Green Synthesis of Silver Nanoparticles Using Trachyspermum Ammi Fruit Extract: Characterization and Antibacterial Activity

INTRODUCTION

Infectious diseases are one of the leading causes of death worldwide, and multidrug-resistant bacterial strains as infectious agents, continue to pose a significant concern for human health. Silver has previously been used to heal wounds or treat infections, but its short duration of effectiveness has limited its recognition as a viable agent in medicine. With the development of nanotechnology, the synthesis of nanoscale metals with enhanced antimicrobial properties has been explored to address this concern [1, 2].

Over the last few decades, nanomaterials have found applications in various fields of sciences including engineering, medicine, environmental, textile, and more, owing to their unique physical, chemical, and biological characteristics [3, 4]. Among the various types of nanoparticles, silver nanoparticles (AgNPs) have gained significant attention in medical science due to their wide-ranging applications in fields such as food preservatives, wound healing, water purification, biosensors, and drug delivery systems [5-8]. Extensive research has demonstrated that AgNPs act as efficient redox catalysts, requiring less activation energy, and possess high surface reactivity due to their elevated surface energy. Their antibacterial properties and tumor cell targeting capabilities have positioned AgNPs as remarkable nanomaterial in the field of nanomedicine. The antibacterial activities of nanoparticles are influenced by their physicochemical characteristics, such as the size, morphology, distribution, chemical composition, including size, morphology, distribution, chemical composition, stability, and the specific type of microorganisms they encounter. The proposed antibacterial mechanism of nanoparticles is closely associated with the generation of reactive oxygen species (ROS) [9-11].  

While various techniques of nanomaterial synthesis have been explored, the growing concern over life-threatening environmental pollution has driven scientists to develop new approaches using natural substances, such as plant extracts or microorganisms, in a process referred to as “green synthesis”. In green synthesis, phytochemical compounds present in the plant extracts serve as reducing, capping, and stabilizing agents [12, 13]. This method is known for its simplicity, cost-effectiveness, and eco-friendliness, as it avoids the use of toxic chemicals. Numerous studies have investigated the synthesis of AgNPs using plant extracts such as Trigonella foenum-graecum [14], Moringa oleifera [15], Ruellia tuberosa [16], Nardostachys jatamansi [17], Cornus mas [18], and Madhuca longifolia [19]. Trachyspermum ammi is a medicinal plant that has antimicrobial, anti-inflammatory, antispasmodic, antipyretic, analgesic, anti-nociceptive, and antioxidant properties [20]. 

In this research, we embarked on a journey to harness the potential of nature’s gifts for a sustainable future. Our study focuses on the synthesis of silver nanoparticles (AgNPs) through both chemical and green methods, utilizing the extract derived from Trachyspermum ammi. These nanoparticles were then evaluated for their antibacterial properties against various bacterial strains. Our research not only contributes to the expanding field of green nanotechnology but also underscores the critical importance of sustainable and eco-friendly solutions in combating bacterial infections. This study paves the way for a hopeful future, where environmentally friendly nanoparticles may revolutionize the landscape of antibacterial treatments.

MATERIALS AND METHODS

Material and Characterization

The fruits of T. Ammi (Fig. 1) were collected from the areas around Birjand, Iran, and were approved by a botanist in the Agriculture Faculty at the University of Birjand. AgNO₃ (Silver nitrate) and Na₃C₆H₅O₇ (Sodium citrate) were obtained from Sigma-Aldrich Company (USA). Five standard bacteria strains, including Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumonia ATCC 9997, Staphylococcus aureus ATCC 25923), and Enterococcus faecalis ATCC 29212 were purchased from Pasteur Institute of Iran (Tehran, Iran). Fourier transform infrared spectroscopy (FT-IR) was employed for the identification of functional groups (Perkin-Elmer, USA), Transmission electron microscopy (TEM) for direct size and morphological observation of products (Zeiss, EM10C, Germany), X-ray diffraction (XRD) for determination of crystal structure of particles (Philips X’pert PRO, Netherlands), and Dynamic light scattering (DLS) analysis for hydrodynamic diameter determination of silver nanoparticles (Nano-ZS, Malvern, UK). 

Preparation of T. ammi extract

Five gram of T. ammi fruits were collected and dried at room temperature conditions in the shade. Subsequently, the fruit powder was mixed with 50 ml of deionized water for 24 hours. Finally, the solution was filtered using the Whatman paper and utilized for synthesis of nanoparticles.

Synthesis of silver nanoparticles

The synthesis of AgNPs was conducted through two methods: (I) chemical route: Initially, 9 mg of AgNO₃ was dissolved at 50 ml of distilled water. Then, 1 ml of sodium citrate (1%) was added to the above solution under vigorous stirring at room temperature. Within 1 hour, the color of the solution changed from colorless to brown, indicating the formation of AgNPs. (II) Green synthesis: In this method, 1.5 ml of T. ammi fruit extract was added to 30 ml of AgNO₃ (1 mM) under intense stirring for 24 h. Following this, the solution was centrifuged at 6000 rpm, washed three times, and then dried. The biosynthesis of AgNPs was monitored using UV-Vis absorption spectroscopy in the range of 300-500 nm. 

