Biogenic synthesis of antibacterial silver chloride ...

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Graphical Abstract

Biogenic synthesis of antibacterial silver chloride nanoparticles using leaf extracts of Cissus quadrangularis Linn.


Q1 V. Gopinath a, S. Priyadarshini a, N. Meera Priyadharsshini a, K. Pandian b, P. Velusamy a a

Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Chennai 603203, India Department of Chemistry, University of Madras, Guindy Campus, Chennai 600025, India

b

Highlights

c AgCl-NPs were synthesized using leaf extract of Cissus quadrangularis for the first time. c Work emphasizes on the chloride ions, present in the leaf extract, is mainly responsible for the synthesis of AgCl-NPs. c By this biological procedure, spherical shaped AgCl-NPs obtained were similar to previous reports. c AgCl-NPs were found to have the highest antibacterial activity than reference drug.

0167-577X/$ - see front matter & 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.matlet.2012.09.102

Please cite this article as: Gopinath V, et al. Biogenic synthesis of antibacterial silver chloride nanoparticles using leaf extracts of Cissus quadrangularis Linn.. Materials Letters (2012), http://dx.doi.org/10.1016/j.matlet.2012.09.102


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Contents lists available at SciVerse ScienceDirect

Materials Letters journal homepage: www.elsevier.com/locate/matlet

Biogenic synthesis of antibacterial silver chloride nanoparticles using leaf extracts of Cissus quadrangularis Linn. Q1

V. Gopinath a, S. Priyadarshini a, N. Meera Priyadharsshini a, K. Pandian b, P. Velusamy a,n a b

Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Chennai 603203, India Department of Chemistry, University of Madras, Guindy Campus, Chennai 600025, India

a r t i c l e i n f o

abstract

Article history: Received 18 May 2012 Accepted 28 September 2012

In this study, we demonstrate a single step method for the synthesis of silver chloride nanoparticles (AgCl-NPs) using leaf extract of Cissus quadrangularis. The AgCl-NPs were characterized by UV–Vis absorption spectroscopy and Fourier transform infra-red spectrum analysis. The crystalline nature and the elemental analysis of the synthesized nanoparticles were analyzed by XRD spectrum, EDAX and HR-TEM. The average size of the AgCl-NPs was found to be 15–23 nm. The antibacterial activity of AgCl-NPs was evaluated against bacterial pathogens of MDR strains by MIC test. Thus, the synthesized AgCl-NPs could be used as an antibacterial agent. & 2012 Published by Elsevier B.V.

Keywords: Biosynthesis XRD FTIR AgCl-NPs

1. Introduction Over the past two decades, there has been increasing interest in synthesis of nanomaterials, due to their attractive applications in electronic, catalytic, optical, physico-chemical properties and drug delivery system. Recently, various inorganic nanoparticles such as metals, metal oxides, metal sulfides and metal chlorides have been successfully synthesized by various methods. Among these, silver chloride is perhaps the most widely recognized and has been extensively used as photographic material [1], catalyst material [2], antibacterial agent [3] and ionic semiconductor material [4]. A number of approaches have been established for the synthesis of silver chloride nanoparticles by chemical [5], host–guest nanocomposite material [6], radiation [7], photochemical methods [8] and electro-spinning [9]. However, in view of the rapid progress of application of nanomaterials in different fields, there is a growing need to develop clean, nontoxic, simple and environment friendly procedures for the synthesis of nanoparticles. Indeed, a number of bacteria, fungi and yeast have been well-known for synthesis of non-toxic noble metal nanoparticles [10]. However, the microbial mediated synthesis of nanoparticles is not industrially feasible as they require expensive medium and maintenance under highly aseptic conditions. Hence, exploration into plant systems has been considered to be a potential bioreactor for synthesis of metal nanoparticles without using toxic chemicals. Keeping the green approach in mind the present study focused on the biosynthesis of antimicrobial AgCl-NPs from leaf extract of C.

quadrangularis is reported. It is well known that the fresh stem and leaf of C. quadrangularis have been used for various medicinal applications like bone fractures, menstruation, asthma, scurvy and hemorrhoids [11]. It is also known to have anti-oxidant, antibacterial, anti-osteoporotic and anti-inflammatory properties [12].

