Nanobiotechnology is a rapidly growing field with numerous applications in the medical field. The discipline combines biological research with various fields of nanotechnology. Nanodevices, nanoparticles, and nanoscale phenomena within nanotechnology are enhanced through nanobiology. Typically, nanoparticles are synthesized using either a top-down or bottom-up approach. However, these chemical methods can be dangerous as they involve handling toxic chemicals and producing toxic by-products. Therefore, researchers have conducted several studies to extract and synthesise nanoparticles from living sources, known as the "Green synthesis of Nanoparticles." Silver nanoparticles are particularly prioritised because they are harmless to the human body but detrimental to microbes even in minimal concentrations. Much effort has been devoted to extracting them from plant sources and microbes. Animal extracts are also potential reducing agents for extracting silver nanoparticles without the use of any toxic substances. It has been discovered that extracts from various animals used in nanoparticle manufacturing contain biomolecules that act as reducing and capping agents. In line with this perspective, the present study focuses on using the telescope snail, Telescopium telescopium (Linnaeus, 1758), as an animal source for producing silver nanoparticles. The extracted TtSN-particles (Telescopium telescopium Silver Nanoparticles) were subjected to various characterisation methods, including Ultra Violet- Visible Spectroscopy (UV Spectroscopy), Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX) and Dynamic Light Scattering Technique (DLS) analyses. The absorption of TtSN-particles was found at 360 nm. The sizes of the TtSN particles visualised in SEM were 98.44 nm, 81.88 nm, and 88.65 nm. The EDX pattern showed a predominance of silver in the colloidal solution of the synthesized TtSN-particles. In DLS, the intensity distribution was observed in diameters of 46.4 nm, 90.4 nm, and 182.8 nm, respectively. Additionally, the sharp peak of the XRD confirmed the crystallinity of the nanoparticles. Further bio-assays were conducted to study the anti-thrombolytic activity of the TtSN-particles. The results showed that the TtSN-particles significantly reduced the formation of blood clots in the veins compared to the control groups. These nanoparticles also exhibited antimicrobial activity against both gram-positive (Streptococcus mutans) and gram-negative bacteria (Escherichia coli). They possessed high antioxidant and anti-inflammatory activities, as confirmed by performing a DPPH assay and Heat hemolysis assay, respectively.
Research Article
Characterization and Biological Application Assessment of Silver Nanoparticles Extracted from Telescopium telescopius (Linnaeus, 1758), the telescope snail - The Green Synthesis Revolution
https://doi.org/10.21203/rs.3.rs-4941846/v1
This work is licensed under a CC BY 4.0 License
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Telescopium telescopium
Silver Nanoparticles
Anti-Microbial
Anti-Oxidant
Anti-Inflammatory
Anti-thrombolysis
The field of medicine needs to incline towards greener solutions for the extraction and preparation of components of drugs. This is the need of the hour due to the harmful side effects of synthetic components on the human system not to mention the deleterious effects of their preparation and extraction on the environment. Here is where nanobiotechnology rises to the challenge and serves as a viable solution. The applications of nanobiotechnology are plenty especially in the field of medicine. Aspects that are enhanced through nanobiology are nanodevices, nanoparticles, and nanoscale phenomena that occur within the discipline of nanotechnology (Abid Haleem at al., 2023). Research in nanobiotechnology in India is still in its infancy. Considering the fact that, biological systems are inherently nano in scale; nanoscience must merge with biology in order to deliver biomacromolecules and molecular machines that are similar to nature. Natural evolution has optimized the "natural" form of nanobiology over millions of years. In the 21st century, humans have developed the technology to artificially tap into this nanobiology (Serjay Sim et al., 2021). There are a lot of promising methods that may rely on Nanobiology in the future. Among the different metals from which the nanoparticles are extracted, Silver is attracting special attention because it is harmless to humans. Usually, the extraction of these nanoparticles is carried out by either a top-down or bottom-up approach. However these chemical methods are very dangerous since they involve handling toxic chemicals as well as the production of toxic by-products. Thus, several attempts and research have been carried out for extracting these nanoparticles from living sources, rightly termed the “Green synthesis of Nanoparticles”. Silver nanoparticles are given priority because they are harmless to the human body but they are detrimental to microbes even in minimal concentrations. Much effort goes into extracting them from the extracts of plant sources and microbes (Pushpa Rani et al., 2023). But animal extract is also a potential reducing agent to extract the silver nanoparticles without the involvement of any toxic substances. It has been discovered that the extracts from a variety of animals utilised in the manufacture of nanoparticles contain biomolecules that act as reducing and capping agents. The reduction and stability of the resulting nanoparticles are facilitated by the proteins found in these biological materials (V. Pushpa Rani et al 2022). In line with this perspective, the present study focuses on using the telescope snail, Telescopium telescopium (Linnaeus 1758), as a source for producing silver nanoparticles. The local people of Sirkazhi, Tamil Nadu, rely on this animal as the primary treatment for piles. The study investigates various applications of these silver nanoparticles, including their antioxidant, cytotoxicity, antimicrobial, anti-inflammatory, and anti-thrombolytic activities.
