards Institute. To determine minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) of AgNPs, tests on 96-well plates were performed. Two species of human bacterial pathogens: G (+) Staphylococcus aureus Newman (Duthie and Lorenz, 1952) and G (−) Pseudomonas aeruginosa PAK (Ramphal et al., 2008), fungi Candida albicans ATCC 90028, and three G (−) plant pathogens: Pectobacterium atrosepticum SCRI 1043, Pectobacterium parmentieri Scc3193 (earlier Pectobacterium wasabie) and Dickeya dadantii 3937, were used throughout our study. All human pathogens were grown overnight in Brain Heart Infusion (BHI) medium (BTL, Poland) at 37 °C, plant pathogens on Lysogeny Broth (LB) medium (Roth, Germany) at 28 °C (Pectobacterium) and at 37 °C (Dickeya). All tested microorganisms are deposited at the IFB, UG & MUG, Poland. Overnight bacterial cultures were diluted to obtain the initial inocula, equivalent to 0.5 McFarland turbidity standard measured by densimeter (DensiMeter II, EMO, Brno). AgNPs from carnivorous plants (in a range of 1–100 µl mL⁻¹ which correspond to 1.7–170 μg nanoparticles/mL) were applied into wells of the 96-well plate and suspended in BHI or LB medium (final volume of the medium was 100 µL). Water extracts (after evaporation) from four species of carnivorous plants were also tested in a range of 1–1000 µl mL⁻¹. The suspension of 0.5 in McFarland scale of bacteria or fungi was diluted (final 1.5 × 10⁵ CFU/mL for bacteria and 1.5 × 10⁴ CFU/mL for fungi) in LB or BHI medium, and 10 µL was added into wells. The plates were incubated at 28 °C or 37 °C for 24 h and the MIC was established. Then, 100 µL of the suspension was plated onto Luria Agar (LA) or BHI Agar medium. Plates were incubated at 28 °C or 37 °C for 24 h and at room temperature for 24 h and the MBC was established. All analyses were performed in triplicate. The in vitro activity of antibiotics: vancomycin – glycopeptide (Sigma-Aldrich), ciprofloxacin – second-generation fluoroquinolone (Sigma-Aldrich), oxacillin – penicillinase-resistant β-lactam (Sigma-Aldrich), cefotaxime – cephalosporin III generation (Ranbaxy, India), piperacillin – β-lactam (Sigma-Aldrich), ceftazidime third-generation cephalosporin (Sigma-Aldrich) and streptomycin – aminoglycoside (Sigma-Aldrich) against bacteria, and fluconazole (Sigma-Aldrich) and amphotericin (Sigma-Aldrich) against fungi was evaluated for comparison purposes. We also tested commercially available silver nanoparticles AgNPs (NanoAmor company, USA) with a purity of 99.9%, 80 nm particle size and density of 10.49 g/cm³ (catalog number 7440-22-4), and AgNPs_COOH (provided by ProChimia Surfaces Co. (Poland) as water-soluble nanoparticles coated with the HS-(CH2)10-COOH⁺ ligand, 5.5 nm in diameter, characterized by a dispersity level of 15%. The initial stock concentration of AgNPs was 10.5 × 10¹⁴ NPs mL⁻¹ which corresponds to 958 µg mL⁻¹ (SPR maximum, λ_max: 420–424 nm).

3.1 Preparation and characterization of AgNPs in water bath or with the use of microwaves and its characterization

After incubation in the water bath the solutions’ colours turned from dark yellow to deep red that is consistent with nanoparticle colloid solutions. In low lighting conditions, the colloid seemed to be clouded. Straight after microwave-assisted synthesis solutions showed a much lighter colour than their counterparts synthesized in the water bath. After cooling down to room temperature the difference in colouration was insignificant.

Transmission and scanning electron microscopy images showed the differences in the morphology of AgNPs (Fig. 2). Depending on the type of extract different size of the nanoparticles were observed. Nanoparticles synthesized using D. muscipula (Fig. 2: A4, B4, A8, B8) and D. indica (Fig. 2: A2, B2, A6, B6) extracts showed high concentration and shape uniformity. We did not observe homogeneity in shape for AgNPs from D. binata. The SEM images revealed the formation of even 300 nm clusters. The sizes of the most antimicrobial active D. indica and D. muscipula nanoparticles are presented in Fig. 3. The water bath synthesized samples showed a considerable fraction of small (∼5–10 nm) nanoparticles (Fig. 2: A1, B1, A2, B2, C1, C2, D1, D2).

