Green synthesis of gold nanoparticles using Pelargonium Graveolens leaf extract: characterization and anti-microbial properties (An in-vitro study)

Microorganism

The study evaluated the antimicrobial effectiveness of the prepared Pelargonium Graveolens AuNPs against clinical isolates of Streptococcus mutans and Candida albicans. These isolates were obtained from the oral cavity of patients in the Pediatric and Preventive Dentistry Department at the University of Baghdad’s College of Dentistry. The laboratory utilized standard biochemical methods to process and identifies these isolates. Following the transfer of the stock cultures onto Mueller-Hinton agar medium, an overnight incubation at 37°C was conducted, followed by storage at 4°C.

Preparation of Pelargonium Graveolens leaf extract and Green synthesis of Pelargonium Graveolens AuNPs

Pelargonium Graveolens leaves collection in Baghdad Governorate, Iraq. The collected leaves were thoroughly cleansed under running water to eliminate traces of chemicals and grime. After a thorough rinsing, the leaves of Pelargonium Graveolens were incubated overnight at 37^oC to dry. After incubation, the completely desiccated leaves were ground into a fine powder for use in the extraction process. A quantity of 100 g of air-dried ground plant material was subjected to extraction using aqueous alcohol as the solvent (methanol, water, ethanol (1:1:3) 80 % v/v) 500 mL the samples were subjected to Soxhlet extraction for a duration of 8 hours on a water bath. The extracts were subjected to solvent removal under reduced pressure at a temperature of 45°C using a rotary evaporator. Subsequently, the desiccated crude concentrated extracts were measured in order to determine the yield. Following this, the powder was diluted to achieve a concentration of 250 μg/ml, which corresponds to MIC of the plant extract, after that, the extract was filtered 50ml of it was taken prepared for the purpose of using it with gold chlorides to act as a reducing agent in the preparation of gold nanoparticles. The gold ion solution was prepared in accordance with the methodology outlined by Ref. 16. with some modification, one gram of HAuCl₄ dissolved by 100 mL of DW to form a 10 mg/ml solution. Gold nanoparticles (Au NPs) were synthesized through a mix of a 1 mg/ml HAuCl₄ gold ion solution and a 50 ml Pelargonium Graveolens extract. The solution was subjected to a magnetic stirrer for duration of 30 minutes while being slightly heated to a temperature range of 35°C to 45°C. As a result, the mixture exhibited a quick alteration in color, transforming into a dark shade of purple/red within a brief time span. The observed change in color can be assigned to the formation of gold nanoparticles (AuNPs) with a concentration of 15.7 (ppm). The manifestation of a purple color following the mixture of the plant extract and HAuCl₄ solution signifies the production of Pelargonium Graveolens AuNPs.

Characterization of Pelargonium Graveolens AuNPs

An X-ray diffract meter (XRD-6000, SHEMADZU-Japan^®,™) used to evaluate the produced nanoparticles characteristics. (Current = 30 mA; voltage = 40 kV). A Cu-Kα incident beam (λ = 1.542 A°) at 2θ = 20°-80° used to determine the patterns in which the particles diffracted. Transmission electron microscopy (PHILIPS CM-120-USA^®,™) is operating at 100 kV to evaluate the size and morphologies of the nanoparticles. Pelargonium Graveolens AuNPs solutions were characterized using a UV-Vis spectrophotometer (SHEMADZU1200i-Japan^®,™) the experiment employed a double beam UV-Vis spectrophotometer to observe the beams under different conditions within the spectral range of 200–1100 nm. An FTIR analysis was conducted utilizing the PerkinElmer Spectrum-USA instrument, covering spectral ranges from 4500 to 500 cm^-1. FTIR analysis was conducted in order to examine the functional groups of biomolecules that are responsible for capping, viable stabilization, and reduction of the prepared Pelargonium Graveolens AuNPs. A Zeta potential test was conducted using a Zetasizer Nano ZS (Malvern Instruments^®,™) at a temperature of 25°C. The (Zetasizer 6.01 software^®,™)was employed for data analysis. The purpose of the zeta-potential measurements was to examine the stability of Pelargonium Graveolens AuNPs in the suspension. Field Emission Scanning Electron Microscopy (FESEM) employed utilizing a (ZIESS, Germany^®,™) Electron Microscopy to identify the morphological features of Pelargonium Graveolens AuNPs. The investigation of elemental composition was carried out using EDX with a (Thermo Scientific TM NORANTM^®,™ System 6 EDS system).

