Investigation of the herbal synthesis of silver nanoparticles using Cinnamon zeylanicum extract
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In the present investigation, biosynthesis of silver nanoparticles (AgNPs) using Cinnamon zeylanicum bark extract was described. The extraction of Cinnamon zeylanicum bark was carried out in various solvents such as aqueous, ethanol, and dimethyl sulfoxide (DMSO) with different polarity. The aldehyde content and antioxidant activity of each extract were estimated. Subsequently, these extracts were used for the synthesis of AgNPs and the growth of nanoparticles was periodically monitored by UV–visible spectroscopy and observed the drastic change in the appearance of the colloidal solutions. The correlation between total aldehyde content, antioxidant activity, and the size of the nanoparticles was investigated. Different shapes and sizes of the nanoparticles in ethanol (spherical), aqueous (polygonal), and DMSO (spherical) were observed by high-resolution transmission electron microscope (HRTEM). The antimicrobial activity of the nanoparticles was investigated against both gram-positive Staphylococcus aureus and gram-negative Escherichia coli bacteria. These results suggest that aqueous extract is appropriate for the synthesis of nanoparticles and optimized for further investigations for the development of infection-resistant materials.
KeywordsSilver nanoparticles Antimicrobial activity Cinnamon zeylanicum Aldehyde content Antioxidant activity
In the past few decades, increasing bacterial resistance towards conventional antibiotic drugs is posing a paramount risk to the healthcare sector. There is enormous interest in the development of safe and effective antimicrobial agents which are capable of killing a wide spectrum of microbes with improved activity compared to conventional drugs . In view of the fact, silver (Ag) is a safe inorganic antibacterial agent with broad-spectrum activity and it minimizes the chances of resistance by bacteria since it targets multiple components in bacterial cells. Among different salts of Ag, silver nanoparticles (AgNPs) have been recognized as the most suitable candidate for defeating pathogens. There are various reports available on the excellent antimicrobial activities of AgNPs [2, 3]. Therefore, AgNPs are used in several biomedical applications as additives and as coating materials for the development of infection-resistant surfaces or materials [4, 5, 6, 7]. As we know that a wide variety of AgNP-based product gains enormous attention but the toxicological and environmental issues should be the point of concern. In a previous report, the antibacterial effect of picosecond laser-generated AgNPs and their toxicity to human cells were investigated. The study concluded that the nanoparticles had broad antibacterial efficiency against microbes but with minimal human cell toxicity .
The conventional method of nanoparticle synthesis involves the use of various chemicals (such as hydrazine, sodium borohydride, aldehydes, and citrates), radiation sources, functional polymers, and many more as a reducing agent which not only cause environmental concerns but also involve high-cost and excessive use of energy and resources [9, 10, 11, 12]. Considering all these factors, the research in the last decade has shifted their focus towards exploring various plant extracts as a potential alternative for the synthesis of metal nanoparticles due to its less toxic nature which makes them safer to human [13, 14, 15]. Plant extracts comprise various biochemicals such as terpenoids, alkaloids, phenolics, aldehydes, proteins, and amino acids, which reduce metal ions to nanoparticles and stabilize them. Plant extracts are easily available and can be easily scaled up for industrial synthesis with less contamination. There have been several reports for the growth of metal nanoparticles by biosynthesis using different plant extracts [16, 17, 18, 19, 20, 21, 22, 23, 24]. The major challenge in design biosynthesis of the nanoparticle is to have proper control over size and morphology of the nanoparticles by controlling the reaction conditions.
Therefore, we used Cinnamon zeylanicum bark for the biosynthesis of AgNPs. The bark is widely used as a spice in cooking and also as medicinal used to treat diarrhea and other problems of the digestive system. The bark extracts contain a high concentration of cinnamaldehyde, eugenol, and linalool with high-antioxidant activity . The aim of this present investigation is to explore the responsible factor in Cinnamon zeylanicum bark extract for morphological control of AgNPs and to investigate the various chemicals involved in the bioreduction [26, 27]. Cinnamon zeylanicum barks were extracted in different solvents having different polarities, i.e., aqueous, ethanol, and DMSO, and then the reduction of silver ions was estimated. Recently, influence of different solvents over the synthesis of cinnamon nanoparticles was investigated and observed that the variation of solvent medium altered the morphology of the nanoparticles . The relationship among antioxidant activity, total phenolic content, and aldehyde content for each solvent extract were investigated. The synthesized AgNPs were characterized by using spectral analysis like UV–vis spectroscopy, HRTEM, and energy-dispersive X-ray analysis (EDX). Furthermore, the biosynthesized AgNPs were tested for their antibacterial activity against E. coli and S. aureus strains. The major finding of this work is related to the identification of specific biochemicals present in the extracts involved in the reduction and stabilization of AgNPs and their antimicrobial activity.
