103

CHAPTER 5

ANTIBACTERIAL EFFECTS OF BIOSYNTHESIZED SILVER

NANOPARTICLES USING AQUEOUS LEAF EXTRACT OF

ROSMARINUS OFFICINALIS L.

5.1 Abstract

In this study, we demonstrate a green approach for the synthesis of silver

nanoparticles (AgNPs) using aqueous leaf extract of Rosmarinus officinalis under ambient

conditions. The uniqueness of this method lies in its rapid synthesis within 15 min. The

synthesized AgNPs were characterized using various analytical tecchniques. The

synthesized particles were found to be 14.20 ̶ 42.42 nm with face centered cubic geometry.

The functional group of flavonoids and terpenoids was largely identified by FTIR which

was found to be responsible for the synthesis and stabilization of the AgNPs. Further,

antibacterial efficacy of the biologically synthesized AgNPs was investigated by the

standard method against Pseudomonas aeruginosa and Staphylococcus aureus.The results

showed that the aqueous leaf extract mediated synthesized AgNPs is an excellent

antibacterial agent against clinical pathogens.

Keywords: Green approach, R. officinalis, AgNPs, TEM, antibacterial activity

5.2 Introduction

Noble metal nanoparticles have drawn the attention of the researchers in the last

two decades because of their unique features and extensive applications in various fields.

104 Among nanoparticles, silver nanoparticles (AgNPs) in particular are known for their

versatile applications in medical industries [1], food processing industries [2], textile

industries [3], consumer goods [4], and for being an efficient antimicrobial agent [5].

Several methods are known to synthesize AgNPs which include chemical reduction [6],

electrochemical reduction [7], Langmuir–Blodgett [8], pulse sonoelectrochemcial method

[9] etc. Studies have shown that the size, morphology, stability, and the chemical–physical

properties of the metal nanoparticles are strongly influenced by the experimental

conditions, the kinetics of the interaction of metal ions with reducing agents, and the

adsorption processes of stabilizing agents with metal nanoparticles [10,11]. Hence, the

design of the synthesis method in which the size, morphology, stability, and properties of

metal nanoparticles are controlled has become a major concern.

Chemical reduction is the most frequently applied method for the preparation of

AgNPs. Some of the common reducing agents like borohydride, citrate, ascorbate,

elemental hydrogen etc. were used for the synthesis of uniform size nanoparticles. The

reduction of silver ions (Ag+) in aqueous solutions generally yields colloidal silver with

particle diameters of several nanometres [12,13]. Initially, the reduction of various

complexes with Ag+ ions leads to the formation of silver atoms, which is followed by

agglomeration into oligomeric clusters. These clusters eventually lead to the formation of

colloidal particles [14]. Ultimately these methods employ toxic chemicals as reducing

agents or non-biodegradable stabilizing agents and are, therefore, potentially dangerous to

the environment and biological systems [15]. Moreover, most of these methods entail

intricate controls or non-standard controls.

In view of the environmental sustainability, there is an urgent need to develop

eco-friendly technologies for synthesis and assembly of nanoparticles and therefore the

focus turned towards the green chemistry approach. The green synthesis of AgNPs

involves three main steps, which must be evaluated based on the green chemistry

105 perspectives, including (a) the selection of the solvent medium, (b) the selection of

environmentally benign reducing agents, and (c) the selection of nontoxic substances for

the stability of AgNPs. Therefore, the green chemistry approach of AgNPs synthesis using

plant extracts has become a major focus for researchers due to simplicity of procedures,

stability, and their potential applications. In recent years, plant materials such as fruit,

bark, fruit peels, root, leaf, and callus have been studied more exclusively in this direction

[16].

In our present study, we describe the biosynthesis of AgNPs using the

commercially available Rosmarinus officinalis leaf as a biomaterial. It has been used since

ancient times for medicinal purposes and is known for its anti-septic, anti-inflammatory,

hepato-protective, anti-cancer, anti-diabetic, anti-ulcerogenic, and anti-depressant effects

[17]. The well known antioxidant compounds are carnosic acid, carnosol, abietanes

diterpenes, rosmarinic acid, hydroxycinnamic acid, caffeic acid, and urosolic acid that are

responsible for various biological activities [18 ̶ 20]. Hence, the present study was aimed at

synthesizing AgNPs using aqueous leaf extract of R. officinalis, and to evaluate its

antibacterial activity towards clinically isolated pathogens.