2.1 Sample Collections
Around 350 specimens of T. telescopium species were collected from the intertidal area of Vellar estuary ,Parangipettai, Cuddalore, Tamil Nadu, India ( coordinates of the exact collection site − 11° 29′N, 79° 46′E). The specimens were washed thoroughly with distilled water Fig. 2.1. and allowed to thrive ( in live condition) in water-sand troughs throughout the experimentation period.
2.2 Identification
The collected Telescopium snails was taken to the Southern regional Centre, Zoological Survey of India and were authenticated by the taxonomist moreover the molecular level of identification was carried out using gene sequencing method using COX1 gene, the BLAST results conforms that the collected samples were Telescopium. The sequenced results were submitted to NCBI-Gen Bank and an unique accession no PP152336 was obtained for the same
2.3 Nomenclature of Telescopium Telescopium
Telescopium telescopium, often known as the telescope snail, is a species of horn snail from the Potamididae family that inhabits mangroves in the Indo-Pacific regions. They are massive snails that can reach 8 to 10 cm (3.1 to 3.9 in) in length and are easily identified by their cone-shaped shell.
Kingdom: Animalia
Phylum: Mollusca
Class : Gastropoda
Order: Stylommatophora
Superfamily: Cerithioidea
Family: Potomididae
Genus: Telescopium
Species: Telescopium Telescopium (Linnaeus, 1758)
2.4 Preparation of Animal Extract
The extraction of the soft body of the molluscan species was done using a shell cutter. The operculum was removed and the whole animal was traced out of its shell. The soft body of the animal was homogenised with 30 mL of deionised water using a mortal and pestle and made into a coarse paste using a mixer. The homogenised extract was then boiled at 45°C for 20 minutes and then filtered using Whatmann No 1 filter paper to obtain the filtrate.
2.5 Extraction of Silver Nanoparticles using Telescopium Extract ( Telescopium telelescopium Silver Nanoparticles -TtSN particles)
Telescopium telescopium Silver Nanoparticles (TtSN-particles) were produced by reducing the silver metal using the animal extract by the action of bioactive molecules present in the extract. The ratio of the homogenised animal extract to the silver nitrate solution was 1:10. Around 40 mL of the homogenised extract was added to 400 mL of 5mM Silver Nitrate solution. The silver nitrate solution was prepared by dissolving 0.849 g of silver nitrate in 1000 mL of deionised water. The animal extract combined with the silver nitrate solution is kept in the dark for 24hours. A colour change from creamy white to brownish black was observed which is indicative of presence of silver nanoparticles. Figure 2.2 26 Extraction of TtSN-particles
The Silver nanoparticles were extracted from the brownish black solution by centrifuging at 13,500 rpm for 15 minutes. The supernatant was discarded and the pellets formed were collected. The pellets were then subjected to an ethanol wash for easy drying and spread on a petridish. After 24 hours, the dried and powdered (now) were collected by scraping the entire petridish. The silver nanoparticles collected, were stored as a colloidal solution and as dry nanoparticle powder (two forms). Figure 2.3
2.3Extraction of TtSN-particles
A) Liquid Colloidal Nano particles B) Crystal form Nanoparticles before scrapping and collection C) Crystal Form TtSN-particles after scraping and collection (in pellet form)
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Liquid colloidal Silver nanoparticles after centrifugation
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Crystal form of Silver Nanoparticles after drying.
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Crystal form TtSN-particles
2.7. Characterization of TtSN-particles
The size, shape, and surface charge of the extracted nanoparticles were evaluated using UV-Visible spectroscopy, SEM, EDX spectrum analysis, X-Ray Diffraction, FTIR and DLS analyses. (Phanjom et al., 2015 ) (R.Anith Jose et al., 2022)
2.7.1 UV-Visible Spectroscopy
Through UV-Visible spectroscopy, it was possible to monitor the progression of silver nitrate reduction to silver nanoparticles over 24 hours. The process involved passing a beam of monochromatic light through the colloidal solution of the pellet extract. The concentration of the solution and the rate at which the radiation intensity decreased in relation to the thickness of the absorbing solution (colloidal solution of the pellet extract) were found to be directly proportional (Tiara Egga Agustina et al., 2021). To obtain the UV graph, PC-Based Double Beam UV-VIS Spectrophotometer 2202 was utilised. The peak value of the colloidal extract was observed against, a quartz cuvette filled with water as a blank. The UV-visible spectrometric values for the colloidal solution ranged from 200 to 800 nm was performed
2.7. 2 Scanning Electron Microscope (SEM)
Scanning electron microscopes produce images of a sample by scanning it with an electron beam.When a sample is extracted from animal tissues and subjected to SEM, the electron beam scans the sample and provides information about the topography of the extract, in this case, the dry pellet of the extracted TtSN-Particles were used. The scanning electron microscopic analysis was carried out using a Hitachi S-4500 SEM. A small amount of sample was dropped onto a copper grid that had been coated with carbon to create a thin layer of extracted TtSN-particles. The sample film on the SEM grid was dried by placing it under a mercury lamp for five minutes and the obtained images were captured .
