Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 257–262
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Biosynthesis of silver nanoparticles using Plectranthus amboinicus leaf extract and its antimicrobial activity B. Ajitha a, Y. Ashok Kumar Reddy b, P. Sreedhara Reddy a,⇑ a b
Department of Physics, Sri Venkateswara University, Tirupati 517502, India Department of Electrical Engineering, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, South Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Nearly spherical Ag NPs are prepared
using P. amboinicus leaf extract. The green synthesis route is simple,
nontoxic, reliable and eco-friendly benign. XRD and SAED patterns attested the crystalline nature of the samples. Biomolecules of the leaf extract play dual role as both reducing and capping agents. It exhibited effective antimicrobial efficacy against E. coli and Penicillium spp.
a r t i c l e
i n f o
Article history: Received 22 October 2013 Received in revised form 20 February 2014 Accepted 23 February 2014 Available online 7 March 2014 Keywords: Green synthesis method Silver nanoparticles Leaf extract Particle size Absorbance Antimicrobial activity
a b s t r a c t This study reports the simple green synthesis method for the preparation of silver nanoparticles (Ag NPs) using Plectranthus amboinicus leaf extract. The pathway of nanoparticles formation is by means of reduction of AgNO3 by leaf extract, which acts as both reducing and capping agents. Synthesized Ag NPs were subjected to different characterizations for studying the structural, chemical, morphological, optical and antimicrobial properties. The bright circular fringes in SAED pattern and diffraction peaks in XRD profile reveals high crystalline nature of biosynthesized Ag NPs. Morphological studies shows the formation of nearly spherical nanoparticles. FTIR spectrum confirms the existence of various functional groups of biomolecules capping the nanoparticles. UV–visible spectrum displays single SPR band at 428 nm indicating the absence of anisotropic particles. The synthesized Ag NPs exhibited better antimicrobial property towards gram negative Escherichia coli and towards tested Penicillium spp. than other tested microorganisms using disc diffusion method. Finally it has proven that the synthesized bio-inspired Ag NPs have potent antimicrobial effect. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Nowadays nanotechnology is gaining profoundness owing to its increase of vital role in most dynamic areas of research in modern materials science. Recently is has broadened the scope of elevating ⇑ Corresponding author. Tel.: +91 877 2289472; fax: +91 877 2249611. E-mail address: [email protected] (P. Sreedhara Reddy). http://dx.doi.org/10.1016/j.saa.2014.02.105 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
research in various scientific disciplines due to their unique properties compared with bulk counterparts. Nanoparticles of noble metals have attracted immense interest incurring applications in catalysis, electronics, optics, environmental and biomedical applications due to their quantum confinement effects, antimicrobial activity and their large reactive surfaces [1–3]. In particular, silver nanoparticles have expunged a considerable attention in present tremendous research due to their exquisite fascinating spectrum
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of physical properties such as catalytic, optical, electrical and antimicrobial applications [4,5] and their significant potential selective activity in a very wide range of applications. They are even being projected as future generation antimicrobial agents [6]. Attribution of small size and high surface to volume ratio of metal nanoparticles enhances its bactericidal property which allows them to have an effect on microorganisms membranes and it is not simply due to release of metal ions in solutions [7]. The prerequisite nature of an ideal antimicrobial agent [8,9], material should have the ability to inhibit the formation of bio-film and cause inactivation/extermination of microbes either through targeting the cell wall for an antimicrobial activity or through direct contact-killing phenomena. Increasing exigency to employ good antimicrobial agent due to ever-increasing resistant bacteria, much attention has been focused on it and researchers found that Ag NPs have significantly displayed all these properties [8–10] serving as a promising candidate in having improved antimicrobial properties. Hence, use of silver nanoparticle coatings in a hospital environment could have a considerable drastic impact on declining the transmission of multidrug resistant bacteria between patients such as methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus is part of the natural flora of the human body and is illustrated as an opportunistic pathogen which can cause serious infections [11]. There are many ways to synthesize silver nanoparticles through physical and chemical methods such as electrochemical [12], c-radiation [13], photochemical [14], laser ablation [15] and chemical reduction [16]. However, physical methods have some disadvantages such as expensive, complicated vacuum techniques and apparatus. Normally, chemical processes are having two main problems. Primarily, high surface energy of nanoparticles enhances reactivity and they often undergo aggregation in liquid dispersions. This aggregation can be prevented during the synthesis [17–19] by the formation of self-assembled monolayers of organic templates, such as polymers, surfactants and DNA on the nanoparticle surface. Another problem is that the reducing reagents, such as citric acid, sodium borohydride, or other organic compounds, may exhibit environmental toxicity or biological hazards [20,21]. Due to these consequences, usage of chemicals is still the subject of paramount concern because they limit the nanoparticles applications in clinical fields. Therefore, biosynthesis of clean, biocompatible, nontoxic and environmental-friendly nanoparticles produced both extracellularly and intracellularly deserves merit [1,22]. Even though there are well established methods, growing need of cost-effective, environmental benign and to produce highly stable nanoparticles still had to be exploited. In this pursuit, researchers have focused on using microorganisms, living plants, plant biomass and plant extracts for the synthesis of bio-inspired metal nanoparticles. Biogenic synthesis is useful and seems to have good strategy not only pertaining to its reduced environmental impact [23,24] compared with physicochemical methods, but also we can produce large quantities of nanoparticles without any contamination and have a well-defined size and morphology [25]. At present, plant extracts usage is emerging as more advantageous and effective for the extracellular synthesis of metal nanoparticles than microbial synthesis in order to avoid elaborated process of culturing and maintaining the cell. The plant extracts itself acts as a reducing and stabilizing agent for nanoparticle synthesis, limiting particle growth and prohibiting agglomeration, resulting in the formation of desired nanoparticles. Mervat et al. [26] have found that Malva parviflora exhibited the best reducing and capping action in the formation of Ag NPs. Susmita et al. [27] have reported green synthesis of Ag NPs using Dillenia indica fruit extract. Recently Das et al. [28] have studied the synthesis of Ag NPs with Sesbania grandiflora leaf extract and observed anti bacterial properties against multi-drug resistant pathogens.
