Green synthesis of silver nanoparticles using Ocimum sanctum Linn. and its antibacterial activity against multidrug resistant Acinetobacter baumannii

Introduction

The synthesis of nanoparticles through the green route is a growing subject in nanotechnology that offers cost-effective and environmentally friendly alternatives to traditional physical and chemical processes (Yeo, Lee & Jeong, 2003). Although nanoparticles are the most promising emerging formulation designs, among these silver nanoparticles are outstanding owing to their all-around pharmacokinetic profiles, lack of human toxicity, and specific antimicrobial properties (AshaRani, Hande & Valiyaveettil, 2009). It is of paramount interest to the pharmaceutical manufacturers that the overall process of producing nanoparticle systems is ecologically balanced while being cost-optimized (Gade et al., 2008; Ouda, 2014). Contrary to the traditional synthetic methods, biological methods of generating nanoparticles are quite adaptive to the environment and also cost-effective (Govindaraju et al., 2010). The most important merit of biologically synthesised nanoparticles is their non-toxic nature and easy biological metabolism. These advantages have made biologically derived nanoparticles one of the most emerging formulation designs widely accepted in the pharmaceutical ecosystem (Das & Smita, 2018).

In recent years, plants have been widely explored for their active principles to treat complex ailments. Novel phytoconstituents derived from plant sources are spanning again around the pharmaceutical markets to develop new drugs, and among these, one of the most commonly used medicinal plants is Holy Basil, that is, Ocimum sanctum L. (O. sanctum). This plant has been used for traditional therapy and also reported as having medicinal significance for anticancer, antimicrobial, cardio-protective, antidiabetic, analgesic, antispasmodic, antiemetic, hepatoprotective, and antifertility actions. Leaves of the O. sanctum L. contain eugenol (1-hydroxy-2-methoxy-4-allylbenzene) as a major active chemical constituent and have been proven for their therapeutic efficacy in various ailments in modern clinical practise (Hemaiswarya, Kruthiventi & Doble, 2008; Raseetha, Cheng & Chuah, 2009). There are various studies on the synthesis of nanoparticles through the green route using parts of plant extracts such as tea leaves, the stem bark of Callicarpa maingayi, Terminalia chebula, Papaver somniferum, and Aloe vera. Additionally, silver nanoparticles have been reported for anti-angiogenesis, anti-inflammatory, anti-platelet, anti-bacterial, and antiviral activity (Bindhani & Panigrahi, 2015).

The misuse of antimicrobials during the last two decades has increased the existence of antibiotic resistance in almost all bacterial strains including A. baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. This has not only made several anti-microbial drugs worthless, but it has also compelled the researchers to explore alternative solutions for fighting deadly microbial infections (Nikaido, 2009). Hence, some recent studies have focused on using silver nanoparticles (AgNPs) as one of the alternatives and have proven the antimicrobial property of silver nanoparticles against both Gram-negative and Gram-positive bacteria without any cytotoxic signs (Biel et al., 2011; Lazar, 2011). Acinetobacter baumannii is a Gram-negative, opportunistic bacterium that causes nearly 2–10% of all hospital-associated infections, particularly among immunocompromised patients (Karlowsky et al., 2003) The organism initially considered of low medical interest, A. baumannii, is now among the top hospital-associated pathogens isolated from the clinical settings, causing a wide array of infections like ventilator-associated pneumonia, septicemia, urinary tract infection, wound, skin, and soft tissue infections and often associated with high morbidity and mortality rates (Karakoc et al., 2013. The major challenges with A. baumannii are its extraordinary abilities to quickly develop resistance against new drugs, to form biofilm on abiotic surfaces, which helps them to survive on hospital equipment for long periods, and also to tolerate the harsh environment for survival (Djeribi et al., 2012). A. baumannii is considered a Red Alert human pathogen and is ranked the number one critical pathogen with high antibiotic resistance by the World Health Organisation for research and new drug discovery (World Health Organization, 2017).

This study describes an easy, fast, and simple method for the biosynthesis of AgNPs from O. sanctum leaf extract. We attempted to characterise the biosynthesized nanoparticles and evaluate their antibacterial activity against multidrug-resistant A. baumannii (MDR-A. baumannii).

Materials and Methods

This collaborative research study involved the preparation, characterization, and evaluation of the antimicrobial potential of biosynthesized AgNPs of O. sanctum leaf extract by adapting a green synthesis process. Figure 1 provides a schematic representation of the steps involved in this study.

