Materials Science and Engineering C 58 (2016) 36–43
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Green synthesis of silver nanoparticles using Coffea arabica seed extract and its antibacterial activity Vivek Dhand a,b,c,⁎, L. Soumya a, S. Bharadwaj d, Shilpa Chakra a, Deepika Bhatt b,1, B. Sreedhar e a
Center for Nanoscience and Technology, IST, JNTUH, Kukatpally, Hyderabad, 500085 AP, India Centre for Knowledge Management of Nanoscience and Technology, 12-5-32/8, Vijayapuri Colony, Tarnaka, 500017 AP, India Department of Mechanical Engineering, College of Engineering, Kyung Hee University, 446-701 Yongin, Republic of Korea d Department of Physics, Gitam Institute of Technology, Gitam University, Vishakapatnam, 530045 Andhra Pradesh, India e I &PC Division, IICT, Tarnaka, Hyderabad 500007, India b c
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 15 May 2015 Accepted 11 August 2015 Available online 15 August 2015 Keywords: Coffee Seed extract Silver nanoparticles UV–vis spectroscopy Antibacterial activity
a b s t r a c t A novel green source was opted to synthesize silver nanoparticles using dried roasted Coffea arabica seed extract. Bio-reduction of silver was complete when the mixture (AgNO3 + extract) changed its color from light to dark brown. UV–vis spectroscopy result showed maximum adsorption at 459 nm, which represents the characteristic surface plasmon resonance of nanosilver. X-ray crystal analysis showed that the silver nanoparticles are highly crystalline and exhibit a cubic, face centered lattice with characteristic (111), (200), (220) and (311) orientations. Particles exhibit spherical and ellipsoidal shaped structures as observed from TEM. Composition analysis obtained from SEM–EDXA conﬁrmed the presence of elemental signature of silver. FTIR results recorded a downward shift of absorption bands between 800–1500 cm−1 indicting the formation of silver nanoparticles. The mean particle size investigated using DLS was found to be in between 20–30 nm respectively. Anti-bacterial activity of silver nanoparticles on E. coli and S. aureus demonstrated diminished bacterial growth with the development of well-deﬁned inhibition zones. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology is a rapidly growing ﬁeld with potential application in ﬁelds ranging from electronics to cosmetics [1,2]. Nanoscience covers the basic understanding of physical, chemical and biological properties in atomic and sub-atomic levels . It has opened the doors to pave way for the rapidly growing technologies involved in the design and development of novel materials which exhibit unique and improved properties. Silver nanoparticles (SNPs) ﬁt very much into this category due to its wide range of commercial applications involving several ﬁelds like optics (for metal-enhanced ﬂuorescence and surface-enhanced Raman scattering), electronics, catalysis, sensors and therapeutics [4–6]. Currently nano-silver is used for a variety of applications in everyday consumer's life such as: nanosilver infused food storage containers , nanosilver coated surfaces of medical devices to reduce nosocomial infections, bandages, footwear and countless household items respectively [8a]. According to Ayurveda (an ancient Indian medical treatise), nanosilver is a popular additive in several Indian health products due to its unique ability to ﬁght infectious diseases ⁎ Corresponding author at: Centre for Knowledge Management of Nanoscience and Technology, 12-5-32/8, Vijayapuri Colony, Tarnaka, 500 017 AP, India. E-mail addresses: [email protected]
(V. Dhand), [email protected]
(D. Bhatt). 1 Late (in memory of).
http://dx.doi.org/10.1016/j.msec.2015.08.018 0928-4931/© 2015 Elsevier B.V. All rights reserved.
[8b, 8c, 8d, 8e]. On the other hand, SNPs are gaining more importance in the medical ﬁeld as antimicrobials, sterilizers and testing tools for diagnosing and detecting sensitive biomolecules. The large surface area of SNPs allows them to be in better contact with microorganisms and thus, impart good antibacterial activity even at lower concentrations. When SNPs enter inside a pathogen, the particle releases silver ions, thereby killing it. Several mechanisms have been proposed to explain the activity of silver ion or SNPs on bacteria like: i) inactivation of respiratory chain, ii) disruption of cell membrane and leakage of its cellular contents, iii) binding to functional group of proteins causing protein denaturation and cell death, iv) blocking of DNA replication, and v) denaturation of enzymes which transport nutrients across bacterial cell membrane . Due to these properties, nanosilver acts as an effective killing agent against a broad spectrum of (Gram-negative and Gram-positive) bacteria, including the antibiotic resistant strains . SNPs can be synthesized using various methods like reduction reaction, chemical and photochemical reaction, thermal decomposition, radiation assisted method, electrochemical process, sono-chemical and microwave assisted synthesis respectively [6,11–13]. Although these methods can successfully produce silver nanoparticles in an efﬁcient manner it usually involves the use of toxic and hazardous chemicals which have several harmful effects on the environment and human health . Again, the ﬁnal product requires more puriﬁcation steps, as some of the chemicals/reducing agents/by-products left behind
V. Dhand et al. / Materials Science and Engineering C 58 (2016) 36–43
during the process get adsorbed on the surface of SNPs and can cause adverse effects during a medical application or treatment. Additionally, these methods use expensive chemicals and usually require stabilizers to prevent agglomeration of SNPs. On the other hand, green chemistry is a widely accepted alternative process for synthesizing SNPs. Green synthesis does not involve the use of any toxic chemicals, it is cost-effective, environment friendly, zero energy based and less time consuming process. Moreover it does not require the use of any kind of stabilizers. Various environmental friendly materials like plant extracts, bacteria, actinomycetes, fungi and enzymes are categorized and used as sources under ‘green synthesis’. SNPs synthesized by green process are highly compatible for pharmaceutical and other biomedical applications . This process can be easily scaled up for the bulk synthesis of SNPs without involving the use of any high pressure, energy and temperature conditions . Various plant resources have been explored by researchers till date to synthesize silver nanoparticles [11,13–19]. The key phytochemicals responsible for converting silver ions into silver nanoparticles are found to be terpenoids, glycosides, alkaloids, phenolics (ﬂavonoids, coumarins, ubiquinones, tannins, etc.) as identiﬁed in IR spectroscopic studies [20,21]. One of the commonest plant products used for daily consumption is the seed (bean) of Coffea arabica fam. Rubiaceae. The bean of this plant was earlier reported to contain good levels of phenolic compounds [20,21] and thus, it could be used as a potential bioreductant to produce SNPs. Based on the above ﬁndings, the present study was carried out to examine the use of dried roasted coffee seed in the form of a hydroalcoholic extract to prepare silver nanoparticles and to prove its efﬁcacy as an effective antibacterial agent.
