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Abstract
Nanotechnology is gaining momentum due to its ability to transform metals into nanoparticles. The synthesis, characterization, and applications of biologically synthesized nanomaterial have become an important branch of nanotechnology. Plant extracts are a cost-effective, ecologically friendly, and efficient alternative for the large-scale synthesis of nanoparticles.In this study, highly stable silver nanoparticles (AgNPs) were synthesized by treating the Caesalpinia pulcherrima leaf extract with aqueous silver nitrate solution at room temperature.
The biosynthesized AgNPs were characterized by ultraviolet-visible spectroscopy, Transmission electron Microscopy, X-ray diffraction and EDX studies. The TEM pattern revealed circular, triangular and cubic geometry of AgNPs with a diameter range of 10-50 nm. The in vitro antimicrobial activity of the synthesized AgNps were investigated against common human pathogens such as Pseudomonas aeruginosa (NCIM 5029), Serratia marcescens(NCIM 2078), Staphylococcus aureus (NCIM 5021), Salmonella typhimurium (NCIM 2501) and Klebsiella pnemoniae (NCIM 2957) by agar well diffusion method.
The AgNps showed potential activity against all of the bacterial strains indicating its potential to be used in the development of value-added products in the biomedical and nanotechnology based industries.
Keywords: Silver nanoparticles, Caesalpinia pulcherrima, one pot green synthesis, Antimicrobial, human pathogens
The field of nanotechnology has grown over the past years and has entered into the field of clinical medicine. Silver Nanoparticles often attract a lot of interests when it comes to their potential application. In fact, silver nanoparticles are imperative as they are effective antibacterial agents against both anaerobic and aerobic bacterial. Nanomaterials have extensive applications for improving human health and the environment.
The first reported use of nanomaterial for human health was over 5,000 years ago in the Indian system of Ayurveda medicine, in which Nano science technology was applied before the term “nano” was even coined. It was only in the 21st century that modern science initiated nanoscience research, and development in this field has been rapidly growing throughout the world. A major outcome of this research is the development of new materials at the nanometer scale, including the development of nanoparticles.
It is imperative to note that apart from being an excellent antibacterial agent, Ag NPs appears to have excellent anti-inflammatory properties as well as what can be described as effect on different synthetic materials. The availability of the different silver nanoparticles has over time ensured that research continues without interruptions and hitches. An early diagnosis to the disease condition is often vital in order to ensure that early treatment is started and this results into an increase chance of getting cured.
This is particularly true when it comes to cancer the silver nanoparticles have an non invasive cancer detection and monitoring. It is of essence to understand that this method is highly promising and this may prove to be important and indispensable tool for the future. When it comes to therapeutics, the nanoparticles play a very important role when it comes to the process of healing. Most studies done have been able to demonstrate the efficacy of the Ag Nps in healing time as ell as the achievement of better cosmetic after healing.
The silver nanoparticles are applicable in purifying drinking water, degrading pesticides and killing human pathogenic bacteria etc.. Nanoparticles have become more significant in recent years and have created much impact in the areas of chemical, electronic, and biological sciences. The silver nanoparticles have reported strong antimicrobial, anti-inflammatory,anti-viral, anti-angiogenesis6 and anti-platelet activity. Recently, the organic and inorganic nanoparticles finding medical applications in an area of continued interest. Based on enhanced effectiveness, the new drugs are synthesized from nanoparticles of polymers, metals or ceramics, which can fight against cancer and also kills human pathogens like bacteria, fungi and viruses. At the present time, phytosynthesis of nanoparticles is gaining more importance due to its simple experimental procedure and eco-friendliness.
Nanoparticles are materials that have overall dimension in the nanoscale, under 100nm. These nanoparticles have been able to grow over the years into important components in modern medicine, and there have been various clinical applications that range from imaging to carriers for gene and drug delivery. There are some instances where the nanoparticles are able to enable the analysis of therapies which cannot be performed otherwise. However, despite of their huge benefit, they also present a myriad of problems as they bring unique environmental as well as societal challenges. One of the problems that they face is regarding to their toxic nature.
