2.1 Preparation of aqueous plant extracts

The aqueous leaf extracts of R. officinalis and E. globulus were prepared as has been described in the previous studies (Dzimitrowicz et al., 2019; Valencia et al., 2014). Briefly, dried leaves of R. officinalis (PRYMAT®, Jastrzebie-Zdroj, Poland) and E. globulus (NAT, Mirkow, Poland) were washed with re-distilled water and finally dried in the dark. A fine powder of each plant was obtained by grounding the dried leaves with a pestle in a marble mortar. 2.0 g of each fine powder was mixed with 200 mL of re-distilled water to get a final concentration of 1% (m/v) (Valencia et al., 2014), heated up to boil and kept boiling for 10 min. Next, the resulting aqueous leaf extracts were filtered using qualitative filter paper discs (Munktell, grade 388). After filtration, the final leaf extracts were collected in glass vials and kept for further studies.

2.2 Preparation of natural essential oil solutions

To provide optimal conditions for AuNPs synthesis using natural essential oils, a 100 μL aliquot of commercial essential oil of E. globulus (SEMIFARM, Gdansk, Poland) or R. officinalis (SEMIFARM, Gdansk, Poland) was mixed with a re-distilled water:ethanol (Avantor Performance Materials, Gliwice, Poland) solution in a 4:1 ratio, as was described by Muniyappan and Nagarajan (2014) for silver nanoparticles production. The final concentration of such natural essential oils were 1% (v/v).

2.3 Green synthesis of AuNPs by using aqueous leaf extracts

For AuNPs synthesis, 10.0 mL of filtered 1% (m/v) aqueous leaf extract of E. globulus or R. officinalis was mixed with HAuCl₄ × 4H₂O (Avantor Performance Materials, Gliwice, Poland) solution, to get a final 50.0 mg L⁻¹ concentration of Au in the reaction mixtures (Dzimitrowicz et al., 2019; Valencia et al., 2014).

2.4 Biosynthesis of AuNPs by utilizing natural essential oils

When using natural essential oils of E. globulus or R. officinalis to produce AuNPs, it was necessary to perform the synthesis at 95 °C, as was previously suggested by Shukla et al. (2008). A heated 1% water-ethanol solution of natural essential oil was mixed with HAuCl₄ × 4H₂O (Avantor Performance Materials, Gliwice, Poland) to get 50.0 mg L⁻¹ final concentration of Au in the reaction mixtures.

2.5 Determination of the optical properties and morphology of AuNPs

All solutions were analyzed to confirm the formation of nanostructures and to define the optical traits and morphology of the generated AuNPs. UV/Vis absorption spectrophotometry was used for evaluating optical properties of AuNPs. The measurements were carried out with a double-beam UV/Vis Specord 210 Plus (Analytik Jena AG, Jena, Germany). Other details are given in the Supplementary Material.

Transmission electron microscopy (TEM) and X-ray energy dispersive spectroscopy (EDX) were applied to determine the morphology of AuNPs and to define the elemental composition of the nanocolloidal fluids. The FEI Tecnai G220 X-TWIN instrument (FEI, Hillsboro, OR, USA) equipped with the EDAX X-ray microanalyzer (FEI) was utilized. Other details are given in the Supplementary Material.

2.6 Qualitative analyses of the chemical composition of aqueous plant extracts and essential oils

Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and gas chromatography-mass spectrometry (GC-MS) were applied to determine the chemical composition of the aqueous leaf extracts and essential oils from both E. globulus and R. officinalis. The ATR-FTIR spectra of 1% (m/v) aqueous leaf extracts and non-diluted essential oils were recorded using a Vertex 70v instrument (Bruker, Bremen, Germany) equipped with a diamond ATR accessory. Other details are given in the Supplementary Material. Identification of chemical compounds present in the investigated aqueous leaf extracts and essential oils was achieved within the mass scanning range of 40–500 Da by comparing the obtained mass spectra to the available spectra libraries including SciFinder (Chemical Abstracts Service) and literature data (Hameed et al., 2015; Song et al., 2009b). Turbo-Mass ver-5.1 software was used.

3.1 Optical properties of synthesized AuNPs

It was previously found that colloidal dispersion of AuNPs lead to a change in the color of nanofluids from yellow to ruby red or bluish due to absorption or scattering of the light that passes through them (Cademartiri and Ozin, 2009). Therefore, the first evidence for AuNPs production is a visible color change of the reaction mixtures. For this reason, visual observations were performed to examine capability of fresh aqueous leaf extracts and natural essential oils to reduce Au(III) ions to the respective Au nanofluids after the addition of 50 mg L⁻¹ of Au.