2. Experimental 2.1. Materials and reagents The experimental material C. arabica dried roasted seeds were purchased from the local market of Hyderabad. Silver nitrate AR grade (AgNO3), nutrient agar, and nutrient broth were purchased from Sigma-Aldrich, and ampicillin was procured from a local chemist shop, and deionized water was used throughout the experimental procedure. The method employed for the synthesis of SNPs was the green synthesis method, where the hydro-alcoholic extract of C. arabica seeds were used to reduce silver nitrate to silver nanoparticles.
2.2. Preparation of hydro-alcoholic extract 10 g of the dried roasted seeds was ﬁnely crushed and stirred with 1:1 ratio of 50 mL de-ionized water and 50 mL ethanol at 60 °C for 1 h and ﬁltered by a Whatman No. 1 ﬁlter paper to get the extract. The ﬁltrate was further used as a reducing agent and also as a stabilizer. The ﬂowchart depicts the stepwise scheme (Fig. 1a) adopted during the synthesis of the extract. 2.3. Synthesis of silver nanoparticles The SNPs were synthesized using 0.02 M, 0.05 M, and 0.1 M silver nitrate solutions and hydro-alcoholic extract of seeds of C. arabica (volume of extract was kept constant for all the three different concentrations). Both the solutions were mixed to initiate the reduction of AgNO3 solution into Ag+ ions. The mixture was then stirred continuously for about 10 min on a magnetic stirrer and the solution was further incubated at room temperature for 2 h. Color changes were observed from light brown to dark brown indicating that the reaction has terminated (Fig. 1b–g). The reduced mixture was then puriﬁed by repeated centrifugation at 4000 rpm for 5 min. The supernatant was transferred to a clean dry beaker for further settlement of particles and repeated process of centrifugation was carried out to purify SNPs. The sample so obtained was dried under an infra-red lamp and was then stored for further characterizations. 3. Characterization Characterization of the samples were carried out using a Bruker AXS D8 Advance X-ray diffractometer (XRD) operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation (1.540 Å) between 2θ° angles of (30°–80°) for analyzing the crystal structure and peak data respectively. UV–vis spectral analysis was carried out using a JASCO V-700 spectrophotometer. UV–Visible spectrums of SNPs were recorded between the range from 300 to 600 nm. Particle size distribution was carried out by a Dynamic Light Scattering (DLS)-Horiba nanoparticle analyzer SZ-100 equipped with a 532 nm laser. Fourier transform infrared spectroscopy (FT-IR) studies were carried out using a Shimadzu FTIR spectrophotometer (FTIR 8400). The obtained samples were prepared using the KBr pellet technique and were analyzed to check the presence of bio-functional moieties of coffee extract and the surface chemistry
Fig. 1. (a) Detailed process ﬂowchart for the synthesis of silver nanoparticles (SNPs) using coffee extract, (b–g) stepwise thematic representation of the synthesis carried out.