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Fig. 2

TEM (A) and SEM (B) images of silver nanoparticles synthesized using: 1, 5 – extracts from D. binata, 2, 6 – extracts from D. indica, 3, 7 – extract from D. spatulata, 4, 8 – extract from D. muscipula. 1–4 AgNPs obtained in water bath, 5–8 AgNPs obtained in microwave.

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Fig. 3

The particle size distribution of the samples (A) AgNPs from D. indica using microwave assisted synthesis, (B) AgNPs from D. muscipula using microwave assisted synthesis assessed by dynamic light scattering.

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Fig. 4 shows an example of EDS analysis of AgNPs obtained from D. muscipula. The EDS spectrum mainly consists of C, O and Ag peaks. The high content of carbon element corresponds to carbon tape, which was used for nanoparticles mounting. The oxygen concentration in amount of 11% is related to the aqueous solution. The EDS analysis and electron diffraction pattern suggest that the nanoparticles are only composed of Ag element. Similar results were obtained for the other AgNPs (data not shown).

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Fig. 4

EDS spectrum for the AgNPs synthesized using microwave assisted synthesis of water extract from D. muscipula. The spectrum consisted of different peaks for silver (Ag), carbon (C), and oxygen.

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The UV–Vis spectra of the silver nanoparticles (AgPNs) formation process with the use of water bath and microwave are presented in Fig. 5 and Fig. 6, respectively. Colour of nanoparticle solutions change from light-yellow to dark-brown through the reaction. The UV–Vis spectra were recorded at different time intervals (15 min, 1 h, 3 h, 6 h, 9 h, 12 h and 24 h) to observe the gradual reduction of silver nitrate in solution after treatment with microwaves of high temperature. The UV–Vis spectra of each AgNPs species recorded after 24 h showed that the absorbance maximum did not change significantly in respect to the 12 h spectrum. The results confirmed that the reaction reached its equilibrium within 24 h. The final spectra for each type of AgNPs showed a characteristic peak at c.a. 420 nm. This peak corresponds to silver nanoparticles appearance in solution and it was present in all tested mixtures. Furthermore, the comparison of UV–Vis spectra (Fig. 7) of carnivorous plant extracts and AgNPs spectra proves that both methods can be used to obtain silver nanoparticles. Additional information about UV–Vis spectral characteristics of other substances used during both synthesis pathways (aqueous solutions of silver nitrate, PVP, plant extracts and PVP/extract solutions) can be found in Supplementary Information, see Fig. S1.

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Fig. 5

Time dependent UV–Vis spectra confirming the AgNPs formation by using water bath method in the presence of: (A) D. binata; (B) D. indica; (C) D. spatulata; (D) D. muscipula; I-IX aqueous solutions of: I – AgNO₃; II, IV, VI, VIII – pure, appropriate plant extracts of Drosera; III, V, VII, IX – adequate Drosera/AgPNs systems obtained.

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Fig. 6

Time dependent UV–Vis spectra confirming the AgNPs formation by using microwave method in the presence of: (A) D. binata; (B) D. indica; (C) D. spatulata; (D) D. muscipula; I-IX aqueous solutions of: I – AgNO₃; II, IV, VI, VIII – pure, appropriate plant extracts of Drosera; III, V, VII, IX – adequate Drosera/AgPNs systems obtained.

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Fig. 7

The spectral comparison of appropriate substrates (pure aqueous Drosera extracts) with products obtained (AgNPs) by microwave and water bath assisted synthesis: (A) D. binata; (B) D. indica; (C) D. spatulata; (D) D. muscipula.

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FT-IR spectra of AgNPs together with spectra of plant extracts used for the synthesis are shown in Fig. 8. It is worth mentioning that the FT-IR spectra of AgNPs obtained by using two independent methods are similar, although the spectra for the microwave method are more explicit aiding in the AgNPs derivatisation identification. FT-IR analysis showed that after AgNPs formation, their binding with compounds from the plant extract is possible and is due to the interactions between the —OH and the C⚌O groups of appropriate polyphenolic compounds in plant extracts. IR spectra carnivorous plant/AgNPs systems illustrate the mentioned phenomenon, with the regions of relevance shown in Fig. 8. The high intensity peaks at 557 and 636 cm⁻¹ indicate the presence of uncoated AgNPs in case of all studied samples. Additionally, there are significant shifts in the —OH and C⚌O peaks, while all the other peaks (mainly aliphatic and aromatic C—H stretching) remain unchanged. The shifts observed for stretch —OH and C⚌O were following: 166 and 87 cm⁻¹ (D. binata extract); 134 and 71 cm⁻¹ (for D. indica extract); 158 and 71 cm⁻¹ (D. spatulata extract); 110 and 63 cm⁻¹ (D. muscipula extract), respectively. These spectral changes may be related to AgNPs coating by compounds from carnivorous plant extracts in studied solutions. Additional information about FT-IR spectral characteristics of other substances used during both synthesis methods (silver nitrate, PVP, plant extracts and PVP/carnivorous plant combinations) can be found in Supplementary Information, Fig. S2.