2. 7.3 Energy Dispersive X-Ray Analysis (EDX)
Energy dispersive X-ray (EDX) is an analytical technique used to analyse different elements of nanoparticles. It works by bombarding a sample with high-energy X-rays, which cause the electrons to drop from a high energy to low energy level. This drop in energy level emits an X-Ray with the same energy as the difference between the two energy levels in the electrons. The energy and intensity of these emitted X-rays provide information about the elements present in the sample. In EDX, the emitted X-rays are detected using a solid-state detector, such as a silicon drift detector, and the resulting signal is processed to produce a spectrum of X-ray energies. The spectrum can be used to identify the elements present in the sample and to determine their relative abundances.
2.7.4 Fourier Transformed Infrared Spectroscopy (FTIR)
FTIR spectroscopy is used to find out the functional groups present in the extracted nanoparticles which is more pronounced in academic and industrial research (Zhang et al., 2020).The functional group of the silver nanoparticles, extracted from animal tissue, was characterized using FTIR. The functional groups in the TtSN particles responsible for reducing, capping and stabilising the silver nanoparticles were determined. The analysis was performed using a JASCO FT-IR-4700 spectrometer and potassium bromide (KBr) pellets. The spectra in the 599–4000 range were obtained using 4 cm-1 resolution,
2.7.5 X-Ray Diffraction (XRD)
X-Ray diffraction analysis (XRD) is a nondestructive technique that provides detailed information about the crystallographic structure, chemical composition, and physical properties of a material. It is based on the constructive interference of monochromatic X-rays and a crystalline sample. X-rays are shorter wavelength electromagnetic radiation that are generated when electrically charged particles with sufficient energy are decelerated. In XRD, the generated X-rays are collimated and directed to a nanomaterial sample, where the interaction of the incident rays with the sample produces a diffracted ray, which is then detected, processed, and counted. The intensity of the diffracted rays scattered at different angles of material are plotted to display a diffraction pattern. Each phase of the material produces a unique diffraction pattern due to the material's specific chemistry and atomic arrangement. The diffraction pattern is a simple sum of diffraction patterns of each phase.. The silver nanoparticles extracted from the animal extract were analyzed using XRD to determine their crystalline structure and diffraction patterns. The analysis was conducted with a PAN analytical XPRT PRO, D-8, advanced Bruker instrument, with measurements taken at a 2θ angle range of 10° to 80°.
2.7.6 Dynamic Light Scattering Technique (DLS)
DLS uses Brownian motion to estimate particle size distribution. However, DLS analyzes all particles in a sample simultaneously and therefore cannot provide information on particle number or concentration. A laser beam passes through a sample enclosed between two polarizing filters before being detected by a sensor. On its way through the sample, the light is scattered by particles. At the detector, the light scattered by the individual particles is subjected to destructive and constructive interference, resulting in a speckled pattern. The interference pattern between the scattered light from the particles in solution changes as they undergo Brownian motion. The distribution of particle size can be estimated by analyzing this changing interference pattern. Although DLS allows precise characterization of particles between 1 nm and 6 µm, it is accurate only for particles within a homogeneous sample and is easily affected by the presence of larger particles. DLS is, therefore, capable of characterizing smaller particles.
2.8 Anti-Microbial activity of the synthesized TtSN-particles
2.8.1 Inoculum Preparation
A loopful of gram-positive bacteria, Streptococcus mutans (Clarke,1924) and gram-negative bacteria Escherichia coli species was inoculated in the sterile nutrient broth and incubated overnight at 37°C.
2.8.2 Well Diffusion Assay
The agar well diffusion assay was used to determine the inhibitory effects of the sample on bacterial growth.
Muller-Hinton agar was prepared and poured into a sterile petridish and allowed to solidify. The gram-positive bacteria, Streptococcus mutans and the gram-negative bacteria E.coli were inoculated using a sterile cotton swab in the Muller-Hinton agar and the prepared petridish was incubated overnight.