V. Dhand et al. / Materials Science and Engineering C 58 (2016) 36–43
of the reduced silver ion. The FTIR spectrums were collected at a spatial resolution of 4 cm− 1 in the transmission mode, between 4000–400 cm−1 respectively. Morphology of the obtained silver nanoparticles was analyzed using electron microscopes (SEM: Hitachi S520: EDXA: Oxford Link ISIS-300 and TEM: Tecnai-12 FEI) along with elemental ﬁngerprinting after sonicating the SNPs for 1 h in ethanol. Antibacterial activities of the bio-synthesized silver nanoparticles were carried out on the E. coli and S. aureus using the standard well diffusion method. The zone of inhibition (ZI) during the sensitivity test was assessed using a standard antibiotic (ampicillin). The growth of the bacterial culture was carried out in a sterilized nutrient agar medium. 3.1. Antimicrobial susceptibility testing Antibiotic susceptibility testing for all the collected samples was done by the Kirby–Bauer method on Mueller–Hinton agar plates (Merck-German) as recommended by the 2011 Clinical and Laboratory Standards Institute guidelines . The bacteria [Staphylcoccus aureus (ATCC 25923); Escherichia coli (ATCC 25922)] were inoculated into nutrient broth medium, and incubated at 37 °C for 2 to 4 h until it reached the turbidity of a 0.5 McFarland standard. Then using a sterile swab they were cultured on a Mueller–Hinton agar plate. Then the surface of the agar was punched with holes (well) using a sterilized cork borer. The prepared wells were then ﬁlled with an equal volume of SNPs mixed in deionized water (50 μL) and standard drug (ampicillin 125 μg/mL) separately. After a period of pre-incubation (for 15 min), the inoculated plates were incubated at appropriate temperature for 24 h. The zone of inhibition (ZI) was measured by a Vernier caliper as a parameter of antibacterial property related to the synthesized silver nanoparticles. The same procedure was repeated for control (sterilized water) respectively. An additional media control plate was also kept aside to monitor any contamination during testing. 4. Results and discussion 4.1. X-ray diffraction studies (XRD) The XRD pattern (Fig. 2a) of the silver nanoparticles obtained after reduction using coffee powder showed four intense peaks at the 2θ angles of 38°, 44°, 64° and 77° respectively in all the samples. The patterns
show good match with four different JCPDS cards # 87-0718; 87-0719, 87-0717 and 03-0921 respectively [23–26]. The details of the 2θ, dspacing and hkl values of obtained silver powders are shown in Table 1. According to the JCPDS card details, the silver powders obtained exhibit a cubic system with an fcc lattice structure. 4.2. Scanning electron microscopy (SEM) The obtained green synthesized silver sample image (Fig. 2b–d) shows the presence of polymorphic shapes like: rocky, ﬂake type, spherical, ellipsoidal and few irregular granulated compact/fused agglomerates of powder with brighter facets. One can also observe that each agglomerate is fused with each other loosely at their ends. The average sizes of these agglomerates are in the range of 3 μm–20 μm respectively. Energy dispersive X-ray analysis (elemental analysis) embedded within the SEM instrument was carried out to detect the composition of the obtained sample (Fig. 2e) which predominantly shows the presence of elemental silver in high quantities with negligible traces of oxygen as impurity. Also no other element was detected during the complete scan rate procedure. 4.3. Transmission electron microscopy (TEM) and selected-area diffraction (SAD) pattern TEM images (Fig. 3a–c) of the silver samples show nanoparticles exhibiting a typical spherical and an ellipsoidal morphology. The image also shows loosely bound particles created due to the effect of sonication treatment. The approximate particle diameter was found to be in the range of 10–40 nm for 0.1 M sample (a); 10–50 nm for 0.05 M sample (b) and 20–150 nm for 0.02 M sample (c) respectively. The respective SAD patterns (Fig. 3d–f) were provided with the diffraction rings along with the spots and the d-spacings indexed as an fcc crystalline structure of silver according to the JCPDS cards just below their respective TEM images with their molar concentrations. Based on the analysis it is clearly evident that the SNPs appear to be of good crystallinity, as the SAD patterns show a strong presence of bright spots along with their crystal orientations appearing within the diffraction rings. The diameter measurement of the pattern from the centre towards the rings was also consistent with the d-spacing and coincided with the fcc phase of silver ions [27,28] as investigated using XRD.
Fig. 2. (a) X-ray diffractograms of silver nanoparticles synthesized using coffee extract, (b–c) scanning electron microscope images of the SNPs synthesized using coffee extract (b = 0.05 M), (c = 0.02 M) and (d = 0.1 M in chronological order), (e) elemental composition of silver nanoparticles analyzed using energy dispersive X-ray analysis (EDXA).
V. Dhand et al. / Materials Science and Engineering C 58 (2016) 36–43 Table 1 XRD details with 2θ, d-spacing and hkl values of the obtained fcc silver powder. S. no.
38.235 44.302 64.481 77.351 38.155 44.252 64.519 77.460 38.133 44.563 64.588 77.386
2.35 2.04 1.44 1.23 2.35 2.04 1.44 1.23 2.35 2.03 1.44 1.23
111 200 220 311 111 200 220 311 111 200 220 311
4.4. Dynamic light scattering (DLS) The size distribution analyses of the SNPs were carried out using a DLS instrument. The analysis was carried out at 25 °C in a standard
monodispersed medium maintained at a viscosity of 0.892 mPa·s. The graphs of the samples are shown in Fig. 3g–i. Image depicts the almost equal size distribution below 50 nm for the silver samples synthesized using 0.05 M and 0.02 M silver nitrate solutions respectively, whereas the size distribution of sample synthesized using 0.1 M shows an increased particle size with a wide distribution till 150 nm. Whereas, the mean particle size calculated for silver nanoparticles synthesized using 0.02 M sample was around 22 nm; for 0.05 M sample 20 nm and for 0.1 M sample it was 16 nm respectively. The obtained nanoparticle size distribution of SNPs using DLS is also in good agreement with the TEM results. 4.5. UV–Visible spectroscopy Silver particles at nano-range exhibit an unusual optical phenomenon called surface plasmon resonance (SPR), due to the cumulative oscillation of the conducting metal surface electrons in resonance with the non-particulate radiation. This property is largely governed and dependent upon the particle type, size, shape and the local chemical ambience. The characteristic ﬁngerprint zone which exhibits this
Fig. 3. (a–c) Transmission electron microscope and SAD pattern images of the SNPs (a, d = 0.05 M), (b, e = 0.02 M) and (c, f = 0.1 M in chronological order), (g) size distribution of the SNPs for all the molar concentrations as analyzed using DLS.
