Silver nanoparticles synthesized using aqueous leaf

Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 391–402

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Silver nanoparticles synthesized using aqueous leaf extract of Ziziphus oenoplia (L.) Mill: Characterization and assessment of antibacterial activity Soumya Soman a, J.G. Ray b,⁎ a b

Laboratory of Ecology and Ecotechnology, School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India -686560 School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India -686560

a r t i c l e

i n f o

Article history: Received 16 September 2015 Available online 31 August 2016 Keywords: Phytosynthesis Leaf extract AgNPs Antibacterial studies Bactericidal activity

a b s t r a c t Biological approach to synthesis of metal nanoparticles using aqueous leaf extract is a highly relevant and recent theme in nanotechnological research. Phytosynthesized AgNPs have better inhibitory and antimicrobial effects compared to aqueous leaf extract and silver nitrate. In the present investigation crystalline silver nanoparticles (AgNPs) with size of 10 nm have been successfully synthesized using aqueous leaf extract (AQLE) of Ziziphus oenoplia (L.) Mill., which act as both reducing as well as capping agent. The particles were characterized using UV Visible spectroscopy, HRTEM-EDAX, XRD, FT-IR and DLS. An evaluation of the anti bacterial activity was carried out using Agar well diffusion method and MIC determination against four bacterial strains, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi; the AgNPs exhibited quite high antibacterial activity. Furthermore, bactericidal studies with TEM at different time intervals after AgNPs treatment showed the presence of AgNPs near cell membrane of bacteria at about 30 min exposure and the bacteriallysis was found completed at 24 h. This gave an insight on the mechanism of bacterial-lysis by direct damage to the cell membrane. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Metallic nanoparticles are known for their unique physico-chemical properties, extremely small size and surface plasmon behavior [1]. Silver nanoparticles (AgNPs) accounts for 55.4% of the total nanomaterial based consumer products such as cosmetics [2,3]. They are also used as anti-microbial agents, biosensor components, supportive in catalysis, optoelectronics and SERS [4–6]. Oligodynamic action [7], broad spectrum killing [8] and low triggering of microbial resistance [9] make them better antimicrobials and components in consumer products like cosmetics, textiles, dietary supplements, food packaging, surgical coatings, medical implants and bacterial filter [10–14]. Biological synthesis has additional advantages in the applications of nanoparticles. Toxicities from chemical and physical means of synthesis are avoided in the biological synthesis. In chemical reduction method Ag+ are reduced to Ag0 using reductants like NaBH4, citrate, ascorbate, polymers and elemental hydrogen [15–17]. Several studies have been employed to synthesize nanoparticles using extracts of different plant parts such as leaf, fruit, latex, petals and cone [18–25]. Plant extracts are good bioreductants, relatively easy to handle, low cost and readily available. Reducing agents in the extracts include secondary metabolites such as phenolic compounds, alkaloids, proteins, flavanoids and

⁎ Corresponding author. E-mail addresses: [email protected] (S. Soman), [email protected] (J.G. Ray).

http://dx.doi.org/10.1016/j.jphotobiol.2016.08.033 1011-1344/© 2016 Elsevier B.V. All rights reserved.

saccharides [23,26]. Since no additional reducing or capping agents are added in the phytosynthesis of nanocrystals, unlike physico-chemical synthesis, the natural process and the nanoparticles produced in the process are environmentally benign. AgNPs either in colloidal or immobilized state is considered as the best choice [10,13,14] for all its applications. Some earlier studies accounts that nanoparticles in 10 nm size range are difficult to obtain [27, 28]. Present work demonstrates a successful synthesis of AgNPs of size 10 nm using the aqueous extract of Ziziphus oenoplia (L.) Mill., which is a well known medicinal plant with cyclopeptide alkaloids [29] in its vegetative parts. Reports on antibacterial, anti diuretic, anti inflammatory and anti cholinergic properties from different parts of the plant are available [30–32]. Therefore, the nanoparticles synthesized using the leaf extracts of the plant are expected to be highly medicinal in nature. Apart from the synthesis of small sized nanoparticles, the present investigation also aimed at characterization of the particles using UV Visible spectroscopy, HRTEM-EDS, XRD, FTIR and DLS. This was to establish the crystalline nature of the particles and to assess the stability of the particles without adding any capping or stabilizing agents. Antimicrobial action based on Zone of Inhibition (ZOI) and Minimum Inhibitory Concentration (MIC) values against four gram negative clinical bacterial strains was an important objective. These values were compared to the raw materials; aqueous leaf extracts (AQLE) and silver nitrate (SN). Bactericidal effect of the particles on E. coli using Transition Electron Microscope (TEM) and Fluorescence Microscopic studies were also carried out.

392

S. Soman, J.G. Ray / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 391–402

The major goal of the research was to develop environmentally benign AgNPs with enhanced antibacterial activity than the raw materials against the selected bacterial strains and to understand the bactericidal effect of particles on bacteria with the help of microscopic analysis.

solution, namely q1 to q3 were prepared in 1:2, 1:9, 1:20 keeping the amount of AQLE at 1 mL and 2 mL, 9 mL and 20 mL of SN respectively.

2. Materials & methods

To understand the effect of reaction time on synthesis of AgNPs in sample s1, UV–visible spectra were recorded after 1 h, 24 h and 48 h of incubation for preparation of AgNPs. The solution with AgNPs obtained was purified by repeated centrifugation at 15,000 rpm for 20 min followed by re-dispersion of the pellet in de-ionized water by sonication. Later the pellet was air-dried and stored in desiccators.