Methods

Plant collection and identification

O. sanctum is a relatively small, erect subshrub that reaches up to 60 cm in height and has reverse green or purple leaves and a hairy stem. The leaves are ovate, up to 5 cm long, toothed, and have a petiole (Pattanayak et al., 2010). For the present study, fresh leaves were collected in September 2021 from the medicinal plant garden of the Delhi Pharmaceutical Science and Research University (DPSRU), New Delhi, and stored in airtight paper bags for further processing.

Preparation of O sanctum leaves aqueous extract

To prepare the aqueous extract of leaves, fresh leaves were collected and placed in a beaker, washed with distilled water until completely free from dust or other residues, and then washed with ultrapure water (Milli-Q^® HX 7000 SD, Merck, Sydney, Australia). A total of 25g of washed O. sanctum leaves were chopped into fine pieces and crushed in 100 mL pure water (Milli-Q^®) using a mortar and pestle. The aqueous extract was ground and then boiled for 10 min at 80 °C in a 250 mL beaker. The aqueous leaf extract was then allowed to cool down to room temperature 25 °C and then filtered with Whatman filter paper with a 1.5 µm pore size (GE Healthcare Life Science, Karnataka, India) (Singh et al., 2018). These filtered leaf extracts were collected and stored at 4 °C, until further use (Rao et al., 2013).

Qualitative phytochemical analysis of O. sanctum extract

The presence of amino acids and proteins was confirmed by boiling the O. sanctum leaf extract with a 0.2% Ninhydrin solution (0.2 g of ninhydrin dissolved in 100 mL ethanol) for five minutes. A change in colour towards violet indicates the presence of amino acids and proteins. The identification of phenols was done by mixing 1 mL of O. sanctum leaf extract with 2 mL of 99.5% ethanol and 3 drops of a 2% ferric chloride (FeCl₃) solution. The reddish-black colour confirmed the presence of phenols. The presence of flavonoids was tested by mixing 0.5 mL of O. sanctum leaf extract with a 2% sodium hydroxide (NaOH) solution and adding four drops of 1% hydrochloric acid (HCl). The colour changes from brown to yellow confirmed the presence of flavonoids (Table 1) (Borah & Biswas, 2018; Saranya, Noorjahan & Siddiqui, 2019).

Table 1:

Phytochemical analysis O. sanctum leaves extract.

DOI: 10.7717/peerj.15590/table-1

Preparation of 1 mM silver nitrate solution

The stock solution was prepared by weighing 170 mg of silver nitrate (99% extra pure) (LobaChemiePvt. Ltd., Mumbai, India) and dissolving it in 10 mL of ultra-pure water (Milli-Q^® HX 7000 SD, Merck, Sydney, Australia). About 1 mL of this stock solution was taken and further dissolved in 100 mL of ultra-pure water. This solution was stored in an amber coloured bottle to prevent the self-oxidation of silver nitrate solution (Saifuddin, Wong & Yasumira, 2009).

Green synthesis of silver nanoparticles (AgNPs)

Silver nanoparticles (AgNPs) were prepared by a single-step synthesis previously reported (Ramteke et al., 2013). In the process, a 90-mL solution of silver nitrate (AgNO₃) at 1 mM concentration was placed in a beaker on a magnetic stirrer (Remi, Mumbai, India) at 400 rpm, and 10 mL of an aqueous extract of O. sanctum leaves was added dropwise for half an hour (30 min). The colour of the solution turned from reddish brown to dark brown, indicating the formation of AgNPs. The preparation was kept at room temperature (25 °C) for 1 h. All procedures were performed in a dark room to prevent the oxidation of silver nitrate. Nanoparticles were separated by the process of centrifugation at 3,000 rpm for 10 min (Remi, Mumbai, India). The synthesised nanoparticles were stored at 4 °C until further use.

Characterization of the synthesized AgNPs of O. sanctum

The biosynthesized AgNPs were characterised by various parameters, as represented in Fig. 1. The process of silver ions reduction and biosynthesis of silver nanoparticles were continuously monitored every 20 min immediately after the blending of plant extract and silver nitrate solution, using an UV-Vis spectrophotometer (Shimadzu, Tokyo, Japan) at 452 nm. The absorption spectra of synthesised AgNPs of O. sanctum and the leaf extract of O. sanctum were also studied in the range of 200–800 nm. In addition, the average particle size and zeta potential were determined by dynamic light scattering, using the Litesizer™ 500 (Anton Paar, Buchs, Switzerland). The morphology of AgNPs in O. sanctum was visualised using a scanning electron microscopy (SEM, Leo 435 VP 501B; Philips, Texas, USA), with accelerated voltage up to 30 kV and magnification efficacy ranges from 10x to 300,000x. Following this, the synthesised nanoparticles were also characterised by Transmission Electron Microscopy (TEM; JEOL, Tokyo, Japan) using a copper grid coated with carbon film and phosphotungstic acid (1% w/v) as a negative stain, and then air dried and allowed to rest at room temperature (25 °C). Next, samples were examined by Fourier-transform infrared spectroscopy (FTIR; PerkinElmer; Waltham, MA, USA). FTIR analysis provides information about the functional groups present.