2. Materials and methods Aqueous extract of C. quadrangularis was prepared using freshly collected leaves (2 g). They were surface cleaned with running tap water, followed by ground with 100 ml of double sterilized deionized water using mortar and pestle. The resulting extract was filtered through Whatman No.1 filter paper and used for further assays. For synthesis of AgCl-NPs, aqueous leaf extract was treated with 1 mM AgNO3 solution and it was incubated in a dark room condition. The reaction progress for the formation of AgCl-NPs was monitored by visual color change and UV–Vis spectral scanning in the range of 200–700 nm. The biologically synthesized nanoparticles solution was lyophilized and used for further analysis by FTIR, XRD, FE-SEM, EDAX and HR-TEM analysis. The antibacterial activity of the AgCl-NPs were determined by MIC test against MDR strains of Gram positive bacterias viz., Staphylococcus aureus and Streptococcus pyogens and Gram negative bacterias viz., Escherichia coli and Proteus vulgaris.

3. Results and discussion n

Corresponding author. Tel.: þ44 27452270; fax: þ 44 27453903. E-mail address: [email protected] (P. Velusamy).

The leaf extract of C. quadrangularis was treated with 1 mM AgNO3 solution. After the incubation period of 18–24 h, the

0167-577X/$ - see front matter & 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.matlet.2012.09.102


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V. Gopinath et al. / Materials Letters ] (]]]]) ]]]–]]]

reaction mixture color was changed pale green to dark brown color. It may be due to the excitation of Surface plasmon resonance (SPR) of the synthesized AgCl-NPs [13]. The progress of this reaction is shown in (Fig. 1a). UV–Vis spectroscopy is a reliable method for studying the presence of metallic nanostructures [14]. The SPR peak at 300 nm was observed due to the active biomolecules present in the leaf extract which are known to interact with silver ions [15]. In addition, an intense peak at 440 nm was corresponding to the formation of AgCl-NPs [7,16]. Gradually, longer incubation time periods (18–24 h), increased the corresponding peak intensities (Fig. 1b, c). On examination of the synthesis mechanism of AgCl-NPs, the chlorine ions present in the leaf extract which was confirmed by EDAX (Fig. 1d) readily reacts with the AgNO3 to form AgCl-NPs (Fig. 2). Hence, the chlorine contents of leaf extract could be most attributed source for the formation of AgCl-NPs. The FTIR spectrum was obtained from both the leaf extract and the synthesized AgCl-NPs (Fig. 3a). The IR spectrum of leaf extract showed that major peaks were observed in the lower frequency regions at 1543, 1652 and 1237 cm 1 which were assigned for CQC, carbonyl ( 4CQO) stretching vibrations of amide I and amide II linkages which is due to the functional group that clearly implies the presence of protein/peptide serves as stabilizing as well as reducing agent for the formation of AgNPs. Infrared active modes attributed to side chain vibrations include C–H stretching symmetric and anti-symmetric modes at 2923 and 2854 cm 1 which corresponds to aliphatic and aromatic compounds, respectively [17,18]. The intense peak absorbance at 3287 cm 1 is the characteristic of the hydroxyl functional group in alcohols and phenolic compounds [19]. The FTIR spectrum of AgCl-NPs showed distinct peak at 1543, 1649, 1232, 2922, 2853 and 3291 cm 1 (Fig. 3b). The comparison of resultant spectrum between the leaf

extract and the synthesized AgCl-NPs slight shift changes were observed in the peak positions as well as the absorption bands [20] and no shift change was observed at 1543 cm 1. From these results, it was concluded that carbonyl, alkyl and hydroxyl groups which are present in the leaf of plant protein have a stronger affinity to bind with metal ions, which facilitate reduction of silver ion into AgCl-NPs. The crystalline nature of the synthesized AgCl-NPs was confirmed using XRD analysis (Fig. 3c). The peaks at 2y ¼28.01, 32.41, 46.37, 54.94, 57.58, 67.53 and 76.781 which corresponds to the (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), (4 0 0) and (4 2 0) planes of the cubic AgCl-NPs (ICDD file no. 00-001-1013). The minor peaks at 38.36, 44.34 and 64.61correspond to the (1 1 1), (2 0 0) and (2 2 0) planes for small amount of cubic Ag (ICDD file no. 00-0870718) can be observed. The broad bottom of peak indirectly proposed the smaller size of nanoparticles. An estimation of the crystallite size was made from the line broadening spectrum of the reflection using the Debye Scherrer formula: D ¼ 0:94k=b cosy