The biosynthesis of nanoparticles is advantageous over chemical synthesis concerning adverse effect of hazardous chemicals on environmental. Therefore, there is need to develop a method that can eliminate such problem; a green process of nanoparticle synthesis may be a solution for that. There are different natural sources like plants, bacteria, fungi and yeast which are used for synthesizing gold and silver nanoparticles11, 12.The synthesis of silver nanoparticles by using plant extracts has been evolved in recent years into the branch of nanotechnology. Although, the use of leaf extract is inexpensive and eliminates the requirement for special culture preparations and maintenance of aseptic condition that are required for microbes13.
Caesalpinia pulcherrima is a species of flowering plant in the pea family. It is a shrub growing to 3 m tall and the flowers are borne in racemes. The leaves Caesalpinia pulcherrima plants are bipinnately compound, fern-like, normally bright green turning red in winter, sometimes leafless in winter14.
The antimicrobial potential of silver nanoparticles is applicable in immense area of biology and medicine to prevent infections in burns and open wounds. The antibacterial activity of silver nanoparticles against common human pathogenic bacteria like Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus,Salmonella typhimurium, and Klebsiella pneumoniae were observed.
Check out how kinetic factors involved in constructing 2-dimensional arrays of metal nanoparticles
Hence, the present study aims to investigate the one pot green synthesis potential of AgNPs from extract of Caesalpinia pulcherrima and their antimicrobial effect on selected human pathogens.
Methods and Materials
Collection of plant material
The analytical chemicals and media components were purchased from Merck Chemicals and Hi-Media, Mumbai, India. All the aqueous solutions were prepared using triple distilled de-ionized water. Fresh leaves of C. pulcherrima collected from Lokmangal College, Wadala (17°52’23” N 75°50’6” E) Solapur, Maharashtra, India.
One pot green synthesis
Leaf weighing 10 g was washed with double distilled water and grinding was done using mortar and pestle. The well-grinded material was mixed with100 mL of double distilled water and then transferred in 500 mL Erlenmeyer flask followed by continuous stirring on magnetic stirrer for 10 min. The content was centrifuged at 10000 rpm for 10 min for the removal of cell debris. 50 mL of aqueous silver nitrate (1 mM) was added with 10 mL of the leaf extract with continuous stirring.
A color change from colorless to yellowish brown, visually confirms the formation ofAgNPs. However, it is imperative to note that the use of silver nanoparticles in many clinical conditions, potential toxicity remains a big concern. In fact, there are studies that have shown that hypersensitivity reactions often report a small proportion of patients who were diagnosed with burns as a result of the ionic silver treatment. The silver nanoparticles have been shown to be safe to use when they are used in low doses.
Nanoparticle characterization
The resulting solution was then diluted by using double distilled water and characterized using UV visible spectroscopy, X ray diffraction and Transmission electron microscopy. The nanoparticles often provide significant improvements when it comes to the traditional biological imaging of cells as well as tissues using fluorescence microscopy and this also includes modern magnetic resonance imaging MRI of various important regions of the body.
UV Visible spectroscopy
UV Visible spectral analysis has been done to know the surface Plasmon resonances band by using Systronics UV-visible absorption spectrophotometer with the resolutionof 1 nm between 200 and 800 nm. The reduction of pure Ag+ ions to form AgNPs using leaf extract was characterized by UV-Visible spectrum of the reaction medium. The same results were obtained up to three years, indicating the excellent stability of these nanoparticles. Nanoparticles are able to stay in the same chemical composition in the exact same shape and size.
They have a small size and consequently the respective Gibbs-Thomson Energy may over time cause dissolution and in most cases the redeposition of ions in the particles surface. The nanoparticles structural and atomic scale transformations in many cases causes particle degradation. In fact, the intra particle atomic ordering changes and the physical and chemical properties differ from those of the initial particles. The stability of the particles is that they are able to settle down while they are in a dispersed state. The stability is important as it directly gives the size shape stability. If there is the existence of aggregation, this leads to a decrease in stability after this, an active form can be checked.
X ray diffraction (XRD)
The biosynthesized AgNPs using Caesalpinia pulcherrima extract was lyophilized to powder. The powdered or dried AgNPs were coated on XRD grid and the spectra were recorded by using by Rich Seifert p 300 instrument. There are many factors that influence the XRD pattern of nanoparticles. Some of the factors include stress, pH value while synthesizing the impurities present change in the original structure of the specimen.