The present study explores the feasibility of utilizing Plectranthus amboinicus leaf extract as a promising material for the synthesis of silver nanoparticles. P. amboinicus spreng belongs to Lamiaceae family and is known as country borage in English. It is commonly grown in Ceylon, Moluccas and throughout India. The leaves are familiarly known to have medicinal values especially for the treatment of cough, sorethrouts and nasal congestion. It has inborn property of protecting against all sorts of bacteria, fungi and viruses. We have synthesized Ag NPs using P. amboinicus leaf extract resulting in reduction of Ag+ to Ag° and investigated its antibacterial and antifungal properties of nanoparticles against different microorganisms.
Experimental Preparation of P. amboinicus leaf extract Fresh P. amboinicus leaves were collected in and around Chittoor district, Andhra Pradesh, India. The plant materials were taxonomically identified and authenticated by the botanical survey of India. The plant leaves were cleaned with tap water and then with double distilled water. The aqueous leaf extract was prepared by collecting thoroughly washed leaves of 20 g, followed by boiling in 100 ml Milli-Q water at 60 °C for 10 min. Then the extract was collected, filtered through Whatman No. 1 filter paper and preserved at room temperature in order to use it for further experiments.
Preparation of silver nanoparticles An aqueous 0.001 M silver nitrate solution was prepared with 50 ml distilled water at room temperature. Then 20 ml of leaf extract was added to the above solution at room temperature while stirring magnetically at 1000 rpm for 5 min. This green synthesis method is effable as simple, efficient, inexpensive and environmental friendly one. Then we had observed color change from colorless to yellowish brown color giving evidence of nanoparticles formation (Fig. 1). The resulted nanoparticles were then further purified by centrifugation and to avoid excess silver ions, the silver colloid was washed with Milli-Q water for three times. Through freeze drying, a dried powder of the nanosized silver was at last obtained.
Fig. 1. Schematic synthesis procedure of green synthesized Ag NPs.
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Characterization of the synthesized Ag NPs
Results and discussion
The green synthesized Ag NPs were subjected to various characterization studies. The structural properties of the prepared nanoparticles were analyzed by Seifert 3003TT X-ray diffractometer, using Cu Ka radiation (k = 0.1546 nm). Elemental compositions of the prepared samples were examined through energy dispersive spectroscopy (EDS) by using Oxford Inca Penta FET x3 EDS instrument attached to Carl Zeiss EVO MA 15 scanning electron microscopy. The morphology of as-synthesized Ag NPs was obtained using a ZEISS, SUPRA 55 field emission scanning electron microscopy (FESEM) measurements. The particle size and structure confirmations were done by Phillips TECHNAI FE 12, transmission electron microscopy (TEM). UV–Vis absorption studies were carried out by a Perkin Elmer Lambda 950 UV– Vis–NIR spectrophotometer with a wavelength resolution better than ±0.2 nm. Photoluminescence spectra were recorded in the wavelength range of 400–700 nm using Horiba Jobin–Yvon Fluorolog-3 Spectrofluorometer (Model FL3-22PTI). Fourier transform infrared spectroscopy (FTIR) spectra of the freeze-dried samples were recorded with ATR-FTIR using Bruker Vertex-80 spectrometer. Raman spectroscopic studies were carried out using a LabRam HR 800 Raman Spectrometer with an excitation of HeANe laser at 632.8 nm. All the measurements were done at room temperature.
Structural analysis
For testing the antimicrobial activity of biosynthesized Ag NPs, we have tested them against different pathogenic microorganisms. The test organisms included the gram-negative Escherichia coli, Pseudomonas spp. and gram-positive Bacillus spp., Staphylococcus spp., regarding antibacterial activity and fungal test organisms include Aspergillus niger, Aspergillus flavus and Penicillium spp. The tested pathogens were collected from microbiology research laboratory.
Antimicrobial activity To determine the antibacterial activity of biosynthesized Ag NPs, a standard Kirby-Bauer disc diffusion assay was employed. The bacterial test organisms were grown in nutrient broth for 24 h and used for further study. Nutrient agar plates are prepared, sterilized and solidified. After solidification, 100 ll over night culture of each organism was spreaded on the petriplates using a sterile glass rod to prepare bacterial lawns. Sterile discs (diameter of 5 mm) are placed on these plates and Ag NPs were loaded at required volumes of 4 ll, 8 ll, 12 ll and 16 ll on respective discs and incubated at 37 °C for 24 h. Distilled water is used as a positive control for the antibacterial assay. After this, zones of inhibitions are observed around the discs. Dose dependent antifungal activity of synthesized Ag NPs was carried out by disc diffusion method. The fungal organisms are grown on potato dextrose broth (PDB) for 72 h and used for further experiment. Lawn culture of the respective organism was prepared by pour plate method with 200 ll of corresponding culture in potato dextrose agar (PDA) media. The discs are then placed on PDA agar plates and loaded with Ag NPs solution at specific volumes of 4 ll, 8 ll, 12 ll and 16 ll respectively on respective discs and plates are incubated for 72 h at room temperature, during which efficacy was evidenced by the zone of inhibition surrounding the discs. The diameter of all such zones is measured using a meter ruler and mean values for each organism were recorded and represented in millimeters.
