Trillium govanianum Wall. Ex. Royle rhizomes extract-medicated silver nanoparticles and their antimicrobial activity

2.1 Extract preparation and synthesis of nano-particles

Extract preparation and green synthesis of silver nanoparticles and their characterization were carried out as described methods [14].

2.2 Antimicrobial activity

Antimicrobial activity of different microbes was determined by Bakht et al. [17] against C. albicans, Curvularia, R. oryzae, A. niger, A. alternaria, Rhizopus, Paecilomyces, B. subtilis, P. aeruginosa, E. coli, K. pneumoniae, S. aureus, and X. campestris.

3.1 Effect of different reaction mixtures on the stability of prepared AgNPs

Green synthesis of the AgNPs and effective reduction of silver from Ag⁺ to Ag⁰ was confirmed by the change in color from colorless to brown in the combination mixture (Figure 1). The changed color of the solution when measured at different wavelength confirmed the production of silver nanoparticles [15]. The reaction mixture (1:1), in which reduction of Ag⁺ ions just occurred, with moderate surface plasma resonance (SPR), band intensities, and wide peak suggested partial reduction of Ag⁺ ions and formation of higher concentration of AgNPs with SPR at 430 nm. At reaction mixture (1:2), the observed intensity of SPR peak was more with small sharpness in the peak when compared with the reaction mixture of 1:1 with SPR at 430 nm. Furthermore, at reaction mixture (1:9), the SPR band intensity and peak were highest indicating a complete reduction of silver ions with SPR at 407.98 nm. Thus, the maximum yield of reduced size of AgNPs observed at reaction mixture (1:9) suggested optimum reaction conditions under room temperature (Figures 2 and 3). Further increase in the ratio of AgNO₃, there was the appearance of a blue shift which is the confirmation of destabilized synthesis and deformed nanoparticles. It may be assumed that it is the result of the saturation of the biomolecules’ functional groups responsible for the synthesis of the nanoparticles with Au ions and less availability of the compounds [16,18]. The UV-Vis spectrum showed that the maximum absorption peak was recorded at the wavelength of 407.98 nm, which is corresponding to the silver nanoparticles absorption range that is 350–450 nm, showing the absorbance of 1.5 crude extract of Trillium govanianum (Figure 4).

Figure 1

Digital optical images of synthesized silver nanoparticles color change of silver nitrate to silver nanoparticles.

Figure 2

Optimization of silver NPs of pure extracts of T. govanianum.

Figure 3

Evaluation of various bioinspired silver nanoparticles spectra prepared by utilizing 1 mL pure plant extract solution with different ratios of silver salt (0.1 mM) solution.

Figure 4

Bioinspired silver nanoparticles spectra prepared by utilizing 1 mL pure T. govanianum extract solution with 9 mL of silver salt (0.1 mM) solution. The sharpest peak of the silver nanoparticles (AgNPs) is at 407.98 nm.

To optimize the synthesis of the bioinspired ecofriendly AgNPs, they were evaluated for various kinds of stability tests, consisting of NaCl concentrations stress, temperature effect, and pH effect using UV-Vis spectrophotometer. These stresses exert a characteristic effect on the fabrication, stability, shape, size, and morphology of AgNPs. Bioinspired silver nanoparticles of the plant extracts indicated that thermal stress can adversely affect the production and reliability of the optimized nanoparticles (Figure 5).

Figure 5

UV-spectra showing the effect of thermal stress on the deformation and synthesized of AgNPs of T. govanianum.

