Accepted Manuscript Lantana camara leaf extract mediated silver nanoparticles: Antibacterial, Green catalyst B. Ajitha, Y. Ashok Kumar Reddy, Syed Shameer, K.M. Rajesh, Y. Suneetha, P. Sreedhara Reddy PII: DOI: Reference:
S1011-1344(15)00183-9 http://dx.doi.org/10.1016/j.jphotobiol.2015.05.020 JPB 10051
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
25 November 2014 11 May 2015 24 May 2015
Please cite this article as: B. Ajitha, Y. Ashok Kumar Reddy, S. Shameer, K.M. Rajesh, Y. Suneetha, P. Sreedhara Reddy, Lantana camara leaf extract mediated silver nanoparticles: Antibacterial, Green catalyst, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.05.020
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Lantana camara leaf extract mediated silver nanoparticles: Antibacterial, Green catalyst B. Ajithaa*, Y. Ashok Kumar Reddyb, Syed Shameerc, K.M. Rajesha, Y. Suneethad, and P. Sreedhara Reddya 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 c Department of Microbiology, Sri Venkateswara University, Tirupati-517502, India d Department of Zoology, Sri Venkateswara University, Tirupati-517502, India ABSTRACT
Silver nanoparticles (AgNPs) have been synthesized by Lantana camara leaf extract through simple green route and evaluated their antibacterial and catalytic activities. The leaf extract (LE) itself acts as both reducing and stabilizing agent at once for desired nanoparticle synthesis. The colorless reaction mixture turns to yellowish brown attesting the AgNPs formation and displayed UV-Vis absorption spectra. Structural analysis confirms the crystalline nature and formation of fcc structured metallic silver with majority (111) facets. Morphological studies elicit the formation of almost spherical shaped nanoparticles and as AgNO3 concentration is increased, there is an increment in the particle size. The FTIR analysis evidences the presence of various functional groups of biomolecules of LE is responsible for stabilization of AgNPs. Zeta potential measurement attests the higher stability of synthesized AgNPs. The synthesized AgNPs exhibited good antibacterial activity when tested against Escherichia coli, Pseudomonas spp., Bacillus spp. and Staphylococcus spp. using standard Kirby-Bauer disc diffusion assay. Furthermore, they showed good catalytic activity on the reduction of methylene blue by L. camara extract which is monitored and confirmed by the UV-Vis spectrophotometer. Keywords: Leaf extract, Green synthesis, AgNPs, Antibacterial activity, Green catalyst *Corresponding author. Tel: +91 877 2289472; Fax: +91 877 2249611. E-mail address: [email protected] (B. Ajitha). 1
1. Introduction Nanotechnology is the latest and one of the most promising areas of research in modern medical science. Nanoparticles are of special group of materials with unique features and extensive applications in diverse fields [1]. Many scientists have great interest in studying these typical features in fact, nanoparticles display completely peculiar properties compared to large size counterparts [2]. Silver nanoparticles (AgNPs) have found tremendous applications in the areas of catalysis, optoelectronics, detection and diagnostic, antimicrobials and therapeutics [3-6] due to their better catalytic, optical and electrical properties. Many attempts have been made to use AgNPs as an anti-cancer agent and they have all turned up positive [7]. To date, various approaches were developed for the synthesis of AgNPs through physical and chemical methods such as electrochemical [8], γ-radiation [9], photochemical [10], laser ablation [11], chemical reduction [12] and recently via green synthesis method [13]. However, most of the reported methods involve more than one steps and leads to unfavorable impact in the medical applications due to usage of unsafe chemicals. There is a rising commercial requirement of eco-friendly and nontoxic nanoparticles. In this regard it has been proven that the green synthesis method is a reliable, simple, low cost and convenient route for the synthesis of AgNPs. Synthesis of gold, silver and bimetallic nanoparticles with different plant extracts have been reported earlier [14]. Lantana camara plant is commonly known as red sage or wild sage or “pulikampa” in Telugu and is a significant weed found throughout India. The essential oil and extracts of the plant are used in herbal medicines for the treatment of various human diseases such as skin itches, leprosy, cancer, chicken pox, measles, asthma, ulcers, tumors, high blood pressure, tetanus, rheumatism etc [15, 16]. Extracts from leaves have been reported to have antifungal, 2
antiproliferative, antibacterial, nematicidal, termicidal, anthelmintic and anticancer activities [1622]. Research on the chemical composition of L. camara plant revealed the presence of terpenoids, steroids, flavonoids and alkaloids as major constituents [16, 23, 24]. In earlier, we studied the synthesis of AgNPs with different leaf extract concentrations using L. camara leaf extract [25]. However, the present work aim is to develop a novel approach for the green synthesis of AgNPs with different AgNO3 concentrations using aqueous leaf extract of L. camara and exploring its catalytic activity in addition to antibacterial activity against some selected gram positive and gram negative organisms. 2. Experimental 2.1 Materials All the reagents purchased were of analytical grade and used as received. Silver nitrate (AgNO3) with ≥ 99.5% purity and methylene blue were purchased from Sigma-Aldrich, India. Fresh leaves of Lantana camara have been harvested from in and around Chittoor district, A.P, India. Milli-Q water was used throughout the experiment. 2.2 Preparation of leaf extract Freshly collected leaves were used for the preparation of Lantana camara leaf extract. The leaves were collected from plant and surface washed at first with running tap water followed by Milli-Q water and then shade dried for 5 days to remove the moisture from leaves completely. Subsequently using motor and pestle, leaves were pulverized and a fine powder was obtained at last. The leaf powder (10 g) was weighted and added to 100 ml of Milli-Q water and heated at 60oC for 15 min. After completion of reaction, extracts were filtered through Whatman No.1 filter paper and filtered extract was stored at room temperature for further studies. 3
2.3 Synthesis and effect of AgNO3 concentration on AgNPs For the synthesis of AgNPs, 50 ml of 0.001 M AgNO3 solution was first prepared in a reaction vessel of 100 ml volume then, 10 ml of aqueous extract of Lantana camara was added and mixed homogeneously on magnetic stirrer. A control setup was also maintained without L. camara leaf extract. Both the reaction solution and control setup were incubated in dark room at 37oC to avoid the photo inactivation of silver nitrate. The stirring was continued for 5 min and cooled to room temperature. Appearance of signatory yellowish brown color confirms the AgNPs formation (Fig. 1). The above mentioned process is reiterated with 0.005 M and 0.01 M AgNO3 concentrations by same leaf extract. Then the colloid solutions was collected and stored at room temperature for further experiments. The obtained AgNPs were further purified by centrifugation at 10,000 rpm for 15 min followed by redispersion of the pellet in deionised water. 2.4 Characterization of the synthesized AgNPs The bio-reduced AgNPs were subjected to various characterization studies. The structural properties of the synthesized nanoparticles were analysed by Seifert 3003TT X-ray diffractometer, using Cu Kα radiation (λ=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 green synthesized AgNPs was observed 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-visible absorption study was carried out by a Perkin Elmer Lambda 950 UV-VisNIR 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 4
