Characterization and Antibacterial Response of Silver Nanoparticles Biosynthesized Using an Ethanolic Extract of Coccinia indica Leaves

The present study was planned to characterize and analyze the antimicrobial activity of silver nanoparticles (AgNP) biosynthesized using a Coccinia indica leaf (CIL) ethanolic extract. The present study included the preparation of CIL ethanolic extract using the maceration process, which was further used for AgNP biosynthesis by silver nitrate reduction. Biosynthetic AgNPs were characterized using UV–Visible spectrometry, zeta potential analysis, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), and energy-dispersive X-ray (EDX) spectrometry. The biogenic AgNP and CIL extracts were further investigated against different bacterial strains for their antimicrobial activity. The surface plasmon resonance (SPR) signal at 425 nm confirmed AgNP formation. The SEM and TEM data revealed the spherical shape of biogenic AgNPs and size in the range of 8 to 48 nm. The EDX results verified the presence of Ag. The AgNPs displayed a zeta potential of −55.46 mV, suggesting mild AgNP stability. Compared to Gram-positive bacteria, the biogenic AgNPs demonstrated high antibacterial potential against Gram-negative bacteria. Based on the results, the current study concluded that AgNPs based on CIL extract have strong antibacterial potential, and it established that AgNP biosynthesis using CIL ethanol extract is an effective process.


Introduction
Evidence shows that humans possess a 1:1 ratio of bacteria and human cells; minor disturbances of this ratio can result in multiple illnesses and diseases [1]. Widespread use of antibiotics results in multiple drug resistance (MDR) against infections and presents a high mortality risk [2]. To resolve the barriers associated with traditional antibiotic preparation, an extensive body of research on metallic nanoparticles has been documented over the past decade. Widespread application of silver nanoparticles (AgNPs) to improve antimicrobials

Extract Preparation
The CIL extract was prepared as per the standard procedure given in the systematic research literature with slight modification [19]. Briefly, 50 g of plant sample was transferred into 250 mL of 99.98% ethanol (1:5 ratio) in a volumetric flask. The conical flask was covered with aluminum foil and placed in an incubator shaker at 180 rpm at 37 • C for 1 week. Next, Whatman No. 1 filter paper was used to filter the plant extract. The extract was then condensed at low temperature (32-40 • C) by evaporation using a rotary evaporator. The concentrated extract was poured into a glass petri plate and left inside a fume chamber overnight for removal of the excess solvent. The petri plate was then sealed with parafilm, protected with foil, and placed in a refrigerator at 4 • C until further use.

Green Synthesis of AgNP
Then, 1 mM of silver nitrate solution was prepared by correctly dissolving 0.0085 g of silver nitrate in 45 mL of autoclaved distilled H 2 O. A magnetic stirrer was used to stir the mixture for 10 min. To the stirred AgNO 3 solution, 5 mL of CIL extract was accurately applied drop by drop until the color changed from colorless to brownish green. The obtained mixture was incubated overnight at room temperature in completely dark conditions. Once the AgNO 3 solution was reduced (color changed to brown), it was then centrifuged at 10,000 rpm for 15 min to separate the AgNP. A few drops of distilled water were mixed with the resulting AgNP pellet. The pellet was scraped out, poured onto watch glass, and kept in air for complete drying. After complete drying, the dried particles were scraped out using a sterile scalpel blade and stored at room temperature [2].

UV-Visible Analysis of AgNP
The success of AgNP biosynthesis was verified using UV-Visible spectrometry. A small aliquot of AgNP was diluted in deionized water. The surface plasmon resonance (SPR) signal was detected by testing the reaction mixture with the UV-Visible spectrometer (Shimadzu UV-VIS-U2800 (Shimadzu, Kyoto, Kyoto, Japan)) at room temperature with a scanning speed of 300 nm/min. The measurements were made between 400 and 800 nm. The decrease in Ag + ions was determined by the UV-visible absorption spectrum of AgNP. At 430 nm, the AgNP solution exhibited an SPR peak.