V. Dhand et al. / Materials Science and Engineering C 58 (2016) 36–43
phenomenon (by the SNPs) predominantly appears in the range of ∼ 400–500 nm respectively [29,30]. The absorption spectra of the samples synthesized using coffee extract are shown in Fig. 4. The spectra shows the characteristic SPR zone of each sample which falls within the reported range (0.1 M = 447 nm; 0.05 M = 459 nm and 0.02 M = 445 nm) respectively. The analysis also shows that as the molar concentration increases the particle size also increases with its movement towards the blue shift of the wavelength. This is also evident with the results obtained using XRD and TEM analyses. Similar instances have earlier been reported by Gavade et al. , with respect to the increase of particle size due to changes in molar concentration. Similar results can also be found in the case of our samples which show that at 0.02 M, the particle size is larger and when the concentration increases to 0.05 M, the particle size gets smaller compared to that of the previous sample. When the concentration is twice the molar ratio that is (0.1 M) the particle size is just smaller than all the previous samples inferring that the change in particle size is concentration dependent. 4.6. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectrum (Fig. 5) of the raw coffee seed extract and the reduced silver nitrate solution with the coffee extract was obtained between the wavenumber ranges of 4000–450 cm−1 respectively. The obtained spectrum of raw coffee seed extract shows the presence of various chemical constituents in detail as listed in Table 2. It is known that during the roasting process (Maillard and Strecker reactions) more soluble solids are formed within the bean along with lesser degradation of chlorogenic acids/aromatic volatiles, and increase in the ﬂavoring agents [31–34]. Much detailed bio-chemical changes occurring within the coffee bean during the roasting can be found in Ref . It is a major challenge in marking all the chemical constituents of coffee within an FTIR spectrum, as several hundreds of complex compounds are evolved with the roasting procedure, hence the present FTIR study highlights the most commonly indexed chemicals for easy understanding. From the spectrum one can ﬁnd the ﬁnger print chemical moieties of coffee in the form of β-type-glycosidic linkages arising from the carbohydrates ranging in the spectrum from 876–880 cm− 1 respectively. Furthermore, Type II-arabinogalactan based carbohydrate peaks can also be observed between the frequencies of 1036–1150 cm−1 respectively. These carbohydrate based peaks are formed during the caramelization of the sucrose and other polysaccharides within the bean during the roasting process [32,33]. The ﬁngerprint signature of stretching mode of caffeine can be observed between 1240–1452 cm− 1 respectively. Certain phenolic groups are
Fig. 5. FT-IR spectroscopy of coffee extract and SNPs synthesized at different molar concentrations.
also observed at 1242 cm−1, this is due to isomerization, epimerization, lactonization and hydrolysis of chlorogenic acids during the roasting process . Chlorogenic acids are a major form of phenolic compounds, that are obtained primarily by esteriﬁcation of trans-cinnamic acids (e.g., ferulic, caffeic and p-coumaric) with quinic acid and melanoidins . The presence of peaks at 1408 and 1603 cm − 1 also represents the deprotonated carboxylic (COO−) groups arising from the changes occurring within the chlorogenic acids. Similarly, peaks arising between 1650–1744 cm− 1 are also attributed to the changes occurring within chlorogenic acids during the roasting process. These changes later add onto the distinct ﬂavor to the coffee. Peaks arising between 2800–3500 cm−1 are attributed to the presence of (–OH) hydroxyl content arising due to the presence of phenol. Parallel within the same range, asymmetrical and symmetrical stretching modes arising from the aliphatic alkane groups (methyl and methylene) are also observed at frequencies of 2925 and 2855 cm−1 respectively [35,36]. The spectrums of bio-synthesized SNPs are displayed below the coffee spectrum. The spectrum shows the changes in the chemistry of coffee solution. The spectrum of the extract also shows several peak shifts and intensity–shape changes of the bands between ﬁngerprint zones of 800–1500 cm− 1. This transition of chemical changes is attributed to the reduction of Ag+ to Ag0 due to the activity of phenolic respectively. 5. Antibacterial activity by the well diffusion method 5.1. Well diffusion method for anti-bacterial activity
Fig. 4. UV–Visible spectroscopy of SNPs.