2.1. Materials In the present study the silver nitrate (SN) of AR grade was procured from SRL. All the chemicals required for the phytochemical studies were purchased from SRL and Hi Media. The stains for fluorescence microscopic studies were obtained from Hi Media. The ingredients of Nutrient agar and Muller Hinton Agar (complete media) were sourced from Hi Media for the bacterial studies. The components of Nutrient agar were (g L− 1); Beef extract 1.0; Peptone 5.0; Yeast extract 2.0; NaCl 5.0; Agar 2.0 and with pH 7. Fresh leaves of Ziziphus oenoplia (L.) Mill. were collected from the MG University campus and were processed in the lab for further studies. 2.2. Phytochemical activity Preliminary phytochemical screening of both aqueous and methanolic leaf extracts Ziziphus oenoplia (L.) Mill. was carried out to check for the presence of alkaloids (Wagner's and Dragendorff's tests), flavanoids (aqueous sodium hydroxide test), terpenoids (Salkowshi's test), carbohydrates (Molisch's test), phenolics and tannins (Ferric chloride test) following the standard protocol [33]. Preparation of aqueous leaf extracts (AQLE). The fresh leaves (5th to 20th from the bud of branches) collected were washed with deionized water to remove the contaminants and were air dried for 3 days under shade. Thereafter, about 5 g of the powdered air dried leaves were heated in 100 mL distilled water in 250 mL Erlenmeyer flask in water bath at 70 °C for 45 min. After cooling, the aqueous leaf extract (AQLE) was collected through filtration using ‘Whatman filter paper No.1’. About 90 mL of the yellow colored AQLE obtained was kept in air tight bottle at 4 °C and was used within seven days for experiments. 2.3. Synthesis of AgNPs a. Effect of metal ion on synthesis:

In order to learn the effect of concentration of SN on synthesis of nanoparticles, three different concentrations of SN were prepared: s1—0.001 M, s2—0.0025 M and s3—0.005 M. 10 mL of the AQLE and 90 mL SN solution were mixed in Erlenmeyer flasks in triplicates for all the three concentrations. AQLE and SN alone were taken as controls. The reaction mixture was mixed thoroughly and heated at 90 °C in the water bath for 45 min. The flasks were incubated in dark to avoid photo inactivation of SN and nanoparticles were allowed to form. b. Effect of temperature on AgNPs synthesis:

The experiment was carried out at three different temperatures namely t1—30 °C, t2—70 °C and t3—90 °C to optimize the temperature for silver nanoparticle synthesis. The reaction mixture of 10 mL AQLE and 90 mL SN (0.001 M) were kept at t1 to t3 in a water bath for about 45 min and afterwards incubated overnight in darkness. c. Effect of volume of AQLE and SN on AgNPs formation:

To optimize the volume of optimum SN concentration (0.001 M) to a fixed volume of the extract, three different ratios of AQLE and the SN

d. Effect of reaction time:

2.4. Characterization of AgNPs The effect of reaction environment for the synthesis of AgNPs, such as metal ion concentration, ratio of AQLE to SN, reaction temperature and reaction time was monitored using Shimadzu UV–Visible spectrophotometer at a resolution of 1 nm; 0.1 mL of sample was taken in quartz cuvette, diluted to 2 mL with deionized water. The infrared spectrum of both AQLE and AgNPs were analyzed to know the possible organic functional groups attached to the surface of the nanoparticles. The FT-IR studies were carried out using Shimadzu FTIR spectrometer in attenuated total reflection mode using a spectral range of 4000– 400 cm−1 and resolution of 4 cm−1. XRD analysis of AgNPs was made from a thin film of solution. X-ray diffraction was carried out on a PANalytical X-ray diffractometer at step size of 0.010° with an operating voltage of 45 kV and a current of 30 mA with Cu Kα radiation (1.5406 A0) monochromatic filter in the range 10– 70°; diffraction intensities were compared with the standard JCPDS files. Average crystallite size of the nanoparticles was calculated using Debye–Scherer equation: D = kλ/βcosθ, where D = Thickness of the nanocrystal, k = Constant, λ = Wavelength of X-rays, β = full width at half maximum and θ = Bragg angle. The size, diffraction ring patterns (SAED), lattice fringes and dspacing were examined in high resolution mode (HR-TEM) using JEOL TEM instrument. A thin film of the nanoparticles from the reaction mixture was coated on to carbon coated-copper grid. The particle size distribution was derived from histogram considering about 100 particles counted using Image J software. Also EDS was taken, which confirms the presence of silver atoms using the EDS instrument attached to JEOL TEM. DLS analysis to measure the hydrodynamic diameter of the particles formed was performed using Malvern Zetasizer. 2.5. Antibacterial screening a. Determination of Zone of Inhibition (ZOI):

Bacterial strains such as Escherichia coli (E.coli), Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhi obtained from pus and urine samples were maintained on nutrient agar slants at 4 °C. The bacteria were identified by standard biochemical tests as per Bergy's Manual of Systematic Bacteriology. The antibacterial activity of AgNPs was assessed by Agar well diffusion method [34,35]. Prior to the start of experiment, all the strains were sub-cultured on Muller Table 1 Phytochemical screening of AQLE of Ziziphus oenoplia (L.) Mill. Sl. no

Phytochemical

Test name

AQLE

MLE

1

Alkaloids

2 3 4 5 6

Carbohydrates Flavanoids Terpenoids Phenolic compounds Tannins

a) Wagner's test b) Dragendorff’ test Molisch's test Aqueous NaOH test Salkowski test Ferric Chloride test Ferric Chloride test

+ + + + + + +

+ + + – – – –


393

Fig. 1. Effect of metal ion (a) UV–visible spectra of s1 to s3 (b) TEM images of s 1 to s 3,shows the particle size of s1 as 9 nm,s2 as 15 nm and s3 as 20 nm.

Hinton Broth and incubated for 2–6 h. Bacteria collected from the log phase of growth were used for the studies. Later Muller Hinton Agar plates were prepared and 6 mm wells were made using gel puncture. Different concentrations of AgNPs ranging from 1.95–500 μg/mL were prepared using serial dilution in deionized water and used for the

studies. The Muller Hinton Agar plates were then swabbed with fresh bacterial broth cultures and approximately 20 μL of test samples (AgNPs) were loaded in the wells. SN and AQLE alone were kept as controls. The culture plates were incubated at 37 °C for overnight. The

Fig. 2. Effect of reaction temperature on AgNPs synthesis.

Fig. 3. Effect of AQLE to SN.