Four wells were made in the agar plate using a sterile cork borer (8-mm diameter). Standard tetracycline (25 µg), 250 µg (TtSNP-), 500 µg (TtSNP-) and 1000 µg (TtSNP-) were each added to one of the four wells and incubated overnight at 37°C. The presence of a zone of inhibition around a well indicated anti-bacterial activity, which was recorded (Du Toi et al 2000 & Athanassiadis et al 2009).
2.9 Anti-Oxidant Activity – 1,1-diphenyl-2-picrylhydrazyl (DPPH) Assay
1,1-diphenyl-2-picrylhydrazyl (DPPH) is a free radical which have hydrogen acceptor capability to antioxidants. Henceforth, this compound usually used in DPPH assay for the determination of antioxidant activity.
.One mL of 0.1 mM DPPH solution in methanol was mixed with 1 mL of various concentrations (50–300 µg/mL) of the TtSN-Particles-. The mixture was then allowed to stand for 30 minutes in the dark. One mL methanol mixed with 1 mL DPPH solution was used as the control (Mensor. L et al 2001). The decrease in absorbance was measured at 517 nm.
The percentage of inhibition was calculated as
2.10 Anti-Inflammatory activity (Anti-haemolysis assay)
2.10.1 Preparation of Red Blood Cells (RBCs) Suspension
Blood was collected from a healthy human volunteer who did not take any NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) for 2 weeks before the experiment and transferred to centrifuge tubes, which were then centrifuged at 3000 rpm for 10 min. Post centrifugation, the tubes with the blood samples were washed three times with equal volumes of normal saline. The volume of blood was measured and reconstituted as 10% v/v suspension with normal saline (Moualek I et al 2016)
2.10.2 Heat-Induced Haemolysis
The reaction mixtures are prepared by using different concentrations of the test samples; concentrations ranging from 50 µg/mL to 300 µg//mL
The reaction mixture (2mL) consisted of a 1 mL test sample (TtSN-particles) of different concentrations (20–120µg/mL) and 200 mL of 10% RBCs suspension, instead of the test sample only saline was added to the control test tube. All the centrifuge tubes containing the reaction mixture were immersed in a water bath at 56°C for 30 minutes. After 30 minutes, the tubes were cooled under running water. The reaction mixtures were centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants were observed at 560 nm. The percentage inhibition of haemolysis of the reaction mixtures were calculated using the follwing formula
% of inhibition = (Abs. control – Abs. sample) X 100 / Abs. Control
2.11 Anti -Thrombolytic Activity
The tip of the finger of a free-willing volunteer was cleaned with alcohol and pricked with a lancet. The blood drop was put on both ends of a clean glass slide and named drop “A”(left-side) and drop “B” (right-side). To blood drop “A”, 10 µl of the extracted nanoparticle colloidal solution was added and blood drop “B” was left undisturbed. This test is done to check if the nanoparticle extract has any anti-thrombolytic and declotting activity ( Maqsood et al 2014)
3.1 UV-Visible Spectroscopy
The characterization of the extracted silver nanoparticles using UV-Vis spectroscopy shows maximum absorption by the nanoparticles at 360.4 nm. Along with the absorption of the wavelength, the curvature of the peak is also considered. Graph 3.1 clearly shows the sharp peak absorbance observed in the extracted silver nanoparticles. The other peaks observed were at 367.8 nm and 393.8 nm.
3.2 Scanning Electron Microscope (SEM)
The distinct border of the aggregates of the silver nanoparticles is seen clearly in the result images. The size of silver nanoparticles are recorded in the range of 80–100 nm. The sizes of the visualized extracted silver nanoparticles are 98.44 nm, 81.88 nm, and 88.65 nm. (Fig. 3.1).
