Antibacterial activity of silver nanoparticles synthesized from latex and leaf extract of Ficus sycomorus

Industrial Crops and Products 62 (2014) 228–234

Contents lists available at ScienceDirect

Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Antibacterial activity of silver nanoparticles synthesized from latex and leaf extract of Ficus sycomorus W.M. Salem a,∗ , M. Haridy b , W.F. Sayed a , N.H. Hassan c a b c

Department of Botany, Faculty of Science, South Valley University, 83523 Qena, Egypt Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, South Valley University, 83523 Qena, Egypt Central laboratory, South Valley university, 83523 Qena, Egypt

a r t i c l e

i n f o

Article history: Received 17 April 2014 Received in revised form 13 August 2014 Accepted 14 August 2014 Keywords: Ficus sycomorus Latex Leaf extract Silver nanoparticles Pathogenic bacteria

a b s t r a c t The development of green synthesis of nanoparticles has received increasing attention due to ease of preparation, less chemical handling, and eco-friendly. In the present study, crystalllization of silver ions to nanosized particles by latex and leaf aqueous extract of Ficus sycomorus through bioreduction process was assessed. Strong plasmon resonance of silver nanoparticles was observed around 435 nm. UV–visible spectroscopy, transmission electron microscope (TEM) and energy dispersive X-ray fluorescence (EDXRF) were performed to examine the formation of silver nanoparticles (SNPs). The antibacterial activity of SNPs was tested against nine human pathogenic Gram −ve bacteria and one Gram +ve bacteria. Silver nanoparticles of extracts showed improved antibacterial activity on all the tested strains than for the extracts alone. This was confirmed either by optical density or zone of inhibition measurements. Silver nanoparticles was more effective in liquid than in solid medium probably because of better contact, for the higher silver content, with bacterial cells. It is concluded that fig (F. sycomorus) leaves and latex extracts can be used for the synthesis of SNPs that is environmentally friendly and cost effective. These preparations can be used for various biotechnology and medical applications for controlling pathogenic bacteria with better dispersion and, consequently, better efficiency in aqueous environment. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The use of various biological entities has received considerable attention in the field of nano-biotechnology (Park et al., 2011). Biosynthesis of nanoparticles is an important area in the field of nanotechnology which has economic and eco-friendly benefits over chemical and physical methods of synthesis (Sun et al., 2014). The uses of inorganic antibacterial agent such as metal have became the new generations for antibacterial materials because of its stability compared to organic one (Sondi and Sondi, 2004). Silver ions and silver-based compounds are highly toxic to microorganisms including 16 major species of bacteria (Zhao and Stevens, 1998). This makes silver an excellent choice for multiple roles in the medical field (Pavagadhi et al., 2014). Silver is generally used in the nitrate form to induce antimicrobial effect, and when nanoparticles are used, there is a huge increase in the surface area to be in contact with microbial cells (Binupriya et al., 2010a; Prabhu and Poulose, 2012; Suganya et al., 2012). Green nanoparticle synthesis from plants is a rapid single-step method, low-cost, eco-friendly, and without using high pressure, ∗ Corresponding author. Tel.: +20 1013065777; fax: +20 96 5211279. E-mail address: wesam [email protected] (W.M. Salem). http://dx.doi.org/10.1016/j.indcrop.2014.08.030 0926-6690/© 2014 Elsevier B.V. All rights reserved.

energy or toxic chemicals (Huang et al., 2007; Sathishkumar et al., 2009a; Binupriya et al., 2010b; Suganya et al., 2012). Therefore, medicinal plants, of well established therapeutic importance, are being widely used for the synthesis of silver nanoparticles (Sathishkumar et al., 2009b, 2010a). Many reports are available on the biosynthesis of silver nanoparticles using several plant extracts, particularly Cymbopogon citratus (Masurkar et al., 2011, 2012), Garcinia mangostana leaves (Ravichandran et al., 2011), and latex of some plants (Patil et al., 2012). The most effectively studied nanoparticles nowadays are those made from noble metals, in particular Ag, Pt, Au, and Pd (Huang et al., 2007; Sathishkumar et al., 2009c, 2010b; Sneha et al., 2011; Zhan et al., 2011; Patil et al., 2012). Ficus sycomorus, a medicinal plant belonging to the family Moraceae, was reported for its inhibitory effect on bacterial growth (Shankar et al., 2004; Salem et al., 2014). Aqueous extract of the plant contained pharmacologically active substances including tannins, saponins, reducing sugars, alkaloids, and flavones aglycones without any hematological, hepatic, or renal toxicities (Kubmarawa et al., 2007). This study is the first report describing the synthesis of silver nanoparticles (SNPs) of F. sycomorus leaves and latex and its antibacterial effect, on some human-pathogenic bacteria.

