3.1. Quantification of phenolic compounds in SFE-LS and preliminary phytochemical screening
The quantification of phenolic compounds was done using Folin-ciocalteu calorimetric assay. The SFE-LS showed 99.31 ± 2.57 mg/gm TPC of dry weight of biomass (DWB). As per the preliminary phytochemical screening done using protocol as mentioned in section 2.3.1 to 2.3.6 the contents of phytochemicals are presented in Table 1.
Table 1
Preliminary phytochemical screening of the SFE-LS
S. no. | | Qualitative test | SFE-LS |
1 | | Alkaloids | + |
2 | | Amino acids | − |
3 | | Carbohydrate | + |
8 | | Tannins | − |
9 | | Saponins | + |
10 | | Gums & mucilage | − |
3.2. UV-vis study
The synthesized silver nanoparticles in the colloidal solution were monitored by UV–vis spectrophotometer analysis. Figure 2A depicts the change in colour from yellow to dark brown that confer the reduction of AgNO3 by phenolic compounds in extract. Figure 2B shows that the absorption spectra of silver nanoparticles formed in the reaction media has an absorbance peak at 430 nm. The increased absorbance at various time intervals (0 to 20 h) and the peak at 430 nm correspond to the surface plasmon resonance of silver nanoparticles. It is reported earlier that absorbance at around 430 nm for silver is a characteristic of these noble metal particles [29]. The λmax range found in the UV-Visible spectrum study is comparable to previous reports for AgNPs synthesized from pomegranate leaves [30]. A comparable analysis yielding essentially identical results was previously conducted in recent studies of AgNP production employing extracts from other plants, including Clerodendrum infortunatum, Azospirillum brasilense, and Allium cepa (onion) [31–33].
3.3. FTIR and XRD studies
Figure 3 show the FT-IR spectra of SFELS-AgNPs that confers the presence of functional groups in Ag nanoparticles. FT-IR spectrum showed the major peak positions at 3334 cm− 1, 2165 cm− 1, 2007 cm-1, 1727 cm-1, 1625 cm− 1, 1461 cm-1, 1349 cm− 1, 1041 cm− 1, 1041 cm− 1, 524 cm− 1 and 455 cm− 1. The peak at 3334 cm-1 intimates OH group in phenolic compounds and carbohydrates [34]. The stretching vibrations of the aliphatic hydrocarbon chains (CH band) observed at 2,925 cm-1, either from the lipids' methyl or methylene groups in the SFE extract. The C = O stretch of aromatic alkenes in the phenolic content showed the band at 1625 cm-1. The band at 1,461 cm-1 is of CH2 and CH3 of aliphatic chains. The band at 1349 cm-1 is attributed to C = C. The stretching vibration of C–O–C is responsible for the prominent peak at 1,041 cm-1. The described FT-IR data suggested that the SFE extracts consist of bioactive compounds with functional groups like OH and COOH. These functional groups serve as stabilising agents in addition to being in charge of the AgNO3 reduction [35, 36].
XRD pattern revealed distinct peaks at 2θ values, which can be attributed to 111, 200, 220 and 311 crystalline planes of silver NPs at 38.02°, 44.16°, 64.34° and 77.26° as shown in Fig. 4. These peaks are associated with the face-centred cubic lattice. Bragg’s reflections of face centre cubic (fcc) structure of metallic silver respectively similar to Joint Committee on Powder Diffraction Standards (JCPDS) file no: ICDD-PDF2, Release 2007, PA, USA, 2007, revealing that synthesized nanoparticles are of pure crystalline silver. The crystalline size of the SFELS-AgNPs formed were calculated using Debye–Scherrer equation (Eq. 1) which was around 13.48 nm, were good in agreement with TEM results.
D= (kλ /β cos θ) ………………………. (1)
Where, D is size of particle, k is Scherrer’s constant, λ is the X-ray wavelength, β is full width at half maximum (FWHM)
3.4. FESEM, HRTEM and EDAX analysis
The morphology of SFELS-AgNPs was analyzed through FESEM image (Fig. 5A). The majority of particles were spherical in shape and there were a few oval AgNPs as well. Biosynthesized nanoparticles had been spread thoroughly in the solution. The size of some selected biosynthesized nanoparticles was around 50 nm according to FESEM images. These results strongly confirmed that SFE-LS extract acts as a reducing and capping agent for the synthesis silver nanoparticles. Figure 5B demonstrated energy dispersive spectrum of the SFELS-AgNPs, which has silver as a ingredient element. Silver nanoparticles generally shows the peak at 3 keV, due to its surface plasmon resonance. The fig also showed the presence of C, O due to the reaction with mixed phenolic groups. This is one of the advantages of synthesis of silver nanoparticles using plant extract. Results of EDAX in this research are conservative with the silver nanoparticles synthesized using Rheum palmatum and Colius aromaticus extract [37, 38].