Biological activities of Pelargonium Graveolens AuNPs

Antibacterial efficacy of Pelargonium Graveolens AuNPs

The antimicrobial effect of Pelargonium Graveolens AuNPs was investigated using the Agar diffusion method. Ten isolates of each S. mutans and C. Albican were applied separately on MH agar plates; the previous approach was explained by Ref. 17. Mueller–Hinton agar media was poured separately into sterile petri dishes and wait until setting. A volume of 0.1ml of activated Streptococcus mutans and Candida Albican was evenly distributed onto Mueller-Hinton agar plates and allowed to incubate at the room temperature for duration of 10 minutes. Subsequently, using a sterile stainless steel Cork borer, uniform and sufficiently deep wells measuring 6mm in diameter were generated in the Mueller-Hinton agar. This process was repeated six times on each plate and for each microorganism. Each well was filled with 0.2 mL of the agent to be tested at varying concentrations of 0.06, 0.12, 0.25, and 0.5 mg/mL. The last well filled with Chlorhexidine 0.12% as a positive control, while the center well filled deionized water as a negative control. Plates left at the room temperature for 10 minutes and then incubated aerobically for 24hrs at 37C. Each zone of inhibition was measured across the diameter of each well. No zone of inhibition indicated complete resistant of S. mutans and C. Albican a to the test agent. Then, we calculated the MIC, MBC, and MFC of Pelargonium Graveolens AuNPs against the individual microorganisms by agar dilution method against S. mutans, and C. albicans. Pelargonium Graveolens AuNPs concentrations of 0.06, 0.12, 0.25, 0.5 mg/ml were serially diluted in Mueller–Hinton agar to make 10 mL of agar and extracts, which were then placed into Petri dishes and allowed to solidify before being inoculated with 0.1 ml of activated isolates of microorganisms. All of these Petri dishes, including the control plates, were incubated at 37°C overnight (the negative control, which contained Mueller-Hinton agar with microbial inoculums without the addition of the extracts, and the positive control plates, which contained MH-A and different concentrations of the Pelargonium Graveolens AuNPs without microbial inoculums). The presence or lack of bacteria growth on the plates is checked. The MIC of Pelargonium Graveolens AuNPs is the concentrations at which microbial growth is totally inhibited. The identical approach was followed for all of the microbial isolates, that had been described previously.¹⁸ The MBC/MFC of Pelargonium Graveolens AuNPs is the concentrations at which microbial growth is totally killed.

Statistical analysis

Statistical Package for Social Research was used for descriptive analysis, and presentation (SPSS Statistical Package for social Science version-22, Chicago, Illinois, USA). The Levene test and Shapiro-Wilk test were conducted on a quantitative variable, which encompassed the minimum, maximum, mean, standard deviation (SD), and standard error (SE). The One-way Analysis of Variance (ANOVA) statistical method was employed, along with Tukey’s Honestly Significant Difference (Tukey’s HSD) and Dunnett’s T3 posthoc tests, to conduct multiple pairwise comparisons among the groups. The results are considered statistically insignificant when the P value is greater than 0.05, while they are deemed statistically significant when the P value is less than 0.05.

Characterization of prepared nanoparticle

Determination of the shape and size

Transmission electron microscopy (TEM) was employed to investigate the characterization of the synthesized nanoparticles (NPs). The morphology of gold nanoparticles (AuNPs) is characterized by a combination of hexagonal, spherical, semi-spherical, and triangular-shaped particles. The size and abundance of gold nanoparticles particles decrease as the concentration of Pelargonium Graveolens leaves extracts increases. The morphology Pelargonium Graveolens AuNPs is depicted in Figure 1-A, where the AuNPs are represented as a core and the Pelargonium Graveolens acts as a shell surrounding the AuNPs surface, and as shown in Figure 1-B the average size of Pelargonium Graveolens AuNPs has been estimated to be 294 nm due to the small concentration of Pelargonium Graveolens leaves extracts is 250 μg/ml (MIC of plant extract), It is considered a good and safe particle size because the smaller size easily to uptake by mammalian cells this is agree by Ref. 19.

