Anticancer and antimicrobial effects of green-synthesized silver nanoparticles using Teucrium polium leaves extract

2.1 Preparation of plant extract

T. polium plant leaves were collected from the market in Riyadh, Saudi Arabia (https://maps.app.goo.gl/HpzkHw8PEzBttyew8?g_st=aw). The plant was identified by Professor Dr. Mohammed Fasil of the Department of Botany and Microbiology at King Saud University in Riyadh, Saudi Arabia, and was deposited at the Herbarium with the reference number King Saud University (KSU) NO-11425. T. polium plant leaves were cleaned with distilled water after being dust-washed multiple times under the water tap. The leaves were then pulverized using a mortar and pestle after being shadow-dried for a week. One day at room temperature was spent with 5 g of leaf powder suspended in 100 mL of distilled water in a beaker. Using Whatman No. 1 filter paper, the extract was filtered in a conical flask and chilled at 4°C. AgNPs were synthesized using this solution.

2.2 Green synthesis of AgNPs from the extract

For the preparation of T. polium AgNPs, 2 mL of the extract from the previous step was mixed with 100 mL of a freshly prepared aqueous AgNO₃ solution at a concentration of 2 mM, using a 250 mL Erlenmeyer flask, following the methodology outlined by Melkamu et al. [19]. The prepared reaction mixture was left undisturbed under the sunlight. The reaction was monitored visually by observing a color change from pale yellow to brown, indicating the formation of nanoparticles (Figure 1). The bioactive compounds in the T. polium extract act as reducing agents, reducing the silver ions (Ag⁺) to form AgNPs. The mixture was centrifuged and filtered to separate the AgNPs from any remaining plant extract or impurities.

Figure 1

Color change during the biosynthesis of T. polium AgNPs.

2.3 Characterization of AgNPs

2.4 Determination of phytochemicals of the extract by GC–MS

The GC–MS 7890B GC system from Agilent Technologies (Santa Clara, CA, USA) autosampler injection system was used to inject 0.9 µL of the diluted T. polium leaves extract, which had been diluted with acetone (1–9 volume ratio). The connected database software was used to identify the items. GC in conjunction with a mass selective detector allowed for the identification of the sample’s constituent parts (GC–MS). The target compounds were separated using an Agilent Technologies DB-5 MS capillary column (30 m in length, 0.25 mm in internal diameter, and 0.25 μm of phase thickness). Helium was used as the carrier gas, with a flow rate of 1 mL·min⁻¹, an inlet temperature of 250°C with a split mode ratio of 50, and an oven temperature range of 50–250°C with a total analysis time of 73 min. The MS detector was configured with the following parameters: MS source temperature of 230°C, acquisition scan type, mass between 40 and 500 g·mol⁻¹, scan speed of 1.56, and solvent delay of 2 min.

2.5 Analysis of total phenolic content (TPC) and total flavonoid content (TFC)

The TPC was determined using the Folin–Ciocalteu method [23]. The TPC was calculated using the standard curve created from the gallic acid standard. TPC content in the T. polium leaf extract was given as mg GAE g^–1 per gram of extract. Each experiment was carried out three times, and the results were reported as mean ± standard deviation (SD).

The amount of TFC was estimated as described previously by using the method aluminum chloride (AlCl₃) colorimetric method which is based on the formation of a complex flavonoid-aluminum having the maximum absorption at 415 nm [24]. A standard curve with quercetin concentrations ranging from 0 to 100 mg·mL⁻¹ was created to quantify TFC. The findings were presented as mean ± SD mg QE g^–1 per gram of extract.

2.6 Anticancer activity

2.7 Screening for antibacterial activity

2.8 Statistical analysis

One-way analysis of variance was used for statistical analysis and the findings were presented as mean  ±  SD. A 5% significance threshold (p < 0.05) was used.