Fluorolog-3 Spectrofluorometer (Model FL3-22PTI). Fourier transform infrared spectroscopy (FTIR) spectra of the freeze-dried samples were recorded with ATR-FTIR using Bruker Vertex80 spectrometer. Zeta potential was measured using Nanopartica (HORIBA). 2.5 Test pathogens The antibacterial activity of bio-reduced AgNPs was tested against different pathogenic bacteria. In order to assess the anti-bacterial activity of synthesized AgNPs, the gram-negative Escherichia coli, Pseudomonas spp. and gram-positive Bacillus spp., Staphylococcus spp. were selected. 2.6 Bactericidal property of silver nanoparticles The bactericidal property of AgNPs was investigated by the standard Kirby-Bauer disc diffusion assay. The bacterial test organisms were grown in nutrient broth for 24 hrs and used for further experiments. Nutrient agar plates were prepared, sterilized and solidified. After this, 100 µl of overnight culture of each organism was spreaded on the petriplates with the help of sterile glass rod to obtain bacterial lawns. Double sterilized paper discs were placed on agar plates and controls were maintained (only with leaf extract and only with silver nitrate). Then, AgNPs (0.001 M) were loaded on to each disc at required volumes of 2 µl, 4 µl, 6 µl and 10 µl in each plate and incubated at 37oC for 24 hrs. After the incubation period, clear zone of inhibition around each disc can be viewed attesting bactericidal property. 2.7 Catalytic activity of bio AgNPs For studying the catalytic activity of as-synthesized AgNPs, the reduction of methylene blue to leucomethylene blue by leaf extract was performed as a probe reaction. Catalytic activity of synthesized AgNPs was evaluated in a standard quartz cuvette of 3.5 ml capacity with 1 cm path length and UV-Vis spectrophotometer was employed in order to monitor the absorbance 5
peaks. The effect of catalyst (AgNPs) on the speed of catalytic reduction was investigated by the comparison of absorbance values of three different reaction mixtures filled in three Falcon tubes. In the first reaction mixture, 1 ml of methylene blue (1×10-4 M) was mixed with 2 ml of Milli-Q water and was monitored by measuring the absorbance intensity. In the second reaction mixtures, 1 ml of methylene blue (1×10-4 M), 0.2 ml of L. camara extract and 1.8 ml of water were taken into second tube and reaction is allowed for 30 min, and then was monitored for absorbance intensity. In the third reaction mixture, 1 ml of methylene blue is mixed with 0.2 ml of extract and 1.8 ml of bioreduced AgNPs and the reaction was monitored at three different time intervals viz., 30 min, 40 min and 50 min. In all the reaction mixtures total volume was made up to 3 ml. After that the reduction was ascertained by comparing the absorbance intensities of the second and third reaction mixtures with that of the pure methylene blue. 3. Results and discussion 3.1 UV-visible analysis UV-Vis spectroscopy is one of the important techniques to determine the formation and stability of metal nanoparticles in aqueous solution. After the addition of leaf extract to control, we can have visual perception of change in color of reaction mixture from watery to yellowish brown indicating the formation of AgNPs, their origin is attributed to the collective oscillation of free conduction electrons results in surface plasmon resonance (SPR) induced by interacting electromagnetic field. Fig. 2(a) shows the UV-Vis spectra of AgNPs depicting single maximum absorption band at 421 nm in supportive to spherical shaped particles. According to Mie theory, two or more SPR bands are expected for prisms and rods formation comprising of transverse surface plasmon band and longitudinal surface plasmon band [26]. The peak at 421 nm was due to strong SPR typical for AgNPs formation. The optical properties of AgNPs are related to 6
excitation of plasmon resonance or inter band transition mainly on the size effect. The result from UV-Vis absorption is in accordance with the earlier report [27]. The effect of AgNO3 concentration on formation of AgNPs was also analyzed using UVVis spectroscopy (Fig. 2(b)). From the spectra it is clear that the formation of AgNPs depends on AgNO3 concentration. At 0.001 M AgNO3 concentration, maximum absorbance occurred at 421 nm which is a characteristic peak of AgNPs and peak observed was narrow which shows narrow size distribution of AgNPs. At 0.005 M AgNO3 concentration, SPR occurred at 427 nm and observed peak was broad which shows wider size distribution of AgNPs. At 0.01 M AgNO3 concentration, peak formed at 435 nm which was broader and showed wider size distribution of AgNPs. Thus, the absorption spectra displayed blue shift with decrease of AgNO3 concentration, attributed to the decrement of particle size. Moreover, it was observed that absorbance intensity increases with increase in AgNO3 concentration which means rate of formation of AgNPs is more at higher concentration of AgNO3 than at lower concentration but due to widening of peak at higher concentration, we may expect particles with larger size. At last, the desired nanoparticles with small size were found to be synthesized with optimum value of 0.001 M AgNO3 concentration and other deep investigations were carried out in order to explore their applicability in biomedical field. The time dependent UV-Vis spectra were recorded to shed light on the processes of nucleation and growth of AgNPs (0.001 M) reduced under the action of L. camara extract. The absorption spectra with respect to time evolution are depicted in Fig. 2(c). It was observed that the appearance of peak at 421 nm corresponds to the absorbance (SPR) intensity and steadily increased as a function of reaction time without any shift in the peak position, which reveals the increase of rate of formation of nano-sized AgNPs. 7
3.2 Structural analysis The XRD profile of green synthesized AgNPs (0.001 M) is shown in Fig. 3(a). The X-ray diffraction peaks appeared at 38.1o, 44.8o, 64.4o and 77.3o indexed as (111), (200), (220) and (311) miller indices, respectively. The AgNPs are crystalline in nature with fcc structure [JCPDS No. 04-0783]. Broadening of the peaks confirms the formation of nanosized particles. DebyeScherrer’s formula is given as [28], D= Kλ/βcosθ
(1)
where K denotes the Scherrer’s constant (K=0.94), λ X-ray wavelength (0.1546 nm), β full-width at half-maximum of diffraction line in radian and θ half diffraction angle. The above formula is used for average particle size calculation and size was estimated to be 20 nm with respect to high intense peak (111). No other impurity peaks were identified, which indicates the purity of AgNPs synthesized. 3.3 Photoluminescence Studies The photoluminescence spectra obtained from the bio-reduced AgNPs (0.001 M) at different excitations is shown in Fig. 3(b). Noble metals exhibits photoluminescence phenomenon due to radiative recombination of Fermi level electrons and sp or d band holes. Fig. 3(b) elicits that the position of PL peaks with increase of excitation wavelength shifted towards the red region in a regular manner. With various excitations of 370, 390, 410 and 420 nm, the peak position shifted to 458, 466, 472 and 476 nm, respectively. Sarker et al. were also reported the red shifting of PL peaks with increasing excitation wavelength in the range of 370−420 nm and speculated this phenomenon due to resonance between the luminescence transition and silver plasmons [29].