ð1Þ

where D is the average crystallite size, k is the X-ray wavelength,

b is the full width at half maximum and y is the diffraction angle. The calculated crystallite size was around 20.12 nm, which has good agreement with the AgCl-NPs estimated by HR-TEM analysis. From the Scherrer equation increase of crystallite size concordantly with the increasing basal space up to its disappearance were shown in Table 1a. The obtained diffraction spectrum results strongly suggest the presence of AgCl-NPs and small amount of silver nanoparticles (AgNPs) in favor of EDAX analysis. FE-SEM images revealed that the particles were spherical in shape and the average size of the particles falls in the range of 15–23 nm (Fig. 4a). Further, the HR-TEM micrographic image

Fig. 1. UV–Vis spectra of C. quadrangularis leaf extract alone (a), and treated with 1 mM aqueous AgNO3 at 18 h (b) and 24 h (c), intervals. Elemental analysis spectrum of leaf extract (d).

Fig. 2. Schematic diagram of possible mechanism for the AgCl-NPs synthesis using leaf extract of C. quadrangularis.


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Fig. 3. FTIR spectrum of C. quadrangularis leaf extract alone (a) and leaf extract reduced AgCl-NPs (b). XRD spectrum of biosynthesized AgCl-NPs (c) and EDAX of AgClNPs (d).

Table 1a Estimation of crystallite size and basal space of biosynthesized nanoparticles. Crystallite size (nm)

Basal space

27.9 26.4 27.7 19.6 6.7

3.19 2.77 1.96 2.35 2.04

(AgCl) (AgCl) (AgCl) (Ag) (Ag)

confirmed that the synthesized AgCl-NPs were spherical in shape and the size of the particles was around 18 nm (Fig. 4b) which well correlates with the SEM images. The elemental composition of the AgCl-NPs was obtained from EDAX measurement (Fig. 3d). Two major peaks with high intensity signals of Ag and Cl were observed, which indicated the presence of AgCl-NPs in the leaf

extract. This is the first report for biogenic synthesis of AgCl-NPs from leaf extracts of C. quadrangularis in our knowledge. The diameter of inhibition zone (DI) and minimum inhibitory concentration (MIC) for the AgCl-NPs, leaf extract and reference drug tetracycline are summarized in Table 1b. It was found that AgCl-NPs had antibacterial activity against both Gram positive and Gram negative bacterias. The DI and MIC data correlated well, the higher DI observed with lower MIC values proved good antibacterial activity. The MIC values of AgCl-NPs were found to be more effective than tetracycline against all the pathogens with very low MIC values at 3–7 mg/mL. Previous study suggests that AgCl-NPs have major inhibitory against E. coli which are depending on their particle size and bioavailability [21]. These results correlated well with several recent reports where AgCl-NPs have been shown to have antibacterial activity [22,23]. The results presented here, the inhibitory effects of microbial growth could have depended on particle size, biocompatibility by AgCl-NPs and synergistic effect with AgNPs. Research is underway to evaluate the fate of AgCl-NPs and AgNPs in antibacterial mechanism.


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Fig. 4. FE-SEM image of AgCl-NPs synthesized using leaf extract of C. quadrangularis showed the average size of 15–23 nm (a) and HR-TEM image of AgCl-NPs (b).

Table 1b Antibacterial activity of AgCl-NPs against bacterial pathogens. Leaf extract

AgCl nanoparticles

References

Tetracycline

Bacteria

DI

MICa

DI

MICa

DI

MICa

1. Gram positive S. pyogens S. aureus 2. Gram negative E. coli P. vulgaris

NI NI NI NI

420 420 420 420

7.777 0.25 8.837 0.26 7.97 0.31 8.47 0.40

4 3 5 7

7.37 0.21 8.17 0.03 7.57 0.15 9.57 0.24

9 12 10 12

DI: Diameter of zone of inhibition (mm) including disc diameter of 6 mm. NI: No inhibitions observed when using the leaf extract alone. a Minimum inhibitory concentration (MIC) mg/mL. The values are means of triplicate with 7SD.

4. Conclusion A novel method has been established for biosynthesis of AgClNPs by employing leaf extract of C. quadrangularis. The formation of AgCl-NPs were visually confirmed by the color changes and followed by UV–Vis spectral characterization. FTIR results reveal that the amide functional groups present in the leaf extract could be responsible for the formation of AgCl-NPs. The synthesized AgCl-NPs were highly stable even after 6 months of storage at room temperature. The AgCl-NPs had excellent antibacterial activity against clinically isolated MDR bacterial pathogens. Thus it can be concluded that C. quadrangularis leaf extract can be used as simple, low cost and environment-friendly biomaterial for synthesis of AgCl-NPs with antibacterial activity.

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