Characterization of AgNP
As described in other research studies, pure AgNPs were used for characterization studies [20,21]. AgNP were routinely washed and centrifuged using deionized water prior to characterization with AgNP characterization data in order to prevent interaction of unbound residual biochemical entities of CIL extract. Different analytical techniques including zetasizer analysis, atomic force microscopy (AFM), energy-dispersive X-ray (EDX), X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to characterize AgNPs. The hydrodynamic diameter of AgNPs was analyzed by a Zeta-PAL zeta potential analyzer (Brookhaven Instruments, Holtsville, NY, USA). To determine the morphology of the AgNPs, TEM, and SEM measurements were carried out. The crystal nature of AgNPs was determined based on the analysis of their XRD spectrum recorded on a PANalytical X'Pert PRO MRD PW 3040/60 X-ray diffractometer (Malvern panalytical, Malvern, UK) using CuKα radiation (λ = 1.5406) at 40 kV to 40 mA in 2θ/θ scanning mode. The EDX spectrum was recorded using an FEI Nova NanoSEM 450 EDX unit (FEI, Hillsboro, OR, USA). AFM assisted in the determination of the presence and size distribution of biosynthesized AgNPs. For AFM analysis, dilute samples (0.05 mg/mL in water) of AgNP were spread over zinc substrate. Evaluation of sample topography (with 1 × 1 µm 2 scanned area) was performed at a set point of 10 nm with a scanning rate of 1 µm/s. The images were analyzed using a Bruker Dimension 3100 with Nanoscope 5 software (Bruker, Dynamostraße 19, Mannheim, Germany).

Disk Preparation
The plant extract (20 mg) and AgNPs (20 mg) were diluted separately in 1 mL of sterile 10% DMSO and mixed well. Next, the 50 µL of prepared plant extract and AgNP solution were separately added drop by drop on the top of 6 layered autoclaved Whatman ® 70 mm Microfiber Filter Paper (Sigma-Aldrich, St. Louis, MI, USA), Grade CF/C disc, which was 6 mm in diameter. The disk was left to dry in a laminar hood at room temperature for 2 h. A ciprofloxacin disc and 10% DMSO were used as positive and negative controls, respectively.

Antibacterial Study
Antimicrobial evaluation of AgNP was based on disk diffusion methodology [22]. The strains of bacteria were swabbed over Mueller-Hinton agar (MHA) using sterile cotton swabs. Disks with ethanolic plant extract, AgNPs, 10% DMSO (negative control), and ciprofloxacin antibiotics (positive control) were applied over MHA agar. After 16 h of incubation, the zone of inhibition (ZOI) diameters (in mm) of each plates were measured.