Immediately after the addition of the solution containing Au(III) ions to the fresh aqueous leaf extracts or natural essential oils, a color change from yellow to ruby red was observed. Based on the above-stated phenomenon of absorption and scattering of the light by AuNPs, the color change of the reaction mixtures was consistent with occurrence of the maximum (λ_max) of the localized surface plasmon resonance (LSPR) absorption band, which for Au nanofluids typically occurs in the range of 520–570 nm (Pal and Kryschi, 2015). To find the position of the λ_max of the LSPR absorption band, UV/Vis absorption spectrophotometry was applied. The λ_max positions of the LSPR absorption band for Au nanofluids synthesized using aqueous plant extracts and essential oils are summarized in Table 2. The area and localization of the λ_max of the LSPR depend on the size and shape of AuNPs, the concentration of the AuNPs precursor, the type of solvent, and the reaction temperature (Lee et al., 2016). Representative absorption spectra for AuNPs obtained using the examined aqueous plant extracts and essential oils are presented in Fig. 1. As can be observed, the absorption spectrum of AuNPs synthesized using aqueous leaf extract of E. globulus is symmetrical, indicating the production of predominantly spherical nanostructures. In contrast, the absorption spectrum of AuNPs produced using E. globulus essential oil included two peaks.

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Figure 1 UV/Vis spectra of AuNPs. AuNPs were synthesized from 50 mg L⁻¹ of Au in the presence of aqueous plant leaf extracts and natural plant essential oils of (A) E. globulus or (B) R. officinalis. Samples were diluted 20-fold prior to analysis.

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The first LSPR band is characteristic of spherical nanostructures and is associated with a single plasmon resonance frequency. The second one, indicative of two plasmon resonance frequencies, is connected with AuNPs aggregation or formation of nanostructures of different shapes. In contrast, only one LSPR absorption band was observed for AuNPs generated by using aqueous leaf extract or essential oil of R. officinalis indicating the production of well-dispersed and spherical AuNPs.

3.2 Morphology of Au nanofluids

In order to determine the size and shape of the produced AuNPs, TEM analyses were performed. Fig. 2 shows that AuNPs produced using aqueous leaf extract of E. globulus have an average size of 12.8 ± 6.3 nm. The generated nanostructures were primarily spherical, but a close-up of the grain sides can also be noticed. A great heterogeneity in shapes of biosynthesized AuNPs was observed when E. globulus essential oil was used for AuNPs bioreduction and stabilization (Fig. 3). In this case, the average size of AuNPs was 42.2 ± 42.0 nm. Using the above-mentioned essential oil, a quite high variety of shapes of AuNPs was observed, mostly including spherical (81%), but also triangular (15%), hexagonal (3%), and the rod-shaped (1%). Fig. 4 presents the particle size distribution of AuNPs synthesized using aqueous leaf extract of R. officinalis. Concerning the above-described nanofluids, the grain sides are located close to each other. The average size of AuNPs produced by utilizing aqueous leaf extract of R. officinalis was 8.7 ± 2.0 nm, while AuNPs produced by using the corresponding essential oil exhibited the average diameter of 60.7 ± 60.6 nm (Fig. 5).

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Figure 2 Morphology of AuNPs synthesized from E. globulus aqueous leaf extract. (A–C) Representative TEM micrographs of AuNPs obtained using 50 mg L⁻¹ of Au and E. globulus aqueous leaf extract. (D) Size distribution of AuNPs was determined on the basis of TEM micrographs.

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Figure 3 Morphology of AuNPs synthesized from E. globulus essential oil. (A, C, D) Representative TEM micrographs of AuNPs obtained using 50 mg L⁻¹ of Au and E. globulus essential oil. (B) Size distribution of AuNPs was determined on the basis of TEM micrographs.

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Figure 4 Morphology of AuNPs synthesized from R. officinalis aqueous leaf extract. (A–C) Representative TEM micrographs of AuNPs obtained using 50 mg L⁻¹ of Au and R. officinalis aqueous leaf extract. (D) Size distribution of AuNPs was determined on the basis of TEM micrographs.

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Figure 5 Morphology of AuNPs synthesized from R. officinalis essential oil. (A, C, D) Representative TEM micrographs of AuNPs obtained using 50 mg L⁻¹ of Au and R. officinalis essential oil. (B) Size distribution of AuNPs was determined on the basis of TEM micrographs.

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In order to determine the elemental composition of the resulting nanofluids, EDX analyses were performed (Fig. 6). In each of the EDX spectra, strong signals of Au were observed, confirming the successful bioreduction of the AuNPs precursor. Besides Au peaks, signals for O, C, and K (only in the case of aqueous leaf extract of E. globulus) were acquired. The presence of above-mentioned elements naturally occurring in aqueous leaf extracts and essential oils can be associated with their binding to the surface of biosynthesized AuNPs. The peak of Cu likely originated from the sample holder, onto which the nanofluids were placed, but it can also be associated with some active centers of plant enzymes, in which Cu ions are fixed, e.g. superoxide dismutase, plastocyanin, tyrosinase (Habtemariam, 2016; Kropat et al., 2015; van Gelder et al., 1997).