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3.4 Compositional analysis EDS spectrum of biologically synthesized AgNPs is depicted in Fig. 4(a). This spectrum shows a high intense major peak of elemental silver at around 3 keV, which is in accordance with the main identification line of metallic silver nanoparticles due to surface plasmon resonance [30]. Less intense peaks of C, O, Cl, S, Fe and P are also arisen due to the formation of capping layer on the surface of nanoparticles by biomolecules of L. camara leaf extract. EDS indicates the reduction of silver ions to elemental silver. Thus, EDS analysis confirmed the formation of AgNPs. Quantitative analysis elucidate that C, O, P, S, Cl, Fe and Ag elements exhibited weight percentages of 6%, 8%, 2%, 0.3%, 7%, 0.2% and 76.5%, respectively and concluding silver in major proportion (Fig. 4(b)). Elemental mapping of silver was depicted in Fig. 4(c), which confirms the uniform distribution of Ag throughout the sample and its prevalent presence. 3.5 Morphological studies FESEM images of bio-reduced AgNPs at different concentrations are represented in Fig. 5(a, b). The morphology of the formed particles consists of nearly spherical morphology with fair agglomeration. As AgNO3 concentration is decreased from 0.01 M to 0.001 M, the average particle size was found to be decreased from 37 nm to 29 nm. The obtained results are further corroborated by TEM observations. TEM measurements also visualized the formation of approximately spherical nanoparticles. As AgNO3 concentration is varied from 0.01 M to 0.001 M, the decrement of the particles size from 30 nm to 23 nm was observed, as shown in Fig. 6(a, b). SAED pattern is shown in Fig. 6(c) with bright circular rings corresponding to the (111), (200), (220) and (311) planes, reveals the particles crystalline nature which is consistent with
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XRD results. The HR-TEM image is shown in Fig. 6(d) and depicts the d-spacing value as 0.235 nm, which is in accordance with the standard data [JCPDS No. 04-0783]. 3.6 FTIR spectroscopy FTIR spectroscopy is used to probe the chemical composition of the surface of the nanoparticles capped by leaf extract biomolecules. FTIR spectrum of green synthesized AgNPs is shown in Fig. 7(a). The strong intense broad band at 3193 cm-1 is assigned to the N−H group vibrational bands from proteins present in the extract [31]. Another intense band at 2347 cm-1 is characteristic of N−H stretching or the C=O stretching vibrations [32]. Also, medium peaks at 1561 and 1328 cm-1 are attributed to –C=C– stretching mode and C−N stretching vibrations of aromatic amines, respectively [33, 34]. Another couple of bands at 1046 and 980 cm-1 implicates with C−OH stretching of secondary alcohols and C−O−C vibrations of proteins/polysaccharides, respectively [33, 34]. Small bands at 1635, 811 and 672 cm-1 corresponds to the carbonyl groups in amide linkages which are involved in AgNPs formation, C−H stretching of alkenes and C−Cl stretching modes of alkyl halides, respectively [32, 33]. The IR peaks for amide I and amide II arise owing to carbonyl stretch and –N–H stretch vibrations in the amide linkages of the proteins. This may be reason for the reduction of metal ions while using the leaf extract for the synthesis of AgNPs [35] indicates the binding of the nanoparticles with proteins. This analysis confirmed that the carbonyl group of amino acid residues and peptides of proteins has a stronger ability to bind metal, so that the proteins could most possibly form a layer covering the metal nanoparticles (i.e. capping of AgNPs) to prevent the agglomeration of the particles and thus, the nanoparticles are stabilized in the medium. This prediction clearly confirms that different bio-constituents of leaf extract have interacted with the surface of the nanoparticles and kept them stable for longer periods. As earlier reports suggested that the 10
phytochemical analysis on Lantana camara reveals the presence of flavonoids, terpenoids and alkaloids [23, 24]. Hence from FTIR spectrum, it is likely to infer that all these biomolecules are responsible for reduction of Ag+ to Ag0 and further proficient stabilization resulting in nanoparticles formation. 3.7 Zeta potential analysis The Zeta potential analysis of synthesized AgNPs is shown in Fig. 7(b). From the graph, the corresponding average zeta value is –36 mV, suggesting very good stability of AgNPs which is reliable with previous report [36]. The larger negative value could be attributed to the capping layer of phyto-constituents present in the leaf extract. The repulsive forces between the negatively charged particles keep away from agglomeration. Thus, results in increase of nanoparticles stability. 3.8 Antibacterial studies In general, smaller nanoparticles with higher surface area interact more with bacteria compared to bigger particles thereby furnishing higher antibacterial activity [37]. The present work elicits that AgNPs synthesized with 0.001 M are found to have less particle size. Meanwhile, AgNPs synthesized by green route are observed to be highly toxic against gram positive and gram negative pathogenic bacteria. The experimental results of this study are presented in Fig. 8 and the resulting zone of inhibition values are given in Table 1. Finally, the current study clearly indicates that the Lantana camara extract mediated AgNPs exhibited excellent antimicrobial activity against gram positive organism of Bacillus spp. and gram negative organism of Pseudomonas spp.