Characterization of Synthesized Nanoparticles
The success of synthesis of AgNPs was based on results of visual inspection and UV-Visible spectrometric analysis. For color shift control, mixtures of AgNO 3 solution and CIL extract were held apart for 60 min at 60 • C. The change in color from yellow to brown after 60 min indicated the formation of AgNPs. The formation of AgNP in brown color solution was further confirmed by UV-Visible analysis, which generated an absorption spectrum comprising curves 1, 2, and 3 ( Figure 1). Curve 1 represented the AgNO 3 , curve 2 represented AgNP, and curve 3 represented CIL extract solution. The presence of an SPR peak at 425 in curve 2 of the UV-Visible spectrum confirmed formation of AgNPs. In the spectrum, curve 3 of the CIL extract did not exhibit a signal near 425 nm.
The AgNP formation was attributed to exposure of AgNO 3 to CIL extract, which reduced Ag + to Ag 0 . The transition in color from yellow to brown as well as the UV-Visible signal at 425 nm were attributed to the property of surface plasmon resonance and stimulated by the possibility of plasmon vibrations [23].
The SEM and TEM derived size distribution histogram data assisted in the determination of the size and shape of AgNPs. The SEM data given in Figure 2A indicated that synthesized AgNP are spherical in shape and poly-dispersed. The average size distribution histogram based on TEM data (given in Figure 2(B1,B2,C), and Table 1) revealed AgNPs to exist in sizes ranging between 1 and 50 nm. The AgNP formation was attributed to exposure of AgNO3 to CIL extract, which reduced Ag + to Ag 0 . The transition in color from yellow to brown as well as the UV-Visible signal at 425 nm were attributed to the property of surface plasmon resonance and stimulated by the possibility of plasmon vibrations [23].
The SEM and TEM derived size distribution histogram data assisted in the determination of the size and shape of AgNPs. The SEM data given in Figure 2(A) indicated that synthesized AgNP are spherical in shape and poly-dispersed. The average size distribution histogram based on TEM data (given in Figure 2B1, B2, C, and Table 1) revealed AgNPs to exist in sizes ranging between 1 and 50 nm. Scanning of AgNPs in tapping mode generated two-dimensional (2D) ( Figure 3A and three-dimensional (3D) ( Figure 3B images. The images confirmed the uniform distribution of AgNPs, as most of the particles sizes were consistent with the SEM and TEM measurements. The EDX analysis by FESEM generated a spectrum, as seen in Figure 4. The spectrum revealed the elemental composition of AgNPs. EDX spectra determined silver (64.05%) as a major constituent element in comparison to chlorine (20.75%) and calcium (15.20%).
Prominent peak at 3 keV confirmed the presence of AgNP elemental silver. The presence of strong signals for silver and other elemental peaks may be attributed to biomolecules bounded to the surface of silver nanoparticles, indicating the reduction in silver ions to elemental silver.
For zeta potential analysis, the AgNP samples (50 µg/mL) were suspended in deionized water and measured in triplicate at 25 • C [24]. Zeta potential assists in determining the stability of AgNP. It is important to note that particles with zeta potential values more positive than +30 mV or more negative than −30 mV are considered to be stable [25]. In the present study the zeta potential of AgNP with −55.46 mV indicated and supported the stability of AgNP biosynthesized using CIL ( Figure 5).
The XRD data specified in Figure 6 exhibited the crystalline nature of AgNP. The spectrum exhibited various diffraction signals at the angle of 2θ ranging from 10 to 90 • . The four distinctive diffraction Braggs signals at 2θ values of 32 • , 46 • , 68 • , and 78 • could be indexed to the 113, 210, 220, and 311 reflection planes, respectively, of face-centered cubic structures of silver. Such a pattern confirmed the crystallinity of AgNPs. In addition to the Bragg peaks representative of AgNPs, some additional peaks were also observed. These peaks were attributed to biomolecules of CIL extract and were responsible for the reduction and stabilization of silver ions into the resultant silver nanoparticles [26,27].
signal at 425 nm were attributed to the property of surface plasmon resonance and stimulated by the possibility of plasmon vibrations [23].
The SEM and TEM derived size distribution histogram data assisted in the determination of the size and shape of AgNPs. The SEM data given in Figure 2(A) indicated that synthesized AgNP are spherical in shape and poly-dispersed. The average size distribution histogram based on TEM data (given in Figure 2B1, B2, C, and Table 1) revealed AgNPs to exist in sizes ranging between 1 and 50 nm.

A B1
Crystals 2020, 10, x FOR PEER REVIEW     The EDX analysis by FESEM generated a spectrum, as seen in Figure 4. The spectrum revealed the elemental composition of AgNPs. EDX spectra determined silver (64.05%) as a major constituent element in comparison to chlorine (20.75%) and calcium (15.20%). Prominent peak at 3 keV confirmed the presence of AgNP elemental silver. The presence of strong signals for silver and other elemental peaks may be attributed to biomolecules bounded to the surface of silver nanoparticles, indicating the reduction in silver ions to elemental silver.
For zeta potential analysis, the AgNP samples (50 µg/mL) were suspended in deionized water and measured in triplicate at 25 °C [24]. Zeta potential assists in determining the stability of AgNP. It is important to note that particles with zeta potential values more positive than +30 mV or more negative than −30 mV are considered to be stable [25]. In the present study the zeta potential of AgNP with −55.46 mV indicated and supported the stability of AgNP biosynthesized using CIL ( Figure 5).   spectrum exhibited various diffraction signals at the angle of 2θ ranging from 10 to 90°. The four distinctive diffraction Braggs signals at 2θ values of 32°, 46°, 68°, and 78° could be indexed to the 113, 210, 220, and 311 reflection planes, respectively, of face-centered cubic structures of silver. Such a pattern confirmed the crystallinity of AgNPs. In addition to the Bragg peaks representative of AgNPs, some additional peaks were also observed. These peaks were attributed to biomolecules of CIL extract and were responsible for the reduction and stabilization of silver ions into the resultant silver nanoparticles [26,27].