3.2 Effect of temperature on the stability of prepared AgNPs

Results revealed that AgNPs of plant extracts indicated a maximum thermal stability in the temperature range of room temperature to 50°C and a most intense and sharp peak was observed. Our results are in accordance with earlier findings of Ganesan et al. [19], who revealed that the synthesis of bioinspired AgNPs is optimal in the temperature range of 20°C to 50°C. The proliferation in temperature amplified the bands of the reaction combination which displayed superior production of AgNPs. Additionally, improved SPR bands were unveiled at this temperature with a slender absorption range demonstrating the severe discrepancy of the manufactured AgNPs. Data in the thermal range from 50°C to 60°C further indicated that heating adversely affect the thermal stability of the green synthesized silver nanoparticles as they were destabilized in this range. The sharpness of the peak in this high-temperature range is low as compared to the low-temperature range revealing the distortion and deterioration of the biological nanoparticles due to thermal stress. Further elevation in the temperature (from 60°C to 100°C) resulted in complete distortion and deterioration of biologically synthesized AgNPs as no peak was observed spectrophotometrically [20].

3.3 Effect of pH on stability of prepared AgNPs

The effect of pH on the consistency and formation of the environment-friendly manufacturing of the AgNPs is shown by the UV spectrophotometer profile. To study the pH effect, one normal sodium hydroxide (1 N NaOH) was used as basic stress while one normal hydrochloric acid (1 N HCl) was used as acidic stress. The spectrophotometric bands of the biologically synthesized AgNPs were assessed for the various levels of pH stresses ranging from pH 3 to 9 (Figure 6). The UV data specified that pH plays an important role in the production and stability of AgNPs by directing the figure, dimension, and crystallinity of the AgNPs [20]. Spectrophotometric data further revealed that the produced sustainable particles were highly stable in the neutral to basic pH because the maximum absorption was recorded by silver nanoparticles in the solution whose pH was modified to 7–8. UV data verified that at pH 3, macro size silver nanoparticles were produced showing complete deterioration, while at higher alkaline/basic pH, more established and nano-size silver nanoparticles were formed. Outcomes of our present study were in harmony with the findings of Amit et al. [21]. Normally, the biogenic noble nanoparticles are supposed to be highly resistant in the basic pH range, as exhibited by the spectrophotometer pattern. The data on the other hand also revealed that green synthesized particles were less resistant to low pH (pH 3–6). The results also confirmed that at the lowest pH such as 3–5, there is complete deformation and deterioration of the majority of the produced nanoparticles as very weak and broad peaks were recorded by the UV-Vis spectrometer. Ashok et al. [22] analyzed various experiments and stated that biogenic silver nanoparticles are more resistant to deformation and deterioration in the alkaline pH [22]. It also appeared to regulate the character, dimension, and crystallinity of the AgNPs [23]. Earlier studies showed that pH had a considerable result on the size, shape, and hence on the reliability of the AgNPs [24]. Their results concluded that at lower pH, there is generally clumping of the synthesized nanoparticles that inversely affect the process of the nucleation process, while at high alkaline pH, chances of nucleation are high instead of clumps formation, which ultimately leads to the synthesis of more stable and appropriate size AgNPs. The UV spectroscopic pattern verified the blue stretch which is the indication of synthesis of smaller-sized Ag nanoparticles. At lower and higher pH, accumulation of AgNPs are more likely to happen which in turn prevent the process of nucleation; on the other hand, in the pH range 5–7, there is more chance of nucleation and no chances of clumping of AgNPs which leads to high intensity and sharp band in the UV profile [25].

Figure 6

UV-spectra showing the effect of pH on the deformation of the synthesized AgNPs of T. govanianum.

3.4 Effect of different salt concentrations on the stability of prepared AgNPs

The stability test for the salt stress on the prepared and optimized AgNPs was performed using various concentrations of salt (NaCl). The results of the UV-Vis spectrophotometer indicated that increasing salt concentration also negatively affects the stability as well as the production of the nanoparticles (Figure 7). The data showed that salt concentration has an inverse relation with the stability of the synthesis of silver nanoparticles. Malarkodi et al. [26] also obtained similar results while performing salts stress on the optimized gold nanoparticles. The data further revealed that silver nanoparticles were highly stable at the lowest concentration that is in 1 mM, moderately stable at the 10 mM, weakly stable at 100 mM, and completely unstable at 1 M concentration of NaCl. The UV spectrum also indicated high saline stress as abrupt distortion and deformation of synthesized particles were noticed at a molar concentration as compared to milli molar concentration of salt. According to the UV pattern, 1 M salt stress have a maximum destabilizing effect on the synthesis of the green preparation of silver nanoparticles and cause deterioration of the AgNPs abruptly [27].