3.3 Energy Dispersive X-Ray Analysis (EDX)
The EDX pattern showed the predominance of silver in the pellet form of the extracted silver nanoparticles. It also shows the percentage fraction of the weight of the elements and the atomic percentage of the element in the extracted silver nanoparticles (Graph 3.2)(Table 3.1)
Table 3.1 List of Elements Present in Tt AgNps in EDX Spectrum
| Elements | Line Type | App.Conc | K.Ratio | Wt% | Wt%Sig. | Std. Label | Factory Standard |
|---|---|---|---|---|---|---|---|
| C | K series | 1.08 | 0.0108 | 13.64 | 0.35 | C Vit | Yes |
| N | K series | 2.58 | 0.0046 | 13.18 | 0.65 | BN | Yes |
| O | K series | 3.28 | 0.011 | 29.5 | 0.38 | SiO2 | Yes |
| Na | K series | 0.5 | 0.0021 | 1.96 | 0.07 | Albite | Yes |
| Mg | K series | 0.41 | 0.0027 | 1.73 | 0.05 | MgO | Yes |
| Cl | K series | 0.76 | 0.0066 | 2.34 | 0.05 | Nacl | Yes |
| Ag | L Series | 9.83 | 0.0983 | 37.65 | 0.39 | Ag | Yes |
| Total | 100 | ||||||
3.4 Fourier Transformed Infrared Spectroscopy (FTIR)
By comparing the peak values found in (Graph 3.3) with the IR chart, the functional groups present in the sample are found in FTIR analysis.(Table 3.2) The functional groups present in the extracted TtSN-particles were aliphatic primary amines, carboxylic acid, amine salt, Thiocyanate, Imine, Nitro compound (nitro group), Aldehyde, Alkyl Aryl Ether, Primary Alcohol, Fluro Compounds and Halo compounds
| Absorption cm-1 | Appearance | Group | Compound Class |
|---|---|---|---|
| 3419.11 | Medium | N-H Stretching | Aliphatic P. Amine |
| 2922.80 | Strong, Broad | O-H Stretching | Carboxylic Acid |
| 2852.53 | Strong, broad | N-H Stretching | Amine Salt |
| 2168.96 | Strong | S-CΞN stretching | Thiocyanate |
| 1642.45 | Medium | C = N stretching | Imine/Oxime |
| 1548.84 | Strong | N-O Stretching | Nitro compound |
| 1384.31 | Medium | C-H Bending | Aldehyde |
| 1228.28 | Strong | C-O Stretching | Alkyl Aryl Ether |
| 1077.42 | Strong | C-O Stretching | Primary Alcohol |
| 1025.14 | Strong | C-F Stretching | Fluro Compound |
| 825.44 | Strong | C-H Bending | 1,4-disubstituted |
| 617.08 | Strong | C-Br Stretching | Halo Compound |
3.5 X-Ray Diffraction
The peak values of the nanoparticles are 28.127°, 32.517°, 38.322°, 46.509°, 55.050°, 57.742°, and 77.124° - corresponding to the particle sizes 3.16995 Å, 2.75136 Å, 2.34688 Å, 1.95105 Å, 1.66682 Å, 1.59536 Å, and 1.23572 Å respectively. Graph 3.4 is the XRD Graph generated from the UXD data.
3.6 Dynamic Light Scattering Analysis
The intensity distribution of the silver nanoparticle starts from a diameter of 1.1 nm to 328.7nm. The 10%, 50% and 90% of intensity distribution are observed in the silver nanoparticles of diameter 46.4 nm, 90.4 nm and 182.8 nm respectively. (Graph 3.5).
3.7 Anti-Microbial Activity
The AgNP solution extract of Telescopium telescopium showed antimicrobial activity against the test microbe Streptococcus mutans with a maximum zone of inhibition of 15 mm at a concentration of 1000 µg/mL. The minimum inhibition concentration was found to be the standard. The results against the test microbe Escherichia coli shows a maximum zone of inhibition of 12 mm at a concentration of 1000 µg/mL These results suggest that the AgNP extract of Telescopium telescopium has moderate to strong antimicrobial activity against the test microbes. The results are tabulated (Table 3.3) and the zones of inhibitions are shown (Fig. 3.3).
Fig 3.3 Anti-Microbial Activity
| S.No | Pathogens | Zone of Inhibition | |||
|---|---|---|---|---|---|
| 250µg/mL | 500µg/mL | 1000µg/mL | Standard (Tetracycline) 25µg/mL | ||
| 1. | Streptococcus mutans | 12 | 13 | 15 | 10 |
| 2. | Escherichia coli | 10 | 11 | 12 | - |
3.8 Anti -Oxidant -DPPH Assay
Upon detailed examination of the data, it can be observed that an extraordinarily high 97.68%inhibition is obtained at 300 µg/mL of the extracted TtSN-particle sample. There exists a strong correlation between the concentration of AgNP solution and the percentage of inhibition. This illustrates the high levels of antioxidant activity found in the AgNPs made from the Telescopium telescopium animal extract (Fig. 3.4) (Table 3.4)
| S.No | Conc. µg/mL | Absorbance @517 nm | %Inhibition |
|---|---|---|---|
| 1. | Control | 0.259 | - |
| 2. | 50 | 0.235 | 6.26 |
| 3. | 100 | 0.195 | 24.71 |
| 4. | 150 | 0.186 | 28.18 |
| 5. | 200 | 0.159 | 38.61 |
| 6. | 250 | 0.138 | 46.71 |
| 7. | 300 | 0.006 | 97.68 |
3.9 Anti-Inflammatory Assay
The AgNp showed significant dose-dependent inhibition of heat-induced haemolysis with 95.45% inhibition at 300 µg/mL. The percentage inhibitions at various concentrations are listed in Table 3.5. These results suggest that the test compound has moderate to high anti-inflammatory activity in the heat-induced haemolysis model. (Fig. 3.4)
Figure 3.5 Anti -Inflammatory activity
| S. No | Conc. (µg/mL) | Absorbance @560nm | % of Inhibition |
|---|---|---|---|
| 1 | Control | 0.022 | - |
| 2 | 50 | 0.020 | 9.09 |
| 3 | 100 | 0.018 | 18.18 |
| 4 | 150 | 0.016 | 27.27 |
| 5 | 200 | 0.010 | 54.54 |
| 6 | 250 | 0.005 | 77.27 |
| 7 | 300 | 0.001 | 95.45 |
3.10 Anti-Thrombolytic Activity
