# Synthesis of Plant-Mediated Silver Nanoparticles UsingDioscorea batatas Rhizome Extract and Evaluation of Their Antimicrobial Activities

Authors: P. C. Nagajyothi; K. D. Lee
Journal: Journal of Nanomaterials
 (2011)

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## Abstract

The eco-friendly synthesis of nanoparticles through various biological means helps to explore various plants for their ability to synthesize silver nanoparticles (AgNPs). Here we have synthesized AgNPs by using rhizome extract ofDioscorea batatas at 80∘C as well as room temperature (25∘C). AgNPs were characterized under UV-vis spectrophotometer, SEM, FTIR, XRD, and EDX. The antimicrobial activity of AgNPs was evaluated on gram positive (B. substilis and S. aureus), gram negative (E. coli), and fungi (S. cerivisae and C. albicans). At room temperature, S. cerivisae and C. albicans were found to be more susceptible to AgNPs than at 80∘C.


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## Body

## 1. Introduction

Biosynthesis provides advancement over chemical and physical method as it is cost effective, environment friendly, and easily scaled up for large-scale synthesis and obviates the need for high pressure, energy, temperature, and toxic chemicals [1, 2]. Large-scale production by chemical and physical methods usually results in particles larger than several micrometers while the biological synthesis can be successfully used for production of small nanoparticles in large-scale operations [3].The use of silver and other metal ions for their sustained antifungal, antibacterial and antiviral effects has been practiced from antiquity. Recent studies have focused on the synthesis of homogenous AgNPs and evaluation of their antimicrobial activities [4, 5]. Silver ion has been known to be effective against a broad range of microorganisms. Antimicrobial activities of AgNPs have been reported by various researchers [6, 7]. Ingle and coworkers [8] found that AgNPs exhibited significant antimicrobial activity against Escherichia coli and multidrug resistant Staphylococcus aureus, and Pal et al. [9] reported that the antibacterial activity of AgNPs against the gram-negative E. coli depends on the shape of the nanoparticles. Kim et al. [10, 11] reported that spherical AgNPs showed potent activity against Trichophyton mentagrophytes, Trichosporon beigelii, and Candida albicans compared with that of commercially available antifungal agents (amphotericin B and fluconazole).Dioscoreagrows extensively on mountains as well as in fields within Korea and Japan. In the present study AgNPs were synthesized using Dioscorearhizome (Figure 1) and characterized with various characterization techniques. The antimicrobial activity of AgNPs was investigated against various pathogenic gram positive and gram negative bacterial and fungal species.Figure 1
Dioscorea batatas rhizome.


## 2. Material and Methods

### 2.1. Synthesis of AgNPs Using Dioscorea Rhizome Extract

Dioscorea rhizomes were collected from Gyeongju-oriental medical college, Gyeongju, South Korea. The rhizomes were air dried for 10 days and then kept in a hot air oven at 60°C for 24 hours. Rhizomes were ground to fine powder. 1 mM silver nitrate (AgNO3) purchased from Sigma-Aldrich Chemical Pvt. Ltd. was added to rhizome extract to make up a final solution 200 mL. A change in the color of solution was observed during the heating process.


### 2.2. UV-Vis Spectrophotometer Analysis

The reduction of pure Ag+ ions was monitored by measuring the UV-vis spectrum of the reaction medium at 5 hours after diluting a small aliquot of the sample into distilled water. UV-vis spectral analysis was done by using UV-vis spectrophotometer (Cary 4000 UV-vis spectro photometer).


### 2.3. SEM and EDX Analysis

The biomass after reaction spontaneously precipitated at the bottom of the conical flasks in 1 hours. After the precipitation, the suspension above the precipitate was sampled for SEM-EDX observations. SEM samples of the aqueous suspension of nanoparticles were fabricated by dropping the suspension onto clean electric glass and allowing water to completely evaporate. Samples were coated by carbon and SEM analyses were performed on a Hitachi s-3500N.


### 2.4. XRD Analysis

The AgNPs solutions were purified by repeated centrifugation at 5000 rpm for 20 minutes followed by redispersion of the pellet of AgNPs into 10 mL of deionized water. After freeze drying of the purified nanoparticles, the structure and composition were analyzed by XRD (Rigaku RINT 2100 series). The dried mixture of AgNPs was collected for the determinutesation of the formation of AgNPs by an X’Pert Pro X-ray diffractometer operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation in a θ-2θ configuration.