W.M. Salem et al. / Industrial Crops and Products 62 (2014) 228–234

2. Materials and methods 2.1. Plant materials and preparation of the extracts Leaves and latex of F. sycomorus were collected from South Valley University campus at Qena city, Egypt. Plant latex was collected by cutting the green stems and receiving the white milky latex in sterile bottles. Crude latex was stored until being used at −20 ◦ C (Singhal and Kumar, 2009). Healthy leaves of F. sycomorus were collected, washed thoroughly with tap water followed by distilled water and air dried on a paper towel for 4–6 days. Dry leaves were ground in a tissue grinder (IKA A10, Germany) to fine powder. Ten grams of the powder were dissolved in 100 ml sterile bi-distilled water and heated for one hour at 80 ◦ C. The obtained extract was filtered through Whatman No. 1 filter paper, the filtrate collected in 250 ml Erlenmeyer flask and then stored at 4 ◦ C until being used (modified from Verastegui et al., 1996). 2.2. Biosynthesis of nano-scale silver particles 2.2.1. From latex extract Stored crude latex was thawed and 3 ml was completed to 100 ml volume using deionized water. Five milliliters of this solution were heated in a conical flask at 60 ◦ C with constant stirring for 15 min in a water bath. For gradual formation of nanoparticles, 50 ml of AgNO3 solution (1 mM) were heated at 60 ◦ C with constant stirring for 15 min in a water bath then mixed with the latex solution and heated at 80 ◦ C for 30–45 min (Wiley et al., 2006). The formation of silver nanoparticles was confirmed by spectrophotometric determination as indicated below. 2.2.2. From leaf extract With constant stirring, 50 ml of AgNO3 solution (1 mM) were added drop-wise to 10 ml of the stored aqueous extract of F. sycomorus leaves at 50–60 ◦ C for the reduction of Ag+ ions. This solution was incubated in the dark at 37 ◦ C until being used. A control solution (without extract) was also incubated under the same conditions (Kumar et al., 2011). 2.3. UV–visible spectra analysis UV–visible spectral analysis, for nanoparticles of both latex and leaves, was carried out by measuring the optical density (OD) using “Shimadzu UV-2401 PC, Japan” scanning spectrophotometer. Measurements were performed between 200 and 800 nm with a resolution of 1 nm and scanning speed of 300 nm/min. The reduction of Ag+ ions was monitored by measuring the UV–vis spectrum of 1 ml aliquots of sample and 2 ml deionized water in quartz cell as indicated earlier (Wiley et al., 2006). Silver nitrate (1 mM) was used to adjust the baseline as a blank.

229

JSX 3222, Japan” EDXRF element analyzer. For complete recovery of nanoparticles from extracts, the mixture was centrifuged as 1000 rpm for 15 min at ambient temperature using “Biofuge Primo R, Heraeus® , Germany” centrifuge. Quantitative analysis of elements was measured as weight percentage (ms%). 2.5. Bactericidal studies in broth Antimicrobial activity of the prepared Ag nanoparticles was tested against 10 human-pathogenic bacteria including the Gram negatives Salmonella typhimurium (ATCC14028), Salmonella typhi (ATCC19430), Shigella flexneri (ATCC12022), Enterococcus faecalis (ATCC29212), Enterobacter cloacae (ATCC13047), Enterobacter aerogenes (ATCC13048), Pseudomonas aeruginosa (ATCC278223), Klebsiella pneumoniae (ATCC13888), Escherichia coli (ATCC25922), and the methicillin-resistant Gram positive Staphylococcus aureus (MRSA, ATCC43300). Bacterial strains were maintained on Mueller–Hinton agar slants and incubated at 37 ◦ C for 24–48 h (Muller and Hinton, 1941). Three replicates of each microorganism were set up. The inocula were spread over Mueller–Hinton agar plates (107 cfu). The nanoparticles of leaves and latex extracts (500 ␮l each) were added into test tubes containing 9 ml of Mueller–Hinton broth medium. Each tube was inoculated with 500 ␮l of the 10 tested bacterial suspensions and incubated at 37 ◦ C for 24 h. Three replicates were set up for each treatment. Control tubes were prepared by adding 500 ␮l of each bacterial suspension without the extract. Chloramphenicol and AgNO3 treatments (as above) were prepared for comparison. Bacterial growth was measured as optical density (OD) at 600 nm using “SPECTRONIC® GENESYSTM 2PC” spectrophotometer, Spectronic Instruments, USA. The percentage of bacterial growth inhibition was calculated as indicated earlier (Banjara et al., 2012) as follows: Percentage of growth inhibition =

OD of control − OD of test OD of control × 100

2.6. Disk diffusion studies Bacterial sensitivity to antibiotics is commonly tested using a disk diffusion test, employing antibiotic impregnated disks (Case and Johnson, 1984). Antibacterial activity was determined against the above pathogenic strains using the paper disk assay method (Bauer et al., 1966). The test was carried out by using a 7 mm filter paper saturated with 50 ␮l of the test solution that was placed over the inoculated medium surface. Chloramphenicol (10 mg ml−1 ) and silver nitrate solution (1 mM) were used as reference antimicrobial agents in the same manner. All plates were incubated at 37 ◦ C for 24–48 h. After the incubation period, the zone of inhibition was measured as an indicator for antibacterial activity compared to chloramphenicol, AgNO3 , and aqueous extracts of leaves and latex.

