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This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.

Synthesis of Silver Nanoparticles using Fagonia

Cretica and their Antimicrobial Activities

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Open Access Article. Published on 04 December 2018. Downloaded on 3/10/2019 6:17:14 PM.

Hina Zulfiqara, Ayesha Zafara, Naveed Rasheedc, Zeshan Alib, Kinza Mehmooda, Abeer Mazherd,

Murtza Hasana,*, Nasir Mahmoode,*

aDepartment of Biochemistry & Biotechnology (Baghdad-ul-Jadeed Campus), The Islamia

University of Bahawalpur, Bahawalpur 63100, Pakistan

bDepartment of Material Science and Engineering, College of Engineering, Peking University,

Beijing 100871, China

cDepartment of Physics (Baghdad-ul-Jadeed Campus), The Islamia University of Bahawalpur,

Bahawalpur 63100, Pakistan

dCSIRO Mineral Resources, Deep Earth Imaging-Future Science Platform, 26 Dick Perry

Avenue, WA, 6151, Australia

eSchool of Engineering, RMIT University, 124 La Trobe Street, 3001 Melbourne, Victoria,

Australia

KEYWORDS: Ag nanoparticles, Fagonia cretica, antimicrobial activities, biological synthesis

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ABSTRACT: Silver nanoparticles (NPs) were synthesized using an efficient bioreducing agent

from Fagonia cretica extract having advantages of eco-friendliness over chemical and physical
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methods. Sharp color change and appearance of representative absorption peak in UV-visible

spectra confirm the quick reduction of Ag salt and evolution of Ag NPs. Morphological and

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structural aspects showed that resulted Ag NPs are well-crystalline with an average size of 16 nm.

Further, compositional analysis of extract confirmed the existence of active bioreducing and

stabilizing agents in Fagonia cretica extract. Furthermore, various concentrations of AgNO3 and

Fagonia cretica extract were employed to obtain higher yield with better stability of Ag NPs. The

resulted Ag NPs showed effective antibacterial activity against Proteus vulgaris, Escherichia coli,

Klebsiella pneumoniae. It is found that the Ag NPs induce maximum reactive oxygen species

(ROS) in Proteus vulgaris as compared Escherichia coli and Klebsiella pneumoniae to induced

cell toxicity, while ROS production in presence of Ag NPs was 30% higher than only plant extract

and control in all three bacterial strains. Thus, present finding show that plant extracts can be a

useful natural resource to prepare functional nanomaterials for targeted applications especially in

the field of biotechnology.

1. INTRODUCTION

Synthesis of functional nanomaterials is getting immense attention of researchers due to their vast

applications like in biomedicine, drugs delivery, cancer treatment, bio-imaging, molecular based

detection etc. But synthesis of nanomaterials via physical and chemical methods have severe

impact on environment and human health as these methods excrete tons of hazardous chemicals

and toxic by-products. [1-5].

Therefore, there is a growing concern to develop clean, efficient, non-toxic, non-lethal,

environment-friendly, cost-effective biological methods for the synthesis of nanoparticles (NPs)

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which have no threat to life. Recently, there are many reports to synthesis Ag NPs by using, root,

leaves and stem extract of plants such as Dracaena Cochinchinensis, Eucommia ulmoides,
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Gloriosa superba, Raphanus sativus and Sargassum polycystum [6-11]. These biological entities

have bioactive compounds in their extracts which have ability to reduce the metal ions into atoms,

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which results nuclei that consequently provide a base for variety of nanomaterials. For example,

Hu et al. investigated that conversion of Ag ions to Ag NPs and their size distribution is totally

dependent on lignin concentration as reducing and capping agent [12]. Similarly, Dragons blood

resin extract containing Loureirin A & B also act as strong reducing and capping agents to produce

Ag NPs [13]. Recent reports have shown that the presence of active functional groups such as

sugar and phenolic acids are responsible for the reduction of metal ions into NPs [14]. Keeping in

mind the above studies, it is highly desirable to find the alternative cheap and easily available

bioreducing sources for the synthesis of various functional nanomaterials to compete with physical

and chemical methods both in terms of cost and yield. It is also interesting to investigate the nature

of biomaterials involves in mechanism of recognition-reduction process and synthesis of NPs.

