Augmented anticancer effect and antibacterial activity of silver nanoparticles synthesized by using Taxus wallichiana leaf extract

Preparation of leaf extract

Fresh leaves of Taxus wallichiana were collected, and washed with distilled water, dried in shade and grinded to fine powder. To prepare leaf extract of Taxus wallichiana, 20 g of leaf powder was taken and added into 200 mL of deionized distilled water. By using magnetic stirrer, the mixture was boiled at 60 °C for 30 min. The extract was then filtered for nanoparticles synthesis (Ahmed et al., 2016).

Synthesis of silver nanoparticles

Stock solution of 10 mM of silver oxide and 5 mM of sodium borohydride were initially prepared. For making nanoparticles, 25 mL of plant extract was taken in the flask to which 25 mL of silver oxide along with sodium borohydride (25 mL) was added. The change in color was observed (Fig. 1), after which the flask was placed on hot plate with continuous stirring for 30 min with the addition of ammonium hydroxide. After 30 min flask was placed at room temperature for 24 h (Rashid et al., 2019). To separate Ag NPs from liquid solution, centrifugation was done at 12,000 rpm for 10 min at 25 °C. The stable Ag NPs were washed three times with the deionized distilled water to enhance Ag NPs refinement. Ag NPs were then dried and stored carefully in a bottle for future analysis (Shah et al., 2019; Shehzad et al., 2018).

Characterization

Anti-bacterial activity

To assess the antibacterial activity of synthesized Ag NPs, aqueous extract of Taxus wallichiana and Ag₂O NPs were used against Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhi (ATCC 6539) and Staphylococcus aureus (KX262679) bacterial strains (Rashid et al., 2019). To evaluate the antibacterial activity of the plant extract, Ag₂O and T. wallichiana synthesized Ag NPs, Agar well diffusion method was used. For bacterial growth, nutrient broth medium (Oxoid, Hampshire, England) was prepared and autoclaved at 121 °C for 15 min. For the preparation of inoculum, 24-h bacteria culture was grown in nutrient broth and 0.5 OD was maintained using spectrophotometer of 600 nm wavelength (BMS UV-1602) (Rashid et al., 2019; Shah et al., 2019).

Nutrient agar (Oxiod, Hampshire, England), plating medium was prepared. Nutrient agar was autoclaved at 121 °C for 15 min and poured in petri plates (Shahzadi et al., 2022). The selected bacterial cultures were inoculated individually on petri plates. For the preparation of well, a 6-mm sterile metallic borer was used. Three different concentrations of Ag NPs were used including 30 (μg/well), 60 (μg/well) and 90 (μg/well) with Ag₂O NPs concentaron of 30 ug/well. Ampicillin 30 (ug/well; Sigma Aldrich, St. Louis, MO, USA) was used as a positive control and water was used as a negative control. The petri plates were kept in incubator for 24 h at 37 °C. The diameter of the clear zones was measured around each well as a zone of inhibition (mm). All tests were performed in triplicate (Shahzadi et al., 2022).

Cell viability assay

Statistical analyses

The data was analyzed by R software (R Core Team, 2022) for antimicrobial and anticancer activity. Triplicate measurements were averaged with standard error (±SE). One-way and Two-way analysis of variance (ANOVA) was performed at p < 0.05 followed by least significant difference (LSD) test for antimicrobial and anticancer activity respectively.

UV-visible spectroscopy

The optical properties of Ag₂O NPs and Ag NPs were determined at room temperature by UV-Visible Spectrophotometer Lambda 25 model (Perkin Elmer, Waltham, MA, USA) between 300 to 800 nm. The UV-vis spectra of Ag NPs and Ag₂O NPs are represented in Fig. 2. The UV absorption peak for Ag₂ONPs appeared at 450 nm which shifted to the lower wavelength after change in a color from yellow to dark brown that confirmed the reduction of Ag₂O NPs to Ag NPs. The characteristic surface plasmon resonance (SPR) peak for Ag NPs appeared at 410 nm. This result is in agreement with the findings reported by Okafor et al. (2013) while studying the plant mediated synthesis of silver nanoparticles and the blue shift further confirmed the successful conversion of Ag₂O to Ag nanoparticles. The presence of bioactive compounds in Taxus wallichiana plant extract were found effective to reduce the silver to it to nano-size. The optical band gap of Ag₂O NPs was calculated by using Tauc’s relation (Sirohi & Sharma, 1999). The band gap of Ag₂O NPs was found 3.87 eV. The band gap energy value of Ag NPs was found more than previously value reported in literature which may be due to quantum confinement (Fig. 3; Das et al., 2016).