Silver nitrate (AgNO3), sodium phosphate, ammonium molybdate, Folin and Ciocalteu’s Phenol (FCP) reagent, ethanol, sodium carbonate (Na2CO3), and sulfuric acid (H2SO4) were purchased from Merck. DMSO was purchased from Qualigens chemicals. Tannic acid and 2,4-dinitrophenylhydrazine (DNPH) were purchased from Central Drug House (CDH) laboratory chemicals, India. Methanol was purchased from Fischer scientific chemicals. Commercially available Cinnamon zeylanicum bark was purchased from local market. Luria broth and agar-agar were obtained from Hi Media Laboratories, India. Bacterial strains of E. coli (ATCC 35218) and S. aureus (ATCC 25923) were provided by AIIMS, India. All chemicals were of analytical grade and were used without any further purification.
2.2 Extraction of Cinnamon Zeylanicum bark
Cinnamon zeylanicum bark was washed with water and acetone to remove impurities and was dried before using for further experiments. Different solvents, ethanol, water, and DMSO were subjected to the extraction process. Cinnamon zeylanicum bark pieces (1 g) were dipped into each solvent (50 mL) for 6 h under stirring. Different composition of bark and solvents were investigated. The extraction in aqueous medium was carried out at 80 °C, while extraction in ethanol and DMSO were carried out at ambient temperature and were filtered and stored under refrigeration. Subsequently, Cinnamon zeylanicum bark extracts were used for the synthesis of AgNPs.
2.3 Estimation of total polyphenol content
The total polyphenolic content of Cinnamon zeylanicum bark extract was estimated using standard Folin-Ciocalteu’s colorimetric method . Tannic acid was used as a reference reagent for the estimation of total phenol content in different extracts. One milliliter of Cinnamon zeylanicum bark extract in different solvent was mixed with 0.5 mL of Folin-Ciocalteu’s reagent and diluted with 5 mL of water. After 5 min of mixing, 1.5 mL of 20% Na2CO3 was added for neutralization and the volume was made up to 10 mL with water. This solution was incubated for 120 min at ambient temperature after which an intense blue color was observed. The data is expressed in milligrams of tannic acid equivalence (TAE). All the experiments were performed in triplicate measurements and values were presented with standard deviation.
2.4 Estimation of total aldehyde content
2.5 Total antioxidant activity
Where Ac is the absorbance of the control and As is the absorbance of the Cinnamon zeylanicum bark extract sample.
2.6 Synthesis of silver nanoparticles
A 25 mL (50% v/v) of each extract solution was used for the reduction process and was added to 25 mL of aqueous AgNO3 solution of 100 mg/L concentration. The solutions were kept at 60 °C during the reduction process and similar conditions were maintained for each extract. The color change of the colloidal solution was monitored by visual inspection. Digital images of the samples were also captured with a digital still camera (Cannon 550D) at an optical zoom of × 4.
2.7 Characterizations of AgNPs
The nanoparticle formation was monitored periodically using a UV–vis spectrophotometer (Shimadzu 4520) at a wavelength in between 300 to 600 nm. The characteristic plasmon resonance peak of monodispersed AgNPs appeared around ~ 420 nm. The morphology and size of the AgNPs were observed under a JEOL JEM-1400. High-resolution transmission electron microscopy (HRTEM) operated at 200 KV equipped with Olympus Soft Imaging Solutions GmbH (software: iTEM; TEM Camera: Morada 4008 × 2672-pixel max) recording system. For HRTEM analysis, the samples were prepared by placing one drop of the colloidal solution on a carbon-coated grid and dried at ambient temperature. Energy-dispersive X-ray spectroscopy (EDAX) studies were also carried out in parallel to find evidence for the presence of Ag in nanoparticles. The structural morphology of the bacteria was studied using STEREOSCAN 360 (Cambridge scientific industries) scanning electron microscope (SEM), with gold coating.
2.8 Antimicrobial studies
Antimicrobial studies of biosynthesized AgNPs in different extracts were carried out by the zone of inhibition method, according to test method AATCC 100–1998 [4, 5, 6, 7, 12]. The antibacterial efficiency of the AgNPs was monitored against gram-positive bacteria S. aureus and gram-negative bacteria E. coli. Freshly obtained colonies of S. aureus and E. coli were suspended in Luria Broth and the turbidity was adjusted to 0.5 McFarland standards. Two hundred microliters of this suspension was spread on Luria-Agar plates to obtain a semi-confluent growth. AgNPs-impregnated disks (0.9 mm diameter) and negative control disks were placed on the inoculated plates. The plates were kept for incubation for 24 h at 37 °C and the inhibition zones were measured using photographic images of the agar plates.