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3.9 Catalytic activity of AgNPs on reduction of methylene blue by L. camara extract Catalytic activity of AgNPs was assessed by using methylene blue. It is well known fact that AgNPs and their composites to their credit show greater catalytic activity in the area of dye reduction and removal also. Kundu et al. reported the reduction of methylene blue by arsine in the presence of silver nano [38], whereas Mallick et al. studied the catalytic activity of AgNPs on the reduction of phenosaffarin dye [39]. Here, we have reported the reduction of methylene blue by the natural green aqueous extract of L. camara mediated AgNPs (Fig. 9). The maximum absorbance value of methylene blue was recorded at 664 nm (Fig. 10). After 30 min of second reaction mixture (extract + methylene blue), it was observed that the absorbance intensity peak of methylene blue is decreased slowly and shifted to higher wavelength side. This decrease of absorbance is attributed to the capability of phytoextract to degrade the methylene blue. But, system containing dye and AgNPs in addition to extract showed remarkable decrease in absorbance peak at the end of 30 min, 40 min and 50 min time interval. The decrease of methylene blue absorbance and increase of SPR peak of AgNPs is observed. This reveals that AgNPs act as an electron transfer mediator because it accepts the electrons from extract and donates to methylene blue by acting as a redox catalyst, which in turn termed as electron relay effect [40]. 4. Conclusions In conclusion, silver nanoparticles were synthesized using Lantana camara leaf extract at room temperature through green route. The bio-reduction of aqueous Ag+ ions by the leaf extract of L. camara has been demonstrated. The major compounds (flavonoids, terpenoids and alkaloids) present in the L. camara leaf extract were responsible for the reduction and stabilization of AgNPs. From the FESEM and TEM analysis the size and shape of the AgNPs 12
was analyzed and variation of particle size with AgNO3 concentration was examined. From the antimicrobial studies it is inferred that the bio-reduced AgNPs through L. camara leaf extract exhibit excellent bactericidal effect against Bacillus spp. and Pseudomonas spp. microorganisms. Additionally, the catalytic activity of nanoparticles was studied and explored that the AgNPs synthesized act as effective green catalyst. Acknowledgements The one of the author B. Ajitha would like to express her gratitude to University Grants Commission (UGC), New Delhi, for awarding UGC-BSR Fellowship in sciences for meritorious students. References [1]
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Table caption: Table 1 The variation of zone of inhibitions towards different pathogenic bacteria at different volumes of AgNPs. Figure captions: Fig. 1. Schematic synthesis procedure of green silver nanoparticles (a) AgNO3 solution, (b) Collected Lantana camara leaves and (c) Biosynthesized AgNPs. Fig. 2. UV-Vis absorbance spectra of (a) L. camara leaf extract and AgNPs, (b) Different AgNO3 concentration and (c) AgNPs at different time intervals. Fig. 3. (a) X-ray diffraction profile of green synthesized AgNPs (b) Photoluminescence spectra of green synthesized AgNPs at different excitation wavelengths using L. camara leaf extract. Fig. 4. (a) Typical EDS spectrum and (b) Quantitative analysis of bio-synthesized silver nanoparticles (c) Elemental mapping of silver. Fig. 5. FESEM images of biosynthesized silver nanoparticles at (a) 0.01 M and (b) 0.001 M concentrations. Fig. 6. TEM images at (a) 0.01 M and (b) 0.001 M concentrations, (c) SAED pattern and (d) HRTEM of silver nanoparticles. Fig. 7. (a) FTIR spectrum and (b) Zeta potential analysis of green synthesized AgNPs using L. camara leaf extract, of synthesized AgNPs. Fig. 8. Disc diffusion assay of bactericidal activity against bacterial organisms at different volumes of AgNPs. Fig. 9. Catalytic activity of AgNPs between L. camara extract and methylene blue (electron relay effect). Fig. 10. UV-Vis spectra of methylene blue reduction by L. camara extract in the presence of AgNPs.
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Figures
Fig. 1. Schematic synthesis procedure of green silver nanoparticles (a) AgNO3 solution, (b) Collected Lantana camara leaves and (c) Biosynthesized AgNPs.
19
(b)
(a)
(c)
Fig. 2. UV-Vis absorbance spectra of (a) L. camara leaf extract and AgNPs, (b) Different AgNO3 concentration and (c) AgNPs at different time intervals.
20
(a)
Intensity (a.u.) 20
(b)
(111)
(200)
(220)
(311)
30
40
50
60
70
80
2θ (degree)
Fig. 3. (a) X-ray diffraction profile of green synthesized AgNPs (b) Photoluminescence spectra of green synthesized AgNPs at different excitation wavelengths using L. camara leaf extract.
21
(a)
(c)
(b)
Fig. 4. (a) Typical EDS spectrum and (b) Quantitative analysis of bio-synthesized silver nanoparticles (c) Elemental mapping of silver.
22
(a)
(b)
Fig. 5. FESEM images of biosynthesized silver nanoparticles at (a) 0.01 M and (b) 0.001 M concentrations.
23
(a)
(b)
(c)
(d)
Fig. 6. TEM images at (a) 0.01 M and (b) 0.001 M concentrations, (c) SAED pattern and (d) HRTEM of silver nanoparticles.
24
(b)
(a)
Transmittance [%]
(3193)
(2347) (1635) (1561) (811) (1328) (980) (1046) (672)
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Fig. 7. (a) FTIR spectrum and (b) Zeta potential analysis of green synthesized AgNPs using L. camara leaf extract, of synthesized AgNPs.
Fig. 8. Disc diffusion assay of bactericidal activity against bacterial organisms at different volumes of AgNPs. 25
Fig. 9. Catalytic activity of AgNPs between L. camara extract and methylene blue (electron relay effect).
26
Methylene blue (MB) MB+Extract (After 30 min) MB+Extract+AgNPs (After 30 min) MB+Extract+AgNPs (After 40 min)
Absorbance (a.u)
MB+Extract+AgNPs (After 50 min)
300
400
500
600
700
800
Wavelength (nm) Fig. 10. UV-Vis spectra of methylene blue reduction by L. camara extract in the presence of AgNPs.