2.4. Characterization of nano-scale silver nanoparticles 2.7. Statistical analysis After the reaction, the biomass had settled at the bottom of the conical flasks and the suspension above the precipitate was sampled for transmission electron microscopy (TEM) observation. The size and shape of extract nanoparticles were observed at 70 kV using “JEOL-2010, Japan” transmission electron microscope (TEM) equipped with digital “Kodak Megaplus® 1.6i camera” and image analysis and processing software (AMT, USA). Sample was prepared by placing a drop of each solution on a carbon-coated copper grid and drying in room temperature as previously described by Sathishkumar et al. (2009b). The size distribution of the resulting nanoparticles was estimated on the basis of TEM micrographs. Energy dispersive X-ray (EDX) analyses were performed on a “JEOL

Data were analyzed using Kruskal–Wallis test followed by post hoc Dunn’s multiple comparisons. Differences were considered significant at P values of ≤0.05. For all statistical analyses, GraphPad Prism version 5 was used. 3. Results 3.1. Biosynthesis of nano-scale silver particles Bioreduction of silver nanoparticles from aqueous AgNO3 solution can be examined using UV–vis spectroscopy indirectly. The

230

W.M. Salem et al. / Industrial Crops and Products 62 (2014) 228–234

sycomorus. The particles of both latex and leaves are sometimes agglomerated and not in a physical contact but are separated by approximately uniform distances. Particle density was higher in case of latex extract compared to leaf, which could be attributed to the possibility of more reductants in latex extract (Fig. 2). 3.2.2. EDXRF analysis of SNPs The particles were initially verified to contain Ag as the major component using energy dispersive X-ray fluorescence (EDXRF) analysis which confirmed the presence of strong signals of elemental silver. In Fig. 3, the peaks around 3 and 3.1 keV are related to the silver elements in the F. sycomorus as shown earlier by (Shameli et al., 2011). Additionally, the EDXRF spectra for the Ag/F. sycomorus confirmed the presence of SNPs in both leaf and latex extraction with some other peaks. From EDAX spectrum, it is clear that F. sycomorus has percent yield of silver content in the particles, was 98.49% for latex nanoparticles and 70.4% for nanoparticles of leaves, respectively as calculated by the instrument (Fig. 3). The results indicate that the synthesized nanoparticles are composed of high purity SNPs. 3.3. Bactericidal studies in broth Fig. 1. UV–vis spectra of silver nanoparticles synthesized using Ficus sycomorus (A) latex extract and (B) leaf extract; initial AgNO3 concentration, 1 mM.

silver nanoparticles (SNPs) exhibited dark brown color with leaf extracts and dark gray with latex extracts after 2 h of the addition of leaf and latex extracts of F. sycomorus to the colorless AgNO3 solution, due to the excitation of plasmon vibrations in the metal nanoparticles. Under UV–vis spectroscopy, silver nanoparticles showed surface plasmon resonance (SPR) with absorption bands at  = 434 nm for latex and  = 435 nm for leaves of F. sycomorus (Fig. 1). 3.2. Characterization of nano-scale silver nanoparticles 3.2.1. Electron microscopy and particle size distribution TEM measurements of the synthesized nanoparticles give us a clear idea of the shape and size of the Ag NPs produced extracellularly by F. sycomorus extracts. As seen in Fig. 2 the majority of the particles are ellipsoidal in shape. Few spherical and irregular silver nanoparticles can also be noticed. The general size of the particles is irregular, and the particles appear to be in different phases of growth. TEM observation revealed that the size of silver nanoparticles is ≤20 nm for leaves and ≤100 nm diameter for latex of F.

Percentage of growth inhibition, on liquid medium, was calculated for plant extracts alone and for its silver nanoparticles, considering the growth on normal media (without extracts) as 100%. Measurements of optical density (OD) were much more decreased when bacterial suspensions were treated with extract nanoparticles than extracts alone (Table 1). Therefore, with both SNPs of latex and leaves, the percentage of growth inhibition, for all the tested bacterial strains, ranged from 92.6 to 100%. For plant extracts alone, only the aqueous extract of latex inhibited S. typhi (ATCC19430) completely (100% inhibition). Percentages of inhibition by SNPs of leaves and latex were 100% for all the tested strains. Exceptions were K. pneumoniae, S. aureus (97.3, 98.4%) for latex SNPs and K. pneumoniae, E. coli, S. aureus (95, 92.6, 99.9%) for leaves SNPs (Table 1). 3.4. Disk diffusion studies Silver nanoparticles, synthesized from both leaves and latex, showed variations in the diameter of inhibition zone, on solid medium, against all the tested bacteria. Maximum zones of inhibition were 19 mm for nanoparticles of leaves on P. aeruginosa (ATCC278223) and 18 mm for latex nanoparticles on S. flexneri

Table 1 Bacterial growth inhibition after treatment with F. sycomorus extracts and SNPs on liquid medium.a Treatments

Growth inhibition (%) measured as ODb

Bacteria

Chloramphenicol (10 mg l−1 )

K. pneumoniae (ATCC13888) E. coli (ATCC25922) S. aureus (ATCC43300) Enterococcus faecalis (ATCC29212) P. aeruginosa (ATCC278223) Enterobacter aerogenes (ATCC13048) Enterobacter cloacae (ATCC13047) Salmonella typhi (ATCC19430) Salmonella typhimurium (ATCC14028) Shigella flexneri (ATCC12022)

96.2 100 99.3 100 100 100 100 100 99.6 100

± ± ± ± ± ± ± ± ± ±

0.6 0.0 0.18 0.0 0.0 0.0 0.0 0.0 0.4 0.0

AgNO3 (1 mM) 93.5 94.2 99.8 100 100 100 100 100 100 100

± ± ± ± ± ± ± ± ± ±

Leaf extract 0.2 0.05 0.17 0.0 0.0 0.0 0.0 0.0 0.0 0.0

11.5** 92.3 69.8* 66.4* 30.55** 90 22.2** 87.02 44.5** 80

± ± ± ± ± ± ± ± ± ±

0.8 4.5 3 0.9 4 0.9 3.2 1.3 2 3

Leaf SNPs 95 92.6 99.9 100 100 100 100 100 100 100

± ± ± ± ± ± ± ± ± ±

1.2 1.4 0.16 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Latex 12.6** 62.3* 73* 45.7** 35.3** 64.7* 9.2** 100 64* 27.6**