### 2.5. FTIR Analysis

To remove any free biomass residue or compound that is not the capping ligand of the nanoparticles, the residual solution of 100 mL after reaction was centrifuged at 5000 rpm for 10 minutes and the resulting suspension was redispersed in 10 mL sterile distilled water. The centrifuging and redispersing process was repeated three times. Thereafter, the purified suspension was freeze dried to obtain dried powder. Finally, the dried nanoparticles were analyzed by FTIR (Bruker model, TENSOR 37).


### 2.6. Antimicrobial Assays

Synthesized AgNPs were tested for antimicrobial activity by agar disc-diffusion method against pathogenic bacteria,B. subtilis, S. aureusand E. coli, and pathogenic fungi C. albicansand S. cerevisiae. Pure cultures of bacterial and fungal pathogens were subcultured on Mueller Hinton and Potato Dextrose Agar (PDA), respectively. The microbial test organisms were grown in nutrient broth at 37°C for 24 hours. A 500 μL aliquot of each organism (1×106 cfu/mL) was spread on agar using a cotton swab and allowed to dry for 10 minutes. Paper discs loaded with 50 μg/mL AgNPs and reference drugs were placed on the surface of each cultured plate and incubated at 37°C for 24 hours after which inhibition zones were measured [12]. Experiments were conducted in duplicate and mean results recorded.


## 2.1. Synthesis of AgNPs Using Dioscorea Rhizome Extract

Dioscorea rhizomes were collected from Gyeongju-oriental medical college, Gyeongju, South Korea. The rhizomes were air dried for 10 days and then kept in a hot air oven at 60°C for 24 hours. Rhizomes were ground to fine powder. 1 mM silver nitrate (AgNO3) purchased from Sigma-Aldrich Chemical Pvt. Ltd. was added to rhizome extract to make up a final solution 200 mL. A change in the color of solution was observed during the heating process.


## 2.2. UV-Vis Spectrophotometer Analysis

The reduction of pure Ag+ ions was monitored by measuring the UV-vis spectrum of the reaction medium at 5 hours after diluting a small aliquot of the sample into distilled water. UV-vis spectral analysis was done by using UV-vis spectrophotometer (Cary 4000 UV-vis spectro photometer).


## 2.3. SEM and EDX Analysis

The biomass after reaction spontaneously precipitated at the bottom of the conical flasks in 1 hours. After the precipitation, the suspension above the precipitate was sampled for SEM-EDX observations. SEM samples of the aqueous suspension of nanoparticles were fabricated by dropping the suspension onto clean electric glass and allowing water to completely evaporate. Samples were coated by carbon and SEM analyses were performed on a Hitachi s-3500N.


## 2.4. XRD Analysis

The AgNPs solutions were purified by repeated centrifugation at 5000 rpm for 20 minutes followed by redispersion of the pellet of AgNPs into 10 mL of deionized water. After freeze drying of the purified nanoparticles, the structure and composition were analyzed by XRD (Rigaku RINT 2100 series). The dried mixture of AgNPs was collected for the determinutesation of the formation of AgNPs by an X’Pert Pro X-ray diffractometer operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation in a θ-2θ configuration.


## 2.5. FTIR Analysis

To remove any free biomass residue or compound that is not the capping ligand of the nanoparticles, the residual solution of 100 mL after reaction was centrifuged at 5000 rpm for 10 minutes and the resulting suspension was redispersed in 10 mL sterile distilled water. The centrifuging and redispersing process was repeated three times. Thereafter, the purified suspension was freeze dried to obtain dried powder. Finally, the dried nanoparticles were analyzed by FTIR (Bruker model, TENSOR 37).


## 2.6. Antimicrobial Assays

Synthesized AgNPs were tested for antimicrobial activity by agar disc-diffusion method against pathogenic bacteria,B. subtilis, S. aureusand E. coli, and pathogenic fungi C. albicansand S. cerevisiae. Pure cultures of bacterial and fungal pathogens were subcultured on Mueller Hinton and Potato Dextrose Agar (PDA), respectively. The microbial test organisms were grown in nutrient broth at 37°C for 24 hours. A 500 μL aliquot of each organism (1×106 cfu/mL) was spread on agar using a cotton swab and allowed to dry for 10 minutes. Paper discs loaded with 50 μg/mL AgNPs and reference drugs were placed on the surface of each cultured plate and incubated at 37°C for 24 hours after which inhibition zones were measured [12]. Experiments were conducted in duplicate and mean results recorded.


