3.1 UV-Visible absorption spectroscopy
It is well known that silver nanoparticles exhibit a yellow-greenish color in aqueous solution because the UV-visible spectrum excitation depends on the particle size [41, 42]. The synthesized silver nanoparticles were confirmed by color formation from a yellow-greenish to a brownish color due to the reduction of silver salt by the presence of reducing agents in the aqueous extract of Launaea procumbens. The UV-visible absorption spectrum of the synthesized silver nanoparticles was obtained at 435 nm (Fig. 1). The broadening of the peak refers to polydispersed silver nanoparticles in the solution. Silver nanoparticles exhibit unique and tunable optical properties on account of their surface plasmon resonance depending on the shape and size distribution of the nanoparticles.
3.2 Fourier Transform Infrared Spectroscopy
FTIR spectra were conducted to classify potential functional classes of the leave biomolecules of Launaea procumbens extract which is responsible for the reduction of silver ions to silver nanoparticles. The phytochemicals analysis of Launaea procumbens reveals the presence of alkaloids, flavonoids, steroids, Saponins, and Phenolic compounds [20, 43-44]. The key functional groups that were present in the leaves of Launaea procumbens and synthesized nanoparticles were identified by an analysis of infrared content. The FTIR spectra of the aqueous extract revealed a broad and strong peak at 3314 cm-1, which could be attributed to the OH starching vibrations of secondary metabolites such as alkaloids, flavonoids, steroids, Saponins, and other Phenolic compounds [20, 44]. This functional group has been modified in to synthesized nanoparticles made of silver. Because of the O-H stretching vibrations and H-bonded alcohol and phenol groups, the wide and strong absorption band was observed at 3381 cm-1 (Fig. 2b). The strong absorption peak at 2972 cm-1 got shifted to 2927 cm-1 assigned to the C–H stretching of aromatic compound. A weak band was observed at 1623 cm-1which corresponds to the 1587 cm-1 thatcould be assigned to carbonyl starching in the carboxyl group (N-H band) of linking proteins present in the leaves [45, 46]. This study shows different stretches of bonds shown at different peaks; 1377, 1225, and 1040 cm-1 (Fig. 2a) can be assigned to the O-H bonds and C-O stretching vibrations band observed. The absorption peaks at 1450 cm-1 could be attributed to the presence of C–H bending. The intense weak band at 1118 and 1033 cm-1 (Fig. 2 b) can be assigned to the C-O and C-N stretching vibrations of aliphatic amines [46, 47]. The FTIR study shows that the groups of the leaves of Launaea procumbens extract hydroxyl, carboxyl, and amine are primarily involved in reducing Ag+ to Ag0 nanoparticles.
3.3 Size Distribution Analysis of Silver Nanoparticles by DLS
Green synthesized silver nanoparticles were analyzed for particle size distribution and the results showed that they were distributed in to different sizes in a polydispersible mode. The particle size in a colloidal solution was measured using DLS. The size of the particles was found to be 223 and 73 nm with diameters of 57.07 and 18.14 nm at the first and second peaks, and the peak strength was found to be 88.3% and 11.7%, respectively. The mean average silver nanoparticles were 378.4 nm in size. It was found that the Polydispersity index was 0.645 (Fig. 3). The ratio of particles of different sizes to the total number of particles is measured by the Polydispersity index. The results obtained from the size distribution from DLS analysis show the larger size of the particles, which is due to the aggregation of the silver nanoparticles [48, 49].
3.4 Field Emission Scanning Electron Microscopy (FE-SEM) and Energy Dispersive X-ray Spectroscopy (EDXS)
The synthesized silver nanoparticles were characterized by standard characterization methods such as field emission scanning electron microscopy. To examine the morphology, structure, scale, shape, and distribution of the nanoparticles produced, a field emission scanning electron microscope was used. The FE-SEM is better and more resolute than the traditional SEM [50]. The synthesized nanoparticles appear larger due to aggregation. Most of the nanoparticles were aggregated, polydispersed, and cluster shaped (Fig.4). The energy dispersive spectrum of the synthesized nanoparticles indicates the presence of silver as an ingredient element. Metallic silver nanoparticles generally show a typically strong signal of the peak at 3 KeV, which is typical for absorption of metallic silver nanoparticles [51, 52]. Figure 5 shows the quantitative information of synthesized silver nanoparticles. The presence of elements such as Ag, O, C, Cl, Na, and Ca is shown in Fig. 5.
3.5 High Resolution Transmission Electron Microscopy (HR-TEM) with SAED Analysis
We have carried out a high-resolution transmission electron microscopic (HRTEM) study to obtain information about the internal structure (size and morphology) of the green synthesized silver nanoparticles as shown in Fig. 6. The HR-TEM images were recorded at different magnifications to find the individual particles. The synthesized silver nanoparticles were observed as almost spherical in shape and nearly monodispersed in nature and the average size of the particles was 24.28 to 31.54 nm. The distance between the lattice fringe spacing of silver nanoparticles (AgNPs) was recorded as 0.23 nm (Fig. 6e) and the selected area diffraction pattern of the silver nanoparticles suggested that face-centered cubic (fcc) crystalline silver was presented by the synthesized silver nanoparticles (Fig. 6f) [50, 53-54].