Moreover, there is need to know about the biomolecules in plant extract which leads to the

formation of metal complexes and ultimately produced corresponding nanomaterials. Further, this

area is getting high attention as these biologically synthesized nanomaterials are highly demanding

for various biomedical and catalytic applications like drug delivery, early disease detection,

antimicrobials, antifungal, and sensing applications [15-19].

Considering the advantages of green synthesis over other methods, here, we have explored new

plant extract named as Fagonia cretica as reducing and stabilizing agent for the synthesis of Ag

NPs due to its large production, low cost as well as no involvement of any bacterial or fungal

species which eliminate all associated clinical concerns, thus, make Fagonia cretica extract unique

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bioreducing source and worth to explore for the mentioned purpose. As-synthesized Ag NPs are

highly crystalline and exhibited significant anti-microbial activities against various strains of gram
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negative bacteria, confirming that biological reactors are better option for the synthesis of

nanomaterials for wide applications in future not only in medical but also in environment and

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energy.

2. EXPERIMENTAL SECTION

Materials & plant extract: Fagonia cretica plant was obtained from Agriculture Department of

The Islamia University of Bahawalpur, Pakistan. After drying the pieces of Fagonia cretica plant,

they were grinded to get fine powdered. Afterwards, 1.5 g of Fagonia cretica powder was mix in

50 mL of in different ethanol to water ratios (30, 50, 70 & 90% ethanol, as shown in Fig. S1) in

250 mL conical flask and shaken overnight at 37 °C. Finally, the extract was filtered using filter

paper and filtrate was kept at 4 °C for further use.

Synthesis of Ag NPs & Physical Characterizations: Initially 0.1 M of aqueous solution of

AgNO3 was prepared and mixed with plant extract in 5:1, respectively. The mixture was stirred on

magnetic stirrer (150 rpm) at 50 °C for time period of 15-120 min. The colour change in reaction

mixture was recorded through visual observation. The bioreduction of Ag ions in aqueous solution

was monitored by periodic sampling of aliquots (150 µL) and UV-vis spectra of the solution was

measured. On completion of reaction, product was collected using centrifuge (20 min at 6000 rpm).

The supernatant was saved in separate falcon tubes and kept at 4 °C for high performance liquid

chromatography (HPLC). Note: various concentrations of AgNO3aqueous solution (0.5 mM, 1

mM and 2 mM), and plant extract (10 mL, 20 mL and 40 mL) were used to determine their impact

on the growth of Ag NPs. The physical characterization and antimicrobial activity of Ag NPs is

given in supporting information.

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3. RESULTS AND DISCUSSION

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Silver NPs synthesized by the bioreducing agents extracted from the Fogonia cretica are of great

interest because of its unique characteristics. The reduction of AgNO3 by the Fagonia cretica

extract is first observed by change in color from light orange to dark red indicating the strong

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absorption of visible light due to the excitation of the NPs surface plasmons (the inset of Fig. 1a),

which depends on the size, shape and concentration of the Ag NPs [20]. Further to confirm the

reduction of Ag ions to metallic Ag NPs, UV-vis spectra was recorded at various time intervals of

15, 30, 60 & 120 min where the maximum absorbance was observed at 440 nm (Fig. 1a). The

control sample does not show any peak due to absence of reducing agent (plant extract) results no

formation of Ag NPs, while sample having plant extract shows maximum intensity for 60 & 120

min assuring that Fagonia cretica extract plays a main role in reducing Ag ions to form Ag NPs.

Size of the NPs is matter of great concern specially for their biological applications as size of NPs

strongly effects the rate of diffusion through the biological membrane. Previously, it has been

observed that small size NPs have more penetration power, but too small size brings the issue of

enhanced toxicity compared to larger size NPs, thus, an appropriate size is highly desirable for

specified biological applications [6, 21]. Therefore, transmission electron microscopy (TEM) was

used to examine the morphology and size of NPs prior to their biological applications. The Fig.