Figure 1. A – TEM of Pelargonium Graveolens AuNPs B – Distribution of particle size of Pelargonium Graveolens AuNPs.

XRD analysis

For further determination of the Pelargonium Graveolens AuNPs, The X-ray diffraction (XRD) pattern was utilized to analyze the diffraction peaks observed at 2θ = 39.9°, 44°, and 65.28°, which can be attributed to the 111, 200, and 220 crystallographic planes of the cubic structure of the gold nanoparticles (Au NPs) as depicted in Figure 2. These results are consistent with the data provided by the Joint Committee on Powder Diffraction Standards (JCPDS) under the reference number 001-1172. The results mentioned above agree with the data presented by Ref. 20. The current data indicates that XRD pattern of the Pelargonium Graveolens AuNPs closely resembles that of pure gold nanoparticles. The mean size of nano crystallites in Pelargonium Graveolens AuNPs is 69.587 nm calculated From the well-known Scherrer formula (D = 0.9λ/β. Cos θ) In this context, the symbol D represents the average size of crystallites, β refers to the line broadening as quantified by the full width at half maximum (FWHM) of a peak, λ represents the wavelength of X-rays used for irradiation, and θ signifies the maximum position value of the peak²¹ as shown in Table 1.

Figure 2. XRD spectra of Pelargonium Graveolens AuNPs.

FTIR spectra of prepared NPs

In the cases of Pelargonium Graveolens AuNPs the FTIR measurements detailed in Table 2 were conducted in an effort to determine the character of the organic protection layer surrounding the AuNPs based on comparable research in the literature.^22,23 The observed significant shifts in the spectra of certain peaks subsequent to the formation of AuNPs are hypothesized to be attributed to the impact exerted by the adjacent metal surface. Hence, it is postulated that the observed displaced peaks, as documented in Table 2, are indicative of the presence of organic matter surrounding the synthesized AuNPs. Figure 3 demonstrates the Fourier Transform Infrared (FTIR) analysis conducted to assess the significant biomolecules involved in the capping and stabilization of Pelargonium Graveolens AuNPs synthesized through a green method. Intense absorptions are observed at 3436.59, 2067.21, 1636.44, and 683.21 cm^-1. IR band at 3436.59 cm^-1 refers to the OH stretching mechanism of the proteins, polyphenols, and carbohydrates. The band noticed at 2067.21 and 683.21 cm^-1 related to the C≡C expanding of the alkynes group and C–N/C–Cl in plane bending, respectively. Furthermore, the IR band at 1636.44 cm^-1 is characteristic of the C=O expanding of the carboxylic group. Hence, it is possible that enzymes/proteins are involved in the process of reducing metal ions through the oxidation of aldehydes to carboxylic acids. Proteins possess the capacity to form links with gold nanoparticles (AuNPs) via carboxylate ions found in amino acid residues or through free amine groups within the protein’s structure.^24,25 In addition, the presence of the C=O stretching mode indicates the presence of the carboxylic acid (-COOH) group in the material that is attached to gold nanoparticles (Au NPs). Hence, the spectral peak observed at a wavenumber of 1636.44 cm^-1 can be attributed to the manifestation of amine groups, signifying a possibility of protein binding onto gold nanoparticles. The provided information holds significant value in informing the design of functionalization procedures, particularly in the context of utilizing particles for drug delivery purposes.

Figure 3. FT-IR details of Pelargonium Graveolens AuNPs.