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Fig. 8

The FT-IR spectra of Drosera/AgNPs systems obtained by using two independent synthetic methods with additional Fourier transform infrared spectra of appropriate plant extracts: (A) D. binata; (B) D. indica; (C) D. spatulata; (D) D. muscipula.

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XPS experiment has been performed to provide insight on valance state of silver nanoparticles in sample obtained from D. muscipula extract using microwave-assisted synthesis method. Exemplary spectra of such samples are presented in Fig. 7. Ag3d_5/2 and Ag3d_3/2 photoelectron peaks of silver have been observed at 368.22 and 374.21 eV respectively. Spin orbit components in all cases were separated by distance of 6 eV which is characteristic for silver. Shape of obtained spectra is comparable to the ones published before (Prieto et al., 2012). However, it is difficult to distinguish between different silver state components of 3d_5/2 peak. Therefore, definition of silver oxidation state on the basis of XPS measurements is challenging and sometimes impossible. Consequently, modified Auger parameter (α′) had to be calculated. In order to acquire it, kinetic energy of silver M4VV Auger line was determined (presented in Fig. 9) at position equaled 357.1 eV. Parameter has been calculated according to the equation:

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Fig. 9

XPS spectra for Ag 3d region (A) and Auger lines (B) of AgNPs synthesized using microwave assisted synthesis of water extract from D. muscipula sample.

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3.2 HPLC analysis

HPLC analysis revealed that main compounds in the water extracts belong to the flavonoids and naphthoquinones groups (Table 1, Fig. 10). The concentration of those compounds differed between carnivorous plant species. From all tested plants D. muscipula tissue was most abundant in secondary metabolites. From the three species of Drosera, the most abundant with secondary metabolites were D. indica extracts. Each of the samples had relatively high concentration of hyperoside and ellagic acid (Fig. 10). The highest concentration of ellagic acid and hyperoside was found in D. musciplula and D. indica respectively. Depending on the tissue, plumbagin (in D. muscipula and D. binata), ramentaceone (in D. spatulata) or plumbagin and ramentaceone (in D. indica) was found. The total concentration of naphthoquinones was similar among tested samples except D. spatulata extract in which the concentration of ramentaceone was 16.397 µg mL⁻¹. Myricetin and isorhamnetin in larger concentrations were only found in D. muscipula tissues (Table 1, Fig. 10).

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Fig. 10

HPLC analysis of water extracts from D. binata, D. muscipula, D. indica and D. spatulata.

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3.3 DPPH radical scavenging assay and study of antimicrobial susceptibility

Plant extracts obtained after the microwave-assisted extraction showed light to dark yellow colour shift. This was due to the solubilization of flavonoids and other water-soluble compounds present in the plant tissue. In order to evaluate the antioxidative activity of water extracts of D. muscipula, D. binata, D. indica and D. spatulata, their ability to scavenge DPPH free radicals was analysed. Both D. muscipula and D. indica possessed similar and the highest radical scavenging properties (IC₅₀ = 0.857 ± 0.023 mg FW mL⁻¹) despite their various secondary metabolite composition (Table 1). DPPH test revealed that D. binata extract possessed the lowest radical scavenging potential out of the four tested extracts, because the IC₅₀ was 5.5 ± 0.049 mg FW mL⁻¹. The IC₅₀ of the water extract for D. spatulata was 1.428 mg ± 0.029 FW mL⁻¹. It is worth mentioning that radical scavenging properties of plants extracts were strongly correlated with the antimicrobial activity of AgNPs obtained using the aforementioned plant extracts.