5.3 Experimental

5.3.1 Chemicals and bacterial pathogens

All the chemicals were purchased from Sigma Aldrich (St. Louis, USA). The

clinical isolates of bacterial strains of Pseudomonas aeruginosa and Staphylococcus

aureus were obtained from SRM Medical College Hospital and Research Centre,

Kattankulathur, Chennai. Double sterilized Milli-Q water was used throughout the

experiments.

106 5.3.2 Synthesis of silver nanoparticles

The leaves of R. officinalis were collected from an agriculture farm in

Chengalpattu, India. The leaf was cut into a small pieces, and thoroughly washed under

running tap water followed by double-distilled water. Leaf extract (LE) was obtained from

10 g of finely cut leaves in a 250 ml Erlenmeyer flask and boiled in 100 ml of double-

distilled water at 60 ºC for 5 min. The boiled solution was filtered through a nylon mesh,

followed by Millipore hydrophilic filter (0.22 μm) and used for further experiments.

In a typical reaction procedure, varying amounts (0.5 ̶ 5.0 ml or 5 ̶ 50% v/v) of the

LE of R. officinalis were added separately to 5 ml solution of 1 mM aqueous AgNO3. The

final volume of the LE solution was adjusted to 10 ml by adding appropriate amount of

double distilled water. This reaction mixture was then heated at 60 ºC, and the solution

became yellowish brown in color after 15 min. UV–vis spectra showed strong SPR band at

436 nm, thus indicating the formation of silver nanoparticles. The AgNPs solution thus

obtained was purified by repeated centrifugation at 15,000 rpm for 20 min followed by re-

suspension of the pellet in deionized water.

5.3.3 Characterization of AgNPs

Surface plasmon resonance (SPR) of AgNPs was characterized using Perkin-

Elmer double beam spectrophotometer, USA at the resolution of 1 nm. Crystalline metallic

silver was examined by X-ray diffraction analysis using X’Pert Pro A Analytical X–ray

diffractometer with a CuKɑ radiation monochromatic filter in the range 30–90º.

Morphological and topographical analysis of the particles were investigated by

transmission electron microscopy (TEM) using JEOL 2100 instrument accelerating

voltage of 80 keV equipped with EDX and atomic force microscopy (AFM) using Model-

Nanosurf easyscan 2. To perform FTIR spectrum analysis, the silver nanoparticles which

were synthesized by using leaf extract of R. officinalis were centrifuged at 12,000 rpm for

107 15 min to remove free proteins or other components present in the solution. The

centrifuged, collected and vacuum dried powder sample was placed in ATR sample holder

of Perkin-Elmer Spectrum-One FTIR instrument for measurement.

5.3.4 Antibacterial experiments

The silver nanoparticles synthesized using R. officinalis leaf extract were tested

for antibacterial activity by broth culture and disc diffusion method against clinical

pathogenic bacteria P. aeruginosa (Gram-negative), and S. aureus (Gram-positive). The

concentration of AgNPs was measured by weighing the freeze-dried AgNPs, and the

particles were diluted in double sterilized water. 100 µl of P. aeruginosaand S. aureus

(OD600 = 1.5) were inoculated into 25 ml LB liquid medium containing AgNPs

concentrations of 0, 5, 10, 15, and 20 µg/ml, and cultured at 37 ºC and 180 rpm. After 4 hr,

the OD600 was measured and the data were recorded. Subsequently antibacterial activity in

solid LB medium was studied by the disc diffusion method. The discs were then loaded

with 30 µl of different concentration of AgNPs (20, 40, 60 and 80 µg/ml) and placed on

LB agar medium which were already swabbed with bacteria (OD600 = 1.2). The plates were

incubated at 37 ◦C overnight. The zone of inhibition was measured in millimeter after the

24 hr of incubation.