The interaction of the AgNPs with bacterial colonies and their mechanism of action against both E. coli and S. aureus were observed by HRTEM. The bacterial cells were treated with colloidal solution of AgNPs for different time durations. The samples were prepared by adding one drop of the sample onto a carbon-coated copper grid and then were dried at 40 °C. The presence of Ag inside the cell and interaction at the cell membrane was confirmed using EDAX.
3 Results and discussion
3.1 Total polyphenol content, total aldehyde content, and antioxidant activity
3.2 AgNPs characterization
HRTEM coupled with EDX analysis was carried out to confirm the presence of pure silver without any other metals. The EDX spectrum shows the distinctive energy peak at ~ 0.2 keV, which is the characteristic peak of carbon and oxygen. Along with characteristic peak, a new sharp peak appeared at 3 keV which corresponds to the presence of silver in the sample. (Figure attached as supplementary information S1).
3.3 Correlation of aldehyde content with metal reduction and particle size
The relationship between the total aldehyde content and the morphology and the size of AgNPs were visualized in different solvents (ethanol, aqueous, and DMSO). As mentioned above, the antioxidant activity of the plant extract is mainly responsible for the bioreduction process. Good correlation was observed between the total aldehyde content and antioxidant activity shown in the previous section. Trend was observed as ethanol > aqueous > DMSO. Identical trend was observed in the case of the size of nanoparticles growing in ethanol extract having the smaller size and narrow distribution, whereas the nanoparticles growing in DMSO extract have larger sizes. Thus, the extract having high concentration of reducing biomolecules tuned the fast reduction of metal ions and also control the growth of nanoparticles. In case of DMSO extract, the reduction rate was slow due to low reducing potential and this extended time provides a suitable time for ionic interaction which leads to the larger size of the nanoparticles. These nanoparticles can then be further studied for enhanced antibacterial properties owing to their respective sizes.
4 Antimicrobial studies
4.1 Zone of inhibition
4.2 Mechanism of bactericidal action
Several factors are involved in the bactericidal and bacteriostatic mechanism of AgNPs. This can be an alteration in the structure of the plasma membrane, and affecting the osmoregulation of the cell or the nanoparticles may penetrate into the cell and cause leakage of the cytoplasmic contents and after internalization, they can affect the replication of DNA, generate ROS, and can affect respiration of the bacterial cell. In case of the gram-negative E. coli cells, a high percentage of the AgNPs were found inside the bacterial cell, as also reported by previous reports . The AgNPs may also cause inhibition of respiratory enzymes and cause ATP and ROS production. Ag nanoparticles can also release Ag+ ions which interact with the thiol groups present in the enzymes necessary for respiration and cause cell death . These mechanisms may occur simultaneous resulting in a quick antibacterial action on the cell.
AgNPs have been synthesized using Cinnamon zeylanicum bark extracts in different polar solvents. The results of this study show a very strong correlation between the aldehyde content, antioxidant activity, and growth of the nanoparticles. It was noticed that different solvent extracts have significant impact on the antioxidant activity and lead to the formation of nanoparticles with different shapes and sizes. The particles grow in different extracts showing size variations from 2 to 50 nm. AgNPs demonstrated both bacteriostatic and bactericidal activity with respect to their size and shape. Microscopic analysis of E. coli and S. aureus cells treated with the AgNPs illustrated their interaction with the cell wall, which facilitated their subsequent entry inside the cells. In conclusion, these nanoparticles could be effectively used for the development of infection-resistant material such as a nano-coating or impregnation for surgical devices, instruments, and wound healing bandages. The subsequent work related to the impregnation of these AgNPs in polymer matrix and their application will be communicated soon.
The authors duly acknowledge the facilities provided by Central Research Facility (CRF) at IIT Delhi. Authors are also thankful to Prof. Arti Kapil, Department of Microbiology, AIIMS, New Delhi for providing bacterial strains.
- 10.D. Malina, A. Sobczak-Kupiec, Z. Wzorek, Z. Kowalski, Dig. J. Nanomater. Biostruct. 7 (2012)Google Scholar
- 12.M. Singh, S. Singh, S. Prasad, I. Gambhir, Dig. J. Nanomater. Biostruct. 3, 115 (2008)Google Scholar
- 27.M. Sathishkumar, K. Sneha, S.W. Won, C.W. Cho, S. Kim, Y.S. Yun, Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity. Colloids Surf. B: Biointerfaces 73, 332–338 (2009). https://doi.org/10.1016/j.colsurfb.2009.06.005 CrossRefGoogle Scholar
- 34.A. Genevieve, M. Laura, L. Amy, E. Janel, ACS Sustain. Chem. Eng. (2014)Google Scholar
- 40.A.R. Shahverdi, A. Fakhimi, H.R. Shahverdi, S. Minaian, Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli. Nanomedicne 3, 168–171 (2007). https://doi.org/10.1016/j.nano.2007.02.001 CrossRefGoogle Scholar