Green Synthesis, Characterization and Antibacterial Activity of Gold and Copper Nanoparticles from Lannea discolor (Sond.) Engl

1. Introduction

Nanotechnology, a globally active research discipline, is rapidly progressing, with nanoparticles being a topic of interest since the 1970s [1]. Nanoparticles (NPs) are microscopic particles with at least one dimension less than 100 nm and can be categorised into different types according to morphology, size, physicochemical properties, and the type of precursor from which they are synthesised [2]. Metal nanoparticles (NPs) are created from metal precursors like gold and copper using synthetic or chemical methods, which may use environmentally unfriendly reducing agents [3]. This has resulted in scientists turning to safer methods of synthesis, by employing biological reductants such as bacterial, fungal and plant material to curb any possible negative effects from nanosynthesis [4]. Gold nanoparticles (AuNPs) are used in various fields such as gene therapy, protein delivery, cancer diagnosis, photodermal and photodynamic therapy, delivery of antitumor agents, DNA detection and catalysis [5]. Ancient Indian healers used them for asthma and arthritis, later, Romans used them for cathedral glassware decoration. Recently, they functioned as photocatalytic air purifiers, gaining significant therapeutic applications [6]. AuNPs are compatible with living tissue, producing no toxic or immunological response, and have been used as nanocarriers due to their small sizes for anti-inflammatory drugs, improving stability and adsorption efficiencies [7,8]. Plant extracts have gained attention as reducing agents for AuNP synthesis due to their low toxicity, eco-friendliness, and simplicity of production [4,9]. Various plants such as Anacardium occidentale [10], Spondias dulcis [11], and Pistacia chinensis [12], have been reported to be effective in reducing gold ions into differently shaped and sized nanoparticles. Nanotechnology has enabled the development of modern techniques for nano-scale copper generation over the past decade [13]. CuNPs are gaining attention due to their ease of availability and economic feasibility, unlike other noble metals that are similar. They are widely utilized in cancer imaging due to their efficient light-to-heat transformation under near-infrared laser irradiation [13]. Other uses include enhancing heat transfer liquids, photonic devices, sensors and electrochemical devices [14,15]. CuNPs, with their unique properties, have gained significant applications in various industries such as cosmetology, agriculture, food, textiles, and construction [16,17]. In several studies, CuNPs providing higher environmental mobility were synthesized using various plant extracts including Cissus vitiginea [17] Zingiber officinalis and Curcuma longa [18] Brassica oleracea [19], Hyptis suaveolens (L.) [20]. Lannea discolor from family Anacardiaceae is a deciduous tree that usually grows up to 15 m on rocky slopes, or on sandy soil. The leaves are discolorous, having a green colored adaxial surface and a grey dense trichome layer on the abaxial surface. Its traditional uses include treatment for diarrhoea, stomach complains, and an array of infections [21]. The pharmacological activities of the plant are purported to be the result of various secondary metabolites such as phenolic flavonoids, alkaloids [22]. Given that CuNPs are a stable substitute for gold, they may serve as low-cost replacement for unattainable precious metals. Conclusively, all NPs were characterized by UV–Vis spectrometry, FT-IR spectroscopy, SEM–EDX and TEM to investigate the optical, morphology and elemental composition as well as the antibacterial activities of nanoparticles, respectively.

2. Results and discussion

2.1. Visual and UV-Vis Spectroscopic Analysis

This study is a simple and sustainable method for synthesizing AuNPs and CuNPs by mixing AuCl4 and CuSO4 solutions with the methanol, acetone and water extracts of Lannea discolor. Following incubation of AuCl4 and CuSO₄ solutions with extracts, solutions went from dark yellow to greyish for the leaves (Figure 1A, C), and intense violet for the stem and root was observed (Figure 1B, D). The extracts when exposed CuSO₄ changed from their usual color to more intensified burnt orange for the leaves (Figure 1E, G) and a brown to a darker brown for the stem and root (Figure 1H).