Results

UV-visible (UV-Vis) spectroscopy

The silver surface plasmon resonance (SPR) was obtained by measuring the peak at 452 nm in the UV-Vis absorption spectrum of the reaction solution, which proved the formation of silver nanoparticles from silver ions. The broad band of UV-Vis absorption spectra is due to the presence of various metabolites of O. sanctum extract presented in the reaction solution that were read in the experimental spectrophotometric range (Figs. 2 and 3).

Particle shape, size, and morphology

SEM images of prepared nanoparticles indicated that particles were almost spherical with smooth surfaces and a size range of 73.24–87.89 nm (Fig. 4). Morphological examination by TEM confirmed the spherical shape of most nanoparticles with a size range of 29–54.9 nm (Fig. 5), while some oval and/or elliptical-shaped nanoparticles were also formed, which is the common feature of most of the biologically synthesised nanoparticles. Lighter edges with a heavier centre were also visible, confirming the capping of protein biomolecules with AgNPs. The mean particle size of nanoparticles was 55 nm, which was fully concordant with results from TEM and SEM analysis. Particles showed a zeta potential of around −27 mV, and an increase in negative values confirmed the repulsion between particles, which also verified the stability of the formulation (Fig. 6A). Furthermore, the particle size distribution of AgNPs from O. sanctum showed 74.95 nm with a 27.9% polydispersity index (Fig. 6B).

Fourier transform infrared spectroscop (FTIR)

Silver nanoparticles (AgNPs) synthesised from O. sanctum are of interest due to their potential use in various biomedical applications. The FTIR spectra of aqueous leaf extract and the biosynthesized AgNPs have been shown in Figs. 7A and 7B. The FTIR spectral analysis revealed strong peaks at 3,619.63 and 3,565.33, 1,741.28 and 1,693.51, and 650.74 and 651.24 cm⁻¹.

The strong bands at 3,619.63 and 3,565.33 cm⁻¹ indicate the presence of alcohols with free OH groups. The vibrational peaks at 1,741.28 and 1,693.51 cm⁻¹ represent the presence of an alkene group. The absorption bands at 650.74 and 651.24 cm⁻¹ are assigned to the aromatic C-H bending. The Fig. 7A represents the FTIR interpretation of O. sanctum leaves extract, and the Fig. 7B represents the FTIR interpretation of AgNPs of O. sanctum.

Antibacterial activity

Agar well diffusion method, MIC and MBC determination

The zone of inhibition determined by the agar-well diffusion method after 24 h of incubation formed by AgNPs of O. sanctum (200 µg/mL) and colistin (16 µg/mL, positive control) was 15 mm and 14 mm, respectively, against the MDR A. baumannii. No zones were observed against distilled water (the negative control) or O. sanctum leaves extract (Fig. 8). The MIC and MBC of AgNPs of O. sanctum determined by the microdilution method against the MDR A. baumannii were 32 µg/mL and 64 µg/mL respectively. The MIC and MBC of positive control colistin were 2 µg/mL and 4 µg/mL respectively.

Effects of biosynthesized silver nanoparticles on A. baumannii cells

Electron micrographs by SEM of A. baumannii cells, untreated and treated with AgNPs, are shown in Fig. 9. SEM images of untreated A. baumannii showed a typical clear surface structure with smooth and intact cell morphology, whereas those of A. baumannii treated with AgNPs showed severely damaged cell structure with ruptures, gaps, an irregular surface, and fragments.

Cytotoxicity test using MTT assay

Cytotoxicity against the human lung adenocarcinoma cell line A549 at concentrations of 500 µg/mL and 250 µg/mL of both AgNPs and O. sanctum extract showed that cells did not remain viable after 24 and 72 h of treatment. However, at all the other concentrations, ranging from 0.97 to 125 µg/mL A549 cells showed viability almost equivalent to that of untreated cells at both time points (Fig. 10).