394


as the lowest concentration of AgNPs that completely inhibited the visible growth in wells. 2.6. Bactericidal effect of AgNPs Transmission Electron Microscopy (TEM) was carried out to examine the bactericidal effect of AgNPs, in particular the morphological changes caused by interaction of AgNPs and E. coli, was studied using JEOL TEM Instrument. TEM analysis was monitored at 30 min and 24 h intervals after the treatment with AgNPs. Untreated E. coli was kept as control. Three different samples were prepared for the analyses:

Fig. 4. Effect of reaction time on AgNP synthesis.

diameter of the zone of inhibition was measured in millimeters (mm). The experiment was repeated for three times to get an average value. b. Determination of Minimum Inhibitory Concentration (MIC): The AgNPs that showed positive inhibitory activities in preliminary well diffusion method were further processed to find out the MIC against the four selected strains. The MIC was determined by standard micro-broth dilution method [36]. About 100 μL test sample (1.95 μg/mL–500 μg/mL of AgNPs) was added to 96 well microtitre plates along with 100 μL broth cultures (mid-log phase) of strains (Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Salmonella typhi). The experiments also included a positive control (MTCC strains) and a negative control (Muller Hinton broth). All the experiments were carried out in triplicate. The wells were mixed thoroughly and were kept at 37 °C for overnight incubation. The MIC was recorded

(a). E. coli cells grown in 100 mL nutrient broth after 12 h was supplemented with 2 mL (125 μg/mL) AgNPs. The flasks were placed in shaker incubator at 37 °C, 200 rpm for continuous agitation for 12 h (b). E. coli cells grown in 100 mL nutrient broth for 24 h were treated with 2 mL (125 μg/mL) AgNPs and kept for 30 min incubation in shaking incubator at 37 °C and agitated at 200 rpm. (c). E. coli cells grown in 100 mL nutrient broth for 24 h (Control).

After the incubation, cells were collected from (a), (b) and (c) by centrifugation at 3000 rpm for 5 min at 40C. They were washed and re-suspended in 1 × PBS buffer solution. A 10 μL sample was taken from each and coated on to carbon coated copper grid for TEM analysis to determine the distribution of AgNPs as well as the morphological changes in E. coli due to the interaction with AgNPs. 2.7. Fluorescence microscopy analysis Live/dead staining was performed using Acridine Orange/Ethidium bromide flurorescent dye to assess the viable bacterial cells after exposure to AgNPs. Smear was prepared for untretaed E.coli , E. coli treated with AgNPs for 30 min and 24 h durations and were allowed to dry. After that 1% Acridine Orange/Ethidium bromide stain was added to the smear. The excess stain was washed off and air dried. Samples

Fig. 5. UV spectra of AQLE, silver nitrate & AgNP.


were viewed after 20 min of incubation with OLYMPUS BX43F Fluorescent microscope [37]. 2.8. Statistical analysis The values of inhibition zones in antibacterial activities of AgNPs synthesized using aqueous extract of Ziziphus oenoplia (L.) Mill. were expressed as mean ± standard deviation (n = 3) for each sample. 3. Results and discussion 3.1. Phytochemical screening Qualitative analysis of aqueous and methanolic leaf extracts (AQLE and MLE) confirmed that the plant, Zizyphus oenoplia (L.) Mill., is rich

395

in active phytochemicals (Table 1) such as alkaloids, flavonoids, phenolics, terpenoids, tannins and carbohydrates. Similar results observed by Yuvarajan et al. [38] for the presence of alkaloids, phenolics, carbohydrates, flavanoids in the aqueous extract of Trichosanthes tricuspidata supports the present study. 3.2. UV –visible analysis 3.2.1. Effect of metal ion concentration Surface plasmon resonance spectra of phytosynthesized AgNPs (Fig. 1.a) revealed that formation of AgNPs depend on SN concentration. Observation of increase in peak absorbance along the metal ion concentration from s1 to s3, formation of larger sized particles towards higher concentration and red shift in the SPR spectra [19,39,40] confirmed the same. Fig. 1.b records the TEM images of s1, s2 and s3. As the

Fig. 6. TEM micrographs of Ag NPs-a to d, HRTEM image with lattice fringes (d-spacing) -e, SAED Pattern -f, and Histogram-g.

396


Fig. 7. EDS spectrum of AgNP.

Fig. 9. Graph–DLS analysis.

concentration of SN increases, the size of particles was found to increase which supports the UV data (Fig. 1.a). Therefore, the lowest concentration, s1was selected for synthesis of optimum sized AgNPs.

3.2.2. Effect of reaction temperature The SPR spectra (Fig. 2) recorded after 2 h of incubation, at three different temperatures (t1—30 °C, t2—70 °C, and t3—90 °C) exhibited an increase in peak sharpness along the temperature gradient, which may be attributed to increase in the reaction rate of synthesis [41]. The sharpness in peak absorbance always reveals the size of synthesized nanoparticles [19,40,42]. Since the sharpest SPR peak is observed at t3, it was selected as the optimum temperature for synthesis of optimum sized nanoparticles. All the prepared AgNPs were stable at room temperature for up to one year, similar to previous observations in this regard [41].

3.2.3. Effect of AQLE to SN Observations of UV spectra (Fig. 3) for synthesis of AgNPs from different ratios of 0.001 M SN with constant volume of AQLE at 90 °C showed that peak absorbance increased with increase in the volume of SN (q1 to q3). There was no peak at q1, which revealed no synthesis of AgNPs at 1:2 ratios of SN and AQLE. However, red shift and broadened SPR peak (458 nm) observed at q3 may correspond to large particle size, as observed in previous reports [41]. Therefore, the q2 ratio of 1:9 was selected as the optimum ratio of AQLE and SN, for synthesis of AgNPs in further studies.

Fig. 8. XRD spectrum of SNP synthesized from AQLE of Ziziphus oenoplia (L.) Mill

Fig. 10. FT-IR spectrum of the plant extract.