## 3. Results and Discussion

### 3.1. UV-Vis Spectro Photometer Study

Bioreduction of aqueous Ag+ ions can easily be followed by UV-vis spectro photometer, and one of the most important features in optical absorbance spectra of metal nanoparticles is surface plasmon band, which is due to collective electron oscillation around the surface mode of the particles. Previous studies have shown that silver exhibits yellowish-brown color due to the excitations of their surface plasmon response (SPR) [13], when dissolved in water. The color of the solution changes from colorless to brownish yellow, within 50 minutes at 80°C and dark yellow; this reaction takes up to 2 hours for completion at room temperature (Figures 2(a)–2(c)). The possible explanation of difference in the reduction time could be due to the difference in their reduction potential for both the metal ions.Color change of rhizome extracts containing AgNPs before and after synthesis: (a) before synthesis, (b) at 80°C, and (c) at 25°C.
(a)(b)(c)Metal nanoparticles such as silver and gold have free electrons, which gives rise to SPR absorption band [14]. The characteristic SPR resonance band of biogenic AgNPs occurred at 420 nm and 416 nm for reaction carried out at 80°C and room temperature, respectively, (Figures 3(a)-3(b)). The reactants are consumed rapidly eventually leading to the formation of smaller nanoparticles [15, 16].UV-vis absorption spectrum of AgNPs (a) at 80°C and (b) at 25°C.
(a)(b)


### 3.2. SEM and EDX Studies

Figures4(a)–4(d) show the SEM images of the synthesized AgNPs. The overall morphology of AgNPs is more clearly seen, and the particles are monodispersed, having circular and flower shapes, respectively, at 80°C and 25°C.SEM images of the biosynthesized of AgNPs showing at different magnifications: (a) and (b) AgNPs at 80°C; (c) and (d) AgNPs at 25°C.
(a)(b)(c)(d)Figures5(a)-5(b) show the EDX spectrum of AgNPs synthesized at 25°C and 80°C. Strong signals from the silver atoms in the nanoparticles were observed, and signals from Si, K, C, and Pt atoms were also recorded. The C and K signals were likely due to X-ray emission from carbohydrates/proteins/enzymes present in the cell wall of the biomass. Metallic AgNPs generally show typical absorption peak approximately at 3 KeV due to SPR [17]. The presence of the elemental silver can be observed in the graph obtained from EDX analysis, which also supports the XRD results.EDX spectrum of AgNPs (a) at 80°C and (b) at 25°C.
(a)(b)


### 3.3. XRD and FTIR Studies

The XRD pattern of the AgNPs is shown in Figures6(a) and 6(b). Various Bragg reflections clearly indicated the presence of (111) and (200) sets of lattice planes and further on the basis that they can be indexed as face-centered-cubic (FCC) structure of silver. Hence from the XRD pattern it is clear that AgNPs formed using Dioscorearhizome broth were essentially crystalline in nature. In addition to the Bragg peaks representative of FCC AgNPs, additional as yet unassigned peaks are also observed suggesting that the crystallization of bio-organic phase occurs on the surface of the nanoparticles.XRD spectrum of AgNPs (a) at 80°C and (b) at 25°C.
(a)(b)FTIR results reveal absorption bands at 3444, 1634, and 694 cm−1 (rhizome extraction) in Figure 7(a); 3438, 1640, and 681 cm−1 (AgNPs at 80°C) in Figure 7(b); 3446, 1645, and 685 cm−1 (AgNPs at 25°C) in Figure 7(c), respectively. The vibrational bands correspond to the bonds such as aminuteso (N–H), –C=C (alkene), and C–Cl (Halogens) which were in the region range of 673–3446 cm−1. The most wide spectrum absorption was observed at 3444 and 13446 cm−1 and it can be attributed to the stretching vibrations of aminuteso (N–H) [18]; absorption peaks centered at 1640 and 1645 cm−1 can be attributed to the stretching vibration of –C=C (alkene) [19].FTIR spectrum of AgNPs: (a)Dioscorea batatas rhizome extract, (b) at 80°C, and  (c) at 25°C.
(a)(b)(c)