Latex SNPs ± ± ± ± ± ± ± ± ± ±

2.9 0.8 0.8 0.2 6.6 1.1 0.06 0.0 1.1 2.6

97.3 100 98.4 100 100 100 100 100 100 100

± ± ± ± ± ± ± ± ± ±

0.04 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0

SNPs – silver nanoparticles. a Data were analyzed using Kruskal–Wallis test followed by post hoc Dunn’s multiple comparisons, calculations were based on considering bacterial growth without treatment as 100% growth. b (OD) optical density measured at 660 nm. * Differences were considered significant at (P ≤ 0.05). ** Highly significant differences (P ≤ 0.01) as compared with control.

W.M. Salem et al. / Industrial Crops and Products 62 (2014) 228–234

231

Fig. 2. TEM images of silver nanoparticles synthesized using Ficus sycomorus (A) latex extract and (B) leaf extract (bar scale: 100 nm for figure (A); 20 nm for figure (B)).

(ATCC12022). The minimum zone of inhibition was 7 mm for both nanoparticles of leaves and latex on S. typhi (ATCC19430) and S. typhimurium (ATCC14028), respectively (Table 2). The inhibitory effect of latex SNPs was on all the tested bacteria except for K. pneumoniae and S. typhimurium where it was the same or lower than extract alone. The same effect was recorded for leaf extract SNPs but only with K. pneumoniae. In general, the inhibition was higher for SNPs of extracts than extracts alone except in few cases as illustrated in Table 2.

4. Discussion Several approaches have been employed to improve the methods for synthesizing silver nanoparticles including chemical and biological methods. Silver nanoparticles were prepared from Aloe vera extract after 24 h of incubation and also synthesized from Acalypha indica leaf extract after only 30 min of incubation (Chandran

et al., 2006; Krishnaraj et al., 2010). Similarly, in the current study, silver nanoparticles were synthesized from leaves and latex extracts of F. sycomorus that started its formation rapidly after 2 h of incubation. The control AgNO3 solution (without extracts) showed no color change. After the addition of leaves and latex extracts, the color of silver nitrate solution turned dark brown and dark gray, for the former and latter, within 30 min of addition. The intensity of colors increased as the time of incubation period increased (Sneha et al., 2010). This may be due to the excitation of Surface Plasmon Resonance (SPR) effect and the reduction of AgNO3 (Mulvaney, 1996). TEM images of the silver nanoparticles produced by F. sycomorus leaf or latex extract are shown in Fig. 2. In general, variations in the shape and size of nanoparticles synthesized by biological systems are common (Binupriya et al., 2010a,b; Babu and Prabu, 2011). But the difference is very meager compared to other reports on biological synthesis of SNPs. This result is very interesting as the attainment of near uniform shape and size of SNPs was believed to be barely possible through bio-route (Binupriya

232

W.M. Salem et al. / Industrial Crops and Products 62 (2014) 228–234

Table 2 Bacterial growth inhibition after treatment with F. sycomorus extracts and SNPs on solid medium.a Treatment

Inhibition zone (mm)

Bacteria

Chloramphenicol (10 mg l−1 )

K. pneumoniae (ATCC13888) E. coli (ATCC25922) S. aureus (ATCC43300) Enterococcus faecalis (ATCC29212) P. aeruginosa (ATCC278223) Enterobacter aerogenes (ATCC13048) Enterobacter cloacae (ATCC13047) Salmonella typhi (ATCC19430) Salmonella typhimurium (ATCC14028) Shigella flexneri (ATCC12022)

20 25 37 14 34 40 37 50 25 12

± ± ± ± ± ± ± ± ± ±

AgNO3 (1 mM)

0.0 5 2.5 4 0.0 0.0 2.5 0.0 5.5 0.5

11 9 11 14 18 15 10 30 13 16

± ± ± ± ± ± ± ± ± ±

0.0 1.5 1.5 0.5 0.0 0.0 2.5 0.0 5 1.5

Leaf extract 7± 12 ± 8± 9± 8± 11 ± 0.0 0.0 12 ± 10 ±

0.0 1.5 0.0 0.5 1 2

1 2

Leaf SNPs 7 14 9 14** ** 19 13 13** 7** 13 12

± ± ± ± ± ± ± ± ± ±

0.0 0.0 0.0 0.5 3.5 2 0.5 0.0 1.5 0.9

Latex 9± 11 ± 12 ± 12 ± 8± 9± 0.0 6± 13 ± 12 ±

Latex SNPs 0.0 2 0.0 3 0.0 0.0 2 0.0 3.2

8 13 ** 15 16** 13** 13** 12** 9* 7** 18**

± ± ± ± ± ± ± ± ± ±

0.0 1.5 3.5 0.0 1.5 0.0 1 0.0 0.0 2

SNPs – silver nanoparticles. a Data were analyzed using Kruskal–Wallis test followed by post hoc Dunn’s multiple comparisons, calculations were based on considering plant extract as control for SNPs. * Differences were considered significant at (P ≤ 0.05). ** Highly significant differences (P ≤ 0.01) as compared with control.