UV–Vis representative absorption spectra for the AuNPs and CuNPs are shown in Figure 2A,B. The absorption peak for the AuNPs from stem and roots extracts, had a maximal absorption at 544 nm, while the leaf extracts NPs solutions peaked at 316 nm. Similar results were observed of the latter, in other studies [23,24]. As much as UV-vis characteristic wavelength of AuNPs has been used to validate the synthesis of NPs, certain peaks may also be attributed to their size and shape, instead of their abundance. The change in color of the NPs solution to grey due to Oswald ripening is a well-understood phenomenon [25]. Similarly, the two-pointed peaks observed in the NPs from the leaf extracts; indicate a different morphology when compared to the stem and roots. Studies show that spherical AuNPs appear red; but visualise blue or grey when they form NFs [26,27].

The CuNPs displayed an absorption peak at ranges of 556 – 564 nm for leaves and 540 -546 nm for stems and roots. As Figure 2A,B show, the increase in the temperature did not significantly reduce or increase the adsorption and synthesis of CuNPs. It is reported in literature, the surface plasmon resonance band of CuNPs provides absorption from 500 to 600 nm [28]. Therefore, CuNPs peaks acquired confirm their formation.

2.2. Morphology of synthesised nanoparticles using Scanning electron microscopy and Energy dispersive X-ray Analysis

SEM analysis shows in Figure 3A, aggregated AuNPs that were present from all leaf extracts, evidently confirming the influence of the leaf extracts in the morphology observed. Fluorescence emission from is observed in Figure 3B inset. Under the light irradiation, AuNPs emit fluorescence, a characteristic suggested to confer phototherapy potential and showing the possible optical properties of the AuNPs [29].

CuNPs were found to be spherical in shape (Figure 4A) and aggregated, which is a result of static tension [30]. Furthermore, there is unexpected sintering observed in Figure 4B after exposure to heat. Sintering involves the joining together of nanoparticles into a solid mass, due to temperature less than 250° C, pressure, and leftover alcohols during evaporation [31]. As such, during both synthesis and drying, the CuNPs undergo sintering, and the well-connected features are observed in Figure 4B. Sintering techniques have been standardised to produce chemically synthesised CuNPs for copper inks used in conductivity [31,32], yet no work has been reported employed sintering temperature as low as 60 ◦C for CuNPs from Lannea discolor extracts. Literature reports that the optical and electronic properties of NPs depend on morphology [32,33], rendering CuNPs as potentials in these types of applications.

The EDX measurements unambiguously confirmed the presence of metallic Au. The EDX profile shows a strong Au signal along with C, O Al, K, and Ca peaks, from the biomolecules on the surface of the AuNPs (Figure 3C, D). These elements may also be present due to the elemental composition of the extracts, from which the AuNPs were not completely purified [34]. The grid, on which the samples were examined, is what causes the presence of Cu. Thus, all measurement techniques confirmed the presence of AuNPs and NFs. Likewise, the EDX profile of the CuNPs shows strong elemental signals of copper, confirming the presence of CuNPs, as shown in Figure 4C - D. The weight percentages of different elements in the CuNPs were below 10 %, as shown in Figure 4C - D. The remaining Wt % were carbon and oxygen present in organic molecules, which act as capping molecules surrounding the nanoparticles as well as the tape used to mount the CuNPs [35].