1b clearly shows that as-synthesized NPs are spherical in their morphology and have very narrow

size distribution where 70% NPs are having size in the range of 11-15 nm, while 22% are ranging

between 16-45 nm and only 1% are of smaller than 5 nm in size (the inset of Fig. 1b). The

abundant NP with a size range of 11-15 nm are highly suitable for biomedical application as the

size of synthesized NP is present within the tolerable range for inducing toxicity within the cell.

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Figure 1. (a) UV-vis spectra of Ag NPs with Fagonia cretica extract at different time points (the

inset shows the colour change before) and after Ag ion reduction) and (b) TEM images of Ag NPs,

while inset shows the particle size distribution.

It is very common that the concentrations of salt precursor (AgNO3) and Fagonia cretica extract

will have high impact on the nuclei formation, growth and yield of Ag NPs. To explore the effect

of AgNO3 and Fagonia cretica extract variable concentrations were employed while keeping the

one constant. The concentration of AgNO3 strongly influence the formation of Ag NPs as sharp

absorption peak for Ag NPs was noticed at 2.0 mM concentration of AgNO3 as compared to 0.5

mM and 1.0 mM as shown in Fig. 2a, which confirm that optimal concentration of Ag+ ions is

required as at lower concentration deficiency of ions and excessive ions at higher concentration

results the formation of improper and/or complex intermediates that ultimately brought poor

conversion efficiencies. Similarly, change in concentration of Fagonia cretica extract also

influenced the growth of Ag NPs at low concentration of extract no peak was observed in UV-vis

spectrum confirming low concentration are unable to provide enough bioreducing molecules for

stable Ag+ ion complex and their conversion to metallic Ag NPs (Fig. 2b). However, increasing

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concentration of Fagonia cretica extract from 10 to 20 mL results in sharp peak confirming the

formation of appropriate metal-organic complex that later on yield Ag NPs as shown in Fig. 2b.
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However, with further increase in Fagonia cretica extract up to 40 mL, we found a reduction in

absorption peak corresponds to poor yield of Ag NPs as well as overall high absorbance due to

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large quantity of plant extract (organic molecules). Regardless, reduction in absorption peak a

change in peak position is also observed might be due to improper metal-organic complex in

presence of higher concentration of organic molecules from Fagonia cretica extract (Fig. 2b).

Thus, confirming that to obtain Ag NPs with better material features appropriate concentrations of

AgNO3 and Fagonia cretica extract are highly required, however, antimicrobial activity was

conducted using Ag NPs prepared under best conditions.

Figure 2. UV–vis absorption spectra of Ag NPs synthesis at different concentrations of (a) AgNO3

and (b) plant extract.

To identify the role of Fagonia cretica extract in the reduction of Ag ions, HPLC studies of

Fagonia cretica extract were conducted before using as reducing agent and after reducing Ag ions

to Ag NPs to confirm its role in reduction as shown in Fig. 3a. A clear difference in absorbance

on basis of retention time was observed before and after the reduction reaction (full spectra are

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shown in Fig. S2). A change in the peak height of about 30% as well as disappearance of shoulder

peak appeared at 2.793 min (pointed by black and red arrows in Fig. 3a) confirm that of Fagonia
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cretica extract is responsible for the reduction of Ag ions to Ag NPs. Hence, proved that Fagonia

cretica extract contains the respective chemical entities that involved in reduction of Ag NPs and

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act as capping agent for the stability and suitability for biomedical applications.

Fourier-transform infrared spectroscopy (FTIR) analysis was carried out to further identify the

prominent bioactive molecules responsible for the stability and capping of the NPs. The FTIR

spectrum of crude Fagonia cretica extract was compared with as-synthesised Ag NPs to

investigate the biomolecules involves in bio-reducing and stability of NPs. FTIR spectrum of

Fagonia cretica extract shows peaks at 3864.44, 3729.37, 3626.26, 3467.80, 2916.00, 1636.56,

1061.73, 800.64, 715.39 and 627.47 cm-1 (Fig. S3a). While in Ag NPs FTIR spectrum peaks at

3739.15, 2358.74, 1472.89, 1401.80, 1114.29, 1050.90, 869.08, 643.89, 600.15 cm-1 are observed

(Fig. S3b). Therefore, the main functional groups involve in bioreducing of Ag NPs from Fagonia

cretica extract are hydroxyl and amino groups. Normally, these are groups with highly

electronegative elements and having lone pairs that can induce the reduction in metallic ions and

create the surface functionalized NPs. It was observed that shift in the absorbance peak from