1.4 1.2

Counts[x1.E+3]

1.1 0.9 0.8 0.6 Ag

0.5

Au Au Au Au Ag

0.3

Ag

Au

0.1

0.0 0.00

5.00

10.00

15.00

20.00 keV

25.00

30.00

35.00

40.00

(A) 1.4 1.2

Counts[x1.E+3]

1.1 0.9 0.8 0.6 0.5 0.3 0.1

La La Fe Ca Fe Ag La Ca Sr

0.0 0.00

5.00

Sr

Sr Ag La La

Ag

10.00

15.00

20.00 keV

25.00

30.00

35.00

La La 40.00

(B) Fig. 3. EDXRF analysis of SNPs of F. sycomorus extracts shows representative EDXRF profile for synthesized silver nanoparticles from Ficus sycomorus (A) latex and (B) leaves extracts. The energy dispersive X-ray analysis reveals strong signal in the silver region and confirms the formation of silver nanoparticles. Other elemental signals are recorded possibly due to elements from enzymes or proteins present within the leaf or latex of Ficus sycomorus.

et al., 2010b). Babu and Prabu (2011) observed similar results with Calotropis procera flower extract and they attributed the various grain sizes of Ag particles due to the agglomeration of Ag in the preparation for TEM analysis. In the current study, TEM figures (Fig. 2) showed that SNPs are ellipsoidal in shape, sometimes spherical with few agglomerated particles. It was also reported earlier that silver nanoparticles should be in the range from 1 to 100 nm in size (Chandran et al., 2006; Sathishkumar et al., 2009b, 2010a; Ponarulselvam et al., 2012; Prabhu and Poulose, 2012). In this study, the nanoparticles of the extracts were in the same range starting form ≤20 and up to 100 nm. Another feature that can be drawn from the TEM micrographs is that the silver nanoparticles seem to be surrounded by a matrix probably organic in nature from the plant extracts. The particle size was found to be between 20 and 100 nm. The nanoparticles are found to be well-separated from each other indicating stability. The separation between the silver nanoparticles seen in the TEM image could be due to capping by proteins and would explain the UV–vis spectroscopy measurements, which is characteristic of well-dispersed silver nanoparticles. The absorption maxima of leaves and latex nanoparticles (Fig. 1) confirm the formation of poly-dispersed silver nanoparticles in the aqueous solution with little agglomeration as illustrated in the TEM images (Fig. 2). The same results were obtained with aqueous extracts of neem and triphala leaves (Asmita et al., 2012). It was also concluded that nanoparticle solutions were stable for more than six months with little signs of aggregation (Ponarulselvam et al., 2012). Energy dispersive X-ray fluorescence (EDXRF) profile for synthesized silver nanoparticles reveals strong signal in the silver region and confirms the formation of silver nanoparticles. There is also a strong signal for other elements that has used as substrate to prepare thin film. Other elemental signals are recorded possibly due to elements from enzymes or proteins present within the leaf or latex of F. sycomorus. Same results were also reported by Shameli et al. (2011), with green biosynthesis of silver nanoparticles using Callicarpa maingayi latex extraction. The main mechanism is plant-assisted reduction due to F. sycomorus phytochemicals including flavones, organic acids, and quinones that are water-soluble and are responsible for the immediate reduction of the ions (Sathishkumar et al., 2009b, 2010a; Prabhu and Poulose, 2012). F. sycomorus leaf extract is rich in quercetin, gallic acid and isoquercitrin (Salem et al., 2013). Polyols such as terpenoids, flavones and polysaccharides in the Cinnamomum camphora leaf were reported to be the main cause of bioreduction of the silver and chloroaurate ions (Huang et al., 2007). Proteins are reported to bind to the nanoparticles either through free amine groups or cysteine residues in the proteins