2.3. TEM, Particle Size and Zeta Potential Analysis

TEM images showed NPs with protruding petal-like features, which are confirmed to be NFs [36]. As observed in Figure 5A, the particles are spherical dots with an average diameter ±10 nm. An in-depth morphological analysis suggests that the formation of AuNFs occurs by initial formation of NPs, followed by rapid anisotropic growth, then assembly into a flower shape by secondary reduction at their interface [36]. The rapid color change from yellow to grey without intermediate colours supports this theory of their progression [36]. Thus, we proposed the formation mechanism to be aggregation-based growth, as illustrated in Figure 6. Their 3-dimensional, gives AuNFs better suspension stability and a greater surface area than spherical AuNPs [37,38]. Also, significant enhancement of the local electrostatic fields and “petals” are advantageous in catalytic reactions, drug delivery and nuclear medicine [38]. The size of the NFs from L. discolor varies from 30 to 97 nm with an average size of 67 nm. Similar sizes were also previously reported by Borah et al using Syzygium cumini [39]. Onmaz et al reported sizes <100 nm NFs using Helichrysum italicum [40]. Other studies suggest that tannin-based polymers enhance reduction [41], explaining the unexpected formation of NFs from L discolor. NFs have a potential in developing biosensors, such that, through surface modification, their interactions to biomolecules of interest can be detected by a color change [41]. Familiar examples of NPs used in sensing is the home pregnancy test based on detection of the hormone pregnancy (β-HCG hormone) [42]. In other studies, the grey suspension of AuNPs was obtained by chemical synthesis, where trisodium citrate and water-soluble polymer were added to HAuCl4 [43,44]. To move to greener synthesis of NFs, using L. discolor leaves may prove to be less tedious and more environmentally friendly. Under the given conditions, spherical AuNPs were formed from stem and root extracts ranging at 10 – 33 and 9.3 –37.5 nm respectively (Figure 5B), with lower sizes (Figure 5D), than those obtained in the leaf samples. Shabestariana et al acquired spherical AuNPs with an average size of 20.83, from Rhuscoriaria L. (Sumac), confirming that significant molecules in the extracts act as ligands which control NPs growth [45,46]. Similarly, Donga et al reported even smaller sized NPs from Mangifera indica ranging at 12.33 – 24.05 nm [47]. Comparably, Pechyen et al reported similar sized spherical AuNPs from Spondias dulcis [11]. Figure 5B insets show a triangular, and hexagonal NPs which have been reported by Song et al to be caused by low concentration of extracts [48]. However, the effect on the size and shape of AuNPs in the present study may be in response to the plant part and solvent used in extraction, indicating the uniqueness of the phytochemicals in the extracts, and their specific requirements for higher temperatures to initiate the reduction process and produce other morphologies [48].

Figure 7A shows the TEM images of spherical CuNPs with particle size in the range 20 - 104 nm (Figure 7B). In both Figure 7A and inset, NPs are similarly sintered as observed in Section 2.2. Authors suggest their large sizes may be more to do with the ability of the present biomolecules to reduce the CuSO₄ and their concentration.

AuNCs and NPs are soluble in water, suggesting high solubility in biofluids and potential as injectables [49]. The corresponding adsorption of these NPs in the body may be improved by their stability, that is measured as zeta potential. In addition, the absolute values of zeta potentials for AuNCs were -14.6, -8.3, -10.8 and - 6.39 mV, indicating their stability, which is due to the larger electrostatic repulsion [2,50]. CuNPs had a stability range of -43.3 to -13.4 mV. Zeta potential greater than 30 mV or less than −30 mV is indicative of stable dispersions of NPs in solutions [50].

2.4. FTIR Analysis

FT-IR measurements of the NPs were carried out to identify the major functional groups of phytochemicals present in L. discolor extract. The typical FT-IR spectra of AuNPs and CuNPs synthesized in the presence of L. discolor extracts are shown in Figure 8. Bands at 2780, 2873,2816, 3015, 3255, 3272,3299, and 3313 cm ^-1 are due to N–H stretching of both amines and amides and/or to O–H stretching of alcohols, phenols, or carboxylic acids. The aromatic stretches (C-H) observed at peaks 1894, 1935, 1938, 1954, 1973, 1994 cm ^-1 in both the AuNPs and CuNPs may be amongst the main capping agents. The binding characteristics of amino groups in the samples could be the cause of the produced CuNPs' aggregation, which resulted in the sintering [51,52].

2.5. Antibacterial Activity of AuNPs and CuNPs

In addition to previously mentioned applications, NPs may be utilized as a potent antibacterial agent. Although extensive literature was devoted to proving the antibacterial activity of other metal-based NPs [53], focus on the antibacterial activity of AuNPs/NFs and CuNPs is limited, as such, the present study contributes hugely. MIC was employed to estimate the inhibition AuNPs/NFs, and CuNPs may have on microorganisms. The MICs for the AuNPs and NFs are summarized in Table. Interestingly, the MIC values of the NPs were similar for the bacterial strains. S. aureus, P. aeruginosa and B. subtilis were the most susceptible to AuNFs from all solvents at both 25 °C and 80 °C and showed the lowest viability compared to K. pneumoniae and E. coli. The authors attributed the activity of the AuNFs to the high-aspect ratio of the elongated petal like surfaces, which induce localised tension and increase reactive oxygen species, thus, causing bacterial membrane rupture [54]. Additionally, the enhancement of L. discolor extracts by NPs, improve their ability to disrupt the bacterial cell membrane [54]. The MIC from stem and root extract NPs were similar and exhibited good antibacterial activity against all the bacteria; this may be due to their similar sizes. Contrastingly, all the crude extracts displayed antibacterial activity at only 1000 µg/ml, explaining the significance of the enhancement from nanoparticles.

