In vitro evaluation of antibacterial activity and associated cytotoxicity of biogenic silver nanoparticles using various extracts of Tabernaemontana ventricosa

2.1 Plant collection, identification, and preparation of extracts

Leaves and stems of mature T. ventricosa plants were collected from the wild at the University of KwaZulu-Natal, Westville Campus, South Africa (29°49′03.3″S, 30°56′32.7″E), during autumn in March 2021. A mix of young and mature plant parts was harvested, taxonomically identified by the herbarium curator, and a voucher specimen (No. 18222) was deposited at the Ward Herbarium, School of Life Sciences. After inspection, samples were air-dried at 23 °C for 3 months, ground into a fine powder using a Mellerware grinder (model 29105), and stored in airtight glass jars in the dark at room temperature for further use.

2.2 Synthesis, confirmation, and characterization of synthesized AgNPs

AgNPs were synthesized using a modified experimental analysis [23]. A 1 mM solution of silver nitrate (AgNO₃) was introduced to 10 mL of extract, heated to 80°C for 3 h, and a negative control of distilled water and AgNO₃. UV–vis spectroscopy was used to verify the use of extracts for AgNP synthesis. The AgNP solution and control were analyzed simultaneously, and optical densities (ODs) were recorded. The solutions were then subjected to centrifugation, purification, and quantification. The final solutions were dried in an oven at 30°C for 7 days, and the dry mass was determined. The dried AgNPs were reconstituted using sterile distilled water for further analysis. The process ensured the purity of the AgNPs [2].

The study used scanning electron microscopy (SEM) and EDX analysis to characterize synthesized AgNPs using various extracts. The samples were sonicated, dried, and coated with gold. The NP size, shape, and distribution were determined using SmartSEM software. Synchronized EDX analyses were conducted using a Zeiss Ultra Plus X-ray spectrometer, and the elemental composition was verified using AZtec analysis software. High-resolution transmission electron microscopy (HRTEM) was used to analyze the morphology and size of synthesized AgNPs. Samples were deposited onto carbon-coated formvar grids, dried, and observed using a JEM 2100 high-resolution transmission electron microscope with Gatan software. Morphology and size were confirmed using ImageJ software. Fourier transform infrared (FTIR) spectroscopy was used to analyze synthesized AgNPs using various extracts, aiming to investigate the surface chemistry and functional groups of capping agents. Data were collected using a bounce diamond-1 ATR and ResolutionPro version 5.0.0.395. The size distribution and zeta potential of AgNPs were evaluated using the nanoparticle tracking analysis (NTA) technique, with a NanoSight NS500 instrument. A solution was prepared, vortexed, and sonicated before analysis. The images were captured and examined using NTA version 3.2 analytical software. The results of characterization are published in our previous study by Naidoo et al. [23].

2.3 Biological assessment of synthesized AgNPs

2.4 Antibacterial screening

The antibacterial activity of different solutions of the synthesized AgNPs was tested against three Gram-positive strains, Bacillus subtilis (ATCC 6653), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300), and S. aureus (ATCC 29213), and two Gram-negative strains, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853).

According to the Clinical and Laboratory Standards Institute (CLSI), [24], an agar disc diffusion method for in vitro antibacterial screening of AgNP solutions was used in this study. Before use, sterile discs were pipetted with the corresponding AgNP solution (20 µL) concentrations and dried for 1 h [6]. Aseptic bacterial strains were cultivated on Mueller–Hinton agar medium for a night at 37°C. After autoclaving and vortexing, a loopful of bacteria was resuspended in test tubes. The OD of the bacterial strains was determined using an Agilent Technologies Cary 60 spectrophotometer. Discs containing AgNP solutions were carefully deposited onto agar, incubated at 37°C, and after 18–24 h, the zones of growth inhibition were investigated to assess the antibacterial activity of the produced AgNP samples. The screening was done in triplicate, and the zones of inhibition were measured, documented, and averaged. The zones of inhibition or resistance to synthetic AgNPs were evaluated using the following standards: no activity = 0 mm, minimal activity = (1–6 mm), moderate activity = >7 or <9 mm, and significant activity = >9 mm. R stands for resistance.

