Antibacterial and photocatalytic degradation efficacy of silver nanoparticles biosynthesized using Cordia dichotoma leaf extract

The chemicals (AgNO₃ and kanamycin) were purchased from Central Drug House, India. The leaf samples of Cordia dichotoma were collected from Central University of Rajasthan campus, Ajmer, India. For the preparation of aqueous extract double distilled water was used.

Healthy leaves of Cordia dichotoma were procured from the campus and were surface sterilized using double distilled water. The clean leaves were then shade dried for a period of 10 days. The aqueous extract of leaves was prepared by boiling 10 g of ground samples in 100 ml of distilled water at 60 °C for 20 min. The extract was further filtered through Whatman filter paper No. 1 and stored at 4 °C.

AgNPs were synthesized by dropwise addition of the aqueous plant extract to the silver nitrate solution of known concentration in an Erlenmeyer flask under stirring followed by centrifugation at 10 000 rpm (Hanil Combi 514R table top refrigerated centrifuge) for 10 min to obtain pellet of AgNPs. The obtained nanoparticles were subjected to washing (thrice) with double distilled water and analysed on UV-vis spectrophotometer (Halo DB-20 Dynamica double beam spectrophotometer).

2.4.1. Time

2.4.2. Ratio of plant extracts and silver nitrate solution

2.4.3. Concentration of silver nitrate solution

2.4.4. Temperature

Initially, the synthesis of AgNPs was monitored by using UV-vis spectrophotometer within the wavelength range of 300–700 nm. Further, Fourier transform infrared spectroscopy (FTIR) analysis was performed with the obtained AgNPs dried under vacuum. The samples were ground with KBr pellets before FTIR analysis. Dynamic light scattering (DLS) was employed to determine the hydrodynamic diameter and polydispersity index using Zetasizer Nano ZS (Malvern Instruments, UK) with 5 mW HeNe laser followed by detection of the scattered light at 173° angle. All the analysis was carried out in an automatic mode and the size of particles were obtained as average value of 13 runs. Morphology and size of the nanoparticles was determined by SEM and TEM. X-ray diffraction studies were done in order to determine the crystalline nature of the biosynthesized nanoparticles.

The antibacterial efficacy of AgNPs was tested against E. coli and P. aeruginosa by performing microbial disc-diffusion assay. Overnight grown bacterial culture was used to streak the agar plate with a density of 10⁵ CFU ml⁻¹. Discs were impregnated with different concentration of AgNPs suspension (5, 10, 20, 50, 100 and 200 μg ml⁻¹) and placed on the plates with positive (Kanamycin, 10 μg/disc) and negative control (distilled water). The microbial culture plates were incubated at 37 °C for 18 to 24 h. Finally, the zone of inhibition was measured and noted as mean ± SD of the duplicate experiment.

The photocatalytic activity of the AgNPs was evaluated by employing methylene blue (10 mg l⁻¹) and Congo red (100 mg l⁻¹) aqueous solution. Thereafter, the experiments (photocatalytic reactions) were conducted outdoor under the sunlight as main energy source. The experiment was set up by preparing a suspension of AgNPs and the respective dye solution. The mixture was kept under stirring for 30 min in dark to bring the AgNPs to constant equilibrium in the mixture. Later, the mixture was kept under sunlight for 5–6 h. The suspension mixture was then measured at regular intervals after centrifugation to ensure photodetoxification of the dye.

All the experiments were done in duplicates, with three separate experiments to demonstrate reproducibility. All the data were presented as mean ± standard deviation (± SD) of all the experiments. Statistical analysis was performed using a Student's t-test. The differences were considered significant for p < 0.05 and p < 0.01 indicative of a very significant difference.