The morphology and size of the synthesized silver nanoparticles were determined by TEM images and they are shown in Fig. 6A and B. The particles formed were spherical in shape. It was determined that the generated nanospheres have a large surface area. The size of the formed nanoparticles ranged from 8 to 20 nm. The average particle size is 14 nm which is share exactly similar results by Saraswathi et al., 2017 [16].
3.5. DLS and zeta potential of SFELS-AgNps
Using the DLS technique, the surface zeta potential of synthesized SFELS-AgNPs in aqueous colloidal solution was measured. The negative zeta potential was determined to be − 25.6 mV in this investigation, with a zeta deviation of 4.63 mV. Figure 7B. The zeta potential values give us the information regarding the surface charge of nanoparticles which appeared to be negative here. The high negative value indicated that synthesized silver nanoparticles did not agglomerate. Additionally, it gives us an idea about the stability of nanoparticles and the zeta potential value obtained for SFELS-AgNPs lays within the stable range, signifying the nanoparticle are stable in aqueous solution [39]. The zeta potential range of ± 30 mV is thought to be the most stable for silver nanoparticles [40]. Figure 7A demonstrates that the average particle size of SFELS-AgNps is around 116 nm with the polydispersity index of 0.244.
3.6. Weight loss and thermal behaviour
Thermogravimetric analysis of SFELS-AgNPs was carried out to examine the influence of temperature on nanomaterial. Figure 8 shows that the nanoparticles lost its first weight at 102.56 ºC. which may be due to the moisture in material. Temperature between 210.96 ºC to 348.18 ºC almost lost 30% of its weight. Nanoparticles almost started degrading after 348.18 ºC, which suggest the threshold of thermal sensitivity of the material.
3.6 Antibacterial activity
The growth inhibition by SFELS-AgNPs against Staphylococcus aureus, Bacillus cereus, Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli was studied using well diffusion method. To describe the expansion of the clean zone, three different concentration 100 µg/mL, 200 µg/mL and 400 µg/mL of silver capped SFE-LS extract and SFE-LS extract itself were used to check the concentration dependent growth inhibition. Results were compared with standard antibacterial drug Chloramphenicol and Gentamycin. SFELS extract showed low clear zone but the SFELS-AgNPs showed significant antibacterial activity (Fig. 9). The zone of inhibition at every sample was counted using a pair of callipers. The results are presented in Table 2.
Table 2
Bactericidal activity (Zone of inhibition in mm) of SFE-LS-NPs against pathogenic bacteria
Sr. No. | Name of samples | Bacterial Zone of inhibition (mm) |
Gram positive | Gram negative |
B. cereus (MTCC1306) | S. aureus (MTCC737) | K. pneumoniae (MTCC109) | P. aeruginosa (MTCC2488) | E. coli |
1 | 100 SFELS-AgNps | 12 | 15 | 20 | 12 | ND |
2 | 200 SFELS-AgNps | 13 | 17 | 22 | 13 | 10 |
3 | 400 SFELS-AgNps | 18 | 16 | 24 | 14 | 12 |
4 | Chloramphenicol/ Gentamycin | 20 | 24 | 22 | 20 | 14 |
5 | 100 SFE-LS | ND | ND | ND | ND | ND |
6 | 200 SFE-LS | ND | ND | ND | ND | ND |
7 | 400 SFE-LS | ND | ND | ND | ND | ND |
8 | Water | ND | ND | ND | ND | ND |
ND: Not determined |
The bacterial cell membrane damages due to electrostatic interference of AgNPs which form pores on the surface, that alters the structure of the bacteria and ultimately leads to cell death. As a result, the zone of inhibition increased with concentration [41]. The AgNPs in prokaryotic bacteria interact with the bacterial cell membrane, bind to the mesosomal cell organelle, impair mesosome function, and enhance the production of reactive oxygen species (ROS). Silver nanoparticles bind with thiol groups in proteins, which inactivates both DNA replication and protein synthesis [42]. Simultaneously, oxygen combines with silver and reacts with the sulfhydryl (-S-H) groups on the bacterial cell wall to eliminate the -H atoms. This prevents the sulphur atoms from forming an R-S-S-R bond and kills the bacterial cells by preventing respiration [43]. The silver nanoparticles are responsible for the disruption of membrane integrity in bacteria and even limits the growth of bacteria by mitigating oxygen, sulphur and nitrogen in essential biological molecules. Despite numerous papers describing the antibacterial action of AgNPs produced by plant materials, the plant extract often exhibited little to no inhibition while the synthesized SFELS-AgNPs displayed minimal to moderate and good to exceptional activity [44]. The results of this study intimated that SFELS-AgNPs are more effective on K. pneumoniae compared to other bacteria.
