Photodynamic cytotoxic and antibacterial evaluation of Tecoma stans and Narcissus tazetta mediated silver nanoparticles

2.4.1 Antibacterial test

The extracts and AgNPs were tested for their antibacterial potential using the well diffusion method (Anburaj et al., 2016). The selected bacterial strains were Staphylococcus aureus ATCC# 6538 (S. aureus), MRSA 10, and MRSA 11. Results were presented as a diameter of zone of inhibition (mm) and MIC (Vacik et al., 1979).

2.4.2 Antioxidant test

DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was conducted to evaluate the antioxidant potential of extracts and AgNPs (Kaviya and Viswanathan, 2011). The sample (20 µl) and DPPH (180 µl) were added to 96 well-plate. The study involved the use of DMSO and ascorbic acid as negative and positive control, respectively. After taking absorbance at 517 nm using microplate reader, percent scavenging was calculated by using below given formula: $$\text{Inhibition}(\%)=(1-A_s/A_c)\times 100$$

Where, A_s and A_c are the absorbances of sample and reagent without sample, respectively. IC₅₀ values were calculated for those samples that showed greater than 50% scavenging potential.

2.4.3 Anticancer test and PDT

MTT (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl) assay was used for the determination of anticancer potential of extracts and AgNPs using rhabdomyosarcoma (RDATCC# CCL-136) cell line (Mativandlela et al., 2006). Dulbecco’s Modified Eagle medium (DMEM; Sterile filtered DMEM (-)L-leucine, (-)L-methionine with 4.5 g/L glucose, 4.0 mM L-glutamine, sodium pyruvate and phenol red) was used for cell culturing. Cell suspension was prepared and 0.2 ml of this cell suspension was added to each well of 96 well plates with density of 1x10⁵ cells/well and stored at 37 °C in 5% CO₂. After incubation of plates, medium was replaced by fresh DMEM along with addition of different samples (50 µl), followed by the re-incubation of plates for 2–3 days at 37 °C in 5% CO₂. From here, the experiment was bifurcated. After incubation, first plate was processed for the removal of medium and washed thrice with PBS. Then, all the wells were treated with MTT solution and the plate was incubated for 200–250 min at 37 °C in 5% carbon dioxide. After incubation, the extracting solution was added and studied using microplate reader at 570 nm after 45 s. Cell Viability (%) was determined using below given formula: $$\text{Cell}\text{Viability}\left(\%\right)=(\text{Mean}\text{OD}/\text{Control}\text{OD})*100$$

The samples which showed more than 50% inhibition were further studied for IC₅₀.

The second plate was utilized for PDT (Fatima et al., 2016). Photo-sense (aluminum chloride phathalocyanine) and light was shined on respective wells, MTT assay was executed and percentage cell viability and IC₅₀ values were calculated.

3.1.1 Antibacterial test

Agar well diffusion approach was used to study antibacterial activity against three strains including S. aureus (ATCC# 6538), MRSA 10 and MRSA 11. Crude extract of T. stans exhibited significantly (p < 0.05) highest activity against S. aureus (ZOI = 24.2 ± 0.9). Crude extract of T. stans showed significant (p < 0.05) higher activity against all bacterial strains with the maximum value of ZOI of 24.2 ± 0.9 mm against S. aureus as compared to the control. The sample of N. tazetta was nearly inactive. AgNPs were prepared with varying concentrations of silver nitrate (1, 5 and 10 Mm) and tested for antibacterial potential. Results showed that AgNPs (5 mM) showed (p < 0.05) higher antibacterial activity than crude extracts. Maximum ZOI of 26.2 ± 0.8 mm was shown by T. stans based AgNPs against S. aureus (Table 1). Minimum inhibitory concentration (MIC) was determined against all three strains. MICs for crude extract was very high, ranging between 512 and 1024 µg/ml, but AgNPs showed low MIC values, especially AgNPs prepared from T. stans leaves (TSLNp) with values of 16 µg/ml, 32 µg/ml and 32 µg/ml against S. aureus, MRSA 10, and MRSA 11, respectively (Table 2).

3.1.2 Antioxidant test

Crude extract of T. stans showed significant (p < 0.05) activity with more than 80% scavenging potential. Maximum scavenging (%) was shown by T. stans leaf extract with a value of 93.5% and IC₅₀ values of 1.19 µg/ml (Table 3). N. tazetta samples exhibited a little antioxidant capability. The scavenging potential of N. tazetta based AgNPs ranged from 40 to 51.5%.

3.1.3 Anticancer test

Extracts of N. tazetta showed good cytotoxicity (IC₅₀ = 16.9 ± 0.6 µg/ml; cell viability (%) = 25.3 ± 1.3), while IC₅₀ value of its AgNPs was 4.79 ± 1.1 µg/ml. The IC₅₀ of T. stans extract based AgNPs was significantly (p > 0.05) lower, i.e. 2.26 ± 0.9 µg/ml (Fig. 1).