Synthesized nanoparticles showed higher antimicrobial activity against plant pathogens in comparison to human pathogens (Table 2). The Minimal Inhibitory Concentration varied depending on the microorganism and was between 5.3 µg mL⁻¹ (for D. dadantii and P. aeruginosa) and 170 µg mL⁻¹ or more (for S. aureus and C. albicans). The highest antimicrobial activity for both pathogens was shown by AgNPs synthesized using the microwave method and extracts from D. muscipula and D. indica because the range of MBC was between 5.3 µg mL⁻¹ and 42.5 µg mL⁻¹ (Table 2). The most resistant to AgNPs was fungi (C. albicans) where MBC was above 170 µg mL⁻¹. Water extracts from carnivorous plants tested in the range from 1 to 1000 µl mL⁻¹ did not show antimicrobial activity against tested microorganisms (data not shown).

4.1 Preparation of AgNPs – Water bath and microwaves-assisted

Already published studies showed that the synthesis of most AgNPs with the use of plant extracts do not require an additional capping agents. While AgNPs were obtained by synthesis based on carnivorous extracts potential and without the use of additional capping agents they occurred unstable and aggregated during the synthesis, forming a grey-silver suspension that precipitated within two hours. Addition of polyvinylpyrrolidone (PVP) as a capping agent in concentration 200 mg 100 mL⁻¹ as previously reported (Banasiuk et al., 2016) enabled the formation of stable colloids in both microwave and water bath assisted synthesis. Polyvinylpyrrolidone is usually used as an additive in the microwave assisted polyol AgNPs synthesis methods (Kim et al., 2006). It is also used as a capping agent for microwave assisted synthesis in water environment with pure/synthetic compounds as reducing agents (Nadagouda et al., 2011). Using capping compounds with plant extracts is usually omitted as the components of the extract can act both as reducing and capping agents. During this study, we tested the influence of PVP on the process. PVP acted only as an end-capping/stabilizing agent. In the control reactions without plant extract, synthesis of the nanoparticles was not observed. This additive is essential for AgNPs formation if the extract possesses high reducing and low capping capabilities. After microwave irradiation, the solution colour had gradually changed over time until the solution reached room temperature. This would suggest that the process continues even after heating of the sample is stopped. It has been previously shown (Bilecka and Niederberger, 2010) that the microwave assisted synthesis is an economical way for delivering heat energy to the reaction vessel. Power consumption during the microwave synthesis was 42 ± 1 Wh per sample, whereas during the water bath synthesis it was 65.25 ± 3 Wh. The value presented for the water bath synthesis does not take into account the energy needed to heat the water bath to the required 70 °C. When considering the whole process, the energy consumption was 249 ± 3 Wh per sample. Taking into consideration the time needed for the process, the use of microwave-assisted synthesis gives an economical advantage for small to medium-scale nanoparticle synthesis processes. On the other hand, large-scale and continuous production of nanoparticles may benefit economically from water/oil bath synthesis since the apparatus is inexpensive and commercially available but the process should be thoroughly optimised. This is due to the nature of the heat distribution in the reaction vessel. Heat distribution using microwaves is more efficient and uniform. Apart from shortening the heating time microwave irradiation can suppress side reactions that would hinder the activity of the final product (Bilecka and Niederberger, 2010). To achieve this in a conventional heating method the reaction vessel should have a capillary diameter.

4.2 Characterization of nanoparticles

We observed a correlation between the DPPH scavenging activity of the used extracts, and the size and number of nanoparticles. Nanoparticles synthesized using D. binata extract formed larger clusters. This might explain their lower antimicrobial activity. Aggregation process has an impact on the surface to mass ratio of AgNPs and thus reduces antimicrobial activity of nanostructures as their surface, binding to the microbial cells, decreases (Morones et al., 2005; Rai et al., 2012). Smaller nanoparticles are usually more desired since they show higher antimicrobial activity (Pal et al., 2007) but this fact is inconsistent with our observed results. We found that larger but similar in size nanoparticles show higher antimicrobial activity rather than those with heterogeneous size distribution.

The samples after the reaction change from light to dark brown in colour (Figs. 5 and 6, insert). For all samples the absorbance maximum was registered at about 420 nm. This indicates the presence of silver nanoparticles. The detailed FT-IR analyses confirmed the presence of AgNPs partially coated with PVP and showed the binding mode between AgNPs and plant extracts components.

4.3 Preparation of plant extract and DPPH radical scavenging assay

The results obtained with DPPH radical scavenging assay for water extracts from carnivorous plants clearly correlated with antimicrobial activity. The most potent AgNPs were synthesized in plant extracts with the highest antioxidant potential (Table 2). The flavonoids from Drosera sp. and Dionaea tissues were crucial in the synthesis of AgNPs. They were responsible for the reduction of silver ions and partially for the stabilization of the obtained nanoparticles. Qin et al. (2010) showed that it is possible to obtain silver nanoparticles by using ascorbic acid (very potent antioxidant) as a reductant. It was shown so far by Krolicka et al. (2009) that the methanol extract (containing flavonoids like quercetin and myricetin) from Drosera aliciae proved to be a very potent antioxidant. The highest concentrations of flavonoids in D. muscipula and D. indica and AgNPs obtained from these tissues were most active against bacteria and fungi. Our research showed that flavonoids from carnivorous plants possess high antioxidant properties and thus are also excellent silver ions bioreductans.