Antibacterial Potential
The antimicrobial potential of CIL extract and AgNPs was evaluated against six Gram-positive bacteria (S. haemolyticus, S. epidermidis, B. subtilis, Lactobacillus, S. aureus, and S. pyogenes) and eight Gram-negative bacteria (E. coli, P. mirabilis, S. typhi, E. cloacae, V. cholerae, P. aeruginosa, A. baumannii, and K. pneumoniae). Tables 2 and 3 present the zones of inhibition (ZOI) exhibited by AgNP and CIL extracts against each bacterial strain. The data are presented in the form of mean (± standard error), p < 0.05. Table 3. Antibacterial activity of CIL ethanolic extract and synthesized AgNPs against Gram-negative bacteria.

Antibacterial Potential
The antimicrobial potential of CIL extract and AgNPs was evaluated against six Gram-positive bacteria (S. haemolyticus, S. epidermidis, B. subtilis, Lactobacillus, S. aureus, and S. pyogenes) and eight Gram-negative bacteria (E. coli, P. mirabilis, S. typhi, E. cloacae, V. cholerae, P. aeruginosa, A. baumannii, and K. pneumoniae). Tables 2 and 3 present the zones of inhibition (ZOI) exhibited by AgNP and CIL extracts against each bacterial strain.  The ZOI was calculated by measuring the zone in millimeters (mm). A clear ZOI indicates the inability of bacteria to grow or multiply around the sample-loaded disk (Tables 2 and 3). The CIL extract inhibited growth of all test microorganisms except Lactobacillus, P. mirabilis, and A. baumannii. Lactobacillus is a non-pathogenic microorganism in which it produces lactic acid as a by-product of glucose metabolism. The bacterial species of the Lactobacillus genus (component of normal flora) are found in the gastrointestinal and genital tract of humans and animals [28]. Therefore, it is beneficial for humans that Coccinia indica plant extract did not exhibit any antimicrobial potential against Lactobacillus. The antimicrobial study of CIL extract against Gram-positive bacteria (GPB) revealed that CIL extract exhibited the maximum ZOI against S. epidermidis and the lowest ZOI against S. pyogenes, whereas the antimicrobial study of CIL extract against Gramnegative bacteria (GNB) revealed that CIL extract exhibited maximum ZOI against V. cholerae and a minimum ZOI against S. typhi and K. pneumoniae.
The antimicrobial activity of CIL extract was compared with the positive control (ciprofloxacin) and negative control (10% DMSO). The ZOIs of ciprofloxacin were larger and more clearly seen in comparison to those created by the CIL extract. A. baumannii did not show any ZOI in the ciprofloxacin disk. The resistance mechanism of A. baumannii is attributed to a single mutation that occurs from serine 83 to leucine in the quinolone resistance determining region (QRDRs) in gyrase subunit A (gyrA) [29].
The C. indica capped AgNPs exhibited substantial antimicrobial activity and ZOI against GPB and GNB. The AgNPs, when tested against GPB, exhibited the largest ZOI against B. subtilis, whereas when tested against GNB, they showed the largest ZOI against V. cholerae. The antimicrobial results revealed that the ZOI formed by AgNPs was larger than that formed by CIL extract. Large surface area per volume and easy penetrating characteristics of AgNPs best explain the antimicrobial activity of AgNPs. Therefore, AgNPs can easily diffuse through the cell walls of bacteria and disrupt microbial cell functions. The CIL extract comprises numerous components and has lesser penetration in comparison to AgNPs, which makes CIL extract less effective in damages bacterial cell walls [5]. Overall, in the present investigation, CIL extract and AgNPs displayed significant antimicrobial potential against GNB and GPB. The findings of present study are supported by previous studies that also reported the antimicrobial potential of C. grandis against K. pneumonia, S. aureus, and B. cereus [17].

Conclusions
The current research investigation shows that green synthesis of AgNP using an ethanolic extract of C. indica leaves is an eco-friendly, fast, and cost-effective method. In the present study, the antimicrobial activity of a 20 mg/mL concentration of CIL extract was shown to be sufficient to inhibit the growth of pathogenic bacteria. This study proves that compared to CIL extract, the ZOI of AgNP is larger against both GPB and GNB. Hence, the present study establishes the broad-spectrum antimicrobial potential of CIL extract derived AgNPs against Gram-negative and Gram-positive bacteria and recommends CIL ethanolic extract as an efficient biomaterial for green synthesis of silver nanoparticles.