D ¼ Kk=b cos h
ð1Þ
e ¼ b=4 tan h
ð2Þ
where K denotes the Scherrer’s constant (K = 0.94), k is the X-ray wavelength, b the full-width at half-maximum of diffraction line in radian and h is half diffraction angle. The size of the silver nanocrystallites was calculated by using above Debye–Scherrer’s formula (Eq. (1)) [29]. The crystallite size, microstrain and lattice parameters of all orientations are calculated and listed in Table 1. The broadening of the diffraction line may be caused by either individual or combined effect of crystallite size and microstrain. From Table 1, we can demonstrate that the broadening of the diffraction lines pertaining to XRD profile is mainly due to the crystallite size, as microstrain values are too small. Compositional analysis Fig. 3(a) elicits the EDS spectrum of synthesized nanoparticles. From this spectrum, the presence of elemental signal of the metallic silver was confirmed. Major emission energy identification peaks for silver displayed and these correspond with peaks in the spectrum at approximately 3 keV, thus attesting that silver has been correctly identified in Ag NPs. Besides the high intense peak, tiny weak signals of O, C, P and Cl were also predicted owing to the bounded biomolecules onto the surface of the Ag NPs. Further the
(111)
Intensity (a.u.)
Test microorganisms
The recorded X-ray diffraction profile for the dried Ag NPs is shown in Fig. 2. The XRD pattern indicates four distinct diffraction peaks at 2h values of 38.1°, 46.2°, 64.5° and 77.3° indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) lattice planes of face centered cubic (fcc) structure of metallic silver and is consistence with JCPDS data [No. 04-0783]. The broadening and strong signals of pattern evinces that the products are nanosized and well crystallized respectively. The peak corresponding to (1 1 1) plane is more intense than other planes suggesting it as a predominant orientation. No extra diffraction peaks in XRD pattern are observed, revealing that synthesized Ag NPs are essentially pure. One can calculate the values of average crystallite size (D) and microstrain (e) from XRD spectrum using the following equations,
(200)
(220)
20
30
40
50
60
(311)
70
2θ (degree) Fig. 2. X-ray diffraction profile of biosynthesized Ag NPs.
80
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Fig. 4. (a) FESEM and (b) TEM images of Ag NPs (inset of the figure shows the SAED pattern of Ag NPs).
Table 1 The variation of crystallite size, lattice parameter and microstrain values of biosynthesized nanoparticles. 2h
Orientation
Crystallite size (nm)
Lattice parameter (nm)
Microstrain
38.1 46.2 64.5 77.3
(1 1 1) (2 0 0) (2 2 0) (3 1 1)
12.8 27.8 15.4 9.3
0.4084 0.4081 0.4013 0.4087
0.00861 0.00330 0.00437 0.00617
size and surface morphology of nanoparticles. Fig. 4(a) shows the FESEM photograph, indicating a high density of Ag NPs synthesized using aqueous P. amboinicus leaf extract confirmed by EDS. The average particle size was found to be 20 nm from FESEM measurements. A transmission electron microscopy (TEM) was employed to analyze size, shape and morphologies of obtained Ag NPs. Fig. 4(b) depicts the TEM micrograph of Ag NPs. TEM analysis proved the formation of nanocrystalline silver particles which are somewhat agglomerated and average particle size was estimated to be 18 nm. The crystallinity of Ag NPs was observed by selected area emission diffraction (SAED). The inset of Fig. 4(b) shows SAED pattern recorded by directing the electron beam perpendicular to nanoparticles. The characteristic fringe array can be indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) of the pure face centered cubic (fcc) lattice structure commonly found for silver crystal, which was also evident from XRD results.
Optical properties
Fig. 3. (a) EDAX spectrum (inset shows the quantitative information) of synthesized Ag NPs using P. amboinicus leaf extract.
inset of Fig. 3(a) shows the quantitative information of biosynthesized Ag NPs. It was attested that Ag, O, C, Cl and P elements exhibited weight percentages of 88.3%, 3.8%, 3.2%, 3.4% and 1.3% respectively. From the selected area elemental mapping (Supplementary file Fig. 3(b)), it was noticed that Ag, O, C, Cl and P elements were present throughout the sample in homogeneous manner, which confirms the uniformity of the sample. Morphological studies Green synthesized Ag NPs are subjected to field emission scanning electron microscopy (FESEM) analysis in order to know the
The optical absorption spectrum of metal nanoparticles is dominated by the surface plasmon resonance (SPR) which exhibits a shift in band position depending upon the particle size, shape and aggregation state. Fig. 5 shows the UV–Vis absorption spectra of bio-synthesized silver solution and P. amboinicus leaf extract. Ag NPs exhibited maximum absorption band at 428 nm and no absorption band for leaf extract. This band can be attributed to the surface plasmon resonance due to collective oscillation of surface electrons [30]. UV–Vis absorption measurements in the range 350–600 nm can provide deep insight into the optical properties of the formed nanosized silver particles. The inset in Fig. 5 shows samples of pure leaf extract and synthesized Ag NPs solution. From this we observed that the color change was due to formation of Ag NPs results in the formation of absorption band. Biosynthesized Ag NPs are found to be photoluminescent. The visible luminescence of silver is intended to an excitation of electrons from occupied d bands into states above the Fermi level. Subsequent electron–phonon and hole–phonon scattering process results in energy loss and consequently photoluminescent radiative recombination of an electron from an occupied sp band with the hole [31,32]. The photoluminescent spectra of biosynthesized Ag NPs at different excitation wavelengths are depicted in Fig. 6. PL spectra enumerate the shifting of peak towards red in a regular manner with increasing excitation wavelength from 350 to 420 nm. Basak et al. [33] had also reported the similar shifting with increasing excitation wavelength in the range 370–550 nm and
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668
837
1259
951
0.6
1373
1454
0.7
1517
b
1681 1659
Transmittance (%)
Absorbance (a.u)
0.8
a a-Leaf extract b-Green synthesized Ag nanoparticles
0.5 1800
300
400
500
600
700
800
@Excitation wavelengths
Intensity (a.u.)
350 nm 370 nm 390 nm 410 nm 420 nm
1200
900
600
Wavenumber (cm-1)
Wavelength (nm) Fig. 5. UV–Vis absorbance spectrum of green synthesized Ag NPs using P. amboinicus leaf extract.