UV-Vis absorbance spectrum

Figure 4 shows the absorbance of the peak of Pelargonium Graveolens AuNPs was at 527 nm. Pelargonium Graveolens AuNPs show a massive peak. The successful synthesis of Pelargonium Graveolens AuNPs was confirmed by the observation of a dark purple color in the reaction mixture and the presence of specific absorption spectra at a wavelength of 527 nm in the UV-vis absorption spectroscopy. This outcome aligns with findings reported in previous studies.¹⁶

Figure 4. UV-visible absorption spectra of Pelargonium Graveolens AuNPs.

Zeta potential

The zeta potentials of the particles present in the sample of Pelargonium Graveolens AuNPs were determined at a temperature of 25°C. Figure 5 shows that the Pelargonium Graveolens AuNPs sample showed positive zeta-potential values between 0 and +50 mV the observed range can be classified as an initial state of stability for colloidal systems that agree with.²⁶ The zeta potential of the Pelargonium Graveolens AuNPs was measured to be 27 mV Figure 5. This value suggests that the nanoparticles were stable and had a reduced propensity to aggregate and form larger particles.

Figure 5. Zeta potential of Pelargonium Graveolens AuNPs.

FESEM-EDX analysis of particle composition

The results clearly presented in Figure 6-B demonstrate that the particle composition unequivocally corresponds to gold. The presence of trace amounts of additional elements, such as oxygen (O), was observed in the elemental composition of the plant extract after the solvent evaporation process. The elemental composition of the plant extracts was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES), revealing the presence of trace amounts of these elements, among others. The data exhibited a strong correlation between the concentrations of different elements present in the plant extracts and the results obtained from Energy-Dispersive X-ray Spectroscopy (EDX), as seen previously in this report,¹⁶ and the findings from field emission scanning electron microscopy (FESEM) analysis, as depicted in Figure 6-A, indicate that the gold nanoparticles (AuNPs) were effectively separated and did not form aggregates. This can be attributed to the presence of Pelargonium Graveolens, which functions as a protective agent, corroborating the observations made through transmission electron microscopy (TEM) and this agree with.²⁷

Figure 6. A SEM image of Pelargonium Graveolens AuNPs, B EDS spectra of Pelargonium Graveolens AuNPs.

Biological studies

Antibacterial activity of Pelargonium Graveolens AuNPs

The antimicrobial efficacy of Pelargonium Graveolens AuNPs was evaluated using Streptococcus mutans and Candida albicans as test organisms. Significant zones of inhibition were observed subsequent to the exposure of the microorganisms to the Pelargonium Graveolens AuNPs that were prepared Figure 7A,B. The findings of the present study demonstrate the effectiveness of the synthesized nanoparticles in inhibiting the growth rate of Streptococcus mutans and Candida albicans following a 12-hour exposure period as shown in Figure 8.

Figure 7. (A) Antibacterial activity of (Pelargonium Graveolens AuNPs) against Candida Albicans A, control negative B, Control positive Chlorhexidine 0.12%. C, 0.06 mg/ml. D, 0.12 mg/ml E, 0.25 microgram/ml. F, 0.5 mg/ml (B) Antibacterial activity of (Pelargonium Graveolens AuNPs) against Streptococcus Mutans, A, control negative B, Control positive Chlorhexidine 0.12%. C, 0.06 mg/ml. D, 0.12 mg/ml. E, 0.25 microgram/ml. F, 0.5 mg/ml.

Figure 8. A-MBC against Streptococcus Mutans, B- MBC against Candida albicans.

Determination of Minimum Bactericidal Concentration (MBC)

The study results indicate that MBC for Candida albicans was 0.5 mg/ml, while MBC for Streptococcus Mutans was 0.12 mg/ml, as presented in the Tables 3 and 4. Subsequent re-culturing on BHI-agar media revealed no growth at these concentrations as shown in Figure 8 indicating that the 0.5 mg/ml concentration had a fungicidal effect on Candida albicans, while the 0.12 mg/ml concentration had a bactericidal effect on Streptococcus Mutans. Also the results presented in the table indicate that the MIC for Candida albicans was 0.25 mg/ml, while for Streptococcus Mutans it was 0.06 mg/ml. However, onto re-culturing on BHI-agar, it was observed that these concentrations exhibited growth, indicating that they had a bacteriostatic effect.