The fingertip of a free-wiling volunteer was cleaned with alcohol and then pricked with a sterile lancet. The resulting blood drop was placed on both ends of a clean glass slide. To blood drop "A", 10 µL of the synthesized nanoparticle solution was added. Blood drop "B" was left untouched. This was done to determine if the nanoparticle possesses any anti-thrombolytic and declotting activity. After a few seconds, it was observed that blood drop “A” did not clot, while drop "B" had clotted. This demonstrates the anti-coagulating activity of the synthesised AgNPs. To blood drop "B", 10 ul of the AgNP solution was added to assess whether the clot would disintegrate. It was observed that drop "B" had liquefied after a few seconds. This indicates the declotting activity of the extracted AgNPs. Figure 3.6
The application of UV spectroscopy to nanoparticles is more complex than for bulk materials due to the fact that nanoparticles exhibit size-dependent optical properties. The absorbance of nanoparticles is dependent on their size, shape, composition, and surrounding medium. In general, as the size of the nanoparticle decreases, the absorbance maximum shifts to shorter wavelengths and the peak broadens. To obtain accurate information about the optical properties of nanoparticles, it is important to carefully control the size, shape, and composition of the nanoparticles and to use appropriate dispersion techniques to ensure that the nanoparticles are well-dispersed in the medium being studied. Additionally, it may be necessary to use more advanced spectroscopic techniques such as time-resolved or surface-enhanced UV spectroscopy to obtain detailed information about the nanoparticle's electronic structure and dynamics. E. P. Ivanova at al, 2020 discussed how UV-Vis Spectroscopy should be used to characterise and discuss results for Nanoparticles. Also, several studies have used UV spectroscopy to characterise nanoparticles. For example, a study published in the Journal of Physical Chemistry by Huang et al. (2010) used UV spectroscopy to investigate the size-dependent optical properties of gold nanoparticles. The results are coherent with this present investigation.
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The surface topology of the extracted TtSN particles was studied using a scanning electron microscope (SEM) at a resolution of 200 nm. The electron micrograph confirmed that the silver nanoparticles (AgNPs) were spherical and well-dispersed. The SEM analysis revealed that the silver nanoparticles interact with the electron beam, producing signals that provide detailed information about the composition, surface topography, and other characteristics, such as electrical conductivity. Individual silver nanoparticles and various aggregates were seen in the SEM image. There are numerous examples of SEM being used for nanoparticle characterization. For instance, a study published in the Journal of Nanoscale by Kriegel et al. (2013) used SEM to investigate the size, shape, and morphology of silver nanoparticles extracted by a wet chemical reduction method. The SEM images revealed that the nanoparticles were spherical in shape, with an average diameter of 100 nm. The authors also observed that the nanoparticles formed aggregates due to van der Waals forces.
Another example is a study published in the Journal of Physical Chemistry by Li et al. (2012), which used SEM to investigate the morphology and size distribution of cobalt ferrite nanoparticles extracted by a hydrothermal method. The SEM images showed that the nanoparticles were uniform in size and shape, with an average diameter of 20 nm. The authors also observed that the nanoparticles had a faceted surface and a smooth surface, which they attributed to the crystal structure of the cobalt ferrite.
Dynamic Light Scattering (DLS) provides information on the hydrodynamic radius of nanoparticles, which is related to their size and shape in solution However, it is important to note that DLS measures the hydrodynamic size of the particles, which may differ from their physical size due to factors such as surface charge, surface hydration, and the presence of bound biomolecules. Additionally, DLS is most effective for measuring nanoparticles in the size range of 1-100 nm, and may not be as accurate for larger particles or aggregates. Overall, DLS is a useful and widely used technique for measuring the size distribution of nanoparticles in solution, but it is important to carefully interpret the results in the context of the specific properties of the nanoparticles being studied. There are numerous examples of DLS being used for nanoparticle characterisation. For instance, a study published in the Journal of Nanobiotechnology by Yang et al. (2019) used DLS to investigate the size distribution and stability of polydopamine-coated gold nanoparticles in different buffer solutions. The DLS results showed that the nanoparticles had an average hydrodynamic diameter of 27 nm and a narrow size distribution. The authors also observed that the zeta potential of the nanoparticles varied with the pH of the buffer solution, which affected their stability.
