WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Hutagalung et al. SJIF Impact Factor 7.421 Volume 7, Issue 6, 1351-1360 Research Article ISSN 2278 4357 GREEN SYNTHESIS OF SILVER NANOPARTICLES USING SEED EXTRACT OF SAUROPUS ANDROGYNUS AND THEIR APPLICATION AS AN ANTI BACTERIAL AGENT Robert Hutagalung 1*, Rustam M. Samual 1, Philipus J. Patty 1 and Synodalia C. Wattimena 2 1 Departemen Fisika Fakultas MIPA - Universitas Pattimura Jl. Ir. M. Putuhena Poka Ambon 97233, Indonesia. 2 Departemen Biologi Fakultas MIPA - Universitas Pattimura Jl. Ir. M. Putuhena Poka Ambon 97233, Indonesia. Article Received on 15 April 2018, Revised on 07 April 2018, Accepted on 28 May 2018, DOI: 10.20959/wjpps20186-11827 *Corresponding Author Robert Hutagalung Departemen Fisika Fakultas MIPA - Universitas Pattimura Jl. Ir. M. Putuhena Poka Ambon 97233, Indonesia. ABSTRACT spectrum of antibacterial activity. This study aims to synthesize silver nanoparticles using seed extract of Sauropus androgynus as a reducing agent and study their antibacterial activity. For this purpose, silver nanoparticles produced were applied on gram negative bacterial strain Escherichia coli, and gram positive bacterial strain Staphylococcus aureus. The application of the silver nanoparticles on both gram negative bacterial strain E coli, and gram positive bacterial strain S. aureus inhibits the growth of the bacteria. These silver nanoparticles show the same effect on both bacterial strains, although gram negative bacteria have thinner cell wall than gram positive bacteria. This suggests that silver nanoparticles synthesized using seed extract of Sauropus androgynus have a broad KEYWORDS: Antibacterial Agent, Silver Nanoparticles, Seed Extract, Surface Plasmon Resonance, Sauropus androgynous. INTRODUCTION Silver nanoparticles (NPs) have been the subject of many studies by many researchers both in academics and in industry. One of the reasons for this fact is their wide range of applications. These include their use in biosensors, medical treatment, water purification, electronics, solar www.wjpps.com Vol 7, Issue 6, 2018. 1351
energy absorption system, and many more. [1,2,3,4,5] The various application of silver NPs is due to their antimicrobial, electronics, and optical properties. The antimicrobial properties of silver NPs have attracted many researchers around the globe. Recently, the antimicrobial properties of silver NPs synthesized using extracts of plants become more popular. Researchers have used extracts of leafs, stems, fruits, flowers, peels, barks, to synthesized silver NPs and study their antimicrobial properties. [6,7,8,9,10,11,12,13] The popularity of silver NPs synthesized using plant extracts compared to those synthesized chemically or physically is due to the fact that the former is cost effective and environmentally friendly. In chemical method, there is an issue of toxicity, while in a physical method a relatively large energy is required. Beside the use of plant extracts in synthesizing silver NPs, researchers also use microorganism, such as algae, bacteria, yeast, and fungi. [14,15] However, the use of plant extract is still more effective. From all extract of different parts of plant used in synthesizing silver NPs, extract of seed is less used. This is in contrary to the availability of the seeds in plants. In this study, we use seeds extract of Sauropus androgynus as a reducing agent in synthesizing silver NPs. We use the NPs to study its ability in combating bacterial strains, gram positive and gram negative bacterial strains. MATERIALS AND METHODS Preparation of Seed Extract Sauropus androgynus seeds were taken from its fruit collected from local garden in Ambon Indonesia. To prepare the extract, the seeds were washed under tap water, followed by distilled water, and were dried. The dried ones were cut into smaller pieces, and 20 g seeds were boiled in a beaker containing 200 ml distilled water for 20 minutes or so. The mixture was cooled and was filtered through a Whatman filter paper No.1. Synthesis of Silver Nanoparticles For synthesis of silver NPs, the seed extract is mixed with 1 mm solution of silver nitrate at volume ratio of extract to silver nitrate of 3:20. This ratio was chosen, since it produces stable silver NPs, where no aggregation was observed. This is done after trying several ratios to check the stability of silver NPs produced. www.wjpps.com Vol 7, Issue 6, 2018. 1352