2.5 MTT cytotoxicity assay

Three cell lines – human embryonic kidney (HEK293), breast adenocarcinoma (MCF-7), and cervical cancer (HeLa) – were acquired from the ATCC, Manassas, USA. All three cell lines were grown to confluency in 25 cm² tissue culture flasks containing Eagle’s minimum essential medium (EMEM), which was made up of 10% (v/v) gamma-irradiated fetal bovine serum (FBS) and 1% antibiotics (100 units/mL penicillin and 100 µg·mL⁻¹ streptomycin). The incubator was a HEPA Class 100 Steri-Cult CO₂ incubator (Thermo Scientific, USA) under conditions of 5% CO₂ and 37°C. The cells were trypsinized, seeded into 96-well clear plates, and allowed to attach at 37°C for the whole night after reaching confluency. The cells were added with 100 µL of fresh complete medium (10% FBS + 1% antibiotics) and EMEM. around 100 µL of every. The cells were then incubated for 36 h at 37°C after being treated with approximately 100 µL of each of the six synthetic AgNP solutions (15, 30, 60, 120, and 240 µg·mL⁻¹) [2]. Only control cells, or cells with 100% vitality, were used as the positive control. Three experiments were conducted in duplicate.

AgNPs were subjected to cytotoxic potential testing utilizing the methods as Daniels and Singh et al. [25] and Mosmann [26]. After 48 h of incubation at 37°C, the growth medium was aspirated and replaced with 100 µL of media containing 10 µL of MTT solution (5 mg·mL⁻¹ PBS), and the cells containing the treatment and negative control were cultured for an additional 4 h at 37°C in a 5% CO₂ atmosphere. Subsequently, 100 µL of dimethylsulfoxide (DMSO) was introduced in place of the medium–MTT combination. To measure the absorbance at 570 nm, a Mindray MR-96A microplate reader (Vacutec, Germany) was utilized. The 50% cell growth inhibition concentration (IC₅₀) was computed using the plots. The viability of the cell lines was closely connected with the absorbance. The proportion of cells that survived was calculated using the following formula:

2.6 Statistical analysis

The study’s results were displayed as mean ± standard deviation, with n = 3. Version 3.6.3 of the R Core Team’s statistical computer program was used to conduct the statistical analyses, which were then followed by Tukey’s honest significant difference range post hoc tests (*P < 0.05; **P < 0.01). To create the graphs, Microsoft Excel 2019 was used.

3.1 Antibacterial assay

Recently, AgNPs synthesized using plant extracts have been extensively utilized in biological applications due to their broad range of bioactivities that include antioxidant, antimicrobial, cytotoxic, antiplasmodial, and anticancer properties [27]. There have been several reports of the potential antibacterial properties of various types of NPs synthesized using Tabernaemontana species such as T. divaricata [10,15,27,28,29,30,31]. However, despite the availability of several reports, the current literature seems to be focused on one species, namely T. divaricata. Therefore, due to the limited or lack of research available on other Tabernaemontana species, the current study aimed to evaluate the antibacterial potential of synthesized leaf and stem AgNPs of T. ventricosa using the agar disc diffusion method. During the antibacterial analyses, six concentrations ranging from 3.125 to 100 mg·mL⁻¹ were tested against five pathogenic bacterial strains, three Gram-positive strains, B. subtilis (ATCC 6653), MRSA (ATCC 43300), and S. aureus (ATCC 29213), and two Gram-negative bacterial strains, E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853).

The zones of inhibition of various AgNPs synthesized using extracts against several strains are presented in Table 1 and Figure 1. Among the synthesized AgNPs, the best-performing extracts were SM, PL, and FL extracts. These consistently exhibited significant antibacterial activity (inhibition zones >9 mm) across most bacterial strains at higher concentrations (50–100 mg·mL⁻¹), particularly against S. aureus, E. coli, and MRSA. The SM extract stood out with the highest zone of inhibition (up to 15.33 mm against B. subtilis), while the PL and FL extracts also showed strong activity, reaching up to 14.33 mm against S. aureus. In contrast, the least-performing extracts were the FS and LM extracts at the lowest concentration (3.125 mg·mL⁻¹), with inhibition zones often just at or below 6 mm, particularly against P. aeruginosa, where no activity (0 mm) was observed in some cases. When compared to the positive control antibiotic (10 µg·mL⁻¹), which produced inhibition zones ranging from 8.67 to 13.00 mm, the AgNPs synthesized from the best extracts often displayed comparable or superior activity, especially at higher concentrations. These results highlight the potential of specific plant-based AgNPs as effective antimicrobial agents, potentially rivaling standard antibiotics in certain contexts. Overall, the results of the current study showed no major differences between AgNPs synthesized using leaf and stem extracts since both types of extracts demonstrated similar levels of inhibition against all bacterial strains.

Figure 1

Diameter of zone of inhibition (mm) of nano extracts (NE) of T. ventricosa at various concentrations (3.125–100 mg·mL⁻¹) against Gram-positive and Gram-negative bacterial strains (n = 3).