3.1 GC–MS analysis of the T. polium leaf extract

The GC–MS analysis of the T. polium leaf extract revealed a variety of phytochemical components. A total of ten compounds were extracted and it was found that the compounds belong to different bioactive chemical classes. The GC–MS results revealed the existence of fatty acid esters (hexanoic acid, 2,7-dimethyloct-7-en-5-yn-4-yl ester), macrolides ((3E,10Z)-oxacyclotrideca-3,10-diene-2,7-dione), monoterpenoids (isolongifolan-8-ol), pyrimidine of barbituric acid derivatives (cyclobarbital), aryl phosphotriesters (1,4-benzenediol,2,6-bis(1,1-dimethylethyl)-), phenylpropanes (1,2-benzenediol, 3,5-bis(1,1-dimethylethyl)-), aromatic heteropolycyclic 2-aminothiophenes (2-amino-4,4,6,6-tetramethylthieno[2,3-c]furan-3-carbonitrile), aromatic monoterpenoids. Steroid esters (pregn-5-ene-3,20-diol-13-carboxylic acid, 3-acetate-18,20-lactone) (56.62%) was found to be the main photochemical component in the aqueous extract; it was followed by sesquiterpenoids of prenol lipids (oplopanone) (16.41%) and (carvacrol, TBDMS derivative) (7.95%) (Table 1 and Figure 2).

Figure 2

GC–MS spectrum of the aqueous extract of T. polium leaf. The GC–MS apparatus was set for 73 min, and an aqueous extract was used for the study. Every detected peak in the spectrum designates a recognized chemical, and the existence of a sizable peak denotes the extract’s major component.

In agreement with our findings, previous studies showed that the GC–MS analysis of the Ariel parts or root methanolic extracts of T. polium revealed variable phytochemicals including germacrene D, β-pinene, carvacrol, bicyclogermacrene, α-thujene, α-pinene, epi-bicyclosesquiphellandrene, t-cadinol, and limonene were the most abundant [34,35]. Some other studies showed that the GC–MS analysis of T. polium essential oils revealed the existence of sesquiterpenes (germacrene D, trans-caryophyllene, and bicyclogermacrene), monoterpenes (β-pinene and linalool), monoterpenoids (geranial and z-citral) compounds [36]. Another study showed that the phytochemical screening of the T. polium ethanolic extract of the aerial parts revealed the existence of many compounds including saponins, alkaloids, polyphenols, flavonoids, tannins, terpenoids, quinines, and coumarins which might explain its potential antioxidant and antibacterial activity against P. aeruginosa, E. coli, S. aureus, and B. subtilis [37]. Such a rich phytochemical profile suggests the potential of T. polium as a source of bioactive molecules with various pharmacological activities.

Several of these chemicals have been previously examined for their biological effects, indicating intriguing areas for further research. In the study conducted by Atallah et al. [38], they revealed that hexanoic acid derivatives found in the black sand of Egypt had significant antibacterial activity against B. subtilis, Salmonella enteritidis, and Pseudomonas aeruginosa. Macrolides are well-known natural antibiotic, antiviral, antiparasitic, antimycotic, anticancer, or immunosuppressant agents [39]. A previous study revealed that (3E,10Z)-oxacyclotrideca-3,10-diene-2,7-dione induced structural alerts and in vitro mutagenicity, which might be due to the ester and phenol groups exist in its chemical structure [40]. Another study demonstrated that some macrolide antibiotics had significant cytotoxic effects against head and neck squamous cancer cell lines under nutrient-deprived conditions [41]. Oplopanone is one of the sesquiterpenoid ketones, which is known for its biological characteristics such as anti-tuberculosis [42] and anti-diabetes effects [43]. Monoterpenoids like isolongifolan-8-ol possess potent antimicrobial properties, inhibiting the growth of both Gram-positive and Gram-negative bacteria [44]. Cyclobarbital is a pyrimidine derivative, which is a well-known anticonvulsant, and hypnotic agent, and has some anti-proliferative properties against Ehrlich ascites carcinoma cells [45,46]. Carvacrol, TBDMS derivative, is an aromatic monoterpenoid that possesses like carvacrol has shown strong antimicrobial, immune response-modulating, antioxidant, anti-inflammatory, anticancer, antidiabetic, and other biological activities [47]. The existence of these chemicals in T. polium leaf extract shows that it has promise as a natural antibacterial and anticancer agent. Further research is needed to understand the mechanisms of action and therapeutic potential of these substances, both individually and in combination.