The antimicrobial activity of the SNP was carried out by the well diffusion method according to the protocol as discussed in the previous section. Fig. 6 shows that SNPs exhibit a good antibacterial activity against both Gram-negative and Gram-positive bacteria. The control (with deionized water) also exhibited zero zone of inhibition (ZI). The plates show that SNPs have higher antibacterial activity against E. coli (Gram-negative) over S. aureus (Gram-positive). From the image we can also observe that, SNP synthesized using 0.02 M did not show any antibacterial activity, whereas the SNPs of 0.05 M and 0.1 M exhibited good ZI in both the cases. When the ZI was compared with the standard drug (ampicillin), it was found that both 0.05 M and 0.1 M SNPs were as strong as the standard drug respectively. Table 3 shows in detail the ZI measurements as calculated using a Vernier caliper along with their respective controls. The controls (deionized water) also did not show any ZI indicating that the procedure was correctly executed. In this study, the lowest concentration of SNPs (0.02 M = 0.107 mg/L) showed
V. Dhand et al. / Materials Science and Engineering C 58 (2016) 36–43
Table 2 Table representing FTIR data of coffee powder and reduced extract with SNP. Chemical signature
Peak position (cm−1)
Characteristic peak remarks
Raw coffee powder
876–880 1036–1150 1242 1240–1452 1408 & 1603 1650–1550 1639 1639 1517 1744 2800–3500 2855 2925 780–1100 1280–1495 1597–1630 1632 1742 2850–2960 3420–3770
Stretching Stretching Stretching Stretching Stretching Stretching Bending Symmetrical stretching Stretching Stretching Stretching Stretching Asymmetrical stretching Stretching Stretching Stretching Bending Stretching Symmetrical and asymmetrical stretching Stretching
β-Type of glycosidic linkages (carbohydrate) Type II-arabinogalactan (carbohydrate) Phenyl-OH Caffeine COO− (deprotonized carboxylic groups) Peptide bonds ν(C_O)/caffeine (N–H) as amide I and amide II C_O/C–C/C–N bands in caffeine and aromatic acids (C_C)/amino groups Esters/lactones –OH C–H C–H Carbohydrates Caffeine C_O N–H C_O C–H –OH
    [31,32] [31,32] [31,32] [31,32] [31,32] [31,32] [31,32]         [31,32]
considerable bacterial lawn growth on the media, indicating that the concentration of SNP is less to induce any antibacterial effect. Whereas, SNPs of 0.05 M (0.2675 mg/L) and 0.1 M (0.535 mg/L) showed high degree of cell death in both the cases respectively. Based on these observations the minimum inhibitory concentration (MIC) of SNPs is ≤ 0.2675 mg/L. Several authors have calculated MIC for SNPs produced by the bio-reduction process of plant extracts. For example, Zeng et al.  synthesized different sized SNPs using macroporous matrix of rice paper plant stem. They found that the MIC of SNPs against E. coli is around 14.1 mg/L. whereas, for Sohail et al.  the MIC of SNPs synthesized using bamboo leaves was found to be around 20 mg/L for E. coli and S. aureus respectively. Similarly, a recent study by Anand and Mandal  showed that the SNPs synthesized using Terminalia bellirica fruit extract exhibited an MIC of 20 mg/mL against both E. coli and S. aureus respectively. Raut et al.  showed that SNPs synthesized using Withania somnifera leaf powder exhibited an MIC of 1.8 ± 0.20 mg/L for E. coli and 1.2 ± 0.14 mg/L for S. aureus respectively. Similarly Kumar et al.  showed that SNPs synthesized using leaf extracts of Paederia foetida exhibited an MIC of 5.394 mg/L for E. coli and 4.454 mg/L for S. aureus respectively. Based on these comparative observations, the present study showed the detrimental effect of low concentrations of SNP against E. coli and S. aureus respectively, where the lowest MIC recorded in our case for both the bacteria is 0.2675 mg/L. 5.2. Plausible mechanism of SNP action The activity of SNPs is suggested to be stronger in the Gram negative bacteria than the Gram positive, due to the difference in the structure of their cell wall. The cell wall of E. coli is composed of a thin layer of peptidoglycan, consisting of linear polysaccharide chains cross-linked by short peptides, thus, forming thinner structures leading to an easy penetration of SNPs as compared to that of S. aureus, where the cell wall possesses thick layer of peptidoglycan. The high bactericidal activity is certainly due to the silver cations released from silver nanoparticles that act as reservoirs for the Ag+ ions as a bactericidal agent. Though the exact mechanism of antibacterial activity of the SNP is still an unknown phenomenon, it is assumed by many researchers that the SNP adheres onto the surface of the bacterial cell and interacts with the sulfur and the phosphorous moieties present within the cell membrane resulting in failure of metabolism and thereby leading to apoptosis/lysis of bacteria . Based on these assumptions, researchers have proposed four possible mechanisms of SNP's antibacterial activity: (1) interference during cell wall synthesis; (2) suppression during