1500
Fig. 7. Fourier nanoparticles.
transform
infrared
spectrum
of
green
synthesized
silver
of nanoparticles. Weak signals at 668 and 1454 cm1 are suggestive to CACl stretching modes of alkyl halides and involvement of aliphatic and aromatic (CAH) plane deformations of methyl, methylene and methoxy groups in the reduction process respectively [35]. It is well known that biological components interact with metal salts and mediate reduction process with these functional groups. FTIR spectrum clearly confirmed the presence of nitro groups, alkenes, amides, alkyl halides, aliphatic and aromatic groups in the leaf extract of P. amboinicus and had ability to perform both capping and reduction functions in Ag NPs formation. Raman spectroscopic analysis
400
450
500
550
600
650
700
Wavelength (nm) Fig. 6. Photoluminescence spectra of biosynthesized Ag NPs at different excitation wavelengths.
speculated this phenomenon as a result of resonance between the luminescence transition and silver plasmons. Excitation of green synthesized Ag NPs by radiation in the range of SPR (420 nm), the absorption of radiation by nanoparticles did not allowed the radiative energy transfer to the emitting particles. This may be the reason for decrease of PL intensity, when excited in SPR range.
The Raman spectroscopy is having capability to provide well-off information about the molecular structure of the sample and to draw conclusions about composition of very complex systems such as biological materials. Fig. 8 displays Raman spectra of green synthesized Ag NPs excited by wavelength of 632.8 nm with HeANe laser. From Fig. 8, we have observed the peaks at 1327, 1582 cm1 and small peak at 1379 cm1 corresponding to the molecules bonded on the surface of the formed Ag NPs. The prominent peak at 1327 cm1 is characteristic of the NO2 stretching mode which falls in the range 1300–1370 cm1 [37]. Small peak at 1379 cm1 is assigned to CH3 symmetric deformation and COOA
1327
* 1379 *
FTIR studies Raman Intensity (a.u.)
In order to determine the possible bio-reducing functional groups involved in reduction process and their unique interactions with Ag NPs, FTIR analysis was performed. The band intensities of infrared spectra of Ag NPs are depicted in Fig. 7. The spectra showed strong absorption peaks at 951, 1259, 1373 and 1517 cm1; weak signals at 668, 837, 1454, 1659 and 1681 cm1. The strong peaks at 1373 and 1517 cm1 are characteristic of NAO stretching of nitro groups [34]. The peaks located at 951, 837 and 1259 cm1 corresponds to stretching vibrations of CAOCH3, CAH stretching of alkenes and CAO stretching aromatic side chains of proteins which have been found to be responsible for reduction of metal ions [35,36]. Besides these, small peaks at 1659 and 1681 cm1 are assigned to C@O stretching vibrations of amides characteristic of plant proteins which acts as surfactants
1582
*
600
800
1000
1200
1400
1600
1800
Raman shift (cm-1) Fig. 8. Raman spectrum of green synthesized Ag NPs using P. amboinicus leaf extract.
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Table 2 The variation of zone of inhibitions at different pathogenic bacteria and fungi as a function of bio-silver volumes. Ag NPs volume (ll)
4 8 12 16
Zone of inhibition of tested pathogenic bacterium (mm)
Zone of inhibition of tested pathogenic fungi (mm)
E. coli
Pseudomonas spp.
Staphylococcus spp.
Bacillus spp.
A. niger
A. flavus
Penicillium spp.
3 5 7 9
3 4 6 7
2 3 5 7
2 3 5 6
3 4 5 6
2 3 4 5
3 5 6 7
symmetric stretching modes of the vibrations and another strong peak at 1582 cm1 corresponds to aromatic CAC stretching [38]. From this discussion it is reasonable to conclude that the surface of prepared Ag NPs are bonded to other biomolecules, which plays important role in nanoparticles formation as evidenced by FTIR results. Antimicrobial studies The synthesized Ag NPs are subjected to antimicrobial activity. The silver nanoparticles exhibited better antimicrobial efficacy by inhibiting the growth and multiplication of pathogenic bacteria like E. coli, Pseudomonas spp., Bacillus spp. and Staphylococcus spp. and fungal pathogens like A. niger, A. flavus and Penicillium spp. Supplementary file Fig. 9 elicits the antibacterial and antifungal activity of green synthesized Ag NPs. E. coli depicted the highest sensitivity to nanoparticles compared to other tested bacterial pathogens. Next to this, nanoparticles are controlling Pseudomonas spp., Staphylococcus spp. and at last showed lower activity against Bacillus spp. The fungal pathogens showed effective activity against Penicillium spp., next towards A. niger and A. flavus. Zone of inhibition values for all tested microbes are listed in Table 2. Conclusions Low cost, non-toxic, proficient and eco-friendly green synthesis of Ag NPs through P. amboinicus leaf extract was successfully prepared. Biomolecules of aqueous leaf extract acts as both reducing and capping agents and hence resulting in reduction of chemical toxicity compared to chemical method. The crystalline nature of biosynthesized silver nanoparticles is evident from XRD diffraction peaks and circular fringes in SAED pattern. The average crystallite size of nanoparticles deduced from XRD results is found to be 16 nm. Morphological studies showed the formation of nearly spherical nanoparticles. The spectroscopic UV–Vis characterization elucidates the formation of stable spherical nanoparticles without any anisotropic presence and shown SPR at 428 nm. PL spectra elucidate red-shift when nanoparticles were excited with different wavelengths. The biosynthesized Ag NPs have displayed high antibacterial and antifungal efficacy against E. coli and penicillium spp. respectively. Finally, the present study emphasizes the use of P. amboinicus for biosynthesis mediated Ag NPs with potent antimicrobial efficacy and is more efficient followed by their usage in biological applications due to the absence of toxic chemicals. Acknowledgements The one of the author B. Ajitha would like to convey her sincere thanks to University Grants Commission (UGC), New Delhi, for awarding UGC-BSR Fellowship in sciences for meritorious students.