3.2 Phenolic and flavonoid content of T. polium leaf extract

TPC and TFC of the aqueous extract of T. polium leaf extract were evaluated. As it is seen the plant is rich in TPC (108.26 ±  1.46 mg GAE g^–1 dry weight of the dry extract) [R ² = 0.997]) and TFC (84 ±  2.14 mg QE g^–1 dry weight of the dry extract [R ² = 0.989]). These phenolic chemicals are essential for scavenging free radicals and avoiding oxidative stress-induced damage to cells and tissues. Also, T. polium extract has a TFC of 84 ±  2.14 mg QE g^–1 dry weight of the dry extract (R ² = 0.989), demonstrating its medicinal potential. Flavonoids have a variety of pharmacological functions, including anticancer, anti-inflammatory, and cardiovascular protection. A previous study showed that T. polium had a TPC of 14.57–157.84 mg of GaA g^–1 and TFC of 6.48–139.87 mg of Ru g^–1 of extract [48]. Another study showed the total flavonoids (20.78 mg equivalent QE g^–1 DW extract) and total polyphenols (227.43 mg equivalent GAE g^–1 DW extract) of the ethanolic extract of T. polium Ariel parts which might explain its potent biological activities [37]. Also, in the study conducted by Benchikh et al., they showed that hydroalcoholic extract was rich in flavonoids (24.30 ± 0.44 μg QE mg^–1) and phenols (36.35 ± 0.294 μg GAE mg^–1), which was reflected in its tested antioxidant, anti-Hyperglycemic, anti-Alzheimer, and anti-inflammatory activities [49]. T. polium’s high TPC value shows that it has the potential to be a natural source of antioxidants, which might help prevent oxidative stress-related disorders. The considerable TFC value emphasizes the presence of flavonoids in the T. polium extract, indicating its potential application in various disease conditions.

3.3 FTIR analysis of the T. polium leaf extract and the synthesized AgNPs

The combined analysis of FTIR spectroscopy and biogenic synthesis of AgNPs using T. polium leaf extract reveals important information about the chemical composition and biophysical properties of this natural extract, as well as its potential applications in nanotechnology and medicine. The FTIR study of T. polium leaf extract revealed four major functional groups: hydroxyl (O–H) stretching at 3,441 cm⁻¹, carbonyl (C═O) stretching at 1,630 cm⁻¹, C–N stretching at 1,115 cm⁻¹, and C–Cl stretching at 595.9 cm⁻¹ (Table 2 and Figure 3). These functional groups indicate the existence of alcohols, alkene compounds, amines, and halogen compounds, respectively. The strong and wide absorption band at 3,441 cm⁻¹ indicates the presence of numerous hydroxyl groups, possibly related to phenolic chemicals in the leaf extract. The identification of these functional groups reveals important information about the chemical composition of T. polium leaf extract, which may be used to guide future research into its biological activity and possible medicinal uses. In the study conducted by Rakhshan et al. [50], the FTIR analysis of T. polium stem extract revealed strong absorption for the functional groups of O–H stretching, C═O asymmetric stretching, and hydroxyl groups of kaolinite. Another study used FTIR spectroscopy and showed that T. polium had seven functional groups for alcohols, alkyl, aliphatic aldehydes, and amides [51].

Figure 3

FTIR spectra of T. polium leaf extract and the synthesized AgNPs.

