Biosynthesis of Silver Nanoparticles by Using Green Tea ( Camellia sinensis ) Extracts

,


Introduction
The study of science, engineering, and technology at the nanoscale, or between 1 and 100 nm, is known as nanotechnology.This cutting-edge technology is employed in a variety of areas, including chemistry, materials science, and others of the same kind.Additionally, numerous varieties of nanoparticles are applied in medicine as imaging agents or medication carriers.Different produced liposome nanoparticles kinds are now employed as vaccination and anti-cancer medication delivery systems.Additionally, gold nanoparticles are utilized in home pregnancy test kits 1,2 .
Chemical procedures are recognized to be riskier than green synthesis.This is so that the latter can produce nanoparticles in a manner that is acknowledged as being more environmentally benign and sustainable (NPs) 3 .Some of the unique green methodologies, such as emerging green nanotechnology, have shown to be crucial in the production of newer nanoparticles.These alternate approaches, which have shown to be more successful in producing NPs are those that incorporate microbes and plant extracts 4 .
Published Online First: October, 2023 https://dx.doi.org/10.21123/bsj.2023.8344P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal Although there are many metals in nature, they are manufactured on a large scale by using a few of them such as gold, silver, palladium and platinum in the form of nanostructures 5 , among the above meta metals, silver nanoparticles have attracted much attention due to their unique properties for use in various applications including pharmacology, agriculture, water detoxification, air purification, textile industries and as a catalyst in oxidation reactions 6 .In addition, the important properties of its antibacterial activity against a wide range of bacteria without any toxicity to animal cells 7 .
Currently, silver nanoparticles are utilized as antiseptics, antibiotics, dental materials; burn wound treatments, and catheters to inhibit the development of germs in a number of applications 8,9 .Aside from that, biological elements like bacteria, fungi, and algae, as well as their enzymes, may be employed to alter the physical, mechanical, and structural properties of AgNPs of various sizes and shapes 10,11 .

Collection of Camellia sinensis L.
Camellia sinensis were obtained from the plantation in Baghdad city, identification as (Camellia sinensis L.) by a professional in the Biology Department/College of Science/University of Baghdad.To obtain Camelia sinensis extract the plant were leaves cleansed and lichens scraped away before drying in the shade on clean drying tables.The plant components were then cut with a knife into smaller pieces and pulverized using an electric laboratory grinder into powder form.Firstly, in order to remove the oil from the leaves, 200 grams of Camellia sinensis leaves powder was macerated with 1 litter of petroleum ether solvent.The residue was collected, air-dried and separated into two batches.Each batch of the defatted plant leaves was individually extracted with water and methanol to prepare aqueous and methanolic extracts.In two separate extraction bottles, 200 g of each powdered plant material was dissolved in 1 liter of sterile distilled de-ionized water and 1 liter (L) of methanol alcohol, and allowed to stand for five days in the dark with occasional daily stirring for homogeneous mixing and extraction.After the crude extract was sieved, the filtrate was concentrated by evaporating over steel pans in a 37°C oven.Evaporation is followed by transfer to tubes, where it is then kept in a refrigerator at 4°C 12 .

Camellia sinensis extracts
Preparation of green silver nanoparticles by Camellia sinensis aqueous and methanolic extracts were done according to Ojha et al. 13 and Krishnadhas et al. 14 with some modifications.In 95 ml of a 10 mM silver nitrate AgNO3 solution, 5 ml of each extract was sprayed dropwise, separately (made by dissolving 1.69 g AgNO3 into 1 L deionized water) under ultrasonic conditions, with an ultrasonic power of 100 W and a frequency of 42 kHz.After 20 minutes of sonication, the solutions were stored at 25°C in opaque bottles.After being stirred at 800 rpm for 30 minutes, After 24 hours, the reaction mixture was centrifuged for 10 minutes at 10,000 rpm to separate the clear supernatant.
The last colloid samples were stored in a refrigerator at 4°C in opaque vials.Over a period of five days, the color of Camellia sinensis silver nanoparticles (CAgNPs) solutions altered, demonstrating the production of silver nanoparticles (AgNPs).