protein bio-synthesis (translation); (3) interference or disruption of transcription process; and (4) disruption of primary metabolic pathways [9,43–45]. Thus the inhibition of bacteria growth is not only limited to structural component but also biochemical components involving deactivation of enzymes, impaired cellular physiology, strong interaction between the ionic charges of the cell components and silver ions resulting in a synergistic action upon the bacteria thereby killing it . Few reports have also suggested that the SNPs induce toxicity effects and kill mammalian cells [46–48]. Studies by various authors also showed that any concentration below ≤ 100 μg/mL can be toxic to human cells which can result in the disruption or reduction of enzymes, cytotoxicity, and genotoxicity induced nucleic acid aberrations [47,49]. Thus inferring that SNPs synthesized using coffee seed extract can also be used effectively for several medical and engineering applications . 6. Conclusions 1. In this study an attempt to synthesize silver nanoparticles using roasted dry C. arabica seed extract based on a green method was demonstrated. This green approach for the SNP synthesis has several advantages for prospective bulk production viz: 1) It is an easy, extremely low energy based, eco-friendly and an economic process, 2) it does not require the use of any kind of hazardous chemicals, reagents for reduction and processing, and 3) there is no need of heating the process and employing the use of other sophisticated apparatus for synthesis. 2. The powder XRD data obtained on silver nanoparticles holds in good agreement with the standard JCPDS cards as reported by the researchers. The XRD pattern revealed that the sample was composed of crystalline face centered cubic (fcc) lattice structures of elemental silver. UV–Visible spectroscopy and DLS results also showed that the size of particles differs as the concentration of AgNO3 increases. 3. The SEM image showed the presence of high density, compact agglomerates of silver particles. The nature of SNPs was found to be highly granulated. The images also showed the formation of different structures which are highly agglomerated with polymorphic shapes like rocky, ﬂake type, spherical, ellipsoidal and few irregular granulated compact/fused agglomerates. Elemental analysis of SNPs conﬁrmed the presence of elemental silver with negligible traces of oxygen as an impurity. Further there are no other elements observed during the EDXA scan, indicating the purity of the synthesized sample. 4. TEM images of different concentrations (0.02, 0.05 and 0.1 M) showed the three dimensional structure of silver nanoparticles
V. Dhand et al. / Materials Science and Engineering C 58 (2016) 36–43
Fig. 6. Antibacterial tests carried out on E. coli and S. aureus in a nutrient agar medium. (a) SNPs inhibiting the growth of E. coli; (b) inhibitory effect of ampicillin antibiotic (standard drug) on the growth of E. coli; (c) SNPs inhibiting the growth of S. aureus; (d) inhibitory effect of ampicillin antibiotic (standard drug) on the growth of S. aureus; (e) control plate after inoculation. (Legends: AP = ampicillin; D/W deionized water as control).
in a spherical and an ellipsoidal shape. SAD pattern showed a definite crystalline ring structure which is in good agreement with the miller indices of poly-dispersed silver nanoparticles. 5. FT-IR results showed, the positive bio-reduction ﬁngerprint zones of coffee phenolics which actively reduced the silver nitrate solution to SNPs. UV–vis spectroscopy conﬁrmed the presence of SNPs
due to a high surface plasmon resonance (SPR) value observed between wavelengths of 400–460 nm respectively. DLS studies showed variable but gradual changes in the particle size based on the increase in molar concentration of the stock solution. 6. Antimicrobial potential of SNPs as a function of its concentration and activity was tested against two different gram stained bacteria like
Table 3 Table representing area of inhibition (cm). S. no.
Zone of inhibition (in cm) for different conc. of SNPsa
Sterilized water (de-ionized)
E. coli S. aureus
0 cm 0 cm
2.3 cm 2.1 cm
3.1 cm 2.7 cm
3.3 cm 2.9 cm
0 cm 0 cm
Amount of SNP in 50 μL of deionized water (mg/L): 0.02 M = 0.107 mg/L; 0.05 M = 0.2675 mg/L; and 0.1 M = 0.535 mg/L.
V. Dhand et al. / Materials Science and Engineering C 58 (2016) 36–43
E. coli (G−ve) and Streptococcus aureus (G+ ve). From the study, SNPs were observed to have strong and almost equal antimicrobial activity when compared with the standard drug “ampicillin”.