W.M. Salem et al. / Industrial Crops and Products 62 (2014) 228–234

(Shankar et al., 2003). A similar mechanism was also reported by Sathishkumar et al. (2010a) as the proteins extracted from the Curcuma longa tuber capped the silver nanoparticles. In our study, it was proposed that the silver ions undergo electrostatic interaction with the proteins present in the F. sycomorus leaf or latex extract which leads to the formation of silver complex. Furthermore, the flexible linkages of proteins and other biomolecules lead to the synthesis of stable nanoparticles. This observation indicates the release of proteins into solution by leaf or latex of F. sycomorus and suggests a possible mechanism for the reduction of the metal ions present in the solution. Thus the water-soluble fractions comprised of complex polyols in the biomass were believed to have played a major role in the bioreduction of silver ions. Also, boiling procedure in silver nanoparticles preparation would have facilitated the release of some reductants which would have played major role in nanoparticle formation in later stages. Ag ions and Ag-based compounds have strong antimicrobial effects (Furno et al., 2004). In our study, we have used nine Gram −ve and one Gram +ve strains. Ag nanoparticles from leaves and latex showed antimicrobial activity against both of the tested Gram −ve and +ve organisms (Tables 1 and 2). This broad spectrum effect was similar to that found earlier (Sondi and Sondi, 2004). On the other hand, both latex and leaves extract SNPs showed no substantial differences in their inhibitory effects from extracts alone on some strains tested on solid medium. On the contrary, there were complete inhibition on liquid medium for the same strains (Table 1). For example, The inhibition zone of E. faecalis was a little higher for both leaf and latex SNPs than extracts alone on solid medium (Table 2) while the percentages of inhibition were 66.4, 45.7% for extract alone and 100% for both SNPs on liquid medium (Table 1). For solid support systems, Sathishkumar et al. (2010a) have argued that increase in silver nanoparticle concentration increased the inhibition zone area on the plate, which directly represents the increase in toxicity. Consequently, Ag+ ions released from the surface of SNPs are responsible for their antibacterial activity. Accordingly, the diffusion of SNPs in agar medium might slightly inhibit the availability of the silver nanoparticles and thereby its toxicity. For aqueous phase systems, the antibacterial test of Ag+ ions is high at the concentration levels reached by releasing, and that the presence of SNPs is very important, which reinforces the idea that the larger the surface area the stronger the antibacterial activity (Prabhu and Poulose, 2012; Rout et al., 2012). This greater effect in liquid medium may be due to better dispersion and contact, of the high silver content, with bacterial cells than in solid medium. In general, the performance of extracts was improved after treatment with its SNPs (Tables 1 and 2). Improving of Eucalyptus chapmaniana leaves extract as antibacterial agent after treatment with its SNPs was also reported by Sulaiman et al. (2013). Furthermore, SNPs synthesized from Ocimum sanctum leaf extract showed higher antibacterial activity compared to leaf extract alone (Rout et al., 2012). Because of their size, SNPs can reach the nuclear content of bacteria and they present a large and impressive surface area, enabling broad contact with bacteria. This could be the reason why they give the best antibacterial effect in liquid media. The exact mechanism that silver nanoparticles employ as an antimicrobial is not clearly known and is a debated topic (Prabhu and Poulose, 2012). It was suggested that antimicrobial activity of silver nanoparticles, against Gram −ve bacteria, is dependent on its concentration and is associated with the formation of “pits” in the bacterial cell wall or membrane (Sondi and Sondi, 2004). The accumulated Ag nanoparticles in the bacterial membrane causes leaks of cell constituents leading to cell death (Sneha et al., 2010). Other workers suggested that irregular pits causes changes in membrane permeability releasing lipopolysaccharides and membrane proteins (Amro et al., 2000; Kim et al., 2007). Our results showed no significant differences between the antibacterial activity of

233

both leaf and latex nanoparticles (Tables 1 and 2). Electron spin resonance spectroscopy studies suggested the formation of free radicals by silver nanoparticles when in contact with bacteria that damage cell membrane by making pores leading to cell death (Danilczuk et al., 2006; Kim et al., 2007). It was also proposed that silver ions are released by the nanoparticles and inactivate many vital enzymes by interacting with its thiol groups (Feng et al., 2008; Matsumura et al., 2003). Moreover, silver is acidic with a natural tendency to react with bases and, therefore, its nanoparticles will react with DNA, that has sulfur and phosphorus as major components, and terminate the microbe by destroying the DNA or causing problems in its replication (Hatchett and Henry, 1996; Matsumura et al., 2003; Morones et al., 2005). 5. Conclusions Phyto-reductive synthesis of nano-sized silver particles using latex and leaves extracts of F. sycomorus yielded ellipsoidal, few spherical and irregular silver nanoparticles. Leaf extracts produce smaller sized particles than latex and both particles are separated by approximately uniform distances. The biosynthesized SNPs are very promising as antibacterial agents for both Gm −ve and Gm +ve bacteria. Applying these findings, in the biomedical and biotechnological fields, may lead to valuable results for controlling harmful bacteria and reduce the incidence of illnesses caused by these bacterial strains. The tested SNPs are more effective against bacteria in liquid media and it is recommended to carry out complementary studies on their suspension and dispersion characteristics. It seems very reasonable to believe that this greener way of synthesizing silver nanoparticles is not just an environmentally viable technique but it also opens up scope to improve their antibacterial properties. References Amro, N.A., Kotra, L.P., Wadu-Mesthrige, K., Bulychev, A., Mobashery, S., Liu, G., 2000. High-resolution atomic force microscopy studies of the Escherichia coli outer membrane: structural basis for permeability. Langmuir 16, 2789–2796. Asmita, J.G., Padmanabhan, P., Suresh, P.K., Suresh, N.J., 2012. Synthesis of silver nanoparticles using extract of neem leaf and triphala and evaluation of their antimicrobial activities. Int. J. Pharm. Bio Sci. 3, 88–100. Babu, S.A., Prabu, G.H., 2011. Synthesis of AgNPs using the extract of Calotropis procera flower at room temperature. Mater. Lett. 65, 1675–1677. Banjara, R.A., Jadhav, S.K., Bhoite, S.A., 2012. MIC for determination of antibacterial activity of di-2-ethylaniline phosphate. J. Chem. Pharm. Res. 4, 648–652. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45, 493–496. Binupriya, A.R., Sathishkumar, M., Soon-Il, Y., 2010a. Myco-crystallization of silver ions to nanosized particles by live and dead cell filtrates of Aspergillus oryzae var. viridis and its bactericidal activity toward Staphylococcus aureus KCCM 12256. Ind. Eng. Chem. Res. 49 (2), 852–858. Binupriya, A.R., Sathishkumar, M., Yun, S.-I., 2010b. Biocrystallization of silver and gold ions by inactive cell filtrate of Rhizopus stolonifer. Colloids Surf. B: Biointerfaces 79 (2), 531–534. Case, C.L., Johnson, T.R., 1984. Laboratory Experiments in Microbiology. Benjumin Cummings Pub. Inc., CA, USA, pp. 126–129. Chandran, S.P., Chaudhary, M., Pasricha, R., Ahmad, A., Sastry, M., 2006. Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotechnol. Prog. 22, 577–583. Danilczuk, M., Lund, A., Saldo, J., Yamada, H., Michalik, J., 2006. Conduction electron spin resonance of small silver particles. Spectrochim. Acta 63, 189–191. Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O., 2008. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. 52, 662–668. Furno, F., Morley, K.S., Wong, B., Sharp, B.L., Arnold, P.L., Howdle, S.M., 2004. Silver nanoparticles and polymeric medical devices: a new approach to prevention of infection. J. Antimicrob. Chemother. 54, 1019–1024. Hatchett, D.W., Henry, S., 1996. Electrochemistry of sulfur adlayers on low-index faces of silver. J. Phys. Chem. 100, 9854–9859. Huang, J., Li, Q., Sun, D., Lu, Y., Su, Y., Yang, X., Wang, H., Wang, Y., Shao, W., Hong, N.J., Chen, C., 2007. Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf. Nanotechnology 18 (10), 105104–105115.