Characterization of the prepared nanoparticles
Characterization measurements (morphological and structural) of silver nanoparticles for identifying AgNPs in this study, were implemented by many different techniques, as follows:

UV-Visible Absorption Spectroscopy
UV-Visible spectroscopy provides that the silver ions in the colloidal solution had been reduced.With pure water used as a reference, a tiny aliquot of AgNPs was placed in a quartz cuvette and monitored for wavelength scanning between 200 and 800 nm.After adding green tea extract to an AgNO3 solution, the UV-Vis absorption spectra of the sample were https://dx.doi.org/10.21123/bsj.2023.8344P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal measured using a Perkin Elmer Spectrophotometer at various times of 5, 10, 15, and 20 minutes 15 .
Fourier Transform Infrared (FTIR) Spectroscopy Analysis FTIR analysis (Shimadzu) was used to study the characterization of functional groups on the AgNPs by plant extracts, and the spectra were scanned in the 4000-400 cm -1 range at a resolution of 4 cm -1 .The samples were produced by spreading them on a glass slide in accordance with the accepted practices.The sample was then looked at after that 16 .

Atomic force microscopy
One of the first methods for seeing, measuring, and modifying materials at the nanoscale is atomic force microscopy (AFM).It offers the capacity to see 3D objects as well as qualitative and quantitative data on a variety of physical characteristics, such as size, morphology, surface texture, and roughness 17 .Each type of nanoparticle sample was applied as a thin layer on a glass slide using 100 μl of the sample, which was then let to dry for five minutes.The slides were then scanned using the AFM 18 .

X-ray diffractometer
The analysis using an X-ray diffractometer (XRD) is useful for knowing the phase structure and purity of synthesized green AgNPs and is generally used as a common technique to study the crystal structure and phase composition of AgNPs.On a glass slide, a thin layer of homogeneous water hung from each type of nanoparticle was created and allowed to dry.The X-ray diffraction (XRD) pattern (operating voltage 40 kV and current 30 mA, Cu K (a) radiation (λ = 1.540) was captured using an X-ray diffractometer 13 .Data were collected for the 2θ range of 10 to 80 degrees with a 0.0200 degree step.The result of the XRD pattern was interpreted using the reference standard for describing AgNPs developed by the Joint Committee on Powder Diffraction Standards (JCPDS card number 04-0783).The Debye-Scherrer equation was used to determine the particle size of the generated samples, and it is as follows: D=0.9 /Β cosƟ In this equation, D stands for the size of the crystal,  is the x-ray wavelength, the diffraction angle (Braggs angle) in radians, and  is the entire width at half maximum of the peak in radians 19 .

Zeta potential analyzer
The produced nanoparticles' stability was assessed using a zeta potential analyzer that can function between -160 mV and +160 mV, and the findings were shown in graph 20 .