3.4 The biogenic attributes of AgNPs synthesized from aqueous extract of T. polium leaf extract

In the present study, the synthesis of AgNPs occurred through the reduction of Ag⁺ to Ag⁰, resulting in a noticeable change in the color of the reaction mixture from light yellow to dark brown. To analyze and identify the synthesized AgNPs, various spectroscopic techniques (UV–vis and EDX) and TEM were employed. In the characterization experiments of the synthesized AgNPs, the prominent peak observed (∼442 nm) in the UV–vis spectrum is characteristic of the surface plasmon resonance (SPR) band of AgNPs, which further confirms their formation. This peak indicates the collective oscillation of conduction electrons in the nanoparticles upon interaction with light, which is a defining feature of metallic nanoparticles (Figure 4). EDX analysis and SEM imaging were used to assess the qualitative and quantitative status of all elements that participated in the synthesis process. As shown in Figure 5a, b, the SEM image showed the morphological characteristics of the synthesized AgNPs. EDX analysis showed a peak of 2.983 keV in the silver region with 8.92% of the mass. That is the typical condition for silver crystal absorption due to their remarkable SPR phenomena. Notably, the two peaks of carbon and oxygen atoms had very weak signals at 0.227 and 0.525 keV, respectively. The TEM analysis in this study confirms the spherical morphology of the AgNPs and provides insight into their size distribution. The average diameter of the AgNPs ranging from 61 to 41 nm suggests the uniformity of particle size and the absence of significant aggregation, which is crucial for various applications requiring precise control over nanoparticle size (Figure 6). In the study conducted by Amini et al., UV–vis spectroscopy of synthesized AgNPs from aqueous extract of T. polium revealed a prominent SPR peak around 430 nm. In the same study, TEM analysis showed that AgNPs were predominantly spherical and homogeneous in size, with an average diameter of 14.3 ± 9.7 nm [52]. In the study conducted by Hashemitabar et al., the AgNPs biosynthesized from the leaf extract of T. polium had a spherical shape and an estimated size of 8–28 nm which was confirmed by the Dynamic light scattering, zeta potential analysis, and TEM [18]. The hydrodynamic diameter in the current study was slightly larger, possibly due to the presence of larger particles impacting light scattering during measurement. Another study showed that AgNPs synthesized from the aqueous extract of T. polium had an SPR of 420 nm. At the same time, the XRD analysis confirmed the presence of pure silver metal, indicating a successful reduction of silver ions to metallic silver by the T. polium leaf extract under the specified reaction conditions, besides, TEM images revealed that the majority of AgNPs observed were spherical, with a small proportion being elongated and ranged in size from 5 to 30 nm [53]. Overall, the combination of spectroscopic and microscopic techniques employed in this study provides a comprehensive characterization of the synthesized AgNPs, offering valuable insights into their optical, elemental, and morphological properties. These findings contribute to the growing body of knowledge in nanotechnology and hold potential for various applications in areas such as catalysis, biomedicine, and environmental remediation.

Figure 4

UV–vis spectroscopy of the synthesized T. polium AgNPs compared to the crude extract.

Figure 5

SEM image (a) and EDX (b) of the synthesized AgNPs.

Figure 6

TEM of the synthesized AgNPs. (a) TEM image and (b) particle size distribution for the sample fitted with a log-normal distribution function.

3.5 Cell cytotoxicity and apoptotic analysis

The toxicity of aqueous T. polium leaf extract and synthesized AgNPs on human cancer cell lines MCF-7 and HepG2 was investigated. The MTT assay results revealed a dose- and time-dependent decrease in cell viability, compared to untreated cells. The IC₅₀ values were 15 ± 3.18 μg·mL⁻¹ and 34.5 ± 3.16 μg·mL⁻¹ in MCF-7 and 12 ± 2.63 μg·mL⁻¹ and 26.5 ± 2.23 μg·mL⁻¹ in HepG2 induced by synthesized AgNPs and the crude extract, respectively (Figure 7a and b). The apoptosis analysis supported these findings. Caspase 3, 8, and 9 activity was considerably higher in cells treated with T. polium leaf extract or synthesized AgNPs than in the control group. The treated cells expressed decreased amounts of anti-apoptotic genes Bcl-xL and Bcl-2 compared to untreated controls (Figure 8a and b). This shows that both therapies induce apoptosis in cancer cells, contributing to their lethal effects. The morphological evaluation of treated cells supported these findings, where the treated cells showed obvious ultrastructural changes. The lethal effects of T. polium and AgNPs on cancer cells are consistent with research demonstrating the promise of plant extracts and nanoparticles in cancer therapy. Previous studies showed the significant anticancer potential of T. polium and AgNPs by triggering apoptosis of MNK45 human gastric cancer cell line [8], NALM-6 acute lymphoblastic leukemia lymphocyte-like cells [52], and VERO (African Green Monkey Kidney) cell lines [6] through elevation of ROS, protein damage, and oxidative stress mechanisms. T. polium AgNPs were found to induce cytotoxic activity for Ntra-2 cancer cells with an IC₅₀ of 106.58 µg·mL⁻¹; besides, the upregulation of caspase 3 and caspase 9 and downregulation of Bcl-2 and genes involved in angiogenesis [10]. An interesting study showed that T. polium L. was able to reduce the carcinogenesis of the liver cells in an animal model, with a remarkable increase in its total antioxidant status [54]. Another study showed that the green-synthesized zinc oxide nanoparticles (TP-ZnO-NPs) from T. polium extract demonstrated significant antiproliferative and anticancer activities against HGC-27 gastric cancer cells, highlighting their potential as a novel therapeutic agent [55]. These studies and our results showed that the observed dose-dependent cytotoxicity and apoptosis demonstrate the therapeutic potential of T. polium and AgNPs in cancer cells.