3.2.4. Effect of reaction time Since the colour change corresponding to formation of AgNPs was observed after about 45 min, UV spectra were recorded at 1 h, 24 h and 48 h intervals (Fig. 4); observations revealed an increase in intensity of the peak along with increase in time. There was no change in peak intensity after 48 h, which suggested completion of reaction by that time. Spectral analysis done after one year showed a red shifted SPR peak, which might be due to Ostwald ripening process [43]. A comparison of the UV spectra of SN, AQLE and AgNPs, and the visual

Fig. 11. FT-IR spectrum of the synthesized AgNPs.


observations in the inset (Fig. 5) is presented as a conclusive proof of optimization of AgNPs synthesis in the present experimentation. Single peak obtained is indicative of spherical nature of the particle which was later confirmed in TEM analysis.

397

biomolecules from the AQLE of Ziziphus oenoplia (L.) Mill. Thus EDS analysis confirmed the formation of elemental silver. 3.5. Structural studies (a). XRD analysis

3.3. Morphological analysis The TEM images (Fig. 6: a–d) revealed spherical nature of synthesized AgNPs, which is in agreement with UV data. The HR-TEM image (6-e) of a single nanoparticle showed crystalline nature of the particles. The d-spacing value of 0.235 nm observed is in agreement with the (111) lattice spacing of face centered cubic (fcc) silver (d111 = 0.2359 nm), which is in accordance with the standard data file (JCPDS No. 04-0783). The bright circular spots in SAED pattern (6-f) indicating the (111), (200) and (220) planes also revealed the crystalline nature of the particles formed, which is consistent with the XRD results obtained. Observations of the histogram (6-g) with variation in particle size suggested the polydisperse nature of phytosynthesized AgNP. The average particle size of 9 nm determined from the histogram in the present experimentation is similar to previous observations in this regard [41,44]. 3.4. Elemental studies with EDS spectrum The EDS spectrum of phytosynthesized AgNPs (Fig. 7) showed an intense optical absorption band peak at 3 KeV. This is typical of the absorption of metallic AgNPs due to SPR [45]. The peaks of Si are from the glass on which sample was coated. The other peaks had arisen from the capping layer on surface or in the vicinity of AgNPs by

The XRD pattern (Fig. 8) obtained for AgNPs synthesized using Ziziphus oenoplia (L.) Mill. gave three peaks at 2 θ = 38.100, 44.040 and 64.460. The three diffraction peaks observed in the 2 θ range 300– 700 can be indexed to the (111), (200), (220) planes of fcc silver (JCPDS file no.04-0783). This is in agreement with SAED pattern obtained [46]. The crystallite size of the AgNPs was calculated using DebyeScherer equation and the average crystallite size was estimated to be 10.28 nm. The X-ray diffraction studies confirmed the crystallite nature of AgNPs. (b). DLS Analysis

The hydrodynamic particle size of the phytosynthesized AgNPs is measured at 110 ± 5 nm in DLS analysis (Fig. 9), where the polydispersity index (PDI) value of 0.219 is indicative for stability of particles. The reaction between SN and AQLE resulted in the conversion of Ag+ to Ag0 by the reducing agents present in the AQLE. The reason for larger particles in DLS analysis may be due to natural nucleation of small particles and their further growth into larger particles with lower surface energy due to Ostwald ripening process [43].

Fig. 12. Agar well diffusion assay; E to H-different concentration of AgNPs, SN—silver nitrate, AQLE—leaf extract.

398


Table 2 ZOI of four bacterial strains. SL no.

1 2 3 4

Test organisms

Zone of Inhibition (ZOI) (mm)

Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli Salmonalla typhi

E (62.5 μg/mL) 18 ± 0 14.3 ± 0.5 12.8 ± 0.3 15 ± 0

F (31.25 μg/mL) 17.6 ± 0.5 12.6 ± 0.5 12 ± 0 14.3 ± 0.5

3.6. FT-IR spectroscopic studies FT-IR spectrum of the AQLE (Fig. 10) and AgNPs (Fig. 11) were examined to identify the possible biomolecules responsible for capping and stabilization of the nanoparticles. The major peaks in FT-IR spectrum of AQLE was observed at 3568 cm− 1, 2920 cm− 1 and 1647 cm−1; the minor peaks at 1544 cm−1 and 1016 cm−1. The peak at 3568 cm−1 corresponds to –O-H stretch which can be assigned to H-bonded \\O\\H stretch and hydroxyl groups. This accounts for the frequencies of alcohol and phenolic groups. The peaks at 1647 cm− 1 and1544 cm− 1 corresponds to \\C_C stretching vibration (alkanes) and\\NO asymmetric stretch (nitro compounds). The peak at 1016 cm−1 corresponds to –CN stretch of aromatic amines. The observed peak at 2920 cm−1 corresponds to\\C\\H asymmetric stretch. The FT-IR spectrum of AgNPs gives a broad peak at 3317 cm− 1 which can be assigned to \\O\\H stretch of alcohols. A strong IR signal at 1633 cm−1 corresponds to\\C_C stretching vibration. All these observations are as per previously established facts in this regard [47]. Images taken after and before reduction concludes that all the chemical functional groups discussed above may be bound to the AgNPs. Reduction in the peak intensity of the FT-IR spectrum of AgNPs at 2920, 1544 and 1016 cm−1 and shifting of peaks at 3568 and 1647 to 3317 and 1633 cm− 1 indicated the positive role of compounds like flavanoids, phenols, alkaloids, terpenoids, carbohydrates and tannins with \\C_C stretching vibrations, \\OH groups, amino groups and nitro compounds in the process of reduction, capping as well as stabilization for the particles formed. 3.7. Antibacterial activity In general, smaller nanoparticles due to higher surface area interact with bacteria more than larger particles leading to higher antibacterial activity [13]. In the current experimentation, different concentrations of the phytosynthesized 10 nm sized AgNPs (7.812–62.5 μg/mL) showed a significantly high anti bacterial activity than the controls AQLE and 0.001 M SN. There are observations of antibacterial activity of phytosynthesized AgNPs using diverse plant materials [34,44,48, 49], but the concentrations of phytosynthesized AgNPs used in the current experimentation is significantly low. The high activity of low concentrations of phytosynthesized AgNPs in the current experimentation can be attributed to the small size of nanoparticles formed. Well diffusion assay (Fig. 12; Table 2) for the phytosynthesized AgNPs against the four bacterial strains showed that there exists no or very low zone of inhibition (ZOI) for the controls, AQLE and SN, respectively. Pseudomonas aeruginosa showed a larger ZOI at all the four Table 3 MIC values of phytosynthesized Ag NPs against different bacterial strains. No