234

W.M. Salem et al. / Industrial Crops and Products 62 (2014) 228–234

Kim, J.S., Kuk, E., Yu, K., Kim, J.H., Park, S.J., Lee, H.J., Kim, S.H., Park, Y.K., Park, Y.H., Hwang, C.Y., Kim, Y.K., Lee, Y.S., Jeong, D.H., Cho, M.H., 2007. Antimicrobial effects of silver nanoparticles. Nanomedicine 3, 95–101. Krishnaraj, C., Jagan, E.G., Rajasekar, S., Selvakumar, P., Kalaichelvan, P.T., Mohan, N., 2010. Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water-borne pathogens. Colloids Surf. B: Biointerfaces 76, 50–56. Kubmarawa, D., Ajoku, G.A., Enwerem, N.M., Okorie, D.A., 2007. Preliminary phytochemical and antimicrobial screening of 50 medicinal plants from Nigeria. Afr. J. Biotechnol. 6, 1690–1696. Kumar, A.M., Parui, S.M., Samanta, S., Mallick, S., 2011. Synthesis of eco-friendly silver nanoparticle from plant latex used as an important taxonomic tool for phylogenetic interrelationship. Adv. Biores. 2, 122–133. Masurkar, S.A., Chaudhari, P.R., Shidore, V.B., Kamble, S.P., 2011. Rapid biosynthesis of silver nanoparticles using Cymbopogon citratus (Lemongrass) and its antimicrobial activity. Nano Microbiol. Lett. 3, 189–194. Masurkar, S.A., Chaudhari, P.R., Shidore, V.B., Kamble, S.P., 2012. Effect of biologically synthesized silver nanoparticles on Staphylococcus aureus biofilm quenching and prevention of biofilm formation. IET Nanobiotechnol. 6, 110–114. Matsumura, Y., Yoshikata, K., Kunisaki, S., Tsuchido, T., 2003. Mode of bacterial action of silver zeolite and its comparison with that of silver nitrate. Appl. Environ. Microbiol. 69, 4278–4281. Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramirez, J.T., Yacaman, M.J., 2005. The bactericidal effect of silver nanoparticles. Nanotechnology 16, 2346–2353. Muller, J.H., Hinton, J., 1941. A protein-free medium for primary isolation of the Gonococcus and Meningococcus. Proc. Soc. Exp. Biol. Med. 48, 330–333. Mulvaney, P., 1996. Surface plasmon spectroscopy of nanosized metal particles. Langmuir 12, 788–800. Park, Y., Hong, Y.N., Weyers, A., Kim, Y.S., Linhardt, R.J., 2011. Polysaccharides and phytochemicals: a natural reservoir for the green synthesis of gold and silver nanoparticles. IET Nanobiotechnol. 5, 69–78. Patil, S.V., Patil, C.D., Salunke, B.K., 2012. Biosynthesis of silver nanoparticles using latex from few Euphorbian plants and their antimicrobial potential. Appl. Biochem. Biotechnol. 167, 776–790. Pavagadhi, S., Sathishkumar, M., Balasubramanian, R., 2014. Uptake of Ag and TiO2 nanoparticles by zebra fish embryos in the presence of other contaminants in the aquatic environment. Water Res. 55, 280–291. Ponarulselvam, S., Panneerselvam, C., Murugan, K., Aarthi, N., Kalimuthu, K., Thangamani, S., 2012. Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities. Asian Pac. J. Trop. Biomed. 2, 574–580. Prabhu, S., Poulose, E.K., 2012. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int. Nano Lett. 2, 32–42. Ravichandran, V., Tiah, Z.X., Subashini, G., Terence, F.W., Eddy, F.C.Y., Nelson, J., Sokkalingam, A.D., 2011. Biosynthesis of silver nanoparticles using mangosteen leaf extract and evaluation of their antimicrobial activities. J. Chem. Soc. 15, 113–120. Rout, Y., Sikha, B., Akshya, K.O., Nayak, P.L., 2012. Green synthesis of silver nanoparticles using Ocimum sanctum (Tulashi) and study of their antibacterial and antifungal activities. J. Microbiol. Antimicrob. 4 (6), 103–109. Salem, M.Z.M., Salem, A.Z.M., Camacho, L.M., Hayssam, M.A., 2013. Antimicrobial activities and phytochemical composition of extracts of Ficus species: an overview. Afr. J. Micro. Res. 33, 4207–4219. Salem, W.M., Sayed, W.F., Haridy, M., Hassan, N.H., 2014. Antibacterial activity of Calotropis procera and Ficus sycomorus extracts on some pathogenic microorganisms. Afr. J. Biotech. 13 (32), 3271–3280.