Furthermore, the general trend of results highlighted that the zone of inhibition of the reference antibiotics ranged from 8.67 ± 0.58 mm to 13.00 ± 2.00 mm, which was slightly lower compared to the AgNPs synthesized using various extracts, which ranged from 0.00 ± 0.00 mm (no activity) to 15.33 ± 0.58 mm. At the lowest concentrations, the AgNPs synthesized using extracts showed very little activity compared to the antibiotics. However, at higher concentrations, the AgNPs synthesized using methanol stems showed substantial activity (higher than the antibiotics) when tested against B. subtilis. Overall, significant differences were noted for the antibacterial analyses of AgNPs across all extract types and concentrations within each bacterial strain (P < 0.05).

Recently, Attri et al. [10] investigated the AgNPs synthesized using leaf ethanolic extracts of T. divaricate, which showed good inhibition against E. coli and B. subtilis. For E. coli, the inhibition zone measured 35 mm, indicating strong antibacterial activity. In contrast, for B. subtilis, the inhibition zone was smaller, measuring 12 mm, suggesting that the AgNPs were less effective against Gram-positive bacteria. It has been suggested that the antibacterial activity of AgNPs using T. divaricate extracts is possibly attributed to the readily available free radicals within the bio-extracts, which reportedly increased the antibacterial activity [10]. In another study by Marathe et al. [2013], the authors evaluated the antibacterial activity of aqueous extracts from the stem bark of T. alternifolia against clinical isolates of MRSA and vancomycin-resistant S. aureus (VRSA), revealing significant activity against these Gram-positive bacteria. The minimum inhibitory concentration (MIC) of the extracts against MRSA ranged from 600 to 800 μg·mL⁻¹, indicating effective bacterial growth inhibition at these concentrations. Two extract types were tested: direct aqueous extract (DAE) and sequential aqueous extract (SAE). Both extracts exhibited antibacterial activity against MRSA, B. subtilis, and Staphylococcus epidermidis, but showed no activity against Gram-negative bacteria such as E. coli and P. aeruginosa.

Considering the similarities recorded in three Tabernaemontana species, it is highly likely that the free radicals present within T. ventricosa likewise influenced the antibacterial activities of the AgNPs in the present study. Moreover, it has been reported that the properties of AgNPs, such as size (<100 nm) and shape (spherical, ovate, and triangular), are well-suited for their effectiveness against biological processes within microorganisms [32]. Studies have confirmed that AgNPs can modify the cell structure and function of bacterial cell walls by a process of denaturation, which leads to the prevention of bacterial respiration, destabilization of the outer membrane, induced depletion of intercellular adenosine triphosphate, and ultimately bacterial cell death [10,14,15]. The AgNPs synthesized using various leaf and stem extracts of T. ventricosa may positively contribute to the inhibition of bacteria. Furthermore, although antimicrobial activity was successfully demonstrated, it would have been more informative if a broad-spectrum antibiotic had been included as a positive control. Future studies will incorporate such standards to provide more robust comparative insights.

3.2 Cytotoxicity assay

As mentioned previously, due to the increased demand for AgNPs in a variety of applications, these NPs must be evaluated for their potential cytotoxicity [2,9,32,33]. The cytotoxic screening of various plant extracts, isolated chemical compounds, novel drugs, and synthesized NPs is often assessed using the MTT assay, which was developed by Mosmann [25]. Previous reports have demonstrated a substantial cytotoxic potential using synthesized NPs of Tabernaemontana species [2,10,28,29]. In the current study, the cytotoxicity of synthesized AgNPs using various extracts of T. ventricosa at different concentrations (15, 30, 60, 120 to 240 µg·mL⁻¹) was conducted using the MTT cell viability assay. One human non-cancerous cell line, HEK293 (human embryonic kidney), and two cancerous cell lines, namely, MCF-7 (human breast adenocarcinoma) and HeLa (human cervical carcinoma), were utilized for the study.

As observed in Figure 2, control 1 (cells only) was viable, and control 2 (cells + 0.1% DMSO) was reduced (72.56–93.12%), possibly due to the conditions or formulation used in the control group. Specifically, the use of certain solvents or medium components at higher concentrations may have introduced stressors that contributed to the observed cytotoxicity. This phenomenon is supported by findings that suggest chemical toxicity can arise from generalized cell stress and cytotoxicity-mediated processes, potentially resulting from factors such as chemical reactivity or physico-chemical disruption of cellular components [34,35].