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Biosynthesis of silver nanoparticles using Plectranthus amboinicus leaf extract and its antimicrobial activity B. Ajitha a, Y. Ashok Kumar Reddy b, P. Sreedhara Reddy a,⇑ a b
Department of Physics, Sri Venkateswara University, Tirupati 517502, India Department of Electrical Engineering, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, South Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Nearly spherical Ag NPs are prepared
using P. amboinicus leaf extract. The green synthesis route is simple,
nontoxic, reliable and eco-friendly benign. XRD and SAED patterns attested the crystalline nature of the samples. Biomolecules of the leaf extract play dual role as both reducing and capping agents. It exhibited effective antimicrobial efficacy against E. coli and Penicillium spp.
a r t i c l e
i n f o
Article history: Received 22 October 2013 Received in revised form 20 February 2014 Accepted 23 February 2014 Available online 7 March 2014 Keywords: Green synthesis method Silver nanoparticles Leaf extract Particle size Absorbance Antimicrobial activity
a b s t r a c t This study reports the simple green synthesis method for the preparation of silver nanoparticles (Ag NPs) using Plectranthus amboinicus leaf extract. The pathway of nanoparticles formation is by means of reduction of AgNO3 by leaf extract, which acts as both reducing and capping agents. Synthesized Ag NPs were subjected to different characterizations for studying the structural, chemical, morphological, optical and antimicrobial properties. The bright circular fringes in SAED pattern and diffraction peaks in XRD profile reveals high crystalline nature of biosynthesized Ag NPs. Morphological studies shows the formation of nearly spherical nanoparticles. FTIR spectrum confirms the existence of various functional groups of biomolecules capping the nanoparticles. UV–visible spectrum displays single SPR band at 428 nm indicating the absence of anisotropic particles. The synthesized Ag NPs exhibited better antimicrobial property towards gram negative Escherichia coli and towards tested Penicillium spp. than other tested microorganisms using disc diffusion method. Finally it has proven that the synthesized bio-inspired Ag NPs have potent antimicrobial effect. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Nowadays nanotechnology is gaining profoundness owing to its increase of vital role in most dynamic areas of research in modern materials science. Recently is has broadened the scope of elevating ⇑ Corresponding author. Tel.: +91 877 2289472; fax: +91 877 2249611. E-mail address: [email protected] (P. Sreedhara Reddy). http://dx.doi.org/10.1016/j.saa.2014.02.105 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
research in various scientific disciplines due to their unique properties compared with bulk counterparts. Nanoparticles of noble metals have attracted immense interest incurring applications in catalysis, electronics, optics, environmental and biomedical applications due to their quantum confinement effects, antimicrobial activity and their large reactive surfaces [1–3]. In particular, silver nanoparticles have expunged a considerable attention in present tremendous research due to their exquisite fascinating spectrum
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of physical properties such as catalytic, optical, electrical and antimicrobial applications [4,5] and their significant potential selective activity in a very wide range of applications. They are even being projected as future generation antimicrobial agents [6]. Attribution of small size and high surface to volume ratio of metal nanoparticles enhances its bactericidal property which allows them to have an effect on microorganisms membranes and it is not simply due to release of metal ions in solutions [7]. The prerequisite nature of an ideal antimicrobial agent [8,9], material should have the ability to inhibit the formation of bio-film and cause inactivation/extermination of microbes either through targeting the cell wall for an antimicrobial activity or through direct contact-killing phenomena. Increasing exigency to employ good antimicrobial agent due to ever-increasing resistant bacteria, much attention has been focused on it and researchers found that Ag NPs have significantly displayed all these properties [8–10] serving as a promising candidate in having improved antimicrobial properties. Hence, use of silver nanoparticle coatings in a hospital environment could have a considerable drastic impact on declining the transmission of multidrug resistant bacteria between patients such as methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus is part of the natural flora of the human body and is illustrated as an opportunistic pathogen which can cause serious infections [11]. There are many ways to synthesize silver nanoparticles through physical and chemical methods such as electrochemical [12], c-radiation [13], photochemical [14], laser ablation [15] and chemical reduction [16]. However, physical methods have some disadvantages such as expensive, complicated vacuum techniques and apparatus. Normally, chemical processes are having two main problems. Primarily, high surface energy of nanoparticles enhances reactivity and they often undergo aggregation in liquid dispersions. This aggregation can be prevented during the synthesis [17–19] by the formation of self-assembled monolayers of organic templates, such as polymers, surfactants and DNA on the nanoparticle surface. Another problem is that the reducing reagents, such as citric acid, sodium borohydride, or other organic compounds, may exhibit environmental toxicity or biological hazards [20,21]. Due to these consequences, usage of chemicals is still the subject of paramount concern because they limit the nanoparticles applications in clinical fields. Therefore, biosynthesis of clean, biocompatible, nontoxic and environmental-friendly nanoparticles produced both extracellularly and intracellularly deserves merit [1,22]. Even though there are well established methods, growing need of cost-effective, environmental benign and to produce highly stable nanoparticles still had to be exploited. In this pursuit, researchers have focused on using microorganisms, living plants, plant biomass and plant extracts for the synthesis of bio-inspired metal nanoparticles. Biogenic synthesis is useful and seems to have good strategy not only pertaining to its reduced environmental impact [23,24] compared with physicochemical methods, but also we can produce large quantities of nanoparticles without any contamination and have a well-defined size and morphology [25]. At present, plant extracts usage is emerging as more advantageous and effective for the extracellular synthesis of metal nanoparticles than microbial synthesis in order to avoid elaborated process of culturing and maintaining the cell. The plant extracts itself acts as a reducing and stabilizing agent for nanoparticle synthesis, limiting particle growth and prohibiting agglomeration, resulting in the formation of desired nanoparticles. Mervat et al. [26] have found that Malva parviflora exhibited the best reducing and capping action in the formation of Ag NPs. Susmita et al. [27] have reported green synthesis of Ag NPs using Dillenia indica fruit extract. Recently Das et al. [28] have studied the synthesis of Ag NPs with Sesbania grandiflora leaf extract and observed anti bacterial properties against multi-drug resistant pathogens.