The effect of temperature also affects the XRD patter of nanoparticles. The grain/particle size, the crystallinity and the defects all have effects on the XRD pattern. The XRD pattern is characterized by the broadening of diffraction peaks. This research utilized high quality XRD patterns of nanosized powders/materials in order to wide the scanning step and lower the scanning speed. This was done in order to increase the intensity of the peaks.
Transmission Electron Microscopy (TEM)
TEM analysis of the AgNPs was recorded by placing a drop of the suspension on carbon-coated copper grids and allowing the water to evaporate.Samples were prepared by drop coating AgNPs solutions on to carbon coated copper TEM grids. The films on theTEM grids were allowed to stand for 2 minutes following which the extra solution was removed using a blotting paper and the grid was allowed to dry, prior to the measurement. The observations of TEM were performedon JEOL 3010 operated at an accelerating voltage of 120KV.
Antimicrobial activity
The one pot green synthesized AgNPs were tested for antimicrobial effect against human
Pathogens such as Pseudomonas aeruginosa (NCIM 5029), Serratia marcescens (NCIM 2078), Staphylococcus aureus (NCIM 5021), Salmonella typhimurium (NCIM 2501) and Klebsiella pnemoniae(NCIM 2957).The organisms were collected from National Chemical Laboratory (NCL), Pure Strains were spread uniformly onto the individual plates using sterile glass spreader. Wells were made on the agar plates using a cork borer to about 10 mm diameter in nutrient agar medium .100 μg of lyophilized AgNPs were added in100 μL of distilled water. 50 μL dispersed solution was added in the well and plain leaf extract was used (C.pulcherremia) as a negative control. After 24 hrs, the diameters of inhibition zones around the wells were measured in millimeter. The size of the circular inhibition zone is directly proportional to the antimicrobial effect of the biosynthesized AgNPs against microbial pathogens.
Results and discussion
One pot green synthesis
In this present study we focused on the one pot green synthesis of AgNPs using a leaf extract of C.pulchemerria which is simple, convenient, rapid, eco-friendly and found to be highly stable up to 3 years. The green synthesis of AgNPs has been investigated as an alternative to chemical and physical ones. A conical flask containing the aqueous silver nitrate (1mM) showed instant and distinct color change (colorless to yellowish brown), within 25 seconds after addition of leaf extract.
This color change was due to excitation of surface Plasmon vibrations in the metal nanoparticles. The silver nanoparticles have their unique physical and chemical properties and it proves to be an alternative for the development of entirely new antibacterial agents. In the solution the silver ions were reduced. They turn from the yellowish after 1,24, and 48 hours of reaction, this indicated the formation of silver nanoparticles. The formation as well as the stability of the silver nanoparticles in aqueous form was confirmed using UV-vis spectrophotometer analysis.
Nanoparticle characterization
UV-Visible analysis
This visible observation indicates the reduction of the Ag+ions and the bio-synthesis of AgNPs. The UV/Vis absorption spectra of the silver nano particles dispersed. This observation was further reconfirmed by UV-visible spectrum and XRD analysis In metal nanoparticles such as in silver, the conduction band and valence band lie very close to each other and through these electrons move freely. These free electrons give rise to a surface Plasmon resonance (SPR) absorption band, occurring due to the collective oscillation of electrons of AgNPs in resonance with the light wave. Classically, the electric field of an incoming wave induces polarization of the electrons with respect to much heavier ionic core of AgNPs. As a result a net charge difference occurs, which in turn acts as a restoring force.
This creates a dipolar oscillation of all the electrons with the same phase. When the frequency of the electromagnetic field becomes resonant with the coherent electron motion, a strong absorption takes place, which is the origin of the observed color, which was yellowish brown in our observation. This absorption strongly depends on the particle size, dielectric medium and chemical surroundings.
The absorption peak (SPR) was obtained in the visible range at 426 nm,the broad spectra is due to the size (10 to 50nm) and shape (spherical) of the biosynthesized AgNPs which were determined by XRD and TEM. Powder X-Ray diffraction (XRD) in this state was used to examine the physic-chemical make-up of unknown solids. The data that is represented in this collected of the single-phase X-ray powder diffraction patterns for more than three intense D values in form of tables, and inter planar spacing.