5.4 Results and discussion

5.4.1 Mechanism involve in the biosynthesis of AgNPs

The present investigation reports the rapid and simple procedure for synthesis of

silver nanoparticles using the aqueous leaves extract of R. officinalis. The possible

mechanism for the synthesis of AgNPs is proposed in Figure 5.1. In this scheme, Ag+ ions

can form an intermediate complex with free radical (2-position of A-ring or 4′-position of

108 ring B) present in semi-quinone structure which subsequently undergoes oxidation to

quinone forms with consequent reduction of Ag+ to AgNPs.

Figure 5.1 Hypothetical mechanism involved in the biosynthesis of AgNPs.

5.4.2 Absorption spectroscopy of AgNPs synthesis

In order to study the effects of LE concentration on the biosynthesis of AgNPs,

we mixed different concentrations of LE (ranging from 5 ̶ 50% v/v) with 1 mM AgNO3

solution. As shown in Figure 5.2, the SPR peak for AgNP concomitantly undergoes a red

Figure 5.2 (a) Absorption spectra of AgNPs synthesized from different concentrations (5 ̶

50 % vol fractions) of leaf extract, and (b) corresponding plot of λmax values against

volume of extract showing red shift with increase in the concentration of extract.

110 results were in accordance with the previous study [25]. It was observed that AgNPs were

stable in the solution and showed very little aggregation even after 4 months.

5.4.3 Crystallographic analysis of biosyanthesized AgNPs

Crystal behavior of the purified solid AgNPs was evaluated using powder XRD.

Powder XRD pattern of AgNPs showed five distinct diffraction peaks at 38.13◦, 44.40◦,

64.55◦, 77.37◦, and 81.68◦ (Figure 5.4), which correspond to crystal facets of (1 1 1), (2 0

0), (2 2 0), (3 1 1), and (2 2 2) face-centered cubic (fcc) of AgNPs, respectively. The (2 0

0), (2 2 0), (3 1 1) and (2 2 2) Bragg reflections are weak and broadened relative to the

intense (1 1 1) reflection. This feature indicates that the nanocrystals are highly anisotropic

[26]. The lattice constant calculated from this pattern was a=4.086 Å which was in good

agreement with reference JCPDF Card No. 03–0921.

Figure 5.4 XRD pattern of biosynthesized AgNPs.

The mean crystallite diameter (D) of the AgNPs formed in the reduction process, is

determined by using Scherrer’s equation D = Kλ/βscosɵ and is estimated to be 31.79 nm

111 (Table 5.1), in which K is the shape dependent Scherrer’s constant (0.94), λ is X-ray

wavelength (1.5406 Å), βs is X-ray line width (FWHM) and ɵ is the Bragg angle.

Table 5.1 XRD peaks corresponding particle size determination.

2ɵ

(Ref.)

2ɵ

(exp.)

FWHM

(exp.)

Miller

indices

Particle

size

Mean

size (nm)

38.09 38.13 0.2460 1 1 1 36.74

44.59 44.35 0.2460 2 0 0 37.30

64.67 64.52 0.3936 2 2 0 25.30 31.79

77.54 76.93 0.2952 3 1 1 36.64

81.49 81.56 0.4800 2 2 2 22.99

5.4.4 Topographic analysis of biosynthesized AgNPs

The topographic features of synthesized AgNPs and the elements present

there were analyzed using FE-SEM coupled with EDX and TEM analysis. FE-SEM image

revealed that the size of many of the AgNPs was in order of 22.90 ̶ 42.42 nm (Figure 5.5

a). The particles appeared to be predominantly spherical in shape. The elemental analysis

for the resultant AgNPs was confirmed by EDX and the spectrum spot profile was made

from the densely populated region of nanoparticles on the slide surface. A strong signal of

Ag peak was observed approximately at 3 keV (Figure 5.5 b) which is typical for the

absorption of metallic silver nanocrystallites due to surface plasmon resonance [27]. Along

with this, weak C, O peaks from the biomolecules which are bound to the surface of the

AgNPs and Cl peaks were also found due to the presence of chloride ions that might be

present in the leaf extract. It has been reported that nanoparticles which are synthesized by

using leaf extract are surrounded by a thin layer of some capping organic material, and are

thus, stable in solution up-to 4 months after synthesis [28]. TEM micrograph of AgNPs

obtained in the aqueous solution under normal conditions shows that the majority of the

112 AgNPs are spherically shaped and well distributed without any aggregation in solution

within the range of 14.20 ̶ 41.67 nm shown in Figure 5.6.