4.4 Study of antimicrobial susceptibility

Bacteria employ various mechanisms to survive in unfavourable environmental conditions. Widespread application of antibiotics caused the emergence of antibiotic-resistant bacterial phenotypes. Therefore, a lot of effort is put into discovering novel classes of antimicrobial compounds, with a mode of action distinct from antibiotics (Singh and Barrett, 2006). Bacterial cell structure is an important factor for the antimicrobial effectivity of compounds. In our research, we have shown that G (−) bacteria, especially plant pathogens, are more susceptible to AgNPs compared to Gram positive bacteria. This is due to the differences in the structure of cell membrane and cell wall between Gram (+) and Gram (−) bacteria (Dakal et al., 2016). Negatively charged bacterial cell membranes and cell wall are the main sites of attachment for positively charged AgNPs. Silver nanostructures aggregate on the bacterial cell surface and therefore disrupt its functions. However, AgNPs are also able to penetrate the bacterial cell membrane and subsequently enter the cytoplasm where highly reactive silver ions are released from their surface. As Ag⁺ ions released intracellularly play a crucial role in bactericidal activity, the rate of membrane penetration renders gram negative bacteria more susceptible to silver nanoparticles.

Ansari et al. (2011) have determined the MBC value for commercial nanoparticles (Nanoparticle Biochem, Inc) for S. aureus reference strain at 25 μg mL⁻¹. This value was very close to the MBC values for AgNPs obtained from the extract of D. indica tissues after microwave synthesis (21.2 μg mL⁻¹). The MBC values of nanoparticles synthesized from D. indica extract and water bath (170 μg mL⁻¹) were 8 times higher than the microwave synthesized AgNPs (21.2 μg mL⁻¹). Khan et al. (2016) found that MBC of AgNPs obtained from the Zizipus nummularia leaf extract was 130 μg mL⁻¹, which is more than the MBC value of any AgNPs obtained in our experiment. Sheikholeslami et al. (2016) determined the MBC value of synthetic silver nanoparticles (NanoLotus Pasargad, Inc) against P. aeruginosa to be 31.25 μg mL⁻¹. Brown et al. (2012), synthesized silver nanoparticles with the use of sodium borohydrate and determined their MBC value at 4 μg mL⁻¹.

We observed that G (−) plant pathogenic bacteria (P. atrosepticum, P. parmentieri and D. dadantii) were more sensitive to AgNPs in comparison to Gram negative P. aeruginosa. This may be related to the fact that plant pathogens have not been subjected to antibiotic pressure for millennia as compared to human pathogens. Certain multiresistant opportunistic species, such as P. aeruginosa and Acinetobacter baumannii, can colonize niches where many other species cannot survive e.g. environments with high concentration of antimicrobial agents (Beceiro et al., 2013). In addition, many of clinical isolates of P. aeruginosa appear to carry the impermeability type of resistance to various antibiotics (Wang et al., 2003). During our study we compared the activity of our nanoparticles to standard antibiotics. We found that in most cases AgNPs were more effective than streptomycin, but less effective than cefotaxime (Table 2). Streptomycin and copper-containing chemicals are the most frequently used for control of plant pathogenic bacteria (Kamysz et al., 2005).

What is more, the addition of carnivorous plant extracts to AgNPs can lower the needed MBC/MFC concentration. Using a checkerboard titration method in our previous study (Krychowiak et al., 2014) we found that the synergistic effect between nanoparticles and plant extracts can drastically (over 50%) lower the effective dose of this antimicrobial agents.

We have compared the activity of our nanoparticles with commercially available nanoparticles (Table 2). AgNPs from NanoAmor synthesized using chemical reduction method did not show activity towards either human or plant pathogens. Nanoparticles with surface modification (HS-(CH2)10-COOH ligand) from Prochimia Surfaces Sp. z o.o. showed activity that was comparable to AgNPs synthesized in D. muscipula extract with the assistance of microwave. Because our approach is a one-pot method with in situ derivatization, it delivers more cost effective way of AgNPs synthesis and modification.