Antibacterial Activity

In this study, the antibacterial activity of T. ammi aqueous extract, chemical AgNPs, and green AgNPs was investigated against E. coli ATCC 25922, P. aeruginosa ATCC 27853, K. pneumonia ATCC 9997, S. aureus ATCC 25923, and E. faecalis ATCC 29212. The minimum inhibitory concentration (MIC) was determined using the microdilution broth method following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [21]. In brief, 100 μl of two-fold serial dilutions of the test compounds in Muller Hinton broth (Merck, Germany) were suspended in sterile polystyrene 96-well microtiter plates, and then 100 μl of the bacterial suspension with a final concentration of 5×10⁵ CFU/ml was added to every well of the plate. Finally, the treated plates were incubated for 18-24 hours at 37°C under ambient air conditions. The bacterial suspension with medium and only the culture medium was used for the positive and negative control, respectively. The antibacterial activity of the test compounds was compared with Tetracycline as an effective antibacterial agent.

RESULTS AND DISCUSSION

Determination of nanoparticle characterization

In our study, the following techniques were employed to determine the properties of synthesized AgNPs.

Dynamic light scattering (DLS) analysis

The stability and average particle size of biosynthesized AgNPs using T. ammi extract were determined using zeta potential and DLS analysis. The average particle size and zeta potential of the AgNPs synthesized through chemical and green processes were approximately 51.73±15.74 nm, 26.78±1.24 nm, -16.35 mV, and -13.96 mV, respectively. The presence of high electrostatic negative charges on the surface of nanomaterials, attributed to Van der Waals force interactions between particles, can play a crucial role in preventing the aggregation of nanomaterials [22]. The negative zeta potential of the AgNPs synthesized using natural extracts has been previously reported in several studies [23-25]. Thus, the obtained zeta potential results confirm that the green synthesized AgNPs using T. ammi extract in our study possess an adequate surface charge for electrostatic stability, effectively preventing aggregation.

Fourier transforms infrared spectroscopy (FT-IR)

FT-IR spectroscopy was performed to investigate the functional groups present in the AgNPs samples synthesized using both the chemical (Fig. 2a) and green (Fig. 2b) routes. The strong broadband at about 3400 cm^-1 was assigned to the O-H stretching vibration group of alcohols [26]. The band about 2900 cm^-1 can be related to the C-H stretching vibration band [27]. In the case of AgNPs synthesized using the extract, the extract is deposited on the surface of the nanoparticles. This phenomenon was clearly observed in the FT-IR spectrum of AgNPs synthesized using T. ammi extract (Fig. 2b). Several peaks at 1652, 1397, and 1076 cm^-1 were associated to -C=O, C-N stretching vibration of amines, and -C-O bending vibrations group, respectively [28, 29]. All FT-IR results revealed that the chemical compounds present in the T. ammi extract were responsible for the biosynthesis of AgNPs. 

Transmission electron microscopy (TEM)

TEM analysis was employed to confirm the formation and morphological characteristics of the synthesized AgNPs using both the chemical (Fig. 3a) and green (Fig. 3b) processes. Oval and spherical morphologies, along with homogeneity were prominent features of the AgNPs. These homogeneous structures may be due to the presence of biomolecules from T. ammi extract, which was the surface covering AgNPs. Furthermore, the various morphologies and sizes of the AgNPs could be due to the presence of reducing agents in the T. ammi extract [30, 31]. A closer examination of the TEM images revealed that the size of nanoparticles synthesized using the chemical and green methods was approximately 75 and 50 nm, respectively. 

X-ray diffraction (XRD)

XRD analysis was employed to determine the crystallinity and structural phase of the samples. The XRD pattern of green-synthesized AgNPs using T. ammi extract is depicted in Fig. 4, and revealing distinct 2theta peaks at values of 38°, 44°, 67°, and 77°. According to the XRD results, the cubic structure of AgNPs exhibited respective planes (111), (200), (220), and (311), which closely matched the standard JCPDS No: 01-087-0717. Furthermore, the XRD pattern of AgNPs showed no evidence of impurities, indicating the presence of a single-phase. The presence of low-intensity peaks may be attributed to the crystallization of bioorganic phases on the surface of the AgNPs [32]. These findings are consistent with prior research [33].

Antibacterial activity

The MIC values for AgNPs synthesized using a chemical reagent and the T. ammi extract are presented in Tables 1 and 2. The results indicate that chemical AgNPs and the T. ammi extract exhibited no antibacterial activity. Notably, AgNPs synthesized using the T. ammi extract displayed significant antibacterial activity compared to those synthesized with a chemical reagent. Also, the results showed a direct relationship between the concentration of nanoparticles and antibacterial activity, so with increasing the concentration of nanoparticles, antibacterial activity increases. As the concentration of AgNPs increased from 1% to 2%, the MIC value was reduced by almost half. In our study, the antibacterial activity of AgNPs synthesized using T. ammi extract seems to be higher against Gram-negative than Gram-positive bacteria. A similar result was obtained in previous studies for AgNPs biosynthesized with various plant extracts [34-36]. These results can be explained by the difference in bacterial cell wall composition and the thickness of the peptidoglycan layer in Gram-positive bacteria, which provide protection against chemical agents. Additionally, the higher electrostatic attraction of AgNPs to Gram-negative bacteria may influence changes in cell wall composition, affecting its permeability [35, 37]. Although the precise mechanism of AgNPs’ antibacterial effect is not yet fully understood, a potential mechanism is depicted in Fig. 5.

CONCLUSION

In this study, T. ammi fruit extract was introduced as an excellent reducing, capping, and stabilizing agent in the green synthesis of AgNPs. Our findings underscore the effective and efficient synthesis of AgNPs by T. ammi extract and its significant antibacterial activities. Based on the study results, it is concluded that AgNPs synthesized using T. ammi extract exhibit significant antibacterial properties, particularly against gram-negative bacteria.

ACKNOWLEDGMENT

This research was supported by Birjand University of Medical Sciences, Birjand, Iran (grant number 455935). 

CONFLICT OF INTEREST

The authors declare that there is no conflict of interests regarding the publication of this manuscript.