Figure 7

UV-Vis spectra showing the effect of NaCl stress on the deterioration and stability of the bioinspired silver NPs of T. govanianum.

3.5 Atomic force microscopic (AFM) analysis

AFM measurement was led to reveal the size, shape, and 3D structure of the biodegradable Ag nanoparticles. AFM is a high-resolution microscopic technique that mainly focuses on the 3D structure of the particles under analysis. The microscopic data generated by AFM measurement validated that various sizes of recyclable AgNPs were synthesized by a green approach. The approximate size of the smallest biogenic gold nanoparticle was found to be 6.5 nm while the size of the biggest AgNPs was calculated to be 65.5 nm. These AFM calculated results are exactly similar to the results of the X-ray diffraction (XRD) pattern analysis, where the size of crystallite was calculated to be 9.9 nm (Figures 8 and 9).

Figure 8

Showing circular and near-circular nanoparticles overlapping each other.

Figure 9

(a) Tapping mode AFM images of SNP synthesized with crude methanol fraction of T. govanianum. The scale bar of 100 nm is labeled in the image. (b) 3 µm × 3 µm AFM image showing a 2D view of silver NPs of T. govanianum. (c) Particle size distribution. The size distribution of synthesized SNP is shown.

3.6 Scanning electron microscope (SEM) analysis

SEM analysis was carried out to establish the size and shape of the green-synthesized AgNPs (Figure 10). The image revealed that biologically prepared silver nanoparticles were distinctive and monodispersed. The scanning micrograph additionally indicated that the prepared particles were almost spherical in morphology. The diameter of the synthesized silver nanoparticles was calculated to be approximately 10 nm with uniform distribution. In the SEM analysis of the fabricated silver nanoparticles, none of them were found to be aggregated and was relatively easy to determine the monodispersed silver nanoparticles and calculate their approximate diameter in the nanometer range. To calculate the approximate size of the monodispersed AgNPs, scanning electron micrograph containing 100 particles was taken and the size was determined. The analysis revealed that these particles were in the range of 10–60 nm with an average size of 10 nm.

Figure 10

SEM image of the AgNPs after bio-reduction by T. govanianum.

3.7 XRD analysis

AgNPs prepared by fabrication with plant extract were face-centered cubic (FCC) silver in nature as determined by XRD analysis (Figure 11). The XRD measurement indicated that in the 2θ range of 20°–80°, there were three sharp peaks at 37.50°(111), 44.13°(200), and 63.91°(220). These bands indicate that the crystallite is face-centered cubic in nature [28]. These results also clearly confirmed the high crystalline nature of AgNPs. The growth direction of the prepared crystallite was determined by the most intense bands at 37.50°, 111 as compared to the rest of the distinct bands. Sherrer’s equation was employed to determine the average size of the obtained AgNPs from the extract. The approximate size of the nanoparticle was calculated based on a half-width full maximum of the most distinct peaks 111, 200, and 220 from the X-ray diffractogram. The approximate size of the crystallite was recorded as 9.99 nm based on Sherrer’s equation [19]. The absence of any additional reflections except the reflections correspondent to the silver crystallite positively exhibited that bioinspired AgNPs were highly pure. While the weak reflections in the X-ray crystallography revealed the fixed nanocrystal in the dry freeze samples of the prepared nanoparticles. While calculating the typical size of the prepared NPs, the reflection of the most distinct peaks was utilized that are supposed to be 111, 200, and 220. The size of the nano-crystallite was revealed to be approximately 11 nm which was in close range to that of AFM size.