Clinical bacteria

MIC values

MIC values

(μg/mL)

(μg/mL)—Control

1 2 3 4

Salmonella typhi Klebsiella pneumoniae Pseudomonas aeruginosa Escherichia coli

31.25 15.62 7.8 7.8

62.5 62.5 62.5 125

G (15.625 μg/mL) 13.3 ± 0.5 12 ± 0 11.8 ± 0.3 13 ± 1

H (7.812 μg/mL) 11 ± 0 11.6 ± 0.5 11.3 ± 0.2 11 ± 1

SN (0.001 M) 12 ± 0.5 11 ± 0 11 ± 0 12 ± 0

AQLE (1 mg/mL) – – – –

concentrations than other strains on comparing with AQLE and SN. Thus, antibacterial activity of the phytosynthesized nanoparticles using leaf extracts of Ziziphus oenoplia (L.) Mill., is successfully established in the current experimentation. 3.8. MIC values The MIC values of the control MTCC strains and clinical strains are given in the Table 3. The MIC values of the AgNPs for the four different strains of clinical bacterial were comparatively lower than MIC values of control MTCC strains (Table 3). Among the four strains, Pseudomonas and E. coli were found to be more sensitive towards AgNPs than Klebsiella and Salmonella. The results suggest that the AgNPs in the present study were more efficient than the same sized AgNPs synthesized using other plants [50,51]. 3.9. Bactericidal studies with TEM analysis: examination of interaction between E. coli and AgNPs In support of the bacterial susceptibility studies of AgNPs, the plausible role of AgNP–E. coli interaction was analyzed using TEM. Transition electron micrographs of untreated E. coli displayed the characteristic bacilli shape (Fig. 13.a), E. coli cells treated with AgNPs for 30 min was seen with ruptured cell wall, condensed cytoplasm (Fig. 13.b), cells with shrinkage and detached flagella (Fig. 13.c). The micrographs showed that on exposure, the AgNPs were present on the cell membrane (Fig. 13.d) and they appear to be attached to the lipopolysacchaaride layer in cell wall of gram negative E. coli as reported in previous studies [52, 53]. The Fig. 13 e-g shows the changes in E. coli cell at 24 h treatment with AgNPs, a completely disintegrated cell was seen (Fig. 13.e) along with AgNPs (Fig. 13 f-g) attached to the membrane. Fig. 13.h&i, depicts a group of untreated E. coli cells with electron dense region in nuclear area and treated E. coli cells at 30 min with electron light region as a result of exposure to AgNPs. Feng et al. had made similar observations [54]. The most possible approach for the AgNPs-bacterial interaction could be due to alteration in structural integrity or physiochemical changes in the cell wall. This might be explained by the change in osmoregulation of the cell which may cause extrusion of intracellular material (Fig. 13.b) and ultimately leading to death [28]. TEM results showed that similar series of changes has occurred in the present study. Various reports have implied that AgNPs are capable of attaching to the bacterial cell membrane as well as entry into cells [14,55,56]. Also it was documented that particles with size 5 nm can damage bacterial cell membrane resulting in leakage of reducing sugars leading to death of bacteria [57]. Evidence for formation of pits and holes in the cell membrane of AgNPs treated E. coli is also available; however the damage to the E. coli was dependent on initial concentration of AgNPs used [55]. In another report investigators have observed that only AgNPs with diameter less than 10 nm were capable of entering E. coli and Pseudomonas [13]thus leading to death. In the light of such observations of the present study, it may be concluded that synthesis of nanoparticles of less than 10 nm size and the interaction of E. coli with AgNPs might be due to initial binding of AgNPs along the cell membrane, change in permeability to membrane resulting in extrusion of intracellular material and thus leading to


399

Fig. 13. TEM images-a) Untreated E. coli b) AgNPs treated E. coli at 30 min seen with ruptured cell wall,condensed cytoplasm,extrusion of intracellular material as shown by arrows c) cell shrinkage and without flagella d) AgNPs attached to cell membrane.

400


Fig. 13 (continued).

death of cells. However it will require extensive studies to elucidate the exact pathway of biocidal effect of AgNP.

synthesized using leaf extracts of Ziziphus oenoplia (L.) Mill. is well established, which opens up its applications as a pharmaceutical and therapeutic agent.

3.10. Evaluation of antibacterial activity by live/dead staining of bacterial cells

4. Conclusions

It is well known that in live/dead staining of treated and untreated E. coli cells, the live cells emit green flurescence whereas the dead cells emit red flurescence [37]. Among the fluorescent microscope images, Fig. 14.a shows only live E. coli cells; Fig. 14.b&c were fluorescent images of E. coli cells after 30 min of exposure to AgNPs, where a mixture of live and dead cells are seen. In the Fig. 14.d, images of bacteria after 24 h of exposure were shown with only dead cells. These observations are supportive to the observations made in TEM imaging of similar samples (Fig. 13.b-d & e-g). Thus the significant antibacterial activity of AgNPs

Phytosynthesis of AgNPs is very well known as an eco-friendly method with additive pharmaceutical and therapeutic capabilities. Current experimentation could standardize the synthesis of AgNPs of 10 nm size using aqueous leaf extracts of Ziziphus oenoplia (L.) Mill. The AgNPs so produced is found to be stable up to one year without the addition of any stabilizers. They are very well characterized using UV visible spectroscopy, TEM-EDS analysis, XRD and DLS studies and FT-IR spectroscopy. The phytosynthesized AgNPs were found to be good antibacterial agents, which are well established on the basis of

Fig. 14. Live/dead cell staining of a—E. coli cells,b & c—treated E. coli cells at 30 min,treated cells—at 24 h.


well-diffusion assay and determination of MIC values. Bactericidal effects of the phytosynthesized AgNPs were also explored using Fluorescent Imaging as well as the TEM. Overall, the present study has illustrated the synthesis of AgNPs using the leaf extract of Ziziphus oenoplia (L.) Mill. at optimum temperature, concentration of SN and the exact ratio of the volume of SN to fixed amount of AQLE, their characterization and establishment of antibacterial efficacy of the free AgNPs against the four gram negative bacterial strains. Also a plausible way of interaction of E. coli cells with AgNPs has been accounted and studied using TEM.