3.7 Evaluation of MIC and MBC
The effectiveness of SFELS-AgNPs was further confirmed by determining the MIC and MBC against the K. pneumonia based on the agar well diffusion assay. The SFELS-AgNPs showed a MIC of 64 µg/ml whereas the MBC was determined as 128 µg/ml. The experiment was repeated three times and each concentration was done in triplicates. Our results showed that SFELS-AgNPs are more successful in suppressing the growth of bacteria, confirming the theory that the antibacterial activity is strongly influenced by the modification of the nanoparticles-bacteria interface [45]. Several studies reported that the major reason of AgNPs stop the growth of microorganisms by interfering with microbial DNA replication, contact death, and the generation of reactive oxygen species [46, 47].
3.8 Effect of SFELS-AgNPS on K. pneumonia biofilm formation
The MIC (64 µg/ml) was used to evaluate the effect of SFELS-AgNPS on bacterial cells. The biofilm formation was assessed in control (Untreated) and treated strains of K. pneumonia as shown in Fig. 10, that revealed cells in the untreated biofilm are congregated and intact. After the treatment, biofilm breaks and cells dispersed and the breakages of cells (red arrow) are also visible. Numerous studies showed the herbal nano formulations has potential to reduce biofilm [48]. The suppression of biofilm formation may be the result of SFELS-AgNPs penetrating the bacterial cell wall, suppressing the synthesis of exo-polysaccharide layers, and interfering with cell adhesion and communication [49]. Mousavi et al., recently studied biofilm reduction in K. pneumonia strains using biologically synthesized silver nanoparticles their results are comparable with this study as they have obtained significant reduction at 128 µg/mL of nanoparticles [50]. Additionally, prior research indicated that these NPs might be effective antibacterial agents against a variety of bacterial infections, such as K. pneumoniae [51].
Although several processes have been hypothesized, the precise mechanism by which AgNPs exert their antibacterial effect remains unknown. The capacity of these NPs to attach the bacterial cell wall and their subsequent entry into the cell, which causes structural alterations in the cellular membrane and causes cell lysis and content leaking, is one of the hypothesized mechanisms [52]. Biofilm-forming microorganisms can cause a wide range of illnesses. In fact, a report from the National Institutes of Health and the Centers for Disease Control states that 65–80% of infections are the cause of these microbes [53]. Therefore, use of biofilm inhibitors is one of the effective ways to reduce the diseases caused by these microorganisms. Numerous studies have reported the effect of AgNPs as biofilm inhibitors against multi drug resistant K. pneumoniae [51]. We discussed how AgNPs may be used to target K pneumoniae biofilm development in the current investigation. The current findings demonstrated that at 64 µg/ml concentration of SFELS-AgNPs, Klebsiella pneumoniae strain was failed to generate biofilms.
3.9. SP-SDS Procedure
Bacterial dilution spotting (10-fold) assay was performed to quantify the growth and sensitivity of K. pneumonia upon treatment with SFELS-AgNPs using MIC. A fixed SFELS-AgNPs concentration (64 µg/ml) was used with varying treatment times (0, 2, 4, 6 & 12 hrs). The sensitivity of K. pneumonia could be correlated with the percentage of cells that survive and form a colony. As evident in the result, with increasing incubation time, the viability decreases at 10 − 4, 10 − 5, & 10 − 6 dilution after 2 hrs treatment; 10 − 3, 10 − 4, 10 − 5, & 10 − 6 dilution after 4 hrs treatment and 10 − 2, 10 − 3, 10 − 4, 10 − 5, & 10 − 6 dilution after 6 hrs treatment and hence there is no or less bacterial colony visible at the spot (Fig. 11). At 12 hours of treatment, complete growth inhibition can be seen at all the dilutions. These results suggests that synthesized nanoparticles are capable of complete inhibition of the k. pneumoniae on 12 hrs of treatment.