Generally, the reduction and stabilization of the AgNPs are aided by the phytochemicals and phenolics present in the plant extracts. The leaves of Cordia dichotoma is known to possess alkaloids, saponins, flavonoids, terpenes and sterols [27] that could probably aid in the synthesis of AgNPs. However, with 1 mM AgNO₃ solution no colour change was observed. Therefore, the experiment was carried out by taking 2 mM solution and the extract was added dropwise to the solution. The AgNPs formation was indicated by the gradual colour change of the solution from light to dark brown (figure 1), and the characteristic surface plasmon resonance (SPR) peak around 430 nm further confirmed the presence of AgNPs in the suspension.

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Synthesis of small sized monodispersed nanoparticles is generally dictated by the optimum conditions such as time, temperature, concentration of AgNO₃ solution and plant extract. Thus, in order to determine the optimum conditions the aforementioned parameters were optimized. Time plays a major factor in the synthesis of nanoparticles. The synthesis of AgNPs was observed after 40 min. The solution turned from light yellow to brown in colour indicating reduction of silver ions. It was also observed that the synthesis of AgNPs increased with increase in time (figure 2(a)). The reaction was performed till 100 min and the AgNPs showed characteristic peak around 430 nm.

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Different concentrations of AgNO₃ solution were also optimized for synthesis of AgNPs. Synthesis of AgNPs started at a concentration of 1.5 mM and showed maximum absorbance at highest (2 mM) concentration. The reaction was performed for 100 min and a characteristic SPR band was observed around 430 nm, indicating efficient formation of AgNPs (figure 2(b)). Similarly, different concentrations of extract were optimized with 2 mM silver nitrate solution. From the graph, it is clear that the yield of AgNPs increased when the concentration of the leaf extract was increased.

The optimum volume of AgNO₃ solution and extract concentration for AgNPs synthesis was taken to be 10:1, as above this concentration the SPR band exhibited red shift (figure 2(c)). Further, temperature of the reaction was also optimized to obtain maximum production of AgNPs. From figure 2(d) it is clear that production of AgNPs was highest at 60 °C and 80 °C, whereas minimal level of AgNPs formation was observed at 25 °C. Thereafter, further reactions were performed with volume ratio of 10:1 for AgNO₃ solution (2 mM) and leaf extract while keeping the reaction temperature as 60 °C for 60/100 min.

UV-vis spectroscopy is widely used for characterizing noble metal nanoparticles exhibiting SPR in the visible range. The SPR property is attributed to the collective oscillation of electrons on the surface of metal nanoparticles excited by external energy source. Thus, the characteristic plasmon peak gives an account of physical nature of the AgNPs. The SPR band mainly depends on the particle size and dielectric medium [28–31]. Generally, a typical sharp band denotes narrow distribution of the AgNPs [31]. The position and number of peaks also varies with the type of nanoparticles, its shape and size. Besides, broadening of the optical spectra denotes broad size distribution and aggregation of nanoparticles, whilst a prominent narrow band indicates monodispersed distribution of the particles [32]. A single SPR band is expected to appear in case of spherical nanoparticles and two or more SPR bands with anisotropic particles [33].

FTIR was performed in order to determine the probable role of biomolecules of leaf extract involved in the reduction of silver ions and capping for AgNPs. The favourable properties could be ascribed to its phytochemical content such as alkaloids, flavonoids, saponins, terpenes and sterols. Leaves are also known to possess quercitin and quercitrin [27].

The FTIR spectra of C. dichotoma leaf extract and AgNPs are shown in figures 3(a) and (b). The FTIR band at 3437 cm⁻¹ indicates OH stretch, depicting the presence of alcohols and phenols. Similarly, bands procured at 2925 cm⁻¹ and 2854 cm⁻¹ also denotes OH stretch of carboxylic acids. The band at 1637 cm⁻¹ and 1459 cm⁻¹ correspond to N-H bend and C-H bend, respectively. From the spectra obtained, it is clearly evident that polyphenols such as flavonoids, tannins, phenolic acids and certain compounds possessing amine groups are responsible for the formation of AgNPs.