Figure 5.5 (a) FE-SEM image of crystal AgNPs, and (b) EDX spectrum of metallic Ag.

Figure 5.6 TEM images of dispersed AgNPs in aqueous solution without aggregation.

114 synthesis. Prominent IR bands are observed at 1012, 1249, 1352, 1446, 1612, 1714, 2922,

and 3340 cm−1 (Figure 5.8). Most of the IR bands are characteristic of flavonoids and

terpenoids present in the leaf extract. The strong absorption bands centered at around

1012, and 1352 cm−1 may arise from ̶ C ̶ O and C ̶ O ̶ C stretching modes of vibration. The

medium absorbtion bands located at 1458, 1612, and 2922 cm−1 correspond to C–N stretch

Figure 5.8 FTIR spectra of (a) leaf extract (LE) alone, and (b) synthesized AgNPs using

R. officinalis LE.

of the aromatic amine group, C=C, and C ̶ H stretching modes of vibration, respectively. In

addition, there was a broad peak located at 3416 cm-1, which could be assigned to the O–H

stretching vibrations, indicating the presence of hydroxyl groups [29–32]. The vibrational

bands corresponding to –C=C, –C=O, –C–O, –C–O–C and –C–N bonds are derived from

the water soluble compounds such as flavonoids, terpenoids and thiamine that are present

in R. officinalis leaf extract . Hence, it may be assumed that these biomolecules apart from

the reduction process, could possibly form a layer covering the AgNPs (i.e., capping of

AgNPs) to prevent agglomeration and thereby stabilizing the silver nanoparticles.

116 aureus (Figure 5.9 a). Moreover, the disc diffusion assay of AgNPs against P. aeruginosa

showed maximum zone of inhibition whereas the S. aureus showed lower zone of

inhibition. This could be due to the presence of thicker peptidoglycan layer in Gram-

positive than Gram-negative bacteria preventing the entry of AgNPs and its antibacterial

activity. The cell wall of the Gram-positive bacteria is composed of a thick layer of

peptidoglycan ~30 nm, consisting of linear polysaccharide chains that are cross-linked by

short peptides; thus they form more rigid structure leading to difficult penetration of the

AgNPs compared to the Gram-negative bacteria where the cell wall possesses thinner layer

of peptidoglycan ~2–3 nm [33]. The high bactericidal activity is certainly due to the silver

cations released from AgNPs that act as reservoirs for the Ag+ bactericidal agent. Big

changes in the membrane structure of bacteria as a result of the interaction with silver

cations lead to the increased membrane permeability of the bacteria [34,35]. Further, Amro

et al. suggested that metal depletion may cause the formation of irregularly shaped pits in

the outer membrane and change membrane permeability, which is caused by the

progressive release of lipopolysaccharide molecules and membrane proteins [36].

McDonnell et al. suggested that silver has a greater affinity to react with sulfur or

phosphorus-containing biomolecules in the cell. Thus, sulfur containing proteins in the

membrane or inside the cells and phosphorus-containing elements like DNA are likely to

be the preferential sites for AgNPs binding which leads to the death of cells [37].

5.5 Conclusion

We demonstrated a simple, rapid and eco-friendly method for silver nanoparticles

synthesis by using R. officinalis leaf extract. The proposed method requires 15 min to

synthesize AgNPs when AgNO3 solution was incubated with R. officinalis leaf extract (5:2

v/v) at 60 ºC. Spherically shaped AgNPs were found within 14.20 ̶ 42.42 nm which were

well supported by XRD, FE-SEM, TEM, and AFM data analysis. FTIR study reveals the

117 presence of flavonoids and terpenoids which are present in the leaf extract might be

responsible for reduction, capping leading to stabilize the AgNPs. Thus the synthesized

AgNPs exhibit superior antibacterial effect against human pathogens.

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