Reports show that the antibacterial activity of AuNPs is associated with their surface modifications, such as shape and the molecules involved in capping the NPs during synthesis [54]. Some bacterial strains, like S. aureus, are considered threatful to humans, hence introducing AuNPs as antibacterial agents is fundamental. Morphological interactions, depend on surface orientations for the enhancement of antibacterial activity [55]. NPs with edges rupture the membrane of bacteria and disrupt them. Many experiments on nanoscale surfaces have observed the morphological interactions of AuNPs/NFs with bacteria, confirming the significance of surface orientation for activity [55,56]. In the present study, the high activity can be attributed to the large surface area and disruptive ability of AuNPs/NFs. Studies have also ascribed the high activity of gold NPs against Gram (-) bacteria to the thin peptidoglycan membrane, and that lower activity is usually observed in gram (+) bacteria with a peptidoglycan membrane with thickness that is 50% higher [57]. However, it is not the case in this study, as activity varies equitably well within both the gram (-) and gram (+) bacteria. Similarly, a few other studies have observed impressive antibacterial activity from AuNPs in both gram (-) and gram (+) bacteria [58,59,60]. The total MIC for different extracts resembling that of AuCl4 confirms that the activity of phytochemicals improves when incorporated into different nano formulations. The outcome displays that the AuNPs/NFs synthesized from Lannea discolor can inhibit the growth of the tested bacteria even at low concentration. The CuNPs in this work showed no activity against tested bacteria, which is ideally indicated by a change in color after addition of Resazurin. This is attributed to the sintered NPs that were unable to interact with the bacterial cells as they are more of a solid mass instead of polydispersed. Other causes may include size, shape, the specific surface area, and surface curvature of the nanoparticles [61]. However, CuNPs in other studies have showed comparable activity in contrast to its counterparts [62]. Possibly, a higher concentration of precursor salts, longer incubation period, may produce CuNPs with better antibacterial activity. Taken together, all these data reported here support the use of the L discolor extracts as an alternative to toxic chemical reductants in the production of AuNPs and NFs and CuNPs that can inhibit the growth of different bacteria.

3. Materials and methods

3.1. Collection of plant material

Lannea discolor material was collected from Vuwani (Vhembe district) (23°09'42.9"S 30°25'22.6"E) in the month of November 2018. The plant species were identified by UR with the aid of the literature, and online herbariums (https://plants.jstor.org/compilation; https://powo.science.kew.org) and voucher specimens (UR 01), were prepared and deposited at the University of Venda Herbarium (UVH) in South Africa. Lannea discolor material reserved for experiments was air-dried, ground with a pestle and mortar and stored for further use.

Extraction

The air-dried material was ground into a fine powder using a grinding mill (NETZSCH, Selb, Germany). The extracts were obtained by maceration for three 3-day periods at a ratio of 1:10 for solvents, methanol, acetone, and water. They were filtered using filter paper (Whatman No. 1), evaporated to dryness, and stored at 4 °C for subsequent tests.

3.2. Synthesis of nanoparticles

Synthesis of AuNPs and CuNPs was carried using Lannea discolor (10mg/ml) extracts. After dissolving 250 mg of gold precursor in 50 mL of distilled water, a stock solution of Gold (III) chloride hydrate (HAuCl4 · aq) that had a concentration of 14 mM. (Millipore, Merck) was prepared. After, the stock solution was diluted to a concentration of 1 mM HAuCl4. The addition of 10 mL of Lannea discolor extract to a mixture containing 40 mL of 1 mM HAuCl4 solution was incubated at 25 °C and 80 °C for an hour then in the dark for twenty-four hours. The hue of the solution changed, indicating AuNPs formation. The mixture was centrifuged for ten minutes at 10,000 rpm after being washed twice with distilled water. AuNPs pellets were gathered, air dried, and kept at 4 °C.