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Fig. 1

Result of anticancer assay of T. stans (A), N. tazetta (B) and IC₅₀ values of extracts and nanoparticles (C). (TSB = T. stans branches, TSL = T. stans leaves, NT = N. tazetta, NP’s = Nanoparticles).

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3.1.4 Photodynamic therapy assay

Both plant extracts and NPs were active in cell line assay and were further used to evaluate their photodynamic potential. It was observed that cell viability was significantly (p < 0.05) decreased when these combinations of extract, NPs, drug and photosensitizer were used. Plant extracts and AgNPs were tested at 40 µg/ml. In case of samples of Tecoma plant extracts, AgNPs showed % cell viability of 53–57% and 40.8–48.9% respectively, while % cell viability significantly (p < 0.05) decreased to 22.4 and 24.9%, respectively, where the combination of plant extract, NPs, drug and photosensitizer was used. The same synergistic behavior was observed with N. tazetta extract and NPs (Table 4). These results were compared to controls.

3.2.1 Visual observation and UV–visible spectroscopy

T. stans and N. tazetta extracts were treated with 1 mM, 5 mM, and 10 mM concentrations of AgNO₃ resulting in the formation of AgNPs, as indicated by the change in solution colour from yellow to brown. UV–vis spectra were recorded by scanning the samples between 300 and 600 nm. Nanoparticles prepared by 5 mM concentration show a sharp peak and lies in the range of 410–420 nm that is characteristic for AgNPs (prepared by chemical reduction). Peaks for 1 mM and 10 mM were not as sharp (Fig. 2).

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Fig. 2

UV spectra of T. stans and N. tazetta nanoparticles.

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3.2.2 FTIR spectroscopy

Different peaks were observed in FTIR spectra of AgNPs. Broader peaks were observed at 3295, 3273 and 3220 cm⁻¹ in the spectra of AgNPs of Tecoma branches, leaves and Narcissus respectively (Fig. 3). Peaks at 2935, 2897 and 2930 cm⁻¹ correspond to –C–H stretching vibration in –CH₂ groups. Peaks appear at 1766, 1730, 1633, and 1680 cm⁻¹ represent C=O stretching vibration. The spectra of T. stans leaves showed a broader peak at 3273, 1633 cm⁻¹. Other peaks were observed at 2897 and 2370 cm⁻¹. Many smaller peaks were also observed at 1730, 1398, 1126, 1033, 976, 948 and 856 cm⁻¹. The spectra of branch extract showed a wider peak at 3295 cm⁻¹. Small peaks were also observed at 2935, 2853, and 2366 cm⁻¹. Narrow peaks were observed at 1766, 1597, 1527, 1386, 1265, 1033 and 826 cm⁻¹. Spectra of N. Tazetta showed broader peaks at 3220 cm⁻¹. Smaller peaks were noted at 1680, 1590, 1539, 1400, 1290, and 1050 cm⁻¹. Small narrow peaks were observed at 1290, 1050, 995, 925, and 789 cm⁻¹ (Fig. 4).

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Fig. 3

FTIR spectra of T. stans leaves (TL) and its branches (TB) and nanoparticles.

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Fig. 4

FTIR spectra of N. tazetta extract and nanoparticles.

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3.2.3 Scanning electron micrograph (SEM) analysis

According to SEM results size of TSBNps in 20–100 nm while the size range of 10–50 nm was observed in case of TSLNps. Same behavior was observed by N. tazetta sample-based AgNPs with variation in size 30–150 nm.

4.1.1 Antibacterial test

Three bacterial strains, S. aureus (ATCC# 6538), MRSA 10 and MRSA 11 were used to evaluate antibacterial potential of samples. MRSA 10 and 11 were resistant strains. As compared to the controls, sample of T. stans showed significantly (p < 0.05) high activity against all selected bacterial strains, while the maximum ZOI (24.2 ± 0.9 mm) was noted against S. aureus. Sample of N. tazetta was almost inactive. These results support the previous findings (Masood et al., 2013). AgNPs were synthesized by using silver nitrate (1, 5 and 10 Mm) and tested for antibacterial potential. It was observed that AgNPs (5 mM) showed significantly (p < 0.05) higher antibacterial activity than crude extracts and these AgNPs were selected for further studies. These results support the previous findings as documented by Ravishakar et al., who found that AgNPs showed strong antibacterial activities (Hammad Aziz et al., 2016). MIC values for crude extract were significantly (p < 0.05) high, ranging between 256 and 1024 µg/ml, but AgNPs showed significantly (p < 0.05) low MIC values, especially AgNPs prepared from T. stans leaves (TSLNp). The MIC of AgNPs synthesized from extract was significantly (p < 0.05) decreased due to decreased particle size and large surface area, which led to enhanced cell permeability and damage of bacterial pathogens (Govindappa et al., 2011).