X ray diffraction
The XRD confirmed thepresence of Ag colloids in the sample. The Braggs reflections were observed in the XRD pattern at 2θ = 26.3 and 30.01. A strong diffraction peak located at 34.50 was ascribed to the (111) facets of Ag. There was also the detection of the particles in different spheres. It is of importance to note that in many cases the X-rays are often less effective when it comes to the ionizing of the AgNps. The reported fraction was proportional to the different actual numbers. This experiment can be said to be a good example of a complimentary principles. This is because there is both the use of wave and particle properties of X-ray radiation in order to make the measurement.
The XRD pattern thus clearly indicated that the AgNps formed in the present synthesis were crystalline in nature. No impurities peaks were observed in the XRD pattern, indicating that the investigated AgNPs were pure. The nanoparticles AgNPs are a special group of materials with unique features and extensive applications when it comes to diverse fields. The AgNPs were chosen as they showed completely unique properties in comparison with other large particles. The chemical reduction is the frequently applied method and it was chosen for this experiment because the Ag nanoparticles as stable in both colloidal dispersions in water solvents and organic solvents. The reduction of the silver nitrate during the exposure to the plant extracts was followed by an increase in color development from the clear to yellow brownish.
TEM analysis
The applications for AgNPs are highly dependent on the chemical composition, shape, size, and monodispersity of the particles. To broaden the potential scope of applications, the AgNPs were characterized using TEM. The samples resulted circular, triangular and cubic geometry of AgNPs with a diameter range of 10-50 nm. The Pseudomonas aeruginosa NCIM 5029 diameter of zone inhibition stood at 16mm[ ]. There was a correlation that existed between the diameter and the different organisms.
The table below shows the difference in diameters between the various organisms. It is noteworthy to note that TEM analysis of nanoparticles dispersions in solution has previously been limited. The measurement of the particle size was difficult to operationalize because of the drying effects that occurred during sample preparation. The TEM route was potentially sensitive route when it came to counting individual nano particles dispersion which also contained nano particle agglomerates.
Antimicrobial activity
In this study, Caesalpinia pulcherrima extract and the AgNps synthesized with Caesalpinia pulcherrima leaf extract were tested for their antimicrobial activities against Pseudomonas aeruginosa (NCIM 5029), Serratia marcescens (NCIM 2078), Staphylococcus aureus (NCIM 5021), Salmonella typhimurium (NCIM 2501), and Klebsiella pnemoniae (NCIM 2957).The AgNPs are extensively used in the pharmaceutical industry and have inhibitory activities on various microorganisms. They have also been used in balms and ointments to avert infections following burns and wounds.
In regards to the environment, the AgNps stable nature means that it is inorganic, however, by putting an organic coating it makes the inorganic AgNps bioactive and biocompatible One of the main toxicological risk associated with the use of AgNps is the exposure of the inorganic core by after the deterioration of the organic layer. Although rare, this process is still possible. Although the existing literature on the toxicity of the equipment is not extensive, there are several reports that highlight the major concerns and they illustrate the need for more work.
The zone of inhibition was found to be as per table:
Serial No Name of organism Diameter of zone of inhibition
( in mm)
1 Pseudomonas aeruginosa NCIM 5029 16
2 Serratia marcescens NCIM 2078 19
3 Staphylococcus aureusNCIM 5021 23
4 Salmonella typhi NCIM 2501 15
5 Klebsiella pnemoniae NCIM 2957 14
Table: Antibacterial activity of Caesalpinia pulcherrima silver nanoparticle extracts as the zone of inhibition in (mm).
Conclusion
The Caesalpinia pulcherrima leaf extract reduced Ag+ metal ions and led to the formation of AgNPs with fairly well-defined dimensions. This “green chemistry” approach for the synthesis of AgNPs has many advantages, such as the ease with which the process can be scaled up and its economic viability. Applications for these eco-friendly nanoparticles in bactericidal, wound healing and other medical and electronic applications signifies that this method has the potential for the large-scale synthesis of other inorganic nanomaterials. The antimicrobial screening demonstrated that the synthesized AgNPs had a high inhibitory effect on bacteria. These observations may serve as a guide for studying the controlled release of these synthesized AgNps, which has potential in the field of infectious diseases. These AgNps may be explored as an option for decreasing the pathogenic potential of infectious bacterial and fungal species.
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Stephen H. Brown, Horticulture Agent, Caesalpinia pulcherrima, Lee County Extension, Fort Myers, Florida (239) 533-7513,
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