The present study explores the feasibility of utilizing Plectranthus amboinicus leaf extract as a promising material for the synthesis of silver nanoparticles. P. amboinicus spreng belongs to Lamiaceae family and is known as country borage in English. It is commonly grown in Ceylon, Moluccas and throughout India. The leaves are familiarly known to have medicinal values especially for the treatment of cough, sorethrouts and nasal congestion. It has inborn property of protecting against all sorts of bacteria, fungi and viruses. We have synthesized Ag NPs using P. amboinicus leaf extract resulting in reduction of Ag+ to Ag° and investigated its antibacterial and antifungal properties of nanoparticles against different microorganisms.
Experimental Preparation of P. amboinicus leaf extract Fresh P. amboinicus leaves were collected in and around Chittoor district, Andhra Pradesh, India. The plant materials were taxonomically identified and authenticated by the botanical survey of India. The plant leaves were cleaned with tap water and then with double distilled water. The aqueous leaf extract was prepared by collecting thoroughly washed leaves of 20 g, followed by boiling in 100 ml Milli-Q water at 60 °C for 10 min. Then the extract was collected, filtered through Whatman No. 1 filter paper and preserved at room temperature in order to use it for further experiments.
Preparation of silver nanoparticles An aqueous 0.001 M silver nitrate solution was prepared with 50 ml distilled water at room temperature. Then 20 ml of leaf extract was added to the above solution at room temperature while stirring magnetically at 1000 rpm for 5 min. This green synthesis method is effable as simple, efficient, inexpensive and environmental friendly one. Then we had observed color change from colorless to yellowish brown color giving evidence of nanoparticles formation (Fig. 1). The resulted nanoparticles were then further purified by centrifugation and to avoid excess silver ions, the silver colloid was washed with Milli-Q water for three times. Through freeze drying, a dried powder of the nanosized silver was at last obtained.
Fig. 1. Schematic synthesis procedure of green synthesized Ag NPs.
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Characterization of the synthesized Ag NPs
Results and discussion
The green synthesized Ag NPs were subjected to various characterization studies. The structural properties of the prepared nanoparticles were analyzed by Seifert 3003TT X-ray diffractometer, using Cu Ka radiation (k = 0.1546 nm). Elemental compositions of the prepared samples were examined through energy dispersive spectroscopy (EDS) by using Oxford Inca Penta FET x3 EDS instrument attached to Carl Zeiss EVO MA 15 scanning electron microscopy. The morphology of as-synthesized Ag NPs was obtained using a ZEISS, SUPRA 55 field emission scanning electron microscopy (FESEM) measurements. The particle size and structure confirmations were done by Phillips TECHNAI FE 12, transmission electron microscopy (TEM). UV–Vis absorption studies were carried out by a Perkin Elmer Lambda 950 UV– Vis–NIR spectrophotometer with a wavelength resolution better than ±0.2 nm. Photoluminescence spectra were recorded in the wavelength range of 400–700 nm using Horiba Jobin–Yvon Fluorolog-3 Spectrofluorometer (Model FL3-22PTI). Fourier transform infrared spectroscopy (FTIR) spectra of the freeze-dried samples were recorded with ATR-FTIR using Bruker Vertex-80 spectrometer. Raman spectroscopic studies were carried out using a LabRam HR 800 Raman Spectrometer with an excitation of HeANe laser at 632.8 nm. All the measurements were done at room temperature.
Structural analysis
For testing the antimicrobial activity of biosynthesized Ag NPs, we have tested them against different pathogenic microorganisms. The test organisms included the gram-negative Escherichia coli, Pseudomonas spp. and gram-positive Bacillus spp., Staphylococcus spp., regarding antibacterial activity and fungal test organisms include Aspergillus niger, Aspergillus flavus and Penicillium spp. The tested pathogens were collected from microbiology research laboratory.
Antimicrobial activity To determine the antibacterial activity of biosynthesized Ag NPs, a standard Kirby-Bauer disc diffusion assay was employed. The bacterial test organisms were grown in nutrient broth for 24 h and used for further study. Nutrient agar plates are prepared, sterilized and solidified. After solidification, 100 ll over night culture of each organism was spreaded on the petriplates using a sterile glass rod to prepare bacterial lawns. Sterile discs (diameter of 5 mm) are placed on these plates and Ag NPs were loaded at required volumes of 4 ll, 8 ll, 12 ll and 16 ll on respective discs and incubated at 37 °C for 24 h. Distilled water is used as a positive control for the antibacterial assay. After this, zones of inhibitions are observed around the discs. Dose dependent antifungal activity of synthesized Ag NPs was carried out by disc diffusion method. The fungal organisms are grown on potato dextrose broth (PDB) for 72 h and used for further experiment. Lawn culture of the respective organism was prepared by pour plate method with 200 ll of corresponding culture in potato dextrose agar (PDA) media. The discs are then placed on PDA agar plates and loaded with Ag NPs solution at specific volumes of 4 ll, 8 ll, 12 ll and 16 ll respectively on respective discs and plates are incubated for 72 h at room temperature, during which efficacy was evidenced by the zone of inhibition surrounding the discs. The diameter of all such zones is measured using a meter ruler and mean values for each organism were recorded and represented in millimeters.