27
Table
Table 1 The variation of zone of inhibitions towards different pathogenic bacteria at different volumes of AgNPs. AgNPs volume (µl)
Zone of inhibition of tested pathogenic bacterium (mm)
2
Bacillus spp. 3
Staphylococcus spp. 3
Pseudomonas spp. 5
4
3
3
6
4
6
4
4
7
4
10
5
4
7
4
28
E. coli 3
Graphical Abstract
29
Research highlights Lantana camara is an excellent bio-source for AgNPs synthesis Biomolecules of L. camara play crucial role in nanoparticles formation Stable, crystalline and nearly spherical nanoparticles are reported Potent antibacterial and catalytic activity are noticed
30
S1011-1344(15)00183-9 http://dx.doi.org/10.1016/j.jphotobiol.2015.05.020 JPB 10051
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
25 November 2014 11 May 2015 24 May 2015
Please cite this article as: B. Ajitha, Y. Ashok Kumar Reddy, S. Shameer, K.M. Rajesh, Y. Suneetha, P. Sreedhara Reddy, Lantana camara leaf extract mediated silver nanoparticles: Antibacterial, Green catalyst, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.05.020
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Lantana camara leaf extract mediated silver nanoparticles: Antibacterial, Green catalyst B. Ajithaa*, Y. Ashok Kumar Reddyb, Syed Shameerc, K.M. Rajesha, Y. Suneethad, and P. Sreedhara Reddya 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 c Department of Microbiology, Sri Venkateswara University, Tirupati-517502, India d Department of Zoology, Sri Venkateswara University, Tirupati-517502, India ABSTRACT
Silver nanoparticles (AgNPs) have been synthesized by Lantana camara leaf extract through simple green route and evaluated their antibacterial and catalytic activities. The leaf extract (LE) itself acts as both reducing and stabilizing agent at once for desired nanoparticle synthesis. The colorless reaction mixture turns to yellowish brown attesting the AgNPs formation and displayed UV-Vis absorption spectra. Structural analysis confirms the crystalline nature and formation of fcc structured metallic silver with majority (111) facets. Morphological studies elicit the formation of almost spherical shaped nanoparticles and as AgNO3 concentration is increased, there is an increment in the particle size. The FTIR analysis evidences the presence of various functional groups of biomolecules of LE is responsible for stabilization of AgNPs. Zeta potential measurement attests the higher stability of synthesized AgNPs. The synthesized AgNPs exhibited good antibacterial activity when tested against Escherichia coli, Pseudomonas spp., Bacillus spp. and Staphylococcus spp. using standard Kirby-Bauer disc diffusion assay. Furthermore, they showed good catalytic activity on the reduction of methylene blue by L. camara extract which is monitored and confirmed by the UV-Vis spectrophotometer. Keywords: Leaf extract, Green synthesis, AgNPs, Antibacterial activity, Green catalyst *Corresponding author. Tel: +91 877 2289472; Fax: +91 877 2249611. E-mail address: [email protected] (B. Ajitha). 1
1. Introduction Nanotechnology is the latest and one of the most promising areas of research in modern medical science. Nanoparticles are of special group of materials with unique features and extensive applications in diverse fields [1]. Many scientists have great interest in studying these typical features in fact, nanoparticles display completely peculiar properties compared to large size counterparts [2]. Silver nanoparticles (AgNPs) have found tremendous applications in the areas of catalysis, optoelectronics, detection and diagnostic, antimicrobials and therapeutics [3-6] due to their better catalytic, optical and electrical properties. Many attempts have been made to use AgNPs as an anti-cancer agent and they have all turned up positive [7]. To date, various approaches were developed for the synthesis of AgNPs through physical and chemical methods such as electrochemical [8], γ-radiation [9], photochemical [10], laser ablation [11], chemical reduction [12] and recently via green synthesis method [13]. However, most of the reported methods involve more than one steps and leads to unfavorable impact in the medical applications due to usage of unsafe chemicals. There is a rising commercial requirement of eco-friendly and nontoxic nanoparticles. In this regard it has been proven that the green synthesis method is a reliable, simple, low cost and convenient route for the synthesis of AgNPs. Synthesis of gold, silver and bimetallic nanoparticles with different plant extracts have been reported earlier [14]. Lantana camara plant is commonly known as red sage or wild sage or “pulikampa” in Telugu and is a significant weed found throughout India. The essential oil and extracts of the plant are used in herbal medicines for the treatment of various human diseases such as skin itches, leprosy, cancer, chicken pox, measles, asthma, ulcers, tumors, high blood pressure, tetanus, rheumatism etc [15, 16]. Extracts from leaves have been reported to have antifungal, 2
antiproliferative, antibacterial, nematicidal, termicidal, anthelmintic and anticancer activities [1622]. Research on the chemical composition of L. camara plant revealed the presence of terpenoids, steroids, flavonoids and alkaloids as major constituents [16, 23, 24]. In earlier, we studied the synthesis of AgNPs with different leaf extract concentrations using L. camara leaf extract [25]. However, the present work aim is to develop a novel approach for the green synthesis of AgNPs with different AgNO3 concentrations using aqueous leaf extract of L. camara and exploring its catalytic activity in addition to antibacterial activity against some selected gram positive and gram negative organisms. 2. Experimental 2.1 Materials All the reagents purchased were of analytical grade and used as received. Silver nitrate (AgNO3) with ≥ 99.5% purity and methylene blue were purchased from Sigma-Aldrich, India. Fresh leaves of Lantana camara have been harvested from in and around Chittoor district, A.P, India. Milli-Q water was used throughout the experiment. 2.2 Preparation of leaf extract Freshly collected leaves were used for the preparation of Lantana camara leaf extract. The leaves were collected from plant and surface washed at first with running tap water followed by Milli-Q water and then shade dried for 5 days to remove the moisture from leaves completely. Subsequently using motor and pestle, leaves were pulverized and a fine powder was obtained at last. The leaf powder (10 g) was weighted and added to 100 ml of Milli-Q water and heated at 60oC for 15 min. After completion of reaction, extracts were filtered through Whatman No.1 filter paper and filtered extract was stored at room temperature for further studies. 3
2.3 Synthesis and effect of AgNO3 concentration on AgNPs For the synthesis of AgNPs, 50 ml of 0.001 M AgNO3 solution was first prepared in a reaction vessel of 100 ml volume then, 10 ml of aqueous extract of Lantana camara was added and mixed homogeneously on magnetic stirrer. A control setup was also maintained without L. camara leaf extract. Both the reaction solution and control setup were incubated in dark room at 37oC to avoid the photo inactivation of silver nitrate. The stirring was continued for 5 min and cooled to room temperature. Appearance of signatory yellowish brown color confirms the AgNPs formation (Fig. 1). The above mentioned process is reiterated with 0.005 M and 0.01 M AgNO3 concentrations by same leaf extract. Then the colloid solutions was collected and stored at room temperature for further experiments. The obtained AgNPs were further purified by centrifugation at 10,000 rpm for 15 min followed by redispersion of the pellet in deionised water. 2.4 Characterization of the synthesized AgNPs The bio-reduced AgNPs were subjected to various characterization studies. The structural properties of the synthesized nanoparticles were analysed by Seifert 3003TT X-ray diffractometer, using Cu Kα radiation (λ=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 green synthesized AgNPs was observed 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-visible absorption study was carried out by a Perkin Elmer Lambda 950 UV-VisNIR 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 4