### 3.4. Antimicrobial Activity of AgNPs

Antimicrobial effect of AgNPs was examinutesed on gram positiveS. aureus and B. substilis, gram negative E. coli, and fungal strains: C. albicans and S. cerevisae. The results consigned in Figures 8(a)–8(d) and 9(a)-9(b) are indicative of the diameters of zones of inhibition due to microbial susceptibility. AgNPs exerted highest toxicity against C. albicans and S. cerevisae intermediary effects on S. aureus and B. substilis and exhibited the lowest effect on E. coli. The order of susceptibility to the metal nanoparticles can be written as fungi > gram positive > gram negative. The AgNPs at 25°C showed more inhibitory activity on microbes compared to AgNPs at 80°C. AgNPs were found to be significantly toxic against the fungal and gram positive microbes and exhibited mild toxicity againstE. coli. AgNPs were more effective against gram positive bacterial strains than the gram negative bacteria strains [18]. Ag+ ions uncouple the respiratory chain from oxidative phosphorylation or collapse the proton-motive force across the dependent on the size and shape of NPs [10]. Morones et al. [20] reported that AgNPs were preferentially bound to the cytoplasmic membrane. In addition to that, the pitting of the AgNPs caused in the bacterial cell wall is also responsible for the death of bacteria. The actual bacteria mechanism of AgNPs is not well known, some researchers agree that silver releases Ag+ ions, and they interact with thiol groups of bacterial proteins affecting the replication of DNA [21].(a)–(d) Zones of inhibition of the growth ofB. substilis (lab culture), E. coli (KCTC 2441), andS. cerevisiae (lab culture) and upon application of reference drug (penicillin). Diagrams show the averaged inhibition-zone diameters for samples. R.D: reference drug; AgNPs 1: at room temperature; AgNPs 2: at 80°C.
(a)(b)(c)(d)(a) and (b) Zones of inhibition of the growth ofS.  aureus (KCTC 1916) and C.  albicans (lab culture), and upon application of reference drug (tetracycline).
(a)(b)


## 3.1. UV-Vis Spectro Photometer Study

Bioreduction of aqueous Ag+ ions can easily be followed by UV-vis spectro photometer, and one of the most important features in optical absorbance spectra of metal nanoparticles is surface plasmon band, which is due to collective electron oscillation around the surface mode of the particles. Previous studies have shown that silver exhibits yellowish-brown color due to the excitations of their surface plasmon response (SPR) [13], when dissolved in water. The color of the solution changes from colorless to brownish yellow, within 50 minutes at 80°C and dark yellow; this reaction takes up to 2 hours for completion at room temperature (Figures 2(a)–2(c)). The possible explanation of difference in the reduction time could be due to the difference in their reduction potential for both the metal ions.Color change of rhizome extracts containing AgNPs before and after synthesis: (a) before synthesis, (b) at 80°C, and (c) at 25°C.
(a)(b)(c)Metal nanoparticles such as silver and gold have free electrons, which gives rise to SPR absorption band [14]. The characteristic SPR resonance band of biogenic AgNPs occurred at 420 nm and 416 nm for reaction carried out at 80°C and room temperature, respectively, (Figures 3(a)-3(b)). The reactants are consumed rapidly eventually leading to the formation of smaller nanoparticles [15, 16].UV-vis absorption spectrum of AgNPs (a) at 80°C and (b) at 25°C.
(a)(b)


## 3.2. SEM and EDX Studies

Figures4(a)–4(d) show the SEM images of the synthesized AgNPs. The overall morphology of AgNPs is more clearly seen, and the particles are monodispersed, having circular and flower shapes, respectively, at 80°C and 25°C.SEM images of the biosynthesized of AgNPs showing at different magnifications: (a) and (b) AgNPs at 80°C; (c) and (d) AgNPs at 25°C.
(a)(b)(c)(d)Figures5(a)-5(b) show the EDX spectrum of AgNPs synthesized at 25°C and 80°C. Strong signals from the silver atoms in the nanoparticles were observed, and signals from Si, K, C, and Pt atoms were also recorded. The C and K signals were likely due to X-ray emission from carbohydrates/proteins/enzymes present in the cell wall of the biomass. Metallic AgNPs generally show typical absorption peak approximately at 3 KeV due to SPR [17]. The presence of the elemental silver can be observed in the graph obtained from EDX analysis, which also supports the XRD results.EDX spectrum of AgNPs (a) at 80°C and (b) at 25°C.
(a)(b)