Another example is a study published in the Journal of Colloid and Interface Science by Wani et al. (2022), which used DLS to investigate the size distribution and stability of iron oxide nanoparticles coated with different types of surfactants. The DLS results showed that the nanoparticles had an average hydrodynamic diameter ranging from 14 to 23 nm, depending on the type of surfactant used. The authors also observed that the stability of the nanoparticles varied with the type of surfactant, with some surfactants leading to agglomeration and precipitation of the nanoparticles.
Since the nanoparticles were synthesized using silver, the Energy Dispersive X-Ray (EDX) analysis was anticipated to reveal a significant presence of silver compared to other elements. As expected, the EDX pattern confirmed a high concentration of silver, showing a peak with 37.6% composition. Additionally, carbon, which was anticipated due to the animal source used in the synthesis, was detected at 13.6%. Other elements, including oxygen, nitrogen, chlorine, sodium, and magnesium, were also present in smaller amounts. EDX is a powerful tool for analyzing the elemental composition of nanoparticles. In this case, it confirms that silver is the predominant element, as expected from the synthesis process. The presence of carbon is also consistent with the use of an animal source, likely due to organic residues or surface capping agents. The detection of additional elements like oxygen and chlorine could indicate the presence of surface oxides, while sodium and magnesium might come from the reaction medium. This comprehensive elemental analysis is crucial for understanding the purity and potential functionalization of the synthesized nanoparticles.(To characterize the crystalline structure and determine the size of the extracted silver nanoparticles (AgNPs), X-ray diffraction (XRD) analysis was performed. The strong diffraction peaks of the synthesized AgNPs are observed at specific 2θ values. By substituting the obtained 2θ values into the relevant equations, along with the constant values, we can accurately calculate the size of the synthesized silver nanoparticles. There are numerous examples of XRD being used for nanoparticle characterisation. For instance, a study published in the Journal of Physical Chemistry by Xu et al. (2019) used XRD to investigate the crystal structure and phase composition of copper oxide nanoparticles extracted using a hydrothermal method. Another example is a study published in the Journal of Materials Science: Materials in Electronics by Liang et al. (2020), which used XRD to investigate the crystal structure and phase composition of tin dioxide nanoparticles extracted using a sol-gel method. The XRD results showed that the nanoparticles were composed of the rutile phase of SnO2, with a crystallite size of about 6 nm.
It is important to note that the interpretation of Fourier Transform Infrared Spectroscopy (FTIR) spectra of nanoparticles can be more complex than for bulk materials, as nanoparticles often exhibit size-dependent optical properties that can affect the peak position and intensity of the absorption bands. Additionally, the presence of surface adsorption and aggregation effects can also influence the FTIR spectrum. FTIR spectroscopy is a powerful technique for the analysis of nanoparticles, but it is important to carefully consider the limitations of the technique and to perform appropriate controls and sample preparation techniques to obtain reliable and accurate results.
The exact mechanism by which silver nanoparticles exert their antimicrobial activity is not fully understood, but it is believed to involve a combination of physical and chemical processes. The small size of the nanoparticles allows them to penetrate the cell wall of microorganisms and interact with internal components, while the release of silver ions can disrupt the membrane potential and interfere with enzymatic processes. Research into the antimicrobial properties of silver nanoparticles is ongoing, and there is growing interest in their potential applications in a variety of fields, including medicine, food packaging, and water treatment. However, it is important to carefully evaluate the safety and efficacy of silver nanoparticles in each specific application, as well as to consider the potential environmental impact of their widespread use. One study published in the Journal of Nanobiotechnology by Durán et al. (2015) investigated the antimicrobial activity of AgNPs against different bacterial strains. The results showed that AgNPs had significant antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multi drug-resistant strains. The authors also found that AgNPs had a synergistic effect when combined with antibiotics, which could potentially reduce the development of antibiotic resistance. Another study published in the journal Nanomedicine: Nanotechnology, Biology, and Medicine by Liu et al. (2019) investigated the antimicrobial activity of AgNPs against the H1N1 influenza virus. The results showed that AgNPs significantly reduced the viral titre of H1N1 In Vitro, and also improved the survival rate of mice infected with the virus In vivo. The authors suggested that AgNPs could be a potential therapeutic option for the treatment of influenza.