Fluorolog-3 Spectrofluorometer (Model FL3-22PTI). Fourier transform infrared spectroscopy (FTIR) spectra of the freeze-dried samples were recorded with ATR-FTIR using Bruker Vertex80 spectrometer. Zeta potential was measured using Nanopartica (HORIBA). 2.5 Test pathogens The antibacterial activity of bio-reduced AgNPs was tested against different pathogenic bacteria. In order to assess the anti-bacterial activity of synthesized AgNPs, the gram-negative Escherichia coli, Pseudomonas spp. and gram-positive Bacillus spp., Staphylococcus spp. were selected. 2.6 Bactericidal property of silver nanoparticles The bactericidal property of AgNPs was investigated by the standard Kirby-Bauer disc diffusion assay. The bacterial test organisms were grown in nutrient broth for 24 hrs and used for further experiments. Nutrient agar plates were prepared, sterilized and solidified. After this, 100 µl of overnight culture of each organism was spreaded on the petriplates with the help of sterile glass rod to obtain bacterial lawns. Double sterilized paper discs were placed on agar plates and controls were maintained (only with leaf extract and only with silver nitrate). Then, AgNPs (0.001 M) were loaded on to each disc at required volumes of 2 µl, 4 µl, 6 µl and 10 µl in each plate and incubated at 37oC for 24 hrs. After the incubation period, clear zone of inhibition around each disc can be viewed attesting bactericidal property. 2.7 Catalytic activity of bio AgNPs For studying the catalytic activity of as-synthesized AgNPs, the reduction of methylene blue to leucomethylene blue by leaf extract was performed as a probe reaction. Catalytic activity of synthesized AgNPs was evaluated in a standard quartz cuvette of 3.5 ml capacity with 1 cm path length and UV-Vis spectrophotometer was employed in order to monitor the absorbance 5
peaks. The effect of catalyst (AgNPs) on the speed of catalytic reduction was investigated by the comparison of absorbance values of three different reaction mixtures filled in three Falcon tubes. In the first reaction mixture, 1 ml of methylene blue (1×10-4 M) was mixed with 2 ml of Milli-Q water and was monitored by measuring the absorbance intensity. In the second reaction mixtures, 1 ml of methylene blue (1×10-4 M), 0.2 ml of L. camara extract and 1.8 ml of water were taken into second tube and reaction is allowed for 30 min, and then was monitored for absorbance intensity. In the third reaction mixture, 1 ml of methylene blue is mixed with 0.2 ml of extract and 1.8 ml of bioreduced AgNPs and the reaction was monitored at three different time intervals viz., 30 min, 40 min and 50 min. In all the reaction mixtures total volume was made up to 3 ml. After that the reduction was ascertained by comparing the absorbance intensities of the second and third reaction mixtures with that of the pure methylene blue. 3. Results and discussion 3.1 UV-visible analysis UV-Vis spectroscopy is one of the important techniques to determine the formation and stability of metal nanoparticles in aqueous solution. After the addition of leaf extract to control, we can have visual perception of change in color of reaction mixture from watery to yellowish brown indicating the formation of AgNPs, their origin is attributed to the collective oscillation of free conduction electrons results in surface plasmon resonance (SPR) induced by interacting electromagnetic field. Fig. 2(a) shows the UV-Vis spectra of AgNPs depicting single maximum absorption band at 421 nm in supportive to spherical shaped particles. According to Mie theory, two or more SPR bands are expected for prisms and rods formation comprising of transverse surface plasmon band and longitudinal surface plasmon band [26]. The peak at 421 nm was due to strong SPR typical for AgNPs formation. The optical properties of AgNPs are related to 6
excitation of plasmon resonance or inter band transition mainly on the size effect. The result from UV-Vis absorption is in accordance with the earlier report [27]. The effect of AgNO3 concentration on formation of AgNPs was also analyzed using UVVis spectroscopy (Fig. 2(b)). From the spectra it is clear that the formation of AgNPs depends on AgNO3 concentration. At 0.001 M AgNO3 concentration, maximum absorbance occurred at 421 nm which is a characteristic peak of AgNPs and peak observed was narrow which shows narrow size distribution of AgNPs. At 0.005 M AgNO3 concentration, SPR occurred at 427 nm and observed peak was broad which shows wider size distribution of AgNPs. At 0.01 M AgNO3 concentration, peak formed at 435 nm which was broader and showed wider size distribution of AgNPs. Thus, the absorption spectra displayed blue shift with decrease of AgNO3 concentration, attributed to the decrement of particle size. Moreover, it was observed that absorbance intensity increases with increase in AgNO3 concentration which means rate of formation of AgNPs is more at higher concentration of AgNO3 than at lower concentration but due to widening of peak at higher concentration, we may expect particles with larger size. At last, the desired nanoparticles with small size were found to be synthesized with optimum value of 0.001 M AgNO3 concentration and other deep investigations were carried out in order to explore their applicability in biomedical field. The time dependent UV-Vis spectra were recorded to shed light on the processes of nucleation and growth of AgNPs (0.001 M) reduced under the action of L. camara extract. The absorption spectra with respect to time evolution are depicted in Fig. 2(c). It was observed that the appearance of peak at 421 nm corresponds to the absorbance (SPR) intensity and steadily increased as a function of reaction time without any shift in the peak position, which reveals the increase of rate of formation of nano-sized AgNPs. 7
3.2 Structural analysis The XRD profile of green synthesized AgNPs (0.001 M) is shown in Fig. 3(a). The X-ray diffraction peaks appeared at 38.1o, 44.8o, 64.4o and 77.3o indexed as (111), (200), (220) and (311) miller indices, respectively. The AgNPs are crystalline in nature with fcc structure [JCPDS No. 04-0783]. Broadening of the peaks confirms the formation of nanosized particles. DebyeScherrer’s formula is given as [28], D= Kλ/βcosθ
(1)
where K denotes the Scherrer’s constant (K=0.94), λ X-ray wavelength (0.1546 nm), β full-width at half-maximum of diffraction line in radian and θ half diffraction angle. The above formula is used for average particle size calculation and size was estimated to be 20 nm with respect to high intense peak (111). No other impurity peaks were identified, which indicates the purity of AgNPs synthesized. 3.3 Photoluminescence Studies The photoluminescence spectra obtained from the bio-reduced AgNPs (0.001 M) at different excitations is shown in Fig. 3(b). Noble metals exhibits photoluminescence phenomenon due to radiative recombination of Fermi level electrons and sp or d band holes. Fig. 3(b) elicits that the position of PL peaks with increase of excitation wavelength shifted towards the red region in a regular manner. With various excitations of 370, 390, 410 and 420 nm, the peak position shifted to 458, 466, 472 and 476 nm, respectively. Sarker et al. were also reported the red shifting of PL peaks with increasing excitation wavelength in the range of 370−420 nm and speculated this phenomenon due to resonance between the luminescence transition and silver plasmons [29].