4.1.2 Antioxidant assay (DPPH free radical scavenging activity)

Antioxidants scavenge free radicals and protect the cells against oxidative stress (Fatima et al., 2016). Extract of T. stans showed more than 80% scavenging. Leaf extract showed maximum scavenging (%) with a value of 93.5% and IC₅₀ values of 1.19 µg/ml (Table 4). These finding correlated with previous reports that flowers and fruits of T. stans showed antioxidant potential due to presence of alkaloids and flavone glycosides (Mariselvam et al., 2014). Samples of N. tazetta showed low antioxidant potential. These results support the previous findings about antioxidant potential of this plant. AgNPs of this plant exhibited a moderate scavenging potential (40–51.5%).

4.1.3 Anticancer assay

MTT assay was done to reveal the anticancer potential. In this assay, extracts of N. tazetta showed good anticancer activity (IC₅₀ value of 16.9 ± 0.6 µg/ml). However, in case of AgNPs, samples of T. stans branches showed lower IC₅₀ value of 2.26 ± 0.9 µg/ml. followed by N. tazetta with a value of 47.9 ± 1.1 µg/ml (Fig. 1C). Both, the methanolic extracts and AgNPs of respective parts showed a dose-dependent decrease in cell viability (%). This trend was in accordance with previous studies (Fu et al., 2016).

4.1.4 Photodynamic therapy

Different combinations of plant extracts and their AgNPs were used to assess photodynamic potential of samples. Tecoma plant extracts and AgNPs showed % cell viability of 53–57% and 40.8–48.9% respectively. The percentage cell viability decreased significantly (p < 0.05) to 22.4 and 24.9% respectively when combination of plant extract, NPs, drug and photosensitizer was used. Same synergistic behavior was observed with N. tazetta extract and NPs. Both extracts and AgNPs were capable of dose and exposure time dependent phototoxicity for rhabdomyosarcoma cell line. This trend was in accordance with the previous studies (Fatima et al., 2016). This is first report about PDT effects of extracts and AgNPs of selected plants.

4.2.1 Visual observation and UV–visible spectroscopy

The synthesis of AgNPs from plants is considered as an important method due to its potential activities in various studies (Ahmed et al., 2016). T. stans and N. tazetta extracts were exposed to metal ions (AgNO3; 1 mM, 5 mM and 10 Mm), and the formation of AgNPs was confirmed by color change i.e., from yellow to brown, due to surface plasmon resonance phenomena. It is also reported by various scientists that biosynthesis may be affected by metal concentration and quantity of plant extract in the reaction medium, which can change the shapes and sizes of AgNPs (Mehrotra et al., 2017). According to UV–vis spectra of the samples recorded between 300 and 600 nm. AgNPs prepared by 5 mM concentration show a sharp peak that lies in the range of 410– 420 nm a diagnostic peak indicative of AgNPs (prepared by chemical reduction). Peak intensities give an idea of the size of AgNPs formed (Liu et al., 2006). It should be noted that the UV absorption peak at 440 nm indicates the formation of larger AgNPs (Jain et al., 2009). It is reported previously that AgNPs are formed and stabilized by phenolics, amino acids, carboxylic acids, alkaloids and proteins which are important secondary metabolites (Varadavenkatesan et al., 2021).

4.2.2 Fourier transform infrared spectroscopy (FTIR)

The FTIR spectra indicated the presence of stretching vibration for –OH (alcohol and phenolic), C=O, C–O– (acid), CH, C=C (aromatic system), C–O–(alcohol) and C–N bonds, as well as the functional group which might belong to phenolic and flavonoid compounds present at the surface of the most active NPs. Broader peaks were observed at 3295, 3273 and 3220 cm⁻¹ in spectra of AgNPs of Tecoma branches, leaves and Narcissus respectively. Peaks at 2935, 2897 and 2930 cm⁻¹ correspond to –C–H stretching vibration in –CH₂ groups. Peaks appear between 1630 and 1766 cm⁻¹ represent the C=O stretching vibration. The flavonoids and phenolic compounds present in the crude extracts are powerful reducing agents which play a significant role in the formation of NPs by the reduction of silver ions. It has already been reported that flavonoids and phenolic compounds present in plant extracts can play a dual function of reducing and capping during NP formation. These findings correlate with previous reports (Ali et al., 2016). Size variation of AgNPs was determined by SEM. AgNPs of Tecoma branches showed size of 20–100 nm, while the size of AgNPs from leaf extract was found between 10 and 50 nm. The same behavior was observed by N. tazetta sample-based AgNPs. Shape of AgNPs is known for its considerable change in optical and electronic properties. Many researchers have reported the size variation of the green synthesized AgNPs (Ali et al., 2016).