Kinetics of Silver NPs Formation For the kinetic formation of silver NPs, we observe qualitatively the change of colour of the sample after mixing seed extract and silver nitrate solution, and quantitatively the absorbance of visible light of the sample. For this purpose, a smartphone was used to record the colour of the mixture in required interval times, and at the same time, Colorimeter Smart 2 LaMotte was used to measure the absorbance. FTIR and UV-VIS Spectroscopy To identify the surface plasmon resonance (SPR) and functional groups of silver NPs, we use UV-VIS spectroscopy (UV-1700 PharmaSpec, Shimattsu Spectrophotometer) and Fourier Transform Infra Red (FTIR) spectroscopy (FTIR spectrometer MB3000), respectively. Antibacterial Assay of Silver NPs The effect of the silver NPs on the growth of bacteria, gram negative bacterial strain E. coli and gram positive bacterial strain S. aureus, was determined by spectrophotometric method. Nutrient Broth (5 ml) was inoculated with 500 µl of each bacterial solution, followed by the addition of the silver NPs (5 ml). The bacterial solutions used were prepared from fresh overnight cultures (OD 620= 0.35). A mixture without silver NPs was used as a control. For the control, the silver NPs was replaced with 5 ml of sterile distilled water. The OD values at 620 nm were recorded after certain incubation time (0, 2, 4, 6, 8, 12, 16, 20, and 24 hours) at room temperature. RESULTS Kinetics of Silver Nanoparticle Formation We use the change of silver NPs colour and the change of its absorbance after mixing the seed extract of Sauropus androgynus and silver nitrate solution as an indicator of the kinetics of silver nanoparticle formation. Figure 1 shows the change of absorbance of the sample in 120 minutes after mixing the seed extract with silver nitrate solution, and its associated change of absorbance of 430 nm wavelength. The choice of 430 nm instead of three other wavelengths available in colorimeter is due to the fact that this wavelength is the closest to the wavelength of the surface Plasmon resonance (SPR). www.wjpps.com Vol 7, Issue 6, 2018. 1353
Figure 1: The change of colour of the sample in 120 minutes after mixing the seed extract of Sauropus androgynus and its associated change of absorbance of 430 nm wavelength. UV-VIS and FTIR Spectroscopy For further indicator of silver nanoparticle formation, we use UV-VIS spectroscopy to observe the wavelength of surface plasmon resonance. Figure 2 shows the spectra of UV-VIS of silver NPs synthesized using seed extract of Sauropus androgynus. The wavelengths in the spectra vary from 300 to 700 nm, and the peak wavelength showing the wavelength of SPR is 428 nm. Figure 2: The spectra of UV-VIS of silver NPs synthesized using seed extract of Sauropus androgynous. We use FTIR spectroscopy to identify functional groups of the silver NPs. Figure 3 shows FTIR spectra of silver NPs synthesized using seed extract of Sauropus androgynus with wave numbers varying 4000 to 500 cm -1. Some features of the spectra observed include: a www.wjpps.com Vol 7, Issue 6, 2018. 1354
broadband peaked at 3449 cm -1, a narrow peak at 1636 cm -1, and additional peaks at 2924 cm - 1, 2852 cm -1, 2029 cm -1, and 1018 cm -1. Figure 3: The spectra of FTIR of the silver NPs synthesized using seed extract of Sauropus androgynous. Antibacterial Activity To study the ability of the silver NPs synthesized using seed extract of Sauropus androgynus as an anti bacterial agent, we applied the NPs against gram negative bacterial strain, E. coli, and gram positive bacterial strain, S. aureus. Figure 4 shows OD of 620 nm wavelength of gram negative bacterial strain, E coli, with and without application of silver NPs in 24 four hour interval time. The bars indicate standard deviations of 3 repeats. We used a two way analysis of variance (ANOVA) to observe the difference in OD between bacterial strain and bacterial strain with applied silver NPs. The result of two way ANOVA analysis is accommodated in letters shown at each data point. The result of the analysis can also apply to the difference between each data point in each sample and between the samples. Figure 4: Optical density of gram negative bacterial strain E. coli with and without application of silver NPs in 24 four hour interval time. www.wjpps.com Vol 7, Issue 6, 2018. 1355
Figure 5 shows OD of 620 nm wavelength of gram positive bacterial strain, S. aureus, with and without application of silver NPs in 24 four hour interval time. The bars indicate standard deviations of 3 repeats. The letters at each data point indicates the result of ANOVA analysis. Figure 5: Optical density (OD) of 620 nm wavelength (in 24 four hour interval time) of gram positive bacterial strain, S. aureus, with and without application of silver NPs. DISCUSSIONS Figure 1 showing the change of absorbance of the sample and its associated colour indicates the formation of silver NPs. The indicator of the silver NPs formation is the change of sample colour to be yellowish brown followed by the measured value of the absorbance upon application of light with proper wavelength. The increase in the absorbance and the change of the colour denotes the reduction of the silver ion becoming silver NPs, thus the increase in the number of silver NPs. In silver NPs, electrons oscillate on the surface collectively known as Plasmon. The frequency of the oscillation determines their colour, yellowish brown for silver NPs. When light with certain wavelength applied on the sample, there will be absorption. The figure suggests the formation of silver NPs took place in less than 20 minutes after mixing the seed extract and silver nitrate solution. The silver NPs formed with this scenario are stable up to few months. We found that the ratio between extract and silver nitrate solution is an important factor for the sample stability. There are some ranges of ratios which can produce silver NPs, outside those ratios we were not able to produce silver NPs. Moreover, we also found that volume ratio of extract to silver nitrate of 3:20, the one we used in this study, produces more stable sample. www.wjpps.com Vol 7, Issue 6, 2018. 1356