[19]

[20]

[21]

[22]

Acknowledgement Authors gratefully acknowledge School of Pure and Applied Physics, Mahatma Gandhi University, Kottayam, Kerala for XRD facility, International and Inter University Centre For Nanoscience and Nanotechnology, M G University for TEM-EDAX and Rubber Research Institute of India for Malvern Zetasizer facility.

[23]

[24]

[25]

References [26] [1] C.N.R. Rao, G.U. Kulkarni, P. John Thomas, P.P. Edwards, Size-dependent chemistry: properties of nanocrystals, Chem. Eur. J. 8 (2002) 28–35, http://dx.doi.org/10. 1002/1521-3765(20020104). [2] S. Asghari, S.A. Johari, J.H. Lee, Y.S. Kim, Y.B. Jeon, H.J. Choi, M.C. Moon, I.J. Yu, Toxicity of various silver nanoparticles compared to silver ions in Daphnia magna, J. Nanobiotechnol. 10 (2012) 14, http://dx.doi.org/10.1186/1477-3155-10-14. [3] M.E. Vance, T. Kuiken, E.P. Vejerano, S.P. McGinnis, M.F. Hochella, D. Rejeski, M.S. Hull, Nanotechnology in the real world: redeveloping the nanomaterial consumer products inventory, Beilstein J. Nanotechnol. 6 (2015) 1769–1780, http://dx.doi. org/10.3762/bjnano.6.181. [4] L. Bois, F. Chassagneux, C. Desroches, Y. Battie, N. Destouches, N. Gilon, S. Parola, O. Stéphan, Electroless growth of silver nanoparticles into mesostructured silica block copolymer films, Langmuir 26 (2010) 8729–8736, http://dx.doi.org/10.1021/ la904491v. [5] X. Dong, X. Ji, H. Wu, L. Zhao, J. Li, W. Yang, Shape control of silver nanoparticles by stepwise citrate reduction shape control of silver nanoparticles by stepwise citrate reduction, J. Phys. Chem. 113 (2009) 6573–6576. [6] Y. Sun, Y. Xia, Shape-controlled synthesis of gold and silver nanoparticles, Science 298 (2002) 2176–2179, http://dx.doi.org/10.1126/science.1077229. [7] J. Thiel, L. Pakstis, S. Buzby, M. Raffi, C. Ni, D.J. Pochan, S.I. Shah, Antibacterial properties of silver-doped titania, Small 3 (2007) 799–803, http://dx.doi.org/10.1002/ smll.200600481. [8] C. Lok, C. Ho, R. Chen, Q. He, W. Yu, H. Sun, P.K. Tam, J. Chiu, C. Che, Proteomic analysis of the mode of antibacterial action of silver research articles, J. Proteome Res. 5 (2006) 916–924, http://dx.doi.org/10.1021/pr0504079. [9] P. Gunawan, C. Guan, X. Song, Q. Zhang, S.S.J. Leong, C. Tang, Y. Chen, M.B. ChanPark, M.W. Chang, K. Wang, R. Xu, Hollow fiber membrane decorated with Ag/ MWNTs: toward effective water disinfection and biofouling control, ACS Nano 5 (2011) 10033–10040, http://dx.doi.org/10.1021/nn2038725. [10] S. Agnihotri, S. Mukherji, S. Mukherji, Antimicrobial chitosan–PVA hydrogel as a nanoreactor and immobilizing matrix for silver nanoparticles, Appl. Nanosci. 2 (2012) 179–188, http://dx.doi.org/10.1007/s13204-012-0080-1. [11] S. Agnihotri, S. Mukherji, S. Mukherji, Immobilized silver nanoparticles enhance contact killing and show highest efficacy: elucidation of the mechanism of bactericidal action of silver, Nanoscale 5 (2013) 7328–7340, http://dx.doi.org/10.1039/ c3nr00024a. [12] Nicola Cioffi, M. Rai, Nano-Antimicrobials, Springer, 2012. [13] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramírez, M.J. Yacaman, The bactericidal effect of silver nanoparticles, Nanotechnology 16 (2005) 2346–2353, http://dx.doi.org/10.1088/0957-4484/16/10/059. [14] S. Pal, Y.K. Tak, J.M. Song, Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia Coli, Appl. Environ. Microbiol. 73 (2007) 1712–1720, http://dx.doi. org/10.1128/AEM.02218-06. [15] Roberto Sato-Berrú, Rocío Redón, J.M.S. América Vázquez-Olmos, Silver nanoparticles synthesized by direct photoreduction of metal salts: application in surface enhanced Raman spectroscopy, J. Raman Spectrosc. 40 (2009) 376–380. [16] L. Rastogi, J. Arunachalam, Sunlight based irradiation strategy for rapid green synthesis of highly stable silver nanoparticles using aqueous garlic (Allium sativum) extract and their antibacterial potential, Mater. Chem. Phys. 129 (2011) 558–563, http://dx.doi.org/10.1016/j.matchemphys.2011.04.068. [17] A.R. Binupriya, M. Sathishkumar, C.S. Ku, S.I. Yun, Biosorption of Procion red MX 5B by Bacillus subtilis and its extracellular polysaccharide: effect of immobilization, Clean: Soil, Air, Water 38 (2010) 775–780, http://dx.doi.org/10.1002/clen. 200900279. [18] M.S. Abdel-Aziz, M.S. Shaheen, A.a. El-Nekeety, M.a. Abdel-Wahhab, Antioxidant and antibacterial activity of silver nanoparticles biosynthesized using Chenopodium

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40] [41]

[42]