3.6 Phytochemical composition of Launaea procumbens
Phytochemical analysis findings revealed the presence of alkaloids, flavonoids, steroids, proteins, saponins, tannin, phenolic compounds and the absence of terpenoids as shown in Table 2. Significant bioactive components in phytochemicals serve as reducers for reactions. Silver ions and thus extracts have been used as a reduction and stabilization agent in the green synthesis of silver nanoparticles [55, 56].
Table 2 Phytochemicals screening of Launaea procumbens leaves extract.
Phytochemicals
|
Results
|
Alkaloids
|
++
|
Flavonoids
|
++
|
Steroids
|
++
|
Terpenoids
|
-
|
Proteins
|
++
|
Saponins
|
++
|
Tannin
|
++
|
Phenolic Compounds
|
++
|
++ = Presence, + = Trace, - =Absence
3.7 Antibacterial activity of silver nanoparticles
The agar well diffusion method was employed for the determination of the antibacterial activity of silver nanoparticles. Four different suspension cultures of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and pseudomonas aeruginosa were spread on nutrient agar medium by the spread plate technique. The plates were incubated at 37 ͦ C for 24 hours. 0.1 ml of silver nanoparticles were inoculated in to one well and 0.1 ml of distilled water was inoculated in to another well on each plate and water served as a control [45, 55]. Antibacterial tests of synthesized silver nanoparticles and Launaea procumbens leaves extract were investigated using a disc diffusion method carried out against four bacteria. The green synthesized silver nanoparticles showed excellent antibacterial activity against Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa. The Gram-positive bacterium Bacillus subtilis showed a maximum zone of inhibition of 20 mm. Staphylococcus aureus showed a zone of inhibition of 19 mm. The gram-negative bacteria Escherichia coli andPseudomonas aeruginosa bacteria showed a zone of inhibition of 13 mm. The mean inhibitory zone of the four replicates of diameter was measured and tabulated in Table 3.
Borago officinalis leaf extract was discovered to be beneficial in the green synthesis of silver nanoparticles. The synthesized AgNPs showed antimicrobial activity against E. coli bacteria with an inhibition zone of 8 mm and no inhibition zone against other bacteria such as S. Typhi, B. subtilis, and S. aureus, and only C. albicans inhibits the inhibition zone of 6 mm while A. flavus is not inhibited in the absence of activity [31].
Because of the antibacterial activities of the plant extract, as well as the nanoparticles high aspect ratio and penetration potential, an additional effect of related phytochemicals by deposition on silver nanoparticles could produce greater antibacterial effects. On the other hand, one of the fundamental mechanisms underlying silver nanoparticles antibacterial activity is the generation of reactive oxygen species (ROS) [57]. The most important aspects of antimicrobial activity discussed were silver ion release, cell membrane damage, DNA interactions, free radical generation, bacterial resistance, and the difference between resistance to silver ions and resistance to silver nanoparticles [58]. The antibacterial activity of silver nanoparticles against S. aureus and E. coli cells was significant. The growth and reproduction of bacterial cells treated with silver nanoparticles was rapidly inhibited. The growth of silver nanoparticles treated cells was unchanged by pH or temperature changes. Silver nanoparticles were found to actively form bactericidal reactive oxygen species (ROS) [58]. Krishnaraj et al. 2010 [59] produced silver nanoparticles from Acalypha indica leaf extract, and 10 µg/ml was determined to be the minimum inhibitory concentration (MIC) against E. coli and V. Cholera. This was explained by the alteration in membrane permeability, which caused the antibacterial activity. Some reports indicated improved antibacterial efficacy, which was most likely due to the reduced size of the AgNPs. For example, using an average mean size of 16 nm and anti bactericidal property at 45 𝜇g/mL on E. coli. Several potential methods for how AgNPs work as antibacterial agents have been postulated, but the specific mechanism is unknown [60]. The antibacterial activity of synthesized silver nanoparticles compared to that of saffron extract and purchased silver nanoparticles against six pathogenic bacteria revealed that the aqueous extract of saffron and purchased nanoparticles had no significant antibacterial effect, but biosynthetic nanoparticles were able to inhibit the total bacteria studied [61]. Espanti et al. 2016 [62] investigated the antimicrobial properties of synthesized silver nanoparticles by Terminal chebula Retz (Myrobalan) against E. coli and Bacillus subtilis in a study performed, and the results showed that silver nanoparticles have a much stronger antibacterial effect than the plant's aqueous extract. Silver nanoparticles kill bacteria by destroying their membranes.
Table 3 Antibacterial activity of green synthesized silver nanoparticles using Launaea procumbens leaves extract.
|
Organisms
|
Zone of inhibition (mm)
|
Synthesis of AgNPs (100µl)
|
(Control) Deionized H2O
(100µl)
|
1.
|
Bacillus subtilis
|
20
|
NZ
|
2.
|
Pseudomonas aeruginosa
|
19
|
NZ
|
3.
|
Staphylococcus aureus
|
13
|
NZ
|
4.
|
Escherichia coli
|
13
|
NZ
|
(NZ: no zone of inhibition)