The anti-thrombolytic activity of the extracted silver nanoparticles is studied and positive results are observed. AgNPs have been shown to have anti-thrombotic activity by inhibiting platelet aggregation and adhesion, which are key events in the formation of blood clots. One study published in the journal ACS Applied Materials & Interfaces by Sharma et al. (2019) investigated the anti-thrombotic activity of AgNPs In Vitro and In Vivo. The results showed that AgNPs significantly inhibited platelet aggregation and adhesion, and also reduced thrombus formation In Vivo in mouse model. The authors suggested that AgNPs could be a potential therapeutic option for preventing thrombotic events. Another study published in the Journal of Nanoparticle Research by Roşca, et al. (2022) investigated the anti-thrombotic activity of AgNPs in a rat model of venous thrombosis. The results showed that AgNPs significantly reduced the formation of blood clots in the veins compared to control groups. The authors suggested that AgNPs could be a potential therapeutic option for preventing venous thrombosis and other thrombotic events.
The antioxidant activity of the silver nanoparticles is observed through the DPPH assay and the percentage of the inhibition activity is noted and given in the tabulated form. The absorbance of the control (methanol extract) is greater than the absorbance of the minimal concentration of the silver nanoparticles. And it is clearly observed that the absorbance of the extracted silver nanoparticles decreases with the increase in concentration. One study published in the Journal of Materials Science: Materials in Medicine by Kim et al. (2014) investigated the antioxidant activity of AgNPs against hydrogen peroxide-induced oxidative stress in human umbilical vein endothelial cells (HUVECs). The results showed that AgNPs had significant antioxidant activity, as they reduced ROS levels and increased cell viability compared to cells treated with hydrogen peroxide alone. The authors suggested that AgNPs could be a potential therapeutic option for diseases associated with oxidative stress. Another study published in the Journal of Nanoscience and Nanotechnology by Bindhu et al. (2016) investigated the antioxidant activity of AgNPs synthesized using the plant extract of Eclipta prostrata. The results showed that AgNPs had significant antioxidant activity, as they inhibited lipid peroxidation and scavenged free radicals In Vitro. The authors suggested that AgNPs synthesized using natural sources could be a safer and more effective alternative to synthetic AgNPs.
The mechanism of action of silver nanoparticles in inflammation is not fully understood, but it is thought to involve the modulation of pro-inflammatory cytokines.a study published in the Journal of Biomedical Materials Research Part A by Pan et al. (2018) investigated the anti-inflammatory and immunomodulatory properties of AgNPs in a mouse model of inflammatory bowel disease. The results showed that AgNPs reduced inflammation and improved immune function compared to control groups. One study published in the journal Nanomedicine: Nanotechnology, Biology and Medicine by Cho et al. (2017) investigated the anti-inflammatory activity of AgNPs in a mouse model of acute lung injury induced by lipopolysaccharide (LPS). The results showed that AgNPs significantly reduced lung inflammation and improved lung function compared to control groups. The authors suggested that AgNPs could be a potential therapeutic option for acute lung injury and other inflammatory diseases. Another study published in the Journal of Biomedical Materials Research Part A by Pan et al. (2018) investigated the anti-inflammatory and immunomodulatory properties of AgNPs in a mouse model of inflammatory bowel disease. The results showed that AgNPs reduced inflammation and improved immune function compared to control groups. The authors suggested that AgNPs could be a potential therapeutic option for inflammatory bowel disease and other inflammatory diseases. The research on the green synthesis of silver nanoparticles (AgNPs) using Telescopium telescopium (TtSN-particles) holds great promise across various fields. In medicine, these nanoparticles could lead to new antimicrobial agents, antioxidant and anti-inflammatory treatments, and anti-thrombolytic therapies, while also serving as effective drug delivery systems. Environmentally, TtSN-particles could be employed in wastewater treatment and bioremediation, offering eco-friendly solutions for contamination and pollution. In agriculture, they could protect crops and improve soil health, while in cosmetics, they might be used in skincare and wound healing products. The food industry could benefit from their use in food preservation and nutritional supplements. Future research should focus on understanding their biological mechanisms, conducting In Vivo studies, and scaling up production, with a strong emphasis on regulatory and safety considerations. Interdisciplinary collaboration and public-private partnerships will be crucial in translating this research into practical applications, potentially leading to significant advancements in medicine, environmental science, agriculture, and other sectors.
Funding
Self funded project
Competing interests
The authors have no relevant financial or non-financial interests to disclose.
Author Contribution
The experiment was carried out by Mr Vignesh P.R and Mr D. Antony Prabhu
The experiment was supervised and guided by Dr. V. Pushpa Rani
The manuscript was revised by Ms Florence Suganya Ravindran
Data Availability
The data is not available elsewhere.
Ethics Approval
Experiment conducted on common animal in an ethical manner.
Consent to publish
Consent to publish acquired from all authors
Corresponding author's email id
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Graphs 3.1 to 3.5 are available in the Supplementary Files section
No competing interests reported.