Discussion

This study was an attempt to synthesise and characterise AgNPs of O. sanctum and further evaluate their potential as an antimicrobial agent against A. baumannii. Preliminary phytochemical screening of the O. sanctum extracts confirmed phenols and flavonoids in the leaf extract, while it was found negative for the presence of proteins. It is well established by previous studies that colour change occurs after addition of O. sanctum extract to silver nitrate solution during the reaction (Pirtarighat, Ghannadnia & Baghshahi, 2019), which indicates the reduction of silver ions to form nanoparticles with the extract metabolites. Further, the colour intensity also increases with respect to the standing time (Fayaz et al., 2010; Kumar, Selvi & Govindaraju, 2013). It is interesting that the ultraviolet and visible (UV-Vis) absorption spectra of the prepared mixture confirmed the silver nanoparticles formation from silver ions, with a peak at 452 nm. This finding, supported by the broad band of UV absorption, is mainly due to the presence of organic metabolites in the O. sanctum- based aqueous extract (Rao et al., 2013). Furthermore, SEM and TEM analysis of biosynthesized nanoparticles (Figs. 4 and 5) demonstrated that nanoparticles are almost spherical in shape with smooth surfaces, have a mean particle size of 55 nm with a −27 mV zeta potential showing stable particles, and their particle size distribution is 74.95 nm. A previous study showed that the average size of silver nanoparticles biosynthesized using the leaf extract of O. sanctum was 42 nm (Rao et al., 2013).

The FTIR spectra depicted some extent of shifting of the AgNPs spectra over that of the aqueous extract, which might be due to the presence of functional groups present in the biosynthesis of plant extract and the capping of nanoparticles. It also exhibited biosynthesized AgNPs and an aqueous extract that differed very slightly in their absorption bands (Figs. 7A and 7B). This may be illustrated by the fact that available biomolecules in plants play a crucial role in the reduction of metal ions and the formation of small nanoparticles (Kandasamy et al., 2013). In addition, peaks at 3,339.30 cm⁻¹ and 1,634.70 cm⁻¹ indicated the binding of proteins, carbohydrates, and nitrogenous compounds on the surface of nanoparticles (Das & Smita, 2018).

The stability of AgNPs of O. sanctum tested at different pH levels showed changes in particle size after pH 5 to 9, and results at different concentrations of NaCl showed that the particle size was reducing, which was in par with a previous study (Wang et al., 2021). It is imperative that the O. sanctum extract play a dual role as a reducing agent as well as a stabilising agent for AgNPs This is therefore highly recommended for more comprehensive studies to justify this association and come up with the final conclusion.

The antimicrobial studies determined that there was no zone of inhibition found in tulsi extract or sterile distilled water, but AgNPs from O. sanctum have promising and effective antibacterial activity on A. baumannii, showing a 15 mm zone of inhibition. The MIC and MBC results also proved that the AgNPs were able to inhibit the growth of MDR A. baumannii. The antibacterial activity can be explained based on nanoparticles interaction with microorganisms (Franci et al., 2015) by the released silver ions being attached to the cell wall of bacteria, modulating the cell membrane permeability, respiration blockage (Dhas et al., 2014; Manjumeena et al., 2014; Muhsin & Hachim, 2014), and destabilisation of the bacterial outer membrane and plasma membrane degradation followed by reduction of intracellular ATP (Ajitha, Reddy & Reddy, 2014; Pirtarighat, Ghannadnia & Baghshahi, 2019). Silver ions also have a great affinity to interact with the sulphur or phosphorus in cell biomolecules, ultimately ceasing bacterial replication (Umashankari et al., 2012). AgNPs might also have affected some of the cellular components and induced the damage of the cell membrane, which finally results in cell decomposition and death (Li, Xie & Shi, 2010).

The bactericidal activities of biosynthesized AgNPs from the extracts of other Ocimum species were also reported. Tailor et al. (2020) observed the antibacterial activity of AgNPs prepared from Ocimum canum against Escherichia coli, with a minimum zone of inhibition of 17 mm at 10 ppm concentration of AgNPs, while the maximum zone of inhibition of 24.5 mm was observed at 30 ppm concentration (Tailor et al., 2020). The susceptibilities of 15 mm, 13 mm, and 12 mm were observed against Bacillus vallisomortis, Bacillus subtilis, and Escherichia coli, respectively, using AgNPs synthesized from Ocimum bacilicum (Pirtarighat, Ghannadnia & Baghshahi, 2019). Using the biosynthesized AgNPs from the extract of O. gratissimum, Das et al. (2017) noted no zone of inhibition in silver nitrate solution alone, but the bio-reduced AgNPs showed considerable growth inhibition against pathogenic Escherichia coli and Staphylococcus aureus. They observed a zone size of eight mm and 12 mm against Escherichia coli using 4 µg/mL (MIC) and 8 µg/mL (MBC) of AgNPs, respectively. Similarly, the zone sizes of 10 mm and 16 mm were observed against Staphylococcus aureus using 8 g/mL (MIC) and 32 µg/mL (MBC). The combined activity of phytochemicals from Ocimum gratissimum and AgNPs demonstrated a beneficial role in reducing the dose required for total microbial growth inhibition.