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Figure 6 Element analysis of reaction mixtures containing biosynthesized AuNPs. EDX spectra are shown for AuNPs biosynthesized with (A) E. globulus aqueous leaf extract, (B) E. globulus essential oil, (C) R. officinalis aqueous leaf extract, (D) R. officinalis essential oil. Elements to which certain peaks correspond to are shown.

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3.3 The chemical composition of aqueous plant extracts and the essential oils

ATR-FTIR spectroscopy was used to identify the chemical compounds present in the applied aqueous leaf extracts and essential oils, as well as to search for differences between them that are responsible for significant variation in the morphological characteristics of the biosynthesized AuNPs. As can be seen in Fig. 7A and C, there is only one striking difference between the spectra of aqueous leaf extracts of E. globulus and R. officinalis, i.e. a more intensive band at 1596 cm⁻¹ (stretching vibrations ν of the C⚌O group) in the case of R. officinalis as compared to the corresponding band at 1607 cm⁻¹ in the case of E. globulus. This may stand for a greater abundance of the compounds containing ketone groups in R. officinalis aqueous leaf extract. Based on the band at 1698 cm⁻¹, corresponding to stretching vibrations ν of the C⚌O group from ketones, it is likely that R. officinalis aqueous leaf extract contains camphor, while the bands at 1263 and 1070 cm⁻¹, corresponding to stretching vibrations ν of the C—O—C group, may indicate the presence of 1,8-cineole (Schulz et al., 2005; Hameed et al., 2015; Sirvaityte et al., 2011). For E. globulus aqueous leaf extract, terpenes such as γ-terpinene and 1,8-cineole were putatively identified according to wagging vibrations ω of the CH₂ group at 919 cm⁻¹ as well as stretching vibrations ν of the C—O—C group at 1043 cm⁻¹ (Schulz et al., 2005; Hameed et al., 2015; Sirvaityte et al., 2011). In both aqueous leaf extracts, broad bands corresponding to stretching vibrations of the O—H group were also observed.

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Figure 7 Identification of the functional groups involved in AuNPs bioreduction and stabilization. ATR-FITR spectra are given for E. globulus aqueous leaf extract (A) and essential oil (B), as well as for R. officinalis aqueous leaf extract (C) and essential oil (D).

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When comparing the spectra of essential oils, they looked similar with two exceptions. As can be noticed in Fig. 7B and D, there is a large band at 1658 cm⁻¹, corresponding to stretching vibrations ν of the C⚌C group, in the spectrum of the E. globulus essential oil that is absent in the spectrum of R. officinalis essential oil. For the latter essential oil, a large band at 1713 cm⁻¹ for stretching vibrations ν of the C⚌O group was noted. These two bands may indicate the presence of camphor in R. officinalis essential oil and α-pinene in E. globulus essential oil. In addition, in the spectrum of R. officinalis essential oil, there were bands at 1368, 1219, 1087, 995, and 852 cm⁻¹, indicating deforming vibrations δ in the CH₃(CO) group, stretching vibrations ν in the C—O—C group, and wagging vibrations ω in the CH₂ group. All of them likely resulted from the presence of eucalyptol (1,8-cineole), which is consistent with the IR data suggesting the occurrence of this compound in the crude plant extract of R. officinalis (Schulz et al., 2005). Finally, a band at 888 cm⁻¹ (wagging vibrations ω of the CH₂ group) could suggest the presence of β-caryophyllene. For E. globulus, wagging vibrations ω of the CH₂ group at 918 cm⁻¹ and 989 cm⁻¹ could be indicative of γ-terpinene and eucalyptol, respectively (Schulz et al., 2005). Not surprisingly, comparison of the absorption bands identified in the spectra of the aqueous leaf extracts and essential oils revealed that most of them were more intensive in the case of analyzed extracts. This points out that aqueous leaf extract contained higher amount of potential reducing agents than the corresponding essential oil. The only exceptions were the intensive bands in the range of 3000–2900 cm⁻¹ in the spectra of the essential oils, corresponding to the saturated aliphatic groups that are the primary components of essential oils.

To confirm the preliminary identification of the chemical composition of aqueous leaf extracts and essential oils by ATR-FTIR, comprehensive GC-MS analyses were performed. The obtained results are included in the Supplementary Material. The following compounds were identified in R. officinalis aqueous leaf extracts and its essential oil: α-pinene, D-limonene, eucalyptol, borneol, camphor and boryl acetate. In R. officinalis aqueous leaf extract ethoxycitronellal and α-thujene were also found. β-phellandrene and caryophyllene were detected in the essential oil of this plant. Similar results were obtained by Hameed et al. (2015), who examined methanolic leaf extract of R. officinalis. Concerning E. globulus essential oil, α-pinene, eucalyptol, D-limonene, α-phellandrene, β-phellandrene, β-ocimene and γ-terpenine were identified and this corroborated suggestions of Sirvaityte et al. (2011). Due to large background noise, ethoxycitronellal was only identified in E. globulus aqueous leaf extract.