Acknowledgment The present work is a self-funded research. Authors acknowledge greatly NCCCM, Hyderabad for SEM analysis and Prof. Ch. Sasikala, HOD, CEN, JNTU for the FTIR analysis. Authors thank IICT, Tarnaka and CNST, JNTU for providing necessary equipments to carry out the SNP synthesis and antibacterial testing. The authors thank Ms. Umme Thahira Khatoon for the help provided during microbiological experiments. The authors sincerely acknowledge our valuable reviewers who with their suggestions and expert advice have helped us in improving the quality of the manuscript. The present paper is also dedicated in memory of my beloved friend (Late) Dr. Deepika Bhatt who has equally contributed and devised the use of coffee extract for this experiment. References  P.V. Kamat, Photophysical, photochemical and photocatalytic aspects of metal nanoparticles, J. Phys. Chem. B 106 (2002) 7729–7744.  D. Manikprabhu, K. Lingappa, Synthesis of silver nanoparticles using Streptomyces coelicolor klmp33 pigment: an antimicrobial agent against extended-spectrum beta-lactamase (ESBL) producing Escherichia coli, Mater. Sci. Eng. C 45 (2014) 434–437.  F.C. Adams, C. Barbante, Nanoscience, nanotechnology and spectrometry, Spectrochim. Acta B 86 (2013) 3–13.  M.K. Shukla, R.P. Singh, C.R.K. Reddy, B. Jha, Synthesis and characterization of agarbased silver nanoparticles and nanocomposite ﬁlm with antibacterial applications, Bioresour. Technol. 107 (2012) 295–300.  T. Som, B. Karmakar, Nano silver:antimony glass hybrid nanocomposites and their enhanced ﬂuorescence application, Solid State Sci. 13 (2011) 887–895.  R. Janardhanan, M. Karuppaiah, N. Hebalkar, T.N. Rao, Synthesis and surface chemistry of nano silver particles, Polyhedron 28 (2009) 2522–2530.  Y. Echegoyen, C. Nerín, Nanoparticle release from nano-silver antimicrobial food containers, Food Chem. Toxicol. 62 (2013) 16–22. [8a] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobials, Biotechnol. Adv. 27 (2009) 76–83; [8b] D. Pal, C.K. Sahu, A. Haldar, Bhasma: The ancient Indian nanomedicine, J. Adv. Pharm. Technol. Res. 5 (2014) 4–12; [8c] A. Chaudhary, Ayurvedic bhasma: nanomedicine of ancient India-its global contemporary perspective, J. Biomed. Nanotechnol. 7 (1) (Feb 2011) 68–69; [8d] Kumar Pal Sanjoy, The Ayurvedic Bhasma: The Ancient Science of Nanomedicine, Recent Pat. Nanomed. 5 (2015) 12–18; [8e] H. Panda, Handbook On Ayurvedic Medicines With Formulae, Processes And Their Uses, ISBN: 978-81-86623-63-3 2004, p. 10.  V.S. Kumar, B.M. Nagaraja, V. Shashikala, A.H. Padmasri, S.S. Madhavendra, B.D. Raju, Highly efﬁcient Ag/C catalyst prepared by electro-chemical deposition method in controlling microorganisms in water, J. Mol. Catal. A 223 (2004) 313–319.  S. Mohanty, S. Mishra, P. Jena, B. Jacob, B. Sarkar, A. Sonawane, An investigation on the antibacterial, cytotoxic, and antibioﬁlm efﬁcacy of starch-stabilized silver nanoparticles, Nanomedicine: NBM 8 (2012) 916–924.  N.M. Shinde, A.C. Lokhande, C.D. Lokhande, A green synthesis method for large area silver thin ﬁlm containing nanoparticles, J. Photochem. Photobiol. B 136 (2014) 19–25.  K.P. Bankura, D. Maity, M.M.R. Mollick, D. Mondal, B. Bhowmick, M.K. Bain, A. Chakraborty, J. Sarkar, K. Acharya, D. Chattopadhyay, Synthesis, characterization and antimicrobial activity of dextran stabilized silver nanoparticles in aqueous medium, Carbohydr. Polym. 89 (2012) 1159–1165.  V. Kathiravan, S. Ravi, S. Ashokkumar, Synthesis of silver nanoparticles from Melia dubia leaf extract and their in vitro anticancer activity, Spectrochim. Acta A 130 (2014) 116–121.  P.U. Rani, P. Rajasekharreddy, Green synthesis of silver-protein (core–shell) nanoparticles using Piper betle L. leaf extract and its ecotoxicological studies on Daphnia magna, Colloids Surf. A 389 (2011) 188–194.  C.K. Tagad, S.R. Dugasani, R. Aiyer, S. Park, A. Kulkarni, S. Sabharwal, Green synthesis of silver nanoparticles and their application for the development of optical ﬁber based hydrogen peroxide sensor, Sensors Actuators B Chem. 183 (2013) 144–149.  A.K. Mittal, Y. Chisti, U.C. Banerjee, Synthesis of metallic nanoparticles using plant extracts, Biotechnol. Adv. 31 (2013) 346–356.  K. Vijayaraghavan, S.P. Kamala Nalini, N. Udaya Prakash, D. Madhankumar, Biomimetic synthesis of silver nanoparticles by aqueous extract of Syzygium aromaticum, Mater. Lett. 75 (2012) 33–35.  S. Venkateswarlu, B.N. Kumar, B. Prathima, K. Anitha, N.V.V. Jyothi, A novel green synthesis of Fe3O4–Ag core shell recyclable nanoparticles using Vitis vinifera stem extract and its enhanced antibacterial performance, Physica B 457 (2015) 30–35.