## 3.3. XRD and FTIR Studies

The XRD pattern of the AgNPs is shown in Figures6(a) and 6(b). Various Bragg reflections clearly indicated the presence of (111) and (200) sets of lattice planes and further on the basis that they can be indexed as face-centered-cubic (FCC) structure of silver. Hence from the XRD pattern it is clear that AgNPs formed using Dioscorearhizome broth were essentially crystalline in nature. In addition to the Bragg peaks representative of FCC AgNPs, additional as yet unassigned peaks are also observed suggesting that the crystallization of bio-organic phase occurs on the surface of the nanoparticles.XRD spectrum of AgNPs (a) at 80°C and (b) at 25°C.
(a)(b)FTIR results reveal absorption bands at 3444, 1634, and 694 cm−1 (rhizome extraction) in Figure 7(a); 3438, 1640, and 681 cm−1 (AgNPs at 80°C) in Figure 7(b); 3446, 1645, and 685 cm−1 (AgNPs at 25°C) in Figure 7(c), respectively. The vibrational bands correspond to the bonds such as aminuteso (N–H), –C=C (alkene), and C–Cl (Halogens) which were in the region range of 673–3446 cm−1. The most wide spectrum absorption was observed at 3444 and 13446 cm−1 and it can be attributed to the stretching vibrations of aminuteso (N–H) [18]; absorption peaks centered at 1640 and 1645 cm−1 can be attributed to the stretching vibration of –C=C (alkene) [19].FTIR spectrum of AgNPs: (a)Dioscorea batatas rhizome extract, (b) at 80°C, and  (c) at 25°C.
(a)(b)(c)


## 3.4. Antimicrobial Activity of AgNPs

Antimicrobial effect of AgNPs was examinutesed on gram positiveS. aureus and B. substilis, gram negative E. coli, and fungal strains: C. albicans and S. cerevisae. The results consigned in Figures 8(a)–8(d) and 9(a)-9(b) are indicative of the diameters of zones of inhibition due to microbial susceptibility. AgNPs exerted highest toxicity against C. albicans and S. cerevisae intermediary effects on S. aureus and B. substilis and exhibited the lowest effect on E. coli. The order of susceptibility to the metal nanoparticles can be written as fungi > gram positive > gram negative. The AgNPs at 25°C showed more inhibitory activity on microbes compared to AgNPs at 80°C. AgNPs were found to be significantly toxic against the fungal and gram positive microbes and exhibited mild toxicity againstE. coli. AgNPs were more effective against gram positive bacterial strains than the gram negative bacteria strains [18]. Ag+ ions uncouple the respiratory chain from oxidative phosphorylation or collapse the proton-motive force across the dependent on the size and shape of NPs [10]. Morones et al. [20] reported that AgNPs were preferentially bound to the cytoplasmic membrane. In addition to that, the pitting of the AgNPs caused in the bacterial cell wall is also responsible for the death of bacteria. The actual bacteria mechanism of AgNPs is not well known, some researchers agree that silver releases Ag+ ions, and they interact with thiol groups of bacterial proteins affecting the replication of DNA [21].(a)–(d) Zones of inhibition of the growth ofB. substilis (lab culture), E. coli (KCTC 2441), andS. cerevisiae (lab culture) and upon application of reference drug (penicillin). Diagrams show the averaged inhibition-zone diameters for samples. R.D: reference drug; AgNPs 1: at room temperature; AgNPs 2: at 80°C.
(a)(b)(c)(d)(a) and (b) Zones of inhibition of the growth ofS.  aureus (KCTC 1916) and C.  albicans (lab culture), and upon application of reference drug (tetracycline).
(a)(b)


## 4. Conclusion

Rapid and green synthesis shows that the environmentally benign and renewable rhizome ofDioscorea batatas can be used as an effective capping as well as reducing agent for the synthesis of AgNPs. The spectroscopic characterizations from UV-vis spectro photometer, FTIR, and SEM support the formation and stability of the biosynthesized AgNPs. Further, the above AgNPs revealed to possess an effective antimicrobial property against C. albicans and S. cerevisae. The present study emphasizes the use of plants medicinal for the synthesis of AgNPs with potent antimicrobial effect. Synthesis of metallic nanoparticles using green resources like Dioscorea batatasrhizome is a challenging alternative to chemical synthesis, since this novel green synthesis is a pollutant free and eco-friendly synthetic route for the synthesis.



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*Source: 573429-2011-08-03.xml*