XRD

The diffractogram of Ag NPs and Ag₂O NPs is represented in Fig. 4. The XRD pattern of synthesized Ag NPs by the reduction of silver ions with aqueous extracts of Taxus wallichiana leaves shows the distinctive peaks in the spectrum with 2θ values ranging from 20°–80°. Indexing of the peaks was carried out by comparing with the standard pattern of cubic Ag (ICSD: 003-0291). The diffraction pattern of Taxus wallichiana leaf extract-based Ag NPs displayed four strong peaks at 2θ values 38.06°, 44.25°, 64.51° and 77.44° which can be indexed to (111), (200), (220), and (311) planes (ICSD: 001-1041). This shows that silver is the main component in Taxus wallichiana based nanoparticles. The crystal structure of synthesized silver nanoparticles was face centered cubic. The size of the AgNPs was found 29 nm as estimated from full width at half maximum (FWHM) of all the four peaks by using Scherer’s formula. The average crystallite size of Ag₂O NPs calculated from XRD data was 26 nm.

SEM/EDX

Figure 5 represents the microscopic structure of synthesized silver nanoparticles and silver oxide nanoparticles. The micrograph displayed that silver oxide particles are nearly spherical shaped, and their size varied from 200–1,000 nm (Fig. 5A). On the other hand, the size of silver particles was found less than 1,000 nm (Fig. 5B). However, due to aggregation, it is difficult to measure the exact size of particle (Sasidharan et al., 2020). Elements composition present in the sample was shown with the help of EDX analysis. The signals thus obtained confirm C, Cu, O, Ca, and Si presence in silver nanoparticles. EDX analysis of silver oxide nanoparticle indicated the presence of silver with a weight percentage of 83.4% (Fig. 5C) while in case of Ag NPs it was 48.9% by weight (Fig. 5D). This indicates the presence of more silver in Ag₂O NPs than Ag NPs which is in accordance with their molecular formula. In both samples, EDX showed strong peaks for silver which confirmed their presence as a major constituent of nanoparticles. The presence of other elements was due to macronutrients such as calcium present in the plant extract. The existence of other elements in the smaller quantities was due to their incorporation during the synthesis process.

FTIR spectroscopy

The presence of functional groups in the Ag NPs as extracted from Taxus wallichiana and Ag₂O NPs were identified and shown in Fig. 6. The FTIR of Ag₂O NPs showed diverse stretching bands that appeared at 442 cm⁻¹, 1,533 cm⁻¹, 2,340 cm⁻¹ and 3,729 cm⁻¹. While, Ag NPs showed peaks at 486 cm⁻¹, 1,309 cm⁻¹, 2,340 cm⁻¹ and 3,714 cm⁻¹ and matched well with the published literature (Rashmi et al., 2020). Presence of aromatic amino group absorption band at 1,309–1,310 cm⁻¹ signifies C-N stretching (Mandal et al., 2021). Whereas a peak at 1,533 cm⁻¹ corresponds to C=C stretching of alkene and aromatic ring (Afreen et al., 2020). A peak appeared at 2,340 cm⁻¹ is assigned to NH₃ structure assigned to amino group stretching (Gopinath et al., 2015). The presence of these functional groups played significant role in the capping of Ag NPs. The characteristic bands obtained in the spectrum of Ag₂O NPs were also found useful in capping and stabilizing the particles (Fig. 6). A broad band in the range 3,200–3,400 cm⁻¹ appeared only in the spectrum of Ag₂O NPs showed that silver in oxide form was more hydrated than bare silver nanoparticles. Peaks in the range of 3,200–3,700 cm⁻¹ were assigned as stretching of N-H group and OH stretching in alcohols and phenolic compounds with strong hydrogen bonds (Helmy et al., 2020). Small intense peaks at 3,714 cm^{− 1} and 3,729 cm^{− 1} denote O-H stretching vibrations. This may denote stretching vibrations of alcohols and phenolic groups from diterpines and lignans in Ag NPs (Ajitha et al., 2016). In plant extract these functional groups are present due to amine group, flavonoids and alcohols that are responsible for the reduction of silver in Ag NPs.

Antibacterial activity of Ag NPs

Pathogenic bacteria are the major cause of infectious diseases that pose a serious threat to human population. Medicinal plants and their various organ parts have been used to counter such pathogenic bacteria from ancient times and in many parts of the world (Sökmen et al., 2017). In this study, Taxus wallichiana Ag NPs were used against bacterial pathogens (Fig. 7). This the first study in which Ag NPs synthesized by using the aqueous leaf extract of Taxus wallichaina were tested against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) Salmonella typhi (S. typhi) and Staphylococcus aureus (S. aureus).