8
3.4 Compositional analysis EDS spectrum of biologically synthesized AgNPs is depicted in Fig. 4(a). This spectrum shows a high intense major peak of elemental silver at around 3 keV, which is in accordance with the main identification line of metallic silver nanoparticles due to surface plasmon resonance [30]. Less intense peaks of C, O, Cl, S, Fe and P are also arisen due to the formation of capping layer on the surface of nanoparticles by biomolecules of L. camara leaf extract. EDS indicates the reduction of silver ions to elemental silver. Thus, EDS analysis confirmed the formation of AgNPs. Quantitative analysis elucidate that C, O, P, S, Cl, Fe and Ag elements exhibited weight percentages of 6%, 8%, 2%, 0.3%, 7%, 0.2% and 76.5%, respectively and concluding silver in major proportion (Fig. 4(b)). Elemental mapping of silver was depicted in Fig. 4(c), which confirms the uniform distribution of Ag throughout the sample and its prevalent presence. 3.5 Morphological studies FESEM images of bio-reduced AgNPs at different concentrations are represented in Fig. 5(a, b). The morphology of the formed particles consists of nearly spherical morphology with fair agglomeration. As AgNO3 concentration is decreased from 0.01 M to 0.001 M, the average particle size was found to be decreased from 37 nm to 29 nm. The obtained results are further corroborated by TEM observations. TEM measurements also visualized the formation of approximately spherical nanoparticles. As AgNO3 concentration is varied from 0.01 M to 0.001 M, the decrement of the particles size from 30 nm to 23 nm was observed, as shown in Fig. 6(a, b). SAED pattern is shown in Fig. 6(c) with bright circular rings corresponding to the (111), (200), (220) and (311) planes, reveals the particles crystalline nature which is consistent with
9
XRD results. The HR-TEM image is shown in Fig. 6(d) and depicts the d-spacing value as 0.235 nm, which is in accordance with the standard data [JCPDS No. 04-0783]. 3.6 FTIR spectroscopy FTIR spectroscopy is used to probe the chemical composition of the surface of the nanoparticles capped by leaf extract biomolecules. FTIR spectrum of green synthesized AgNPs is shown in Fig. 7(a). The strong intense broad band at 3193 cm-1 is assigned to the N−H group vibrational bands from proteins present in the extract [31]. Another intense band at 2347 cm-1 is characteristic of N−H stretching or the C=O stretching vibrations [32]. Also, medium peaks at 1561 and 1328 cm-1 are attributed to –C=C– stretching mode and C−N stretching vibrations of aromatic amines, respectively [33, 34]. Another couple of bands at 1046 and 980 cm-1 implicates with C−OH stretching of secondary alcohols and C−O−C vibrations of proteins/polysaccharides, respectively [33, 34]. Small bands at 1635, 811 and 672 cm-1 corresponds to the carbonyl groups in amide linkages which are involved in AgNPs formation, C−H stretching of alkenes and C−Cl stretching modes of alkyl halides, respectively [32, 33]. The IR peaks for amide I and amide II arise owing to carbonyl stretch and –N–H stretch vibrations in the amide linkages of the proteins. This may be reason for the reduction of metal ions while using the leaf extract for the synthesis of AgNPs [35] indicates the binding of the nanoparticles with proteins. This analysis confirmed that the carbonyl group of amino acid residues and peptides of proteins has a stronger ability to bind metal, so that the proteins could most possibly form a layer covering the metal nanoparticles (i.e. capping of AgNPs) to prevent the agglomeration of the particles and thus, the nanoparticles are stabilized in the medium. This prediction clearly confirms that different bio-constituents of leaf extract have interacted with the surface of the nanoparticles and kept them stable for longer periods. As earlier reports suggested that the 10
phytochemical analysis on Lantana camara reveals the presence of flavonoids, terpenoids and alkaloids [23, 24]. Hence from FTIR spectrum, it is likely to infer that all these biomolecules are responsible for reduction of Ag+ to Ag0 and further proficient stabilization resulting in nanoparticles formation. 3.7 Zeta potential analysis The Zeta potential analysis of synthesized AgNPs is shown in Fig. 7(b). From the graph, the corresponding average zeta value is –36 mV, suggesting very good stability of AgNPs which is reliable with previous report [36]. The larger negative value could be attributed to the capping layer of phyto-constituents present in the leaf extract. The repulsive forces between the negatively charged particles keep away from agglomeration. Thus, results in increase of nanoparticles stability. 3.8 Antibacterial studies In general, smaller nanoparticles with higher surface area interact more with bacteria compared to bigger particles thereby furnishing higher antibacterial activity [37]. The present work elicits that AgNPs synthesized with 0.001 M are found to have less particle size. Meanwhile, AgNPs synthesized by green route are observed to be highly toxic against gram positive and gram negative pathogenic bacteria. The experimental results of this study are presented in Fig. 8 and the resulting zone of inhibition values are given in Table 1. Finally, the current study clearly indicates that the Lantana camara extract mediated AgNPs exhibited excellent antimicrobial activity against gram positive organism of Bacillus spp. and gram negative organism of Pseudomonas spp.