Figure 2

In vitro cytotoxicity activity of synthesized AgNPs using LM, SM, FL, FS, PL, and PS extracts from T. ventricosa. Control 1 = cells only; Control 2 = cells + DMSO. Percentage cell survival of (a) HEK293, (b) MCF-7, and (c) HeLa cell lines. *P < 0.05 and **P < 0.01 are considered significant.

The present investigation indicated that all AgNPs synthesized using various extracts of T. ventricosa showed significant cytotoxicity across all cell lines, including the normal cell line, HEK293. This suggests that AgNPs do not possess any selectivity between noncancerous and cancerous cell lines [34,35]. According to Baharara et al. [2015], AgNPs often exhibit selective cytotoxicity, showing fewer effects on normal cell lines due to differences in cellular uptake, redox state, and membrane composition. Cancer cells typically have higher metabolic rates, elevated ROS, and increased nanoparticle internalization, making them more susceptible to oxidative stress and apoptosis induced by AgNPs. Normal cells possess more efficient antioxidant systems and membrane integrity, which protect them from AgNP-induced damage. Moreover, the surface chemistry of biosynthesized AgNPs may enhance biocompatibility, further minimizing toxicity in normal cells [35]. However, most recognized anticancer drugs, such as 5-fluorouracil, are known for their antiproliferative effects against both cell types (normal or cancerous) [34]. The results further revealed that the synthesized AgNPs using FL and FS extracts displayed remarkable cytotoxic activity (lowest percentage cell survival) across three cell lines at 15 and 240 µg·mL⁻¹, respectively (Figure 2), except for AgNPs synthesized using FS extracts at 15 µg·mL⁻¹ for the MCF-7 cell line which showed a relatively high percentage cell survival compared to other treatments (Figure 2). AgNPs synthesized using LM and PS extracts showed moderately high cell survival across cell lines (Figure 2). These results confirm previous findings by Devaraj et al. [2] and Sivaraj et al. [28] since the leaf extracts of T. divaricata in both studies successfully inhibited the growth of cells by more than 90%. Overall, significant differences were observed for the cytotoxic analyses of AgNPs across all concentrations within each extract type (P < 0.05).

Most significantly, an evident relationship between the AgNP concentrations and percentage cell survival was noted, suggestive of dose-dependent cytotoxicity. These results correspond to reports by Devaraj et al. [2], where T. divaricata leaf extracts were evaluated for their cytotoxic activity, which similarly displayed dose-dependent cytotoxicity on MCF-7 cells. Likewise, Preetam Raj et al. [29] investigated the anticancer activity of gold NPs synthesized using T. divarcata extracts, which also demonstrated good dose-dependent association. Furthermore, Manasa et al. [31] evaluated the cytotoxicity of zinc oxide NPs using A549 cell lines, which likewise displayed dose-dependent cytotoxicity.

The IC₅₀ values of AgNPs synthesized using various extracts of T. ventricosa showed moderate (IC₅₀ > 50–100) to significant (IC₅₀ < 50) cytotoxic activity, with none of the extracts displaying low antiproliferative activity (IC₅₀ > 100) (Table 2). AgNPs synthesized using FL extracts exhibited significant activity with IC₅₀ values of 1.40, 6.50, and 0.30 µg·mL⁻¹ for cell lines HEK293, MCF-7, and HeLa, respectively. The weakest IC₅₀ values were observed for AgNPs synthesized using PL (66.00 µg·mL⁻¹), PS (60.60 µg·mL⁻¹), and LM (50.10 µg·mL⁻¹) extracts, respectively, across all cell lines. Overall, the most significant IC₅₀ value was observed for AgNPs synthesized using FL extracts (0.39 µg·mL⁻¹). The IC₅₀ values reported in the present study are like those reported by Sivaraj et al. [28], who observed a moderate IC₅₀ value of 30.65 µg·mL⁻¹ for zinc oxide NPs using T. divaricata leaf extracts. Similarly, Devaraj et al. [2] reported a significant IC₅₀ value at 20 µL·mL⁻¹. However, Manasa et al. [31] investigated the synthesis of zinc oxide particles from T. heyneana, which showed a weak IC₅₀ value between 89.47 and 185.80 µg·mL⁻¹. These variations are possible due to the different types of NPs synthesized. Although the current study focused on evaluating the general cytotoxic effects of AgNPs, future investigations will include detailed molecular analyses, such as the assessment of apoptotic gene expression (Bcl-2, Bax, and caspase-3) and quantification of apoptosis and necrosis through flow cytometry. Incorporating these assays will enhance understanding of the mechanisms underlying NP-induced cell death and support the development of safer biomedical applications [36,37,38].