The killing kinetic assay showed time- and dose-dependent action against MDR A. baumannii. The bactericidal activity was gradual, and complete killing was observed within 24 h using MBC and higher concentrations of AgNPs. A statistically significant result was observed in comparison to the killing action of the antibiotic colistin. The time kill curve analysis by Das et al. (2017) using AgNPs against Escherichia coli and Staphylococcus aureus showed bacterial killing activity that increases with time of exposure of bacteria to AgNPs at their respective MBC concentrations, and complete bactericidal results were obtained. The bacterial exposure with AgNPs demonstrated a rapid dose and time-dependent killing leading to an early stationary phase. This rapid bactericidal activity of AgNPs could significantly decrease the bacterial mechanism to induce resistance development. Therefore, AgNPs may be a promising alternative to significantly reduce the development of drug resistance in bacteria and an effective antimicrobial agent for human use after the strong clinical trials (Thammawithan et al., 2021).

Various studies have reported that the bactericidal activity of AgNPs depends on their size and shape. The smaller the size, the higher would be the antibacterial property compared to the big size particles (Panacek et al., 2006). This result could be due to the higher penetration ability of smaller nanoparticles (Morones et al., 2005). This evidence of small size nanoparticles showed good results against bacterial inhibition, but studies also reported the adverse effects and health issues of nanoparticles due to their nano size. (Basavaraja et al., 2008; El-Ansary & Al-Daihan, 2009). This small size of nanoparticles makes them mobile both in the human body and in the external environment as well (Braydich-Stolle et al., 2005). Pal, Tak & Song (2007) demonstrated that truncated triangular AgNPs revealed the highest bactericidal activity against Escherichia coli when compared with rod- and spherical-shaped nanoparticles. The same result was also shown by Sharma, Yngard & Lin (2009).

Apart from the antibacterial efficacy against MDR A. baumannii, cytotoxicity testing to the mammalian cell is also crucial to the development of novel antimicrobials. The optimum features that support the efficacy of a new antimicrobial agent require properties like potent antimicrobial activity and a low cytotoxicity level (Thammawithan et al., 2021). Our finding with the bio-synthesized AgNPs of O. sanctum showing antibacterial activity against MDR A. baumannii at a MBC of 64 µg/mL and cytotoxicity against the human A549 cell only above the concentration of 250 µg/mL clearly indicates that AgNPs are less to moderately toxic against human cells compared to their effect on bacterial cells. This study shows a good ray of hope for the development of novel antibiotics using nanoparticles with more intense research and clinical trials.

Conclusion

This study focused on the single-step green approach for the biosynthesis of silver nanoparticles from an aqueous leaf extract of O. sanctum. One of the most important benefits of this method is that it is eco-friendly and reduces traces of organic solvents that are hazardous to human health. Silver nanoparticles were successfully synthesized and confirmed by the colour change. The various evaluation parameters supported the nano-sized range with stable silver nanoparticles owing to the presence of biomolecules present in leaf extract that may act as surface-active stabilising agents supporting the formulation of silver nanoparticles. The antibacterial studies revealed their efficacy against clinically isolated MDR A. baumannii, and the cytotoxic activity of AgNPs and O. sanctum extract against mammalian cells showed moderate action. This effective bactericidal activity of AgNPs could lead to useful alternative treatment strategies to minimise bacterial resistance. Therefore, AgNPs may be a promising way to decrease the number of antibiotic-resistant bacteria and an alternative antimicrobial agent for human use after strong clinical trials. Further studies would be performed to prove its efficacy more effectively.

This study is a part of a PhD thesis entitled “Molecular characterization, detection of carbapenem resistance genes, and effect of natural products using nanotechnology against multidrug-resistant Acinetobacter baumannii isolated from various clinical specimens from Central Referral Hospital, Sikkim, India”, Walailak University, Thailand.

Supplemental Information