Figure 11

XRD patterns of silver NPs from T. govanianum showing the intense reflections with 2θ valves.

3.8 FT-IR analysis

Comparative analysis of the IR peaks of the biogenic nanoparticles and pure crude extract identified that absorption stretch at 1455.32 cm⁻¹ in nanoparticles IR profile vanished completely which validate that the aromatic amines having –C–N functional are responsible for the stabilization and fabrication of nanoparticles (Figures 12 and 13). FTIR profile revealed that silver nanoparticles were synthesized by aromatic amines. Similarly on careful comparative analysis of the combined FT-IR profile of the plants’ pure methanolic extract and prepared green nanoparticles additionally discovered a shift of approximately ±1 to 100 wavenumbers in the IR reflections. Peaks shifts were also observed in the broad spectrum range for pure AgNPs and plant extract as mentioned 3341.67–3340.34, 2925.77–2925.17, 1736.80–1740.76, 1634.95–1636.23, 1377.37–1377.98, 1046.01–1026.06 cm⁻¹ [20,21]. The FTIR spectra of silver NPs reveals that peak at 3340 cm⁻¹ might be attributed to the O–H bonds in OH-functional groups. C–H stretching of alkanes resulted in peaks at 2,925 cm⁻¹. Similarly, the peak noticed at 1,736 cm⁻¹ might correspond to carbonyl groups. A distinct peak recorded in the region of 1,636 cm⁻¹ may be related to C═O stretching frequency. An obvious peak at 1,377 cm⁻¹ could be related to the stretching vibrations of alcohols, ethers, esters and carboxylic acids, and amino groups. One peak noticed in the region of 1,037 cm⁻¹ might be attributed to N–C bond stretching of aliphatic amine groups. The results of our study are in agreement with previous studies on silver nanoparticles synthesized using different plant extracts [29].

Figure 12

FTIR spectra of the crude methanol extract of Trillium govanianum.

Figure 13

Comparative FTIR spectra of T. govanianum crude methanol extract and silver NPs

3.9 Antimicrobial potentials of the prepared biogenic AgNPs using T. govanianum extracts

Similarly, the antimicrobial potentials of the biogenic AgNPs prepared by using plant extracts were also determined against various bacterial strains in the current study (Figure 14). Experimental results revealed that in the case of crude methanol AgNPs from the rhizome, the active zone of inhibition was observed by AgNPs at 18 µL disc⁻¹ against X. campestris (53.74%) while the less effective zone of inhibition was confirmed at 6 µL disc⁻¹ against E. coli (19.00%). Results also suggested that X. campestris, P. aeruginosa, and S. aureus displayed a maximum response to AgNPs, B. subtilis and K. pneumoniae displayed reasonable response while E. coli exhibited the least sensitiveness to AgNPs [30,31,32]. It was also concluded in the present work that the zone of inhibition depends on the dose of the bioinspired AgNPs. Bacterial strains display maximum growth retardation and inhibition to a high concentration of nanoparticles. Our results are in complete harmony with the conclusions of already documented literature [33,34]. Similarly, the antifungal activity of AgNPs of T. govanianum and against various fungal strains was carried out. It was identified that in the case of T. govanianum, crude methanol extract prepared AgNPs from the rhizome, maximum zone of inhibition was shown by AgNPs at 18 µL disc⁻¹ against A. alternaria (56.76%) while C. albicans did not exhibit any response at all concentration and were resistant (Figure 15). It also revealed that no zone of inhibition was observed for any of the remaining tested strains at any applied concentration of the disc diffusion assay.

Figure 14

Antibacterial activity with a standard deviation of AgNPs of crude methanolic extract of T. govanianum against different bacterial strains by disc diffusion assay.

Figure 15

Antifungal activity with a standard deviation of AgNPs of pure ethanol extract of T. govanianum against different fungal strains.