Biosynthesis and characterization of nanoparticles
Methanolic and aqueous extracts of Camellia sinensis were used to make the silver nanoparticles.Compared to other bio reductants, the production of metallic nanoparticles utilizing plant extracts is easier and more successful 21 .It is widely known that phytochemicals not only convert Ag+ into Ag 0 but also cap the Ag + to create these very stable nanoparticles 22,23 .In this study, the formation of silver nanoparticles was monitored depending on color change and UV spectroscopy absorption.The colors of the green silver nanoparticle solutions were changed for Camellia sinensis silver nanoparticles (CAgNPs) from dark green to light brownish green, with the addition of Camellia sinensis methanolic and aqueous extracts, respectively, to silver nitrate solution.The color began to change after 24 hours, and after 48 hours the color changed to the final color.The presence of active molecules in the methanolic and aqueous extracts of Camellia sinensis implies the synthesis of silver nanoparticles (AgNPs) by the reduction of silver metal ions Ag+ into silver nanoparticles Ag 0 .The stability and transformation of metallic silver into AgNPs depend on compounds including phenols, terpenoids, alkaloids, flavonoids, proteins, and carbohydrates 13 .By lengthening the incubation period, it is possible to further accelerate the rate of particle formation and decrease it, which causes the color's intensity to rise as the reaction time increases 24  hues in solutions 25 .The activation of the metal nanoparticles' surface plasmon resonance is what causes the color shift (SPR).Silver nanoparticles' fascinating optical properties are directly tied to localized surface plasmon resonance, which is greatly influenced by the form of the nanoparticles 26 .This outcome is in agreement with Saliem et al. 24 and Thamer 27 , who demonstrated the possibility of color change following the reduction of silver ions into silver nanoparticles following contact with plant extracts.As a result, AgNP characterization is crucial for assessing the functional properties of the produced particles.Other researchers had also chosen Camellia sinensis leaf extract as a reducing biomaterial 12,15 UV-Visible spectroscopy UV-Vis spectroscopy is one of the primary techniques for identifying and quantifying the production of NPs.UV-Vis spectroscopy was utilized to confirm the stability of the synthesized AgNPs since the plasmon band of Ag is sensitive to the size and shape of the generated NPs 28 .The elements in the plant extract cause the silver ions to be reduced to silver atoms 29 .
UV-visible spectroscopy is an important step in confirming the synthesis of AgNPs and the color change.When the Camellia sinensis extract was mixed with an aqueous solution of AgNO3, this resulted in a change of color.This change in color is a result of the collective oscillation of free electrons of silver nanoparticles in resonance with the light wave in silver nanoparticle synthesis and this oscillation gives a typical peak value.UV-visible spectra of the plant extracts without AgNO3 solution and with it were shown in Fig. 1 and 2. The existence of many chemical compounds known to interact with silver ions is indicated by the faint absorption peak at 200 nm 26 .The type, size, and morphologies of the NPs generated, the dielectric constant of the medium and temperature, as well as their interparticle distances, all have a remarkable impact on the surface plasmon resonance absorbance 30,31 .The absorption spectrum was recorded between 200 nm and 800 nm.It is observed that the silver surface plasmon resonance band centered at 263 nm in the (CAgNPs) aqueous extract and 270 nm in the methanolic (CAgNPs) extract, in comparison with UV Test for Camellia sinensis methanolic and aqueous extract 271 and 272 nm respectively.AgNPs made via biological processes were monitored for more than a year for stability, and an SPR peak at the same wavelength was seen using UV-vis spectroscopy 32 .Fourier transformation infrared spectroscopy (FTIR) analysis was employed to identify functional groups that may be responsible for the reduction/ bioreduction of AgNO3 to Ag-NPs and their stabilization.FTIR spectroscopy is a technique used to measure the vibration frequencies of the bonds in molecules.It is used to confirm the presence of the functional groups of the active components in the synthesized AgNPs based on the band value in the region of the infrared radiation 33 .The dual role of the plant extract as a bioreduction and capping agent was confirmed by FTIR analysis of the prepared AgNPs of Camellia sinensis leaves extract.
The presence of a functional group in the synthesized AgNPs was similar to that reported by Waris et al. 34 where the FTIR spectrum showed the carbonyl group formed amino acid residues and this finding is in agreement with other researchers 13,35 who also found that proteins have stronger affinity for binding metal, suggesting that the proteins may form metal nanoparticles (i.e., cap silver nanoparticles) to prevent agglom.Das et al. 36 mentioned the changes in the functional groups in active biomolecules might suggest their involvement in the synthesized of (AgNPs).-------------C-N   The CAgNPs aqueous and methanolic extracts were spherical in form, either singly or in aggregates, according to the results of the AFM study, in both the two-dimensional and three-dimensional views.The average particle size for the two types of extracts was also revealed by the AFM investigation was 108.3 nm and 84.76 nm, for CAgNPs aqueous and methanolic extract respectively Figs. 5 and 6.The finding was in agreement with Silver nanoparticles that were produced by biosynthesis were virtually spherical, solitary 25-50 nm, or found in clumps 100 nm, according to Bhat et al. 37 .According to Githala et al. 38 who used an atomic force microscope to measure the size and shape of partials, silver particles had an irregular polygonal form and had diameters ranging from 1.0 to 130 nm.The average nanoparticular dimension was 63.3 nm.While the particles of Ag and ZnO were irregular, LiO2 appeared elongated and irregular, even though their diameters varied from 1.8 to 2.24 nm, respectively 39 .