D ¼ Kk=b cos h
ð1Þ
e ¼ b=4 tan h
ð2Þ
where K denotes the Scherrer’s constant (K = 0.94), k is the X-ray wavelength, b the full-width at half-maximum of diffraction line in radian and h is half diffraction angle. The size of the silver nanocrystallites was calculated by using above Debye–Scherrer’s formula (Eq. (1)) [29]. The crystallite size, microstrain and lattice parameters of all orientations are calculated and listed in Table 1. The broadening of the diffraction line may be caused by either individual or combined effect of crystallite size and microstrain. From Table 1, we can demonstrate that the broadening of the diffraction lines pertaining to XRD profile is mainly due to the crystallite size, as microstrain values are too small. Compositional analysis Fig. 3(a) elicits the EDS spectrum of synthesized nanoparticles. From this spectrum, the presence of elemental signal of the metallic silver was confirmed. Major emission energy identification peaks for silver displayed and these correspond with peaks in the spectrum at approximately 3 keV, thus attesting that silver has been correctly identified in Ag NPs. Besides the high intense peak, tiny weak signals of O, C, P and Cl were also predicted owing to the bounded biomolecules onto the surface of the Ag NPs. Further the
(111)
Intensity (a.u.)
Test microorganisms
The recorded X-ray diffraction profile for the dried Ag NPs is shown in Fig. 2. The XRD pattern indicates four distinct diffraction peaks at 2h values of 38.1°, 46.2°, 64.5° and 77.3° indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) lattice planes of face centered cubic (fcc) structure of metallic silver and is consistence with JCPDS data [No. 04-0783]. The broadening and strong signals of pattern evinces that the products are nanosized and well crystallized respectively. The peak corresponding to (1 1 1) plane is more intense than other planes suggesting it as a predominant orientation. No extra diffraction peaks in XRD pattern are observed, revealing that synthesized Ag NPs are essentially pure. One can calculate the values of average crystallite size (D) and microstrain (e) from XRD spectrum using the following equations,
(200)
(220)
20
30
40
50
60
(311)
70
2θ (degree) Fig. 2. X-ray diffraction profile of biosynthesized Ag NPs.
80
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Fig. 4. (a) FESEM and (b) TEM images of Ag NPs (inset of the figure shows the SAED pattern of Ag NPs).
Table 1 The variation of crystallite size, lattice parameter and microstrain values of biosynthesized nanoparticles. 2h
Orientation
Crystallite size (nm)
Lattice parameter (nm)
Microstrain
38.1 46.2 64.5 77.3
(1 1 1) (2 0 0) (2 2 0) (3 1 1)
12.8 27.8 15.4 9.3
0.4084 0.4081 0.4013 0.4087
0.00861 0.00330 0.00437 0.00617
size and surface morphology of nanoparticles. Fig. 4(a) shows the FESEM photograph, indicating a high density of Ag NPs synthesized using aqueous P. amboinicus leaf extract confirmed by EDS. The average particle size was found to be 20 nm from FESEM measurements. A transmission electron microscopy (TEM) was employed to analyze size, shape and morphologies of obtained Ag NPs. Fig. 4(b) depicts the TEM micrograph of Ag NPs. TEM analysis proved the formation of nanocrystalline silver particles which are somewhat agglomerated and average particle size was estimated to be 18 nm. The crystallinity of Ag NPs was observed by selected area emission diffraction (SAED). The inset of Fig. 4(b) shows SAED pattern recorded by directing the electron beam perpendicular to nanoparticles. The characteristic fringe array can be indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) of the pure face centered cubic (fcc) lattice structure commonly found for silver crystal, which was also evident from XRD results.
Optical properties
Fig. 3. (a) EDAX spectrum (inset shows the quantitative information) of synthesized Ag NPs using P. amboinicus leaf extract.
inset of Fig. 3(a) shows the quantitative information of biosynthesized Ag NPs. It was attested that Ag, O, C, Cl and P elements exhibited weight percentages of 88.3%, 3.8%, 3.2%, 3.4% and 1.3% respectively. From the selected area elemental mapping (Supplementary file Fig. 3(b)), it was noticed that Ag, O, C, Cl and P elements were present throughout the sample in homogeneous manner, which confirms the uniformity of the sample. Morphological studies Green synthesized Ag NPs are subjected to field emission scanning electron microscopy (FESEM) analysis in order to know the
The optical absorption spectrum of metal nanoparticles is dominated by the surface plasmon resonance (SPR) which exhibits a shift in band position depending upon the particle size, shape and aggregation state. Fig. 5 shows the UV–Vis absorption spectra of bio-synthesized silver solution and P. amboinicus leaf extract. Ag NPs exhibited maximum absorption band at 428 nm and no absorption band for leaf extract. This band can be attributed to the surface plasmon resonance due to collective oscillation of surface electrons [30]. UV–Vis absorption measurements in the range 350–600 nm can provide deep insight into the optical properties of the formed nanosized silver particles. The inset in Fig. 5 shows samples of pure leaf extract and synthesized Ag NPs solution. From this we observed that the color change was due to formation of Ag NPs results in the formation of absorption band. Biosynthesized Ag NPs are found to be photoluminescent. The visible luminescence of silver is intended to an excitation of electrons from occupied d bands into states above the Fermi level. Subsequent electron–phonon and hole–phonon scattering process results in energy loss and consequently photoluminescent radiative recombination of an electron from an occupied sp band with the hole [31,32]. The photoluminescent spectra of biosynthesized Ag NPs at different excitation wavelengths are depicted in Fig. 6. PL spectra enumerate the shifting of peak towards red in a regular manner with increasing excitation wavelength from 350 to 420 nm. Basak et al. [33] had also reported the similar shifting with increasing excitation wavelength in the range 370–550 nm and
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668
837
1259
951
0.6
1373
1454
0.7
1517
b
1681 1659
Transmittance (%)
Absorbance (a.u)
0.8
a a-Leaf extract b-Green synthesized Ag nanoparticles
0.5 1800
300
400
500
600
700
800
@Excitation wavelengths
Intensity (a.u.)
350 nm 370 nm 390 nm 410 nm 420 nm
1200
900
600
Wavenumber (cm-1)
Wavelength (nm) Fig. 5. UV–Vis absorbance spectrum of green synthesized Ag NPs using P. amboinicus leaf extract.