11
3.9 Catalytic activity of AgNPs on reduction of methylene blue by L. camara extract Catalytic activity of AgNPs was assessed by using methylene blue. It is well known fact that AgNPs and their composites to their credit show greater catalytic activity in the area of dye reduction and removal also. Kundu et al. reported the reduction of methylene blue by arsine in the presence of silver nano [38], whereas Mallick et al. studied the catalytic activity of AgNPs on the reduction of phenosaffarin dye [39]. Here, we have reported the reduction of methylene blue by the natural green aqueous extract of L. camara mediated AgNPs (Fig. 9). The maximum absorbance value of methylene blue was recorded at 664 nm (Fig. 10). After 30 min of second reaction mixture (extract + methylene blue), it was observed that the absorbance intensity peak of methylene blue is decreased slowly and shifted to higher wavelength side. This decrease of absorbance is attributed to the capability of phytoextract to degrade the methylene blue. But, system containing dye and AgNPs in addition to extract showed remarkable decrease in absorbance peak at the end of 30 min, 40 min and 50 min time interval. The decrease of methylene blue absorbance and increase of SPR peak of AgNPs is observed. This reveals that AgNPs act as an electron transfer mediator because it accepts the electrons from extract and donates to methylene blue by acting as a redox catalyst, which in turn termed as electron relay effect [40]. 4. Conclusions In conclusion, silver nanoparticles were synthesized using Lantana camara leaf extract at room temperature through green route. The bio-reduction of aqueous Ag+ ions by the leaf extract of L. camara has been demonstrated. The major compounds (flavonoids, terpenoids and alkaloids) present in the L. camara leaf extract were responsible for the reduction and stabilization of AgNPs. From the FESEM and TEM analysis the size and shape of the AgNPs 12
was analyzed and variation of particle size with AgNO3 concentration was examined. From the antimicrobial studies it is inferred that the bio-reduced AgNPs through L. camara leaf extract exhibit excellent bactericidal effect against Bacillus spp. and Pseudomonas spp. microorganisms. Additionally, the catalytic activity of nanoparticles was studied and explored that the AgNPs synthesized act as effective green catalyst. Acknowledgements The one of the author B. Ajitha would like to express her gratitude to University Grants Commission (UGC), New Delhi, for awarding UGC-BSR Fellowship in sciences for meritorious students. References [1]
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Table caption: Table 1 The variation of zone of inhibitions towards different pathogenic bacteria at different volumes of AgNPs. Figure captions: Fig. 1. Schematic synthesis procedure of green silver nanoparticles (a) AgNO3 solution, (b) Collected Lantana camara leaves and (c) Biosynthesized AgNPs. Fig. 2. UV-Vis absorbance spectra of (a) L. camara leaf extract and AgNPs, (b) Different AgNO3 concentration and (c) AgNPs at different time intervals. Fig. 3. (a) X-ray diffraction profile of green synthesized AgNPs (b) Photoluminescence spectra of green synthesized AgNPs at different excitation wavelengths using L. camara leaf extract. Fig. 4. (a) Typical EDS spectrum and (b) Quantitative analysis of bio-synthesized silver nanoparticles (c) Elemental mapping of silver. Fig. 5. FESEM images of biosynthesized silver nanoparticles at (a) 0.01 M and (b) 0.001 M concentrations. Fig. 6. TEM images at (a) 0.01 M and (b) 0.001 M concentrations, (c) SAED pattern and (d) HRTEM of silver nanoparticles. Fig. 7. (a) FTIR spectrum and (b) Zeta potential analysis of green synthesized AgNPs using L. camara leaf extract, of synthesized AgNPs. Fig. 8. Disc diffusion assay of bactericidal activity against bacterial organisms at different volumes of AgNPs. Fig. 9. Catalytic activity of AgNPs between L. camara extract and methylene blue (electron relay effect). Fig. 10. UV-Vis spectra of methylene blue reduction by L. camara extract in the presence of AgNPs.
18
Figures
Fig. 1. Schematic synthesis procedure of green silver nanoparticles (a) AgNO3 solution, (b) Collected Lantana camara leaves and (c) Biosynthesized AgNPs.
19
(b)
(a)
(c)
Fig. 2. UV-Vis absorbance spectra of (a) L. camara leaf extract and AgNPs, (b) Different AgNO3 concentration and (c) AgNPs at different time intervals.
20
(a)
Intensity (a.u.) 20
(b)
(111)
(200)
(220)
(311)
30
40
50
60
70
80
2θ (degree)
Fig. 3. (a) X-ray diffraction profile of green synthesized AgNPs (b) Photoluminescence spectra of green synthesized AgNPs at different excitation wavelengths using L. camara leaf extract.
21
(a)
(c)
(b)
Fig. 4. (a) Typical EDS spectrum and (b) Quantitative analysis of bio-synthesized silver nanoparticles (c) Elemental mapping of silver.
22
(a)
(b)
Fig. 5. FESEM images of biosynthesized silver nanoparticles at (a) 0.01 M and (b) 0.001 M concentrations.
23
(a)
(b)
(c)
(d)
Fig. 6. TEM images at (a) 0.01 M and (b) 0.001 M concentrations, (c) SAED pattern and (d) HRTEM of silver nanoparticles.
24
(b)
(a)
Transmittance [%]
(3193)
(2347) (1635) (1561) (811) (1328) (980) (1046) (672)
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Fig. 7. (a) FTIR spectrum and (b) Zeta potential analysis of green synthesized AgNPs using L. camara leaf extract, of synthesized AgNPs.
Fig. 8. Disc diffusion assay of bactericidal activity against bacterial organisms at different volumes of AgNPs. 25
Fig. 9. Catalytic activity of AgNPs between L. camara extract and methylene blue (electron relay effect).
26
Methylene blue (MB) MB+Extract (After 30 min) MB+Extract+AgNPs (After 30 min) MB+Extract+AgNPs (After 40 min)
Absorbance (a.u)
MB+Extract+AgNPs (After 50 min)
300
400
500
600
700
800
Wavelength (nm) Fig. 10. UV-Vis spectra of methylene blue reduction by L. camara extract in the presence of AgNPs.
27
Table
Table 1 The variation of zone of inhibitions towards different pathogenic bacteria at different volumes of AgNPs. AgNPs volume (µl)
Zone of inhibition of tested pathogenic bacterium (mm)
2
Bacillus spp. 3
Staphylococcus spp. 3
Pseudomonas spp. 5
4
3
3
6
4
6
4
4
7
4
10
5
4
7
4
28
E. coli 3
Graphical Abstract
29
Research highlights Lantana camara is an excellent bio-source for AgNPs synthesis Biomolecules of L. camara play crucial role in nanoparticles formation Stable, crystalline and nearly spherical nanoparticles are reported Potent antibacterial and catalytic activity are noticed
30