3729.37 to 3739.15 cm-1, with increase in band intensity due to presence of hydroxyl group on

NPs surface. The peak at 1061.73 cm-1 in crude plant extract represents the C=H bending

vibrations for ether linkages is shifted to1050.89 cm-1 which may help in attaching the carboxyl

groups on the surface of Ag NPs. Another differential peak in plant extract of Fagonia cretica and

Ag NPs was compassion and it was found that the peak absorbance for C-H plan bending at 627.47

cm-1 is shifted to 643.89 cm-1 from plant extract to Ag NPs. The existence of these peaks indicate

that Ag NPs growth is based on the reducing molecules of Fagonia cretica which not only reduce

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the Ag ions but also attached the several functional group e.g. hydroxyl and secondary amines on

the surface of as-synthesized Ag NPs that stabilize the particles and assist in their better biological
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activity [13, 22]. These findings are well supported by the literature where ketone and hydroxyl

groups are reported to be responsible for the reduction of silver ions to Ag NPs, which has better

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stability due to naturally existing capping agents [23-27].

The crystallinity of Ag NPs was characterized by x-ray diffractometer (XRD) that delineates its

face centered cubic (fcc) structure (Fig. 3b). The XRD spectrum of biologically synthesized Ag

NPs showed distinct peaks at 2θ of 38°, 45°, 65 and 77° corresponds to lattice planes of (111),

(200), (220) and (311), respectively, in a good agreement with standard PDF No. 04-0783 while

chemically synthesized Ag NPs showed diffraction peaks on 38°, 45°and 77°. However, the

presence of more and sharp diffraction peaks confirms the better crystallinity of biologically

synthesized Ag NPs, showing that biological synthesis is an effective way not only for biological

applications also for many other fields as it offers to produce large amounts in easy ways with

possibility to creat various functionalities. The Scherrer's equation (D=kλ/β cosθ) is applied to

determine the crystallite size of Ag particles which is 1.66 nm (biological) and 1.19 nm

(chemically), the smaller crystallite size is might be due to presence of capping agent in the

Fagonia cretica extract that control the growth to smaller size and provide high stability. Further,

Fig. S4 shows the stability of colloidal solution of Ag NPs at pH 4, where it is clear that suspension

showed high stability even after one month. A slight change in color after one month shows little

sedimentation of NPs, however, no obvious aggregation of Ag NPs was observed in one-month

old solution which represents their high stability and suitability for the biomedical application.”

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Figure 3. (a) Chromatographic profiling of Fagonia cretica extract before and after reduction and

(b) XRD spectrum of Ag NPs synthesized using Fagonia cretica extract and chemical method.

Combative antimicrobial activities of as-synthesized Ag NPs and standard ciprofloxin against

strain of Proteus vulgaris, Escherichia coli and Klebsiella pneumonia have been investigated.

These microbes cause infections mainly in people with an impaired immunity system, and most

all of them may be a source of complicated urinary tract and wound infections as well as

nosocomial infections. Our result showed that antimicrobial activity of synthesized Ag NPs is

significant against the growth of Proteus vulgaris, Escherichia coli and Klebsiella pneumoniae

strains with different concentrations while Fagonia cretica extract (tested as to eliminate its impact

and validity of Ag NPs) with all different concentrations has not shown any significant

antimicrobial activity Fig. 4a-c. It is found that growth of all three bacterial strains (Proteus

vulgaris, Escherichia coli, Klebsiella pneumoniae) were significantly suppressed by treating with

Ag NPs and ciprofloxin compared to control experiment (Table S1, S2 & S3). Furthermore, Ag

NPs has shown maximum antimicrobial activity for Proteus vulgaris while minimum in

Escherichia coli for all concentrations (5, 10 & 20 μg) and similar trend is observed for Fagonia

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cretica extract but with much lower activity as shown in Fig. 4d&e. While, ciprofloxin showed

better antimicrobial activity against Escherichia coli and Klebsiella pneumoniae based on different
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concentration and comparatively lower activity against Proteus vulgaris (Fig. 4e). However, Ag

NPs shows much better antimicrobial activity for all three strains as compared to the commercial

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ciprofloxin and Fagonia cretica extract that shows as-synthesized Ag NPs are more effective than

commercially available ciprofloxin.