X-ray diffractometer
An effective method for assessing crystalline materials is the X-ray diffractometer (XRD), which offers data on structures, phases, preferred crystal orientations, and other structural characteristics including average grain size, crystallinity, strain, and crystal defects 40 .
By reducing Ag+ ions with Camellia sinensis extracts, AgNPs were generated, as was evident from the XRD pattern found in crystals.Some unassigned peaks were found, which may have been caused by the bio-organic phase metalloproteins that were present on the surface of the silver nanoparticles or by the plant extracts lower concentration of biomolecules that function as stabilizing agents like enzymes or proteins 12 .
The average crystallite sizes according to Debye-Scherrer equation calculated are found to be 99.66 and 61.24 nm for CAgNPs methanolic and aqueous extracts respectively.The finding agrees with the study by Ssekatawa et al. 12  Interestingly, most of the researchers 34,40,42,43 that synthesized the nanoparticles using plant extracts seem to obtain a similar crystal structure.Whereas several reports state that the NPs produced https://dx.doi.org/10.21123/bsj.The stability of colloidal dispersions is largely determined by the zeta analysis.A measure of how strongly neighboring similarly charged particles are attracted to one another electrostatically in dispersion is expressed by the magnitude of the zeta potential.A strong zeta potential will confer stability to sufficiently small molecules and particles, implying that the solution or dispersion will resist aggregation.Colloids with high zeta potentials (positive or negative) are electrically stable, but those with low zeta potentials coagulate or flocculate because the dispersion might shatter and flocculate if attractive forces outweigh the repulsion 45 .In general, the nanoparticles' zeta potential should be more than +30 mV or less than -30 mV 46 .
The finding agrees with Surega 47 , who discovered that the zeta analysis of green produced AgNPs was -41.7, -27.9, and -37.2 mV using plant extracts of Tridax procumbens, Euphorbia hirta, and Azardirachta indica, respectively.The Zeta potential distributed with wide range of -41.7 mV indicated the highly stable nature of AgNPs synthesized using T. procumbans extract.Anandalakshmi and Venugobal 48 synthesized AgNPs using Vitex negundo leaf extract, zeta potential value was -13.5mV which was incipient instability.
Many of the physiologically active substances included in natural extracts may be to blame for both the stability of the generated nanoparticles and the drop in silver ions.By transforming silver ions into AgNPs, phytochemicals such as phenolics, coumarins, terpenoids, glycosides, alkaloids, and tannins may function as bio reductants in this green synthesis technique.Furthermore, it's possible that the peptides and proteins in turmeric and cinnamon extracts will aid in the production of silver nanoparticles and will lessen the number of silver ions in silver 49 .
Additionally, because proteins' carbonyl groups have a strong affinity for bonding to metal nanoparticles, they can deposit a coating layer on the surface of AgNPs.As a result, the generated nanoparticles are less likely to aggregate and are more stable in aquatic environments 50 .

Conclusion
This study concluded that the aqueous and methanolic extracts of Camellia sinensis leaves contain phenolic compounds and the presence of a variety of active compounds in addition to the safe handling and low costs of this can be used as a good reductant for the non-toxic or green synthesis of metallic silver nanoparticles.
2023.8344 P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal by reacting AgNO3 with biological solutions are face-centered cubic with slight variations in peak values based on the kind of extract, metabolites present, and binding characteristics 44 .

Figure 10 .
Figure 10.The zeta potential value of CAgNPs methanolic extract.

-
Conflicts of Interest: None.-I hereby confirm that all the Figures and Tables in the manuscript are mine.Furthermore, any Figures and images, that are not mine, have been included with the necessary permission for republication, which is attached to the manuscript.-Ethical Clearance: The project was approved by the local ethical committee in University of Baghdad.