401

murale leaf extract, J. Saudi Chem. Soc. 18 (2014) 356–363, http://dx.doi.org/10. 1016/j.jscs.2013.09.011. H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Green synthesis of silver nanoparticles using latex of Jatropha curcas, Colloids Surf. A Physicochem. Eng. Asp. 339 (2009) 134–139, http://dx.doi.org/10.1016/j.colsurfa.2009.02.008. S.P. Dubey, M. Lahtinen, M. Sillanpää, Tansy fruit mediated greener synthesis of silver and gold nanoparticles, Process Biochem. 45 (2010) 1065–1071, http://dx.doi. org/10.1016/j.procbio.2010.03.024. R. Manikandan, B. Manikandan, T. Raman, K. Arunagirinathan, N.M. Prabhu, M. Jothi Basu, M. Perumal, S. Palanisamy, A. Munusamy, Biosynthesis of silver nanoparticles using ethanolic petals extract of Rosa indica and characterization of its antibacterial, anticancer and anti-inflammatory activities, Spectrochim. Acta A Mol. Biomol. Spectrosc. 138 (2015) 120–129, http://dx.doi.org/10.1016/j.saa.2014.10.043. J.R. Nakkala, R. Mata, A.K. Gupta, S.R. Sadras, Biological activities of green silver nanoparticles synthesized with Acorous calamus rhizome extract, Eur. J. Med. Chem. 85C (2014) 784–794, http://dx.doi.org/10.1016/j.ejmech.2014.08.024. Y. Park, Y.N. Hong, a. Weyers, Y.S. Kim, R.J. Linhardt, Polysaccharides and phytochemicals: a natural reservoir for the green synthesis of gold and silver nanoparticles, IET Nanobiotechnol. 5 (2011) 69, http://dx.doi.org/10.1049/iet-nbt.2010.0033. B. Ajitha, Y.A. Kumar, S. Shameer, K.M. Rajesh, Y. Suneetha, P.S. Reddy, Journal of Photochemistry and Photobiology B: Biology Lantana camara leaf extract mediated silver nanoparticles: antibacterial , green catalyst, J. Photochem. Photobiol. B Biol. 149 (2015) 84–92, http://dx.doi.org/10.1016/j.jphotobiol.2015.05.020. P. Velmurugan, J. Park, S. Lee, J. Jang, K. Lee, S. Han, S. Lee, M. Cho, B. Oh, Journal of Photochemistry and Photobiology B: biology synthesis and characterization of nanosilver with antibacterial properties using Pinus densiflora young cone extract, J. Photochem. Photobiol. B Biol. 147 (2015) 63–68, http://dx.doi.org/10.1016/j. jphotobiol.2015.03.008. K.L. Niraimathi, V. Sudha, R. Lavanya, P. Brindha, Biosynthesis of silver nanoparticles using Alternanthera sessilis (Linn.) extract and their antimicrobial, antioxidant activities, Colloids Surf. B: Biointerfaces 102 (2013) 288–291, http://dx.doi.org/10.1016/ j.colsurfb.2012.08.041. J. Yang, H. Yin, J. Jia, Y. Wei, Facile synthesis of high-concentration, stable aqueous dispersions of uniform silver nanoparticles using aniline as a reductant, Langmuir 27 (2011) 5047–5053, http://dx.doi.org/10.1021/la200013z. S. Agnihotri, S. Mukherji, S. Mukherji, Size-controlled silver nanoparticles synthesized over the range 5–100 nm using the same protocol and their antibacterial efficacy, RSC Adv. 4 (2014) 3974, http://dx.doi.org/10.1039/c3ra44507k. D.B.R. Ramalingam, B. Madhavi Bindu, A. Ravinder Nath, N. Duganath, E. Udaya Sri, In vitro denaturatuion and antibacterial activities of Ziziphous oenoplia, Scholarsresearchlibrary.com. 2 87–93 (2010)http://scholarsresearchlibrary.com/ ABR-vol1-iss2/ABR-2010-1-2-87-90.pdf. P.S. Udayan, S. George, K.V. Tushar, I. Balachandran, Some common medicinal plants used by the Nayaka community, Savandurga forest of Magadi taluk, Bangalore rural district, Karnataka, India, J. Nat. Remedies. 5 (2005) 35–40. N. Rajakaruna, C.S. Harris, G.H.N. Towers, Antimicrobial activity of plants collected from serpentine outcrops in Sri Lanka antimicrobial activity of plants collected from serpentine outcrops in Sri Lanka, Pharm. Biol. 40 (2002) 235–244, http://dx. doi.org/10.1076/phbi.40.3.235.5825. M.L. Eswari, R.V. Bharathi, N. Jayshree, Preliminary phytochemical screening and heavy metal analysis of leaf extracts of Ziziphus oenoplia ( L ) Mill. Gard, 5 (2013) 38–40. S. Sasidharan, Y. Chen, D. Saravanan, K.M. Sundram, L. Yoga Latha, Extraction, isolation and characterization of bioactive compounds from plants' extracts, Afr. J. Tradit. Complement. Altern. Med. 8 (2011) 1–10, http://dx.doi.org/10.4314/ajtcam.v8i1. 60483. D. MubarakAli, N. Thajuddin, K. Jeganathan, M. Gunasekaran, Plant extract mediated synthesis of silver and gold nanoparticles and its antibacterial activity against clinically isolated pathogens, Colloids Surf. B: Biointerfaces 85 (2011) 360–365, http:// dx.doi.org/10.1016/j.colsurfb.2011.03.009. R. Mariselvam, a J. a Ranjitsingh, A. Usha Raja Nanthini, K. Kalirajan, C. Padmalatha, P. Mosae Selvakumar, Green synthesis of silver nanoparticles from the extract of the inflorescence of Cocos nucifera (family: Arecaceae) for enhanced antibacterial activity, Spectrochim. Acta. A. Mol. Biomol. Spectrosc. 129 (2014) 537–541, http://dx. doi.org/10.1016/j.saa.2014.03.066. M.B. Coyle (Ed.), Manual of Antimicrobial Susceptibilty Testing, American Society for Microbiolgy, 2005. B.M. Alphonsa, P.T. Sudheesh Kumar, G. Praveen, R. Biswas, K.P. Chennazhi, R. Jayakumar, Antimicrobial drugs encapsulated in fibrin nanoparticles for treating microbial infested wounds, Pharm. Res. 31 (2014) 1338–1351, http://dx.doi.org/10. 1007/s11095-013-1254-6. R. Yuvarajan, D. Natarajan, C. Ragavendran, R. Jayavel, Journal of Photochemistry and Photobiology B: biology photoscopic characterization of green synthesized silver nanoparticles from Trichosanthes tricuspidata and its antibacterial potential, J. Photochem. Photobiol. B Biol. 149 (2015) 300–307, http://dx.doi.org/10.1016/j. jphotobiol.2015.04.032. J.J. Mock, M. Barbic, D.R. Smith, D.A. Schultz, S. Schultz, Shape effects in plasmon resonance of individual colloidal silver nanoparticles, J. Chem. Phys. 116 (2002) 6755–6759, http://dx.doi.org/10.1063/1.1462610. P. Mulvaney, Surface plasmon spectroscopy of nanosized metal particles, Langmuir 12 (1996) 788–800, http://dx.doi.org/10.1021/la9502711. D. Philip, Biosynthesis of Au, Ag and Au\ \Ag nanoparticles using edible mushroom extract, Spectrochim. Acta. A. Mol. Biomol. Spectrosc. 73 (2009) 374–381, http:// dx.doi.org/10.1016/j.saa.2009.02.037. S. Lee, S. Chang, Y. Lai, C. Lin, C. Tsai, Y.-C. Lee, J.-C. Chen, C.-L. Huang, Effect of temperature on the growth of silver nanoparticles using plasmon-mediated method