Figure 4. Antibacterial activity of synthesis of (a) Ag NPs, (b) Ciprofloxin and (c) Fagonia cretica

extract via disc method. A plot of concentration of (d) Ag NPs, (e) Ciprofloxin and (f) Fagonia

cretica extract vs. zone of bacterial inhibition.

the oxidation of Ag NPs into Ag+ ions which can diffuse into the biological system or put oxidative

stress to produce reactive oxygen species (ROS) that change the permeability of membranes,

damage the DNA/RNA, proteins and lipids and finally induce the cytotoxicity in prokaryotic cells

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[28-29]. Therefore, investigation of ROS magnitude in all three bacterial strains (Proteus vulgaris,

Escherichia coli, Klebsiella pneumoniae) in response to various concentrations (5, 10 & 20 μg) of
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Ag NPs, ciprofloxin and plant extract are very important to quantify the level of toxicity in

comparison to control experiments as presented in the Fig. 5a-c. The ROS generation was totally

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dependent on dose of treatment as can be seen from Fig. 5a that as the concentration is increased

from 5 μg/mL to 20 μg/mL of Ag NPs the production of ROS is also increases to its maximum in

all bacterial strain. However, ROS calculations have shown that Ag NPs are comparatively more

effective towards Proteus vulgaris as compared to Escherichia coli and Klebsiella pneumoniae.

On the other hand, variation in ciprofloxin concentration did not influence much to production of

ROS, however, ciprofloxin shows maximum production of ROS in Escherichia coli as compared

to Klebsiella pneumoniae and Proteus vulgaris as shown in Fig. 5b. The plant extract induces

very small production of ROS, therefore, its concentration did not induce much variation on ROS

production as well as no specificity for any bacterial strain was found as shown in Fig. 5c.

In short, the production of ROS in presence of Ag NPs is 30% higher than positive control of all

concentration in three bacterial strains. These results suggested that the interaction Ag NPs with

different bacterial cells cause ROS production, which induce toxicity in terms of oxidative stress

in the cells, leading to cell death as shown schematically in Fig. 5d. The Ag NPs strongly affect

the protein synthesis process in cell which induces structural changes and ultimately death of the

cell a basic principal behind the antibacterial activity of the Ag NPs [30-34].

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Figure 5. Production of ROS in various bacterial strains under the subject of different

concentrations of (a) Ag NPs (b) ciprofloxin and (c) plant extract after 24 h exposure time. (d)

Schematic description of the possible mechanism of action of Ag NPs for their antibacterial

activity.

4. CONCLUSIONS

In cessation, we have successfully synthesized Ag NPs using Fagonia cretica extract in ethanol

which is highly environment benign. The morphological and structural analysis shows that as-

synthesized Ag NPs are highly crystalline with an average particles size of 16 nm. Further, when

employed as antimicrobial agent, the crystalline Ag NPs showed significant antibacterial activity

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against Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae by producing 30% more ROS

than control. The HPLC and FTIR analysis revealed that Fagonia cretica extract has ability to
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reduce the Ag ions to Ag atoms which construct Ag NPs and consequently coat them with hydroxyl

and amine groups for high stability. Hence, easy and fast production of functional Ag NPs makes

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Fagonia cretica extract promising candidate for the preparation of nanomaterials for various

applications e.g. in biomedicine, pharmacokinetics, pharmaceutical and bio-nanotechnology.

ASSOCIATED CONTENT

Supporting Information. The supporting information contains part of experimental section,

FTIR and chromatography spectra as well as three Tables describing the details of Zones of

inhibition.

AUTHOR INFORMATION

Corresponding Author

1. Dr. Murtza Hassan (murtaza@[Link])

2. Dr. Nasir Mahmood ([Link]@[Link])

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval

to the final version of the manuscript.

ACKNOWLEDGMENT

The authors would like to acknowledge the financial and technical support provided by The

Islamia University of Bahawalpur, Pakistan; Peking University, China and RMIT University,

Australia.

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