Sathishkumar, M., Mahadevan, A., Vijayaraghavan, K., Pavagadhi, S., Balasubramanian, R., 2010b. Green recovery of gold through biosorption, biocrystallization, and pyro-crystallization. Ind. Eng. Chem. Res. 49 (16), 7129–7135. Sathishkumar, M., Sneha, K., Kwak, I.S., Mao, J., Tripathy, S.J., Yun, Y.-S., 2009a. Phyto-crystallization of palladium through reduction process using Cinnamomum zeylanicum bark extract. J. Hazard. Mater. 171, 400–404. Sathishkumar, M., Sneha, K., Won, S.W., Cho, C.W., Kim, S., Yun, Y.S., 2009b. Cinnamomum zeylanicum bark extract and powder-mediated green synthesis of nano-crystal silver particles and its bactericidal activity. Colloid Surf. B: Biointerfaces 73 (2), 332–338. Sathishkumar, M., Sneha, K., Yun, Y.-S., 2009c. Palladium nanocrystal synthesis using Curcuma longa tuber extract. Int. J. Mater. Sci. 4 (1), 11–17. Sathishkumar, M., Sneha, K., Yun, Y.-S., 2010a. Immobilization of silver nanoparticles synthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity. Bioresour. Technol. 101 (20), 7958–7965. Shameli, K., Ahmad, M.B., Zargar, M., Wan Yunus, W.M.Z., Ibrahim, N.A., Shabanzadeh, P., Ghaffari-Moghadam, M., 2011. Synthesis and characterization of silver/montmorillonite/chitosan bionanocomposites by chemical reduction method and their antibacterial activity. Int. J. Nanomed. 6, 271–284. Shankar, S.S., Ahmad, A., Sastry, M., 2003. Geranium leaf assisted biosynthesis of silver nanoparticles. Biotechnol. Prog. 19, 1627–1631. Shankar, S.S., Rai, A., Ahmad, A., Sastry, M., 2004. Rapid synthesis of Au, Ag, and bimetallic Au core–Ag shell nanoparticles using neem (Azadirachta indica) leaf broth. J. Colloid Interface Sci. 275, 496–502. Singhal, A., Kumar, V.L., 2009. Effect of aqueous suspension of dried latex of Calotropis procera on hepato renal functions in rat. J. Ethnopharmacol. 122, 172–174. Sneha, K., Sathishkumar, M., Lee, S.Y., Bae, M.A., Yun, Y.-S., 2011. Biosynthesis of Au nanoparticles using cumin seed powder extract. J. Nanosci. Nanotechnol. 11 (2), 1811–1814. Sneha, K., Sathishkumar, M., Mao, J., Kwak, I.S., Yun, Y.-S., 2010. Corynebacterium glutamicum-mediated crystallization of silver ions through sorption and reduction processes. Chem. Eng. J. 162 (3), 989–996. Sondi, I., Sondi, S.B., 2004. Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram negative bacteria. J. Colloid Interface Sci. 275, 177–182. Suganya, R.S., Priya, K.B., Roxy, S., 2012. Phytochemical screening and antibacterial activity from Nerium oleander and evaluate their plant mediated nanoparticle synthesis. Int. Res. J. Pharm. 3, 285–288. Sulaiman, G.M., Wasnaa, H.M., Thorria, R.M., Ahmed, A.A., Abdul Amir, H.K., Abu Bakar, M., 2013. Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles using Eucalyptus chapmaniana leaves extract. Asian Pac. J. Trop. Biomed. 3 (1), 58–63. Sun, Q., Xiang, C., Jiangwei, L., Zheng, M., Chen, Z., Yu, C.-P., 2014. Green synthesis of silver nanoparticles using tea leaf extract and evaluation of their stability and antibacterial activity. Colloids Surf. A: Physicochem. Eng. Asp. 444, 226–231. Verastegui, M.A., Sanchez, C.A., Heredia, N.L., Santos, G.A.J., 1996. Antimicrobial activity of extracts of three major plants from the Chihuahuan desert. J. Ethnopharmacol. 52, 175–177. Wiley, B.J., Im, S.H., Li, Z.Y., Mclellan, J., Siekkenen, A., Xia, Y., 2006. Maneuvering the surface plasmon resonance of silver nanostructures through shape-controlled synthesis. J. Phys. Chem. 110, 15666–15675. Zhan, G., Huang, J., Du, M., Abdul-Rauf, I., Ma, Y., Li, Q., 2011. Green synthesis of Au–Pd bimetallic nanoparticles: single-step bioreduction method with plant extract. Mater. Lett. 65, 2989–2991. Zhao, G., Stevens, J., 1998. Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion. Biometals 11, 27–32.