Figure 7

The effect of aqueous T. polium leaf extract and synthesized AgNPs on the viability of MCF-7 (a) and HepG2 cells (b) was examined using the MTT assay. Cells were treated with T. polium leaf extract and synthesized AgNPs (0–400 μg·mL⁻¹) for 24 h. Mean ± SD is presented from three independent experiments (*p < 0.05 compared to non-treated cells (control)).

Figure 8

Effect of aqueous T. polium leaf extract (35 μg·mL⁻¹) (a) and synthesized AgNPs (15 μg·mL⁻¹) (b) on MCF-7 and HepG2 cell lines. Synthesized AgNPs treated MCF-7 and HepG2 cells showed an increased percentage of the apoptotic population (caspase 3, 8, and 9 and Bax) after 24 h incubation. The results are represented as the mean ± SD of three independent experiments (*p < 0.05 compared to non-treated cells [control]).

3.6 Antibacterial effects

In the current study, the antibacterial effect of T. polium leaf extract and synthesized AgNPs against bacterial strains was investigated against Gram-positive bacteria (S. aureus, S. epidermidis, and B. subtilis) and Gram-negative bacteria (K. pneumoniae and P. aeruginosa). In comparison to the positive control of 25 µg·mL⁻¹ ciprofloxacin, the synthesized AgNPs demonstrated robust antibacterial efficacy. The MIC values for T. polium leaf extract ranged from 31.25 ± 0.00 and 62.5 ± 0.00 μg·mL⁻¹, while the MIC values for synthesized AgNPs ranged from 5.85 ± 2.76 to 31.25 ± 0.00 μg·mL⁻¹ (Tables 3 and 4). In agreement with these findings, a previous study revealed that the 4-hydroxybenzoic acid and catechol fractions of the T. polium ethyl acetate and ethanolic extracts had significant antibacterial activities against S. aureus, S. typhi, P. aeruginosa, K. pneumoniae, and E. coli with ZOI ranged from 19 to 23 mm [56]. In the study conducted by Alreshidi et al. [6], the methanolic extract of T. polium had high zoon ranged from 11 to 19.33 mm for Acinetobacter baumannii, Proteus mirabilis, P. aeruginosa, Streptococcus pyogens, and Enterobacter cloacae and lowered effects against E. coli, K. pneumoniae, Staphylococcus sciuri, P. aeruginosa, S. aureus, and Sphingomonas paucimobilis with ZOIs ranged from 6 to 7 mm. In multiple studies of the essential oil extracted from T. polium, the GC–MS analysis revealed that the phytochemical composition of 3-carene, γ-muurolene, α-pinene, α-phellandrene, carvacrol, and caryophyllene increased its antibacterial activity against S. aureus and A. baumannii [57,58,59]. Another study showed that the methanolic extracts of T. polium aerial parts and root had more antibacterial activities against Gram-positive bacteria (B. cereus and S. aureus) than Gram-negative bacteria (Shigella flexneri and E. coli) with an MBC value ranged from 150 to 600 µg·mL⁻¹ in the extracts from the root parts [60]. A recent study demonstrated that the AgNPs fabricated from T. polium extracts had stronger antibacterial activities than the crude extracts [18]. In a clinical trial, a toothpaste containing T. polium extract significantly reduced the number and growth of Streptococcus mutans in the mouth [61]. Overall, these results support the potential use of T. polium and synthesized AgNPs as effective antibacterial agents.