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DLS technique was used to determine the hydrodynamic size distribution of the particles. In this process, the size is measured by illuminating the particles in Brownian motion by laser beam. The scattered light from the particles is then analysed by the auto-correlator. The mean particle hydrodynamic size of the AgNPs was found to be 90.42 nm with polydispersity index (PDI) 0.3 (figure 4(a)).

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On the basis of obtained peaks, the crystalline nature of the AgNPs is confirmed. The diffraction peaks 38°, 44°, 64°, 77° and 82° corresponds to (111), (200), (220), (311) and (222) planes of fcc crystal structure that ultimately determines the fcc structure of the AgNPs (figure 4(B)).

SEM was performed in order to determine the size and morphology of the AgNPs. The image showed small sized spherical AgNPs (∼20 nm) uniformly distributed throughout the sample (figure 4(c)). Further, the AgNPs characterized on TEM showed average particle size of ∼10 nm (figure 4(d)).

The zone of inhibition obtained against E. coli and P. aeruginosa clearly depicts the potent nature of AgNPs synthesized. A considerable difference in the diameter of zone of inhibition was observed with different concentration of AgNPs as displayed in the graph (figure 5). In case of both pathogens it was observed that the AgNPs exhibited its potential activity from a concentration of 10 μg ml⁻¹ up to a concentration of 200 μg ml⁻¹. Several mechanisms of action of AgNPs have been proposed [34–36]. It is noted that AgNPs of 10 nm size or below display activity by itself owing to its smaller particle size and proper cell interaction. Whereas, toxicity caused by 20–80 nm AgNPS is attributed to the release of silver ions inside the cell. Besides, AgNPs react with the thiol groups of proteins and hinders in DNA replication leading to bacterial inactivation [34–36].

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Photocatalytic activity of the AgNPs was determined using detoxification of MB and CR under sunlight for a particular period of time. Initially, the catalytic degradation of the dyes in the presence of AgNPs was observed visually by the change in colour. The intensity of the colour gradually decreased with time from dark blue/orange to light blue/orange marking an effective catalytic degradation activity of AgNPs in the presence of sunlight. Thereafter, the results were confirmed by obtaining absorption band of the dye solution.

MB, an aromatic cationic dye, is widely deployed in textile industries for various purposes. Exposure to the contaminated wastewaters might lead to eye irritation, gastrointestinal tract and skin irritation [37]. Generally, the maximum absorption band of MB aqueous solution is observed at 665 nm owing to the n-π* transition of the MB [38, 39]. The photocatalytic degradation of the MB solution could be determined by the decreasing intensity of the absorption band with respect to time while exposed to sunlight. Thus, SPR property of the AgNPs could be responsible for the decrease in the peak intensity. A similar type of work was performed, where the maximum degradation of MB was attained after 72 h [40]. Whereas in present case, the MB solution was exposed for a period of 6 h that showed significant decrease in the peak intensity (figure 6(a)).

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CR, a secondary diazo anionic dye, is most often used dye in industries. A carcinogenic metabolite, benzidine of CR is known to cause bladder cancer among humans. It is also studied that the effluents with CR are highly coloured and possess high chemical oxygen demand along with high amount of dissolved solids [41]. The photocatalytic degradation of the dye was monitored spectrophotometrically. The characteristic peak of CR is obtained around 500 nm. The degradation of CR dye was confirmed by the gradual decrease of peak intensity at concentration of 0.5 mg ml⁻¹ AgNPs. The complete degradation of dye was observed within 20 min from bright orange colour to light yellow colour (figure 6(b)). The probable mechanism of degradation could be attributed to the SPR effect where the excited surface electrons might interact with the dissolved oxygen molecules and ultimately produce hydroxyl radicals while allowing Ag⁺ ions to interact with the anionic dye [42, 43]. Hence, it is evident that AgNPs synthesized from Cordia dichotoma leaf extract is highly potential photocatalytic agent for dye degradation in the presence of sunlight.