To synthesise CuNPs, 1 mM copper (II) sulphate (CuSO4) was made in distilled water, after which, 10 mL of L discolor extracts was added to 40 mL copper sulphate solution (1 mM) and incubated at 25 °C and 80 °C for 1 hour. The color of the mixtures changed, indicating formation of CuNPs. After 24 hrs, the nanoparticles were rinsed twice with distillation water, and centrifuged at a speed of 12,000 rpm for 15 min. CuNPs pellets, were dried at 60 °C overnight, then stored at 4 °C for future use.

3.3. Characterisation

3.3.1. Physicochemical Characterization of AuNPs and CuNPs

UV–Vis Spectroscopy Analysis recorded the absorbance of AuNPs and CuNPs in 96 well plates containing 160 µl of NPs using a SpectraMax M3 spectrophotometer (Molecular Devices, California, USA) at wavelengths of 200–800 nm and scan speed of 200 nm/min.

3.3.2. Scanning electron microscopy analysis and energy dispersive x-ray microanalysis

The AuNPs and CuNPs were sonicated for 20 min, after which about 40 μL of each sample was mounted on a glass coverslip attached to a brass stub with adhesive carbon tape and dried under a mercury lamp for about 60 min. The NPs were made conductive with a layer of gold (ca.25mm) using an automated Quorum (Q15OR ES) Module sputter coater (vacuum of 0.1 Torr for 2.5 min) and examined at varying magnification, using a Zeiss Ultra-Plus FEG-SEM at an acceleration voltage of 5 kV. The images were captured with a NIS-D image software, while elemental analysis was done with an energy dispersive X-ray spectrometer (EDX) coupled to an Astronomical Thermal Emission Camera (Aztec) version 1.2 at 20 kV.

3.3.3. Transmission Electron Microscopy analysis and Measurements of Zeta Potential

Carbon coated 200 mesh copper grids were used to scoop a portion of the AuNPs and CuNPs, and dried under a mercury lamp for about 30 min before analysis. The morphology and size of the nanoparticles were determined using a JEOL JEM-2100 TEM at an acceleration voltage of 200 kV.

Zeta analysis was used to measure the surface charge of the NPs using a Zetasizer Nano-Zs90 (Malvern International Ltd, MPT-Z, UK). For each sample, the mean diameter ± SD for ten runs was calculated by applying a multimodal animation.

3.3.4. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

The ATR-IR FTIR (Alpha 1; Bruker, Germany, Europe) was used to describe the chemically possible compound interactions. The samples were developed and evaluated at a scan range of 4000–400 cm−1. Interpretation of the spectra was achieved by comparing the spectral data with references from identification of functional groups existing in literature [63].

3.4. Antibacterial Assay

The antibacterial activity of the NPs, gentamicin, crude extracts, HAuCl₄ and CuSO₄ solutions were studied against Staphylococcus aureus and Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus subtilis using the minimum inhibitory concentration assay (MIC). A 96 well microtiter plate (Tarsons, Kolkata, India) was prepared by transferring 100 µL of Mueller–Hinton broth under laminar air flow chamber. A volume of 100 µL of NPs, extract and gentamicin was added into the first row of the plate, followed by 10 µL of bacterial inoculum. After which, serial dilutions with concentration 1000 – 7.8 μg/ mL, were performed, and the plates were placed for 24 hrs at 37 °C for incubation. Upon removal, 10 µL of Resazurin solution as per Madikizela et al was added to each well followed by further 30 min incubation to determine the MIC value by the observed color change [64].

4. Conclusions

In the current study, a straightforward AuNPs NFs and CuNPs production was reported for the first time employing Lannea discolor leaves, stem and roots extracts in a more cost-effective, green technique. The ability of the functional groups in the phytochemicals of L discolor to function as a reducing and capping agent is linked to the formation of AuNFs and NPs as well as spherical CuNPs. TEM micrographs provided evidence towards the morphology of AuNPs with several shapes including flowers, spherical, triangular, and hexagonal. The sintered CuNPs can potentially find a unique position in many technological applications such as sensors, flexible devices, and conductivity due to their merged surface area. Despite AuNPs and NFs bacterial activity, more study into their intricate cytotoxicity profiles is still required. On the other hand, there may be a need to optimise techniques to produce AuNFs and sintered CuNPs from L discolor.