Based on our results, Taxus wallichiana leaf based Ag NPs were found to be significant effective against E. coli and S. typhi (Fig. 7) where these nanoparticles gave maximum antibacterial activity against S. typhi with 19 mm zone of inhibition with 90 ug/well concertation compared to 30ug/well Ampicillin based on statistical analysis (R Core Team, 2022). It was also observed that S. aureus gave a maximum zone of inhibition of 16 mm with 90 ug/well concentration which indicated that Taxus wallichiana leaf extract Ag NPs works equally well for both gram negative and gram-positive bacteria. A different study based on Taxus yunnanensis Ag NPs gave significant antibacterial activity against clinical bacteria strains of S. aureus, B. subtilis, E. coli and S. paratyphi B that was attributed to combined effect of silver and plant-based callus extract (Xia, Ma & Wang, 2016). The cell wall of gram-negative bacteria is composed of lipopolysaccharide in addition to peptidoglycan whereas gram positive bacteria cell wall mainly consists of peptidoglycan. Similar observations for both types of bacteria may be due to the adsorbed bioactive compounds on Ag NPs surfaces and Ag NPs large surface to small size ratio to cause cell wall damage. The key underlying mechanism to induce toxic effects as reported by other researchers is the production of reactive oxygen species (ROS) such as superoxide radical (O₂^−•), hydroxyl radical (OH^−•) and hydrogen peroxide (H₂O₂) once these Ag NPs are inside the cell. Excessive ROS disrupt a number of biologically important life process, such as fatty acids radical generation in lipid bilayer resulting in lipid peroxidation, protein-enzymes inactivation and their interference with DNA molecule that ultimately leads to bacterial cell death (Fig. 8; Park et al., 2009).

Effect of silver nanoparticles on cell viability and morphology

Taxol is a major therapeutic compound extracted from Taxus wallichiana that belongs to family of diterpenes and is used to treat various sort of cancers. Taxus wallichiana is regarded as an endangered tree species by IUCN due to increased demands for Taxol, therefore alternate effective approaches specially based on silver nanoparticles for effective drug delivery can be utilized to compensate for it’s over exploitation (Sharma & Garg, 2015).

Anticancer activity of silver nanoparticles extracted from leaves of Taxus wallichiana was performed against U251 cells, derived from a human malignant GBM. It is one of the most aggressive forms of brain cancers and the current therapeutic effects of the approved drugs are not satisfactory especially in terms of resistance development. The major reason for developing resistance and not having the desired therapeutic effects is the presence of blood brain barrier (BBB). There is growing evidence that nano carrier-based therapeutics is a promising approach for the treatment of brain cancer. Thus, based on this, our study is the first novel therapeutic strategy based on Ag NPs adsorbed anticancer compounds (leaf extract of Taxus wallichiana) against brain cancer was tested in current study using brain cancer cell line U251.

The cytotoxicity or anticancer activity indicate that after 48 h of treatment with different formulations, Ag NPs showed a significantly better cytotoxicity as compared to leaf extract and Ag₂O NPs (Fig. 9A). Significant results were obtained with 2.5 and 5 ug/mL of Ag NPs with 42% and 50% cell viability followed by 10% at 10 and 20 ug/mL Ag NPs concentrations based on two-way ANOVA followed by LSD at 0.05 significance level (R Core Team, 2022). This indicated that cytotoxicity or anticancer activity against U251 cells increased in a dose dependent manner and increasing concentration of Ag NPs resulted in decreased cell viability. The cytotoxic potential were also tested post 72 h of treatment with Ag NPs. Our results showed non-significant difference between treatments at 1.25, 2.5 and 5 ug/mL concentrations of Ag NPs however the cytotoxic potential of the formulation decreased significantly at 10 and 20 ug/mL of Ag NPs as compared to Ag₂O NPs and Taxus wallichiana leaf extract based on statistical analysis (Fig. 9B; R Core Team, 2022).

Overall, our results are indicative of the fact that Taxus wallichiana Ag NPs can be a potential anticancer therapeutic targeted against brain cancers. Similarly, cell morphologies after the treatment of cells with Ag NPs showed clumping together and cells death as a sign of cytotoxicity with increasing Ag NPs concentrations in a time dependent manner at 48 and 72 h of incubation (Fig. 10).

Ag NPs from Taxus brevifolia extract showed cytotoxic effect on human breast cancer cell line MCF-7 with 25 mM that showed 75% mortality rate (Sarli, Kalani & Moradi, 2020). Silver nanoparticles synthesized with Taxus baccata extract showed Caov-4 cells mortality up to 50% after 48 h with 2.5 ug/mL concentration and complete mortality with 10 μg/mL concentrations Our results also indicate 50% mortality with 2.5 μg/mL and maximum mortality with 10 μg/mL after 48 h incubation. Although complete cell mortality was observed after 72 h similar to Taxus baccata but with different concentrations (Kajani et al., 2016). These somewhat similar findings may be due to intrinsic features such as related metabolites associated with Taxus tree species (Sharma & Garg, 2015). Interestingly, a recent report showed that paclitaxel (brand name Taxol found in Taxus wallichiana) loaded Ag₂O NPs decreased tumor growth of GMB xenografts compared to normal control mice (Chen & Wen, 2022). Similarly, the expression of Beclin1, LC3I, LC3II and glutathione peroxidase 4 (GPX4), marker proteins associated with autophagy induced cell ferroptosis increased significantly in U251 cells treated with paclitaxel Ag₂O NPs (Chen & Wen, 2022; Wen et al., 2021). Autophagy results in the degradation of iron coated nanoparticles that release iron ions coated with metabolites such as diterpenes and flavonoids causing synergistic effect, that leads to ROS production and eventually causing ferroptosis of GBM cancer cells. This could also be in our case with Taxus wallichiana coated Ag NPs treated U251 cancer cells which requires further study.