 Y.S. Rao, V.S. Kotakadi, T.N.V.K.V. Prasad, A.V. Reddy, D.V.R. Sai Gopal, Green synthesis and spectral characterization of silver nanoparticles from Lakshmi tulasi (Ocimum sanctum) leaf extract, Spectrochim. Acta A 103 (2013) 156–159.  R. Mariselvam, A.J.A. Ranjitsingh, A.U.R. Nanthini, K. Kalirajan, C. Padmalatha, P.M. Selvakumar, Green synthesis of silver nanoparticles from the extract of the inﬂorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity, Spectrochim. Acta A 129 (2014) 537–541.  C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water borne pathogens, Colloids Surf. B 76 (2010) 50–56.  V. Pereira, C. Lopes, A. Castro, J. Silva, P. Gibbs, P. Teixeira, Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal, Food Microbiol. 26 (2009) 278–282.  V. Gopinath, S. Priyadarshini, N.M. Priyadharsshini, K. Pandian, P. Velusamy, Biogenic synthesis of antibacterial silver chloride nanoparticles using leaf extracts of Cissus quadrangularis Linn, Mater. Lett. 91 (2013) 224–227.  D. Rajesh, C.S. Sunandana, XRD, optical and AFM studies on pristine and partially iodized Ag thin ﬁlm, Results Phys. 2 (2012) 22–25.  K. Dhanapal, T.A. Revathy, M. Anand Raj, V. Narayanan, A. Stephen, Magnetic anisotropy studies on pulsed electrodeposited Ni/Ag/Ni trilayer, Appl. Surf. Sci. 313 (2014) 698–703.  S. Duhan, N. Kishore, P. Aghamkar, S. Devi, Preparation and characterization of sol– gel derived silver–silica nanocomposite, J. Alloys Compd. 507 (2010) 101–104.  G.K. Podagatlapalli, S. Hamad, M.A. Mohiddon, S. Venugopal Rao, Effect of oblique incidence on silver nanomaterials fabricated in water via ultrafast laser ablation for photonics and explosives detection, Appl. Surf. Sci. 303 (2014) 217–232.  N.L. Gavade, A.N. Kadam, M.B. Suwarnkar, V.P. Ghodake, K.M. Garadkar, Biogenic synthesis of multi-applicative silver nanoparticles by using Ziziphus jujuba leaf extract, Spectrochim. Acta A 136 (Part B) (2015) 953–960.  N. Venugopal, A. Mitra, Inﬂuence of temperature dependent morphology on localized surface plasmon resonance in ultra-thin silver island ﬁlms, Appl. Surf. Sci. 85 (2013) 357–372.  G. Kaur, R.K. Verma, D.K. Rai, S.B. Rai, Plasmon-enhanced luminescence of Sm complex using silver nanoparticles in polyvinyl alcohol, J. Lumin. 132 (2012) 1683–1687.  P. Capek, E. Paulovičová, M. Matulová, D. Mislovičová, L. Navarini, F.S. Liverani, Coffea arabica instant coffee—chemical view and immunomodulating properties, Carbohydr. Polym. 103 (2014) 418–426.  N. Wang, L.T. Lim, Fourier transform infrared and physicochemical analyses of roasted coffee, J. Agric. Food Chem. 60 (2012) 5446–5453.  R.J. Clarke, Coffee: green coffee/roast and ground, second ed., Encyclopaedia of Food Science and Nutrition, 3 2003, pp. 1487–1493.  A. Farah, Coffee constituents, in: Yi-Fang Chu (Ed.), Coffee: Emerging Health Effects and Disease Prevention, John Wiley & Sons Inc., Blackwell Publishing Ltd, 2012 (Chapter 2).  D. Pujol, C. Liu, J. Gominho, M.À. Olivella, N. Fiol, I. Villaescusa, H. Pereira, The chemical composition of exhausted coffee waste, Ind. Crop. Prod. 50 (2013) 423–429.  G. Suresh, P.H. Gunasekar, D. Kokila, D. Prabhu, D. Dinesh, N. Ravichandran, B. Ramesh, A. Koodalingam, G.V. Siva, Green synthesis of silver nanoparticles using Delphinium denudatum root extract exhibits antibacterial and mosquito larvicidal activities, Spectrochim. Acta A 127 (2014) 61–66.  F. Zeng, C. Hou, S. Wu, X. Liu, Z. Tong, S. Yu, Silver nanoparticles directly formed on natural macroporous matrix and their antimicrobial activities, Nanotechnology 18 (2007) 055605–055613.  Y. Sohail, L. Liu, J. Yao, Biosynthesis of silver nanoparticles by bamboo leaves extract and their antimicrobial activity, J. Fiber Bioeng. Inform. 6 (2013) 77–84.  K.K. H. Anand, B.K. Mandal, Activity study of biogenic spherical silver nanoparticles towards microbes and oxidants, Spectrochim. Acta A 135 (2015) 639–645.  R.W. Raut, V.D. Mendhulkar, S.B. Kashid, Photosensitized synthesis of silver nanoparticles using Withania somnifera leaf powder and silver nitrate, J. Photochem. Photobiol. B 132 (2014) 45–55.  S. Kumar, M. Singh, D. Halder, A. Mitra, Mechanistic study of antibacterial activity of biologically synthesized silver nanocolloids, Colloids Surf. A 449 (2014) 82–86.  A. Rawani, A. Ghosh, G. Chandra, Mosquito larvicidal and antimicrobial activity of synthesized nano-crystalline silver particles using leaves and green berry extract of Solanum nigrum L. (Solanaceae: Solanales), Acta Trop. 128 (2013) 613–622.  F.C. Tenover, Mechanisms of antimicrobial resistance in bacteria, Am. J. Infect. Control 34 (2006) S3–S10.  S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao, D. Dash, Characterization of enhanced antibacterial effects of novel silver nanoparticles, Nanotechnology 18 (2007) 225103–225111.  M.R. Bindhu, M. Umadevi, Antibacterial and catalytic activities of green synthesized silver nanoparticles, Spectrochim. Acta A 135 (2015) 373–378.  P. Dubey, I. Matai, S.U. Kumar, A. Sachdev, B. Bhushan, P. Gopinath, Perturbation of cellular mechanistic system by silver nanoparticles toxicity: cytotoxic, genotoxic and epigenetic potential, Adv. Colloid Interf. Sci. (2015), http://dx.doi.org/10.1016/ j.cis.2015.02.007 (in press).  P.V. Asharani, G. Low Kah Mun, M.P. Hande, S. Valiyaveettil, Cytotoxicity and genotoxicity of silver nanoparticles in human cells, ACS Nano 3 (2009) 279–290.  Y.H. Hsin, C.F. Chen, S. Huang, T.S. Shih, P.S. Lai, P.J. Chueh, The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells, Toxicol. Lett. 179 (2008) 130–139.  H. Arakawa, J.F. Neault, H.A. Tajmir-Riahi, Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis, Biophys. J. 81 (2001) 1580–1587.