402

[43]

[44]

[45]

[46]

[47]

[48]

[49]

S. Soman, J.G. Ray / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 391–402 under the irradiation of green LEDs, Materials (Basel) 7 (2014) 7781–7798, http:// dx.doi.org/10.3390/ma7127781. M. Chen, Y.G. Feng, X. Wang, T.C. Li, J.Y. Zhang, D.J. Qian, Silver nanoparticles capped by oleylamine: formation, growth, and self-organization, Langmuir 23 (2007) 5296–5304, http://dx.doi.org/10.1021/la700553d. U.B. Jagtap, V.a. Bapat, Green synthesis of silver nanoparticles using Artocarpus heterophyllus Lam. seed extract and its antibacterial activity, Ind. Crop. Prod. 46 (2013) 132–137, http://dx.doi.org/10.1016/j.indcrop.2013.01.019. M. Puchalski, P. Dabrowski, W. Olejniczak, P. Krukowski, P. Kowalczyk, K. Polański, The study of silver nanoparticles by scanning electron microscopy, energy dispersive X-ray analysis and scanning tunnelling microscopy, Mater. Sci. 25 (2007). G. Singhal, R. Bhavesh, K. Kasariya, A.R. Sharma, R.P. Singh, Biosynthesis of silver nanoparticles using Ocimum sanctum (Tulsi) leaf extract and screening its antimicrobial activity, J. Nanopart. Res. 13 (2011) 2981–2988, http://dx.doi.org/10.1007/ s11051-010-0193-y. H. Schulz, M. Baranska, Identification and quantification of valuable plant substances by IR and Raman spectroscopy, Vib. Spectrosc. 43 (2007) 13–25, http://dx.doi.org/ 10.1016/j.vibspec.2006.06.001. M.R. Bindhu, M. Umadevi, Spectrochimica Acta Part A: molecular and biomolecular spectroscopy synthesis of monodispersed silver nanoparticles using Hibiscus cannabinus leaf extract and its antimicrobial activity highlights, Spectrochim. Acta A Mol. Biomol. Spectrosc. 101 (2013) 184–190, http://dx.doi.org/10.1016/j.saa. 2012.09.031. M. Zargar, A.A. Hamid, F.A. Bakar, M.N. Shamsudin, K. Shameli, F. Jahanshiri, F. Farahani, Green synthesis and antibacterial effect of silver nanoparticles using Vitex negundo L, Molecules 16 (2011) 6667–6676, http://dx.doi.org/10.3390/ molecules16086667.

[50] V.K. Sharma, R. a Yngard, Y. Lin, Silver nanoparticles: green synthesis and their antimicrobial activities, Adv. Colloid Interf. Sci. 145 (2009) 83–96, http://dx.doi.org/ 10.1016/j.cis.2008.09.002. [51] S. Maiti, D. Krishnan, G. Barman, S. Ghosh, J. Laha, Antimicrobial activities of silver nanoparticles synthesized from Lycopersicon esculentum extract, J. Anal. Sci. Technol. 5 (2014) 40, http://dx.doi.org/10.1186/s40543-014-0040-3. [52] M.E. Samberg, P.E. Orndorff, N.A. Monteiro-riviere, Antibacterial efficacy of silver nanoparticles of different sizes, surface conditions and synthesis methods, Nanotoxicology 5 (2011) 244–253, http://dx.doi.org/10.3109/17435390.2010. 525669. [53] X.N. Xu, W.J. Brownlow, S.V. Kyriacou, Q. Wan, J.J. Viola, Real-time probing of membrane transport in living microbial cells using single nanoparticle optics and living cell imaging †, Biochemistry 43 (2004) 10400–10413. [54] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, A Mechanistic Study of the Antibacterial Effect of Silver Ions on Eshcerichia coli and Staphylococcus aureus, Vol. 6018, John Wiley Sons, 2000 662–668. [55] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci. 275 (2004) 177–182, http://dx.doi.org/10.1016/j.jcis.2004.02.012. [56] A. Dror-ehre, H. Mamane, T. Belenkova, G. Markovich, A. Adin, Journal of colloid and interface science silver nanoparticle–E. coli colloidal interaction in water and effect on E. coli survival, J. Colloid Interface Sci. 339 (2009) 521–526, http://dx.doi.org/ 10.1016/j.jcis.2009.07.052. [57] C.R., J. Li, K. Rong, H. Zhao, F. Li, Z. Lu, Highly selective antibacterial activities of silver nanoparticles against Bacillus subtilis, J. Nanosci. Nanotechnol. 13 (10) (Oct 2013) 6806–6813 13 (2013) 6806–13.