1500
Fig. 7. Fourier nanoparticles.
transform
infrared
spectrum
of
green
synthesized
silver
of nanoparticles. Weak signals at 668 and 1454 cm1 are suggestive to CACl stretching modes of alkyl halides and involvement of aliphatic and aromatic (CAH) plane deformations of methyl, methylene and methoxy groups in the reduction process respectively [35]. It is well known that biological components interact with metal salts and mediate reduction process with these functional groups. FTIR spectrum clearly confirmed the presence of nitro groups, alkenes, amides, alkyl halides, aliphatic and aromatic groups in the leaf extract of P. amboinicus and had ability to perform both capping and reduction functions in Ag NPs formation. Raman spectroscopic analysis
400
450
500
550
600
650
700
Wavelength (nm) Fig. 6. Photoluminescence spectra of biosynthesized Ag NPs at different excitation wavelengths.
speculated this phenomenon as a result of resonance between the luminescence transition and silver plasmons. Excitation of green synthesized Ag NPs by radiation in the range of SPR (420 nm), the absorption of radiation by nanoparticles did not allowed the radiative energy transfer to the emitting particles. This may be the reason for decrease of PL intensity, when excited in SPR range.
The Raman spectroscopy is having capability to provide well-off information about the molecular structure of the sample and to draw conclusions about composition of very complex systems such as biological materials. Fig. 8 displays Raman spectra of green synthesized Ag NPs excited by wavelength of 632.8 nm with HeANe laser. From Fig. 8, we have observed the peaks at 1327, 1582 cm1 and small peak at 1379 cm1 corresponding to the molecules bonded on the surface of the formed Ag NPs. The prominent peak at 1327 cm1 is characteristic of the NO2 stretching mode which falls in the range 1300–1370 cm1 [37]. Small peak at 1379 cm1 is assigned to CH3 symmetric deformation and COOA
1327
* 1379 *
FTIR studies Raman Intensity (a.u.)
In order to determine the possible bio-reducing functional groups involved in reduction process and their unique interactions with Ag NPs, FTIR analysis was performed. The band intensities of infrared spectra of Ag NPs are depicted in Fig. 7. The spectra showed strong absorption peaks at 951, 1259, 1373 and 1517 cm1; weak signals at 668, 837, 1454, 1659 and 1681 cm1. The strong peaks at 1373 and 1517 cm1 are characteristic of NAO stretching of nitro groups [34]. The peaks located at 951, 837 and 1259 cm1 corresponds to stretching vibrations of CAOCH3, CAH stretching of alkenes and CAO stretching aromatic side chains of proteins which have been found to be responsible for reduction of metal ions [35,36]. Besides these, small peaks at 1659 and 1681 cm1 are assigned to C@O stretching vibrations of amides characteristic of plant proteins which acts as surfactants
1582
*
600
800
1000
1200
1400
1600
1800
Raman shift (cm-1) Fig. 8. Raman spectrum of green synthesized Ag NPs using P. amboinicus leaf extract.
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Table 2 The variation of zone of inhibitions at different pathogenic bacteria and fungi as a function of bio-silver volumes. Ag NPs volume (ll)
4 8 12 16
Zone of inhibition of tested pathogenic bacterium (mm)
Zone of inhibition of tested pathogenic fungi (mm)
E. coli
Pseudomonas spp.
Staphylococcus spp.
Bacillus spp.
A. niger
A. flavus
Penicillium spp.
3 5 7 9
3 4 6 7
2 3 5 7
2 3 5 6
3 4 5 6
2 3 4 5
3 5 6 7
symmetric stretching modes of the vibrations and another strong peak at 1582 cm1 corresponds to aromatic CAC stretching [38]. From this discussion it is reasonable to conclude that the surface of prepared Ag NPs are bonded to other biomolecules, which plays important role in nanoparticles formation as evidenced by FTIR results. Antimicrobial studies The synthesized Ag NPs are subjected to antimicrobial activity. The silver nanoparticles exhibited better antimicrobial efficacy by inhibiting the growth and multiplication of pathogenic bacteria like E. coli, Pseudomonas spp., Bacillus spp. and Staphylococcus spp. and fungal pathogens like A. niger, A. flavus and Penicillium spp. Supplementary file Fig. 9 elicits the antibacterial and antifungal activity of green synthesized Ag NPs. E. coli depicted the highest sensitivity to nanoparticles compared to other tested bacterial pathogens. Next to this, nanoparticles are controlling Pseudomonas spp., Staphylococcus spp. and at last showed lower activity against Bacillus spp. The fungal pathogens showed effective activity against Penicillium spp., next towards A. niger and A. flavus. Zone of inhibition values for all tested microbes are listed in Table 2. Conclusions Low cost, non-toxic, proficient and eco-friendly green synthesis of Ag NPs through P. amboinicus leaf extract was successfully prepared. Biomolecules of aqueous leaf extract acts as both reducing and capping agents and hence resulting in reduction of chemical toxicity compared to chemical method. The crystalline nature of biosynthesized silver nanoparticles is evident from XRD diffraction peaks and circular fringes in SAED pattern. The average crystallite size of nanoparticles deduced from XRD results is found to be 16 nm. Morphological studies showed the formation of nearly spherical nanoparticles. The spectroscopic UV–Vis characterization elucidates the formation of stable spherical nanoparticles without any anisotropic presence and shown SPR at 428 nm. PL spectra elucidate red-shift when nanoparticles were excited with different wavelengths. The biosynthesized Ag NPs have displayed high antibacterial and antifungal efficacy against E. coli and penicillium spp. respectively. Finally, the present study emphasizes the use of P. amboinicus for biosynthesis mediated Ag NPs with potent antimicrobial efficacy and is more efficient followed by their usage in biological applications due to the absence of toxic chemicals. Acknowledgements The one of the author B. Ajitha would like to convey her sincere thanks to University Grants Commission (UGC), New Delhi, for awarding UGC-BSR Fellowship in sciences for meritorious students.
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