Spectra of UV-VIS shown in Figure 2 denote that the absorbance peaks at 428 nm wavelength. This means that frequency associated with 428 nm wavelength is the same frequency of the surface Plasmon: thus surface Plasmon resonance (SPR). Spectra of FTIR was explained based on the chapter by John Coates. [16] The broadband peaked at 3449 cm -1 indicates O-H stretching vibration, suggesting a presence of hydroxyl groups in silver NPs. The narrow peak at 1636 cm -1 is due to C=O stretching vibration, suggesting the presence of carbonyl groups in silver NPs. Additional peaks at 2924 cm -1 and 2852 cm -1 indicates the asymmetric and symmetric C-H stretching, respectively. This FTIR spectra is similar the FTIR spectra of silver NPs synthesized using extracts of leaf and stem of Anredera cordifolia. [6,7] Figures 4 and 5 describe the effect of silver NPs on the growth of E coli and S. aureus, respectively. The increase in the OD shows the growth of bacteria. So, the effect of silver NPs on the growth of bacteria can be seen on the OD difference between the samples of bacteria and of bacteria with silver NPs in 24 hours. From both figures, we found that by applying silver NPs on the bacteria, the growth of the bacteria ceased. This applies to both E. coli and S. aureus. We used two way ANOVA to analyze OD difference between samples of bacteria and of bacteria with silver NPs. Figure 4 shows that although OD of E. coli is higher than that of E. coli with silver NPs, the significant difference between them just occurred 16 hours after application of silver NPs. This is also the case for S. aureus shown in Figure 5. These results suggest that silver NPs synthesized using seed extract can cease the growth of both gram negative and gram positive bacteria. We found that these silver NPs have the same antibacterial ability on gram negative and gram positive bacteria, although gram negative bacteria have thinner cell wall than gram positive bacteria. Hence, silver NPs synthesized using seed extract of Sauropus androgynus have a broad spectrum of antibacterial activity. Previous studies of silver NPs using other plant materials also show that silver NPs have the same antibacterial ability on gram negative and gram positive bacteria, thus a broad spectrum of antibacterial activity. [6,7,9,17] Other studies show that antibacterial activity of silver NPs is more effective on gram negative bacterial strains than on gram positive bacterial strains. [12,18,19] The ability of silver NPs to combat cell bacteria is due to their large surface area, which enables them to have appropriate contact with cell membrane. It has been reported that this www.wjpps.com Vol 7, Issue 6, 2018. 1357
ability is related to the disruption of the cell membrane by their produced free radicals, which leads to the leakage of protein. [20] CONCLUSIONS Silver Nanoparticles have been synthesized using seed extract of Sauropus androgynus and the products were applied on gram negative bacterial strain E coli, and gram positive bacterial strain S. aureus. The formation of silver nanoparticles occurred in less than 20 minutes after mixing the extract and silver nitrate solution. This is shown by the change of the sample colour to be yellowish brown and the sample absorption upon light applied. The wavelength at which surface Plasmon resonance takes place was 428 nm. The application of the silver nanoparticles on both gram negative bacterial strain E coli, and gram positive bacterial strain S. aureus inhibits the growth of the bacteria. These silver nanoparticles show the same effect on both bacterial strains, although gram negative bacteria have thinner cell wall than gram positive bacteria. Hence, silver NPs synthesized using seed extract of Sauropus androgynus have a broad spectrum of antibacterial activity. REFERENCES 1. Sotiriou, G.A., Sannomiya, T., Teleki, A., Krumeich, F., Voros, J., Pratsinis, S.E. Nontoxic dry coated nanosilver for plasmonic biosensors. Adv. Funct. Mater., 2010; 20: 4250-4527. 2. Becker RO. Silver ions in the treatment of local infections. Metal-based drugs, 1999; 6: 311-314. 3. Jain P, Pradeep T. Potential of silver nanoparticles coated polyurethane foam as an antibacterial water filter. Biotechnol. Bioengg, 2005; 90: 59-63. 4. Chen D., Qiao X, Qiu X, Chen J. Synthesis and electrical properties of uniform silver nanoparticles for electronic applications. Journal of Materials Science, 2009; 44(4): 1076-1081. 5. Liu K, Qu S, Zhang X, Tan F, and Wang Z. Improved photovoltaic performance of silicon nanowire/organic hybrid solar cells by incorporating silver nanoparticles. Nanoscale Research Letters, 2013; 8(1): 1 6. 6. Wattimena SC and Patty PJ. Antibacterial Properties of Silver Nanoparticles Synthesized Using Leaf Extract of Anredera cordifolia as a Reducing Agent. WJPPS, 2017; 6(12): 1673-1683. www.wjpps.com Vol 7, Issue 6, 2018. 1358
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