Green synthesis, characterization of silver nanoparticles using Rhynchosia capitata leaf extract and their biological activities

: Green production of silver nanoparticles ( AgNPs ) using biological samples is the most cost - e ﬀ ective and environment friendly method. Plants and other biological resources might be exploited to create biologically active AgNPs. Rhynchosia capitata ( an endangered species ) leaf extract acted as reductant in fabrication of AgNPs in the current study; while, the fabricated particles have been characterized by UV - visible spectrophotometry, scanning electron microscopy ( SEM ) , and thermal gravimetric ana - lysis ( TGA ) . UV - visible spectroscopy was utilized to con ﬁ rm the fabrication of the nanoparticles ( NPs ) via bioreduction. SEM revealed the formation of round and spherical AgNPs with sizes ranging from 2 to 60 nm. According to TGA, the synthesized R. capitata AgNPs were not much stable and high mass loss was observed at temperature from 40 to 80°C. The antioxidant potential was higher as estimated through 2,2 - diphenyl - 1 - picrylhydrazyl - hydrate assay with IC 50 value of 60 µg/mL rather than 2,2 - azinobis -[ 3 - ethyl - benzthiazoline ]- 6 - sulfonic acid assay ( IC 50 120 µg/mL ) . The antibacterial potential against the selected bacterial strains for NPs was high as compared to aqueous extract, determined through agar well di ﬀ usion, minimum inhibitory concentration, and minimum bactericidal concentration methods. These ﬁ ndings demonstrated that R. capitata - based NPs had greater antibacterial and antioxidant properties than plant extract and it should be potentially used as antibac - terial and antioxidant


Introduction
Nanotechnology being a new research field is concerned with the processing of developing materials at the atomic level to give them unique properties that may be suitably tuned for the desired usage and applications [1].The controlled size and composition of nanoparticles (NPs) has attracted the interest of scientists and engineers because they provide solutions to technical and environmental challenges in the fields of catalysis, water purification, medicine, and solar energy conversion.As a result, creating and applying nanostructures between 1 and 100 nm is a viable area of research [2][3][4].Chemical and physical techniques [5], photochemical reactions (in reverse micelles) [6], electro-chemical methods [7], and, more recently, green chemistry have all been utilized to manufacture and stabilize metal NPs [8].Green chemistry/chemical processes (being associated with less environmental hazards), are rapidly being incorporated into research and industry for long-term growth and development [4].
The synthesis of nanomaterial utilizing a green method has emerged as a growing subject in nanotechnology during the last few decades due to its applications in chemistry, physics, medicine, and biology.Plants have grabbed scientists' interest as a rapid, cost-effective, ecologically benign, and one-step solution for the biosynthesis process [9].The development of eco-friendly materials synthesis methods is critical for expanding their biological applications.Nowadays, several technologies have been used to synthesize green NPs with well-defined chemical composition, size, and shape, and their applications in many innovative technological sectors have been investigated [10,11].NPs can be used for medication and gene delivery, pathogen and protein detection, magnetic resonance imaging, tissue engineering, and biological molecular purification.The use of plants in the production of NPs has grabbed the interest of researchers due to its simplicity and one-step biological process.Plants has become the ideal and best alternative as a safe, non-toxic technique of NP production since cheap natural capping agents are more in such samples that can also be easily extracted from plants [12].
Nanotechnology is an emerging field that has greater potential to alter the current and conventional agricultural practices in the production of crops [13].It has the potential to bring the revolution in agriculture and food production.Bioprocesses-mediated nanotechnology has turned the agricultural and food wastes into energy and usable byproducts [14].Metal oxide NPs have demonstrated effects in promoting plant development and yield [15].
Biological green synthetic techniques are the most convenient ways to solve the disadvantages associated with synthetic chemical approaches in use.The greener approach is simple, cost-effective, reliable, and environment friendly in synthesizing metal NPs including silver nanoparticles (AgNPs).Another benefit is the convenience of solvent medium selection in this kind of procedures.The manufacturing of AgNPs with smallest possible sizes and forms has received special attention nowadays.Plant extracts and microorganisms such as fungus (as well as tiny biomolecules such as vitamins and amino acids) are employed as an alternate source of reducing metal ions and as capping agents [13][14][15][16][17][18][19].
As mentioned, the use of plants in NPs synthesis has attracted researchers interest due to simplicity of method and presence of a variety of natural capping agents in the form of primary and secondary metabolites in parental plant material that might assist in reduction of the metal ions, which make plants the preferable solution for fabrication of NPs with safe and non-toxic nature [12].As bio-reductants, various plant extracts such as lemongrass soup, alfalfa, capsicum annum, geranium leaves, and aqueous geranium extract have been employed to synthesize NPs [19,20].Plant extracts, bacteria, fungi, and algae are utilized as reducing agents in the biogenic method of metal NP synthesis [21] as they contain a variety of capping and reducing agents, the biomolecules, particularly primary and secondary metabolites including polysaccharides, vitamins, amino acids, enzymes, proteins, flavonoids, polyphenolics, etc. [21][22][23].
Because of their unique physical and chemical characteristics, morphology and distribution, shape, large surface area, and size, AgNPs are widely employed in a variety of disciplines including food, medicine, healthcare, and industrial applications.They have demonstrated improved properties in optical, electrical, and thermal devices with excellent electrical and heat conductivity, as well as a wide range of organic chemistryrelated applications.They are used in optical sensors, medical device coatings, cosmetics, and other pharmaceutical and food business sectors.It is also worth mentioning their usage as anti-inflammatory, anticancer, and antibacterial agents, in diagnostics, theragnostic, and in the delivery of medication [20,24,25].
Rhynchosia capitata also known as Glycine capitata is an annual prostrate twining plant, regarded as weed.This plant is indigenous to India, Sri Lanka, and Pakistan.This plant belongs to the family Fabaceae [26].R. capitata was first analyzed for the presence of phytochemicals by gas chromatography-mass spectrometry followed by reverse phase-high performance liquid chromatography analysis for the occurrence of medicinally important phenolic compounds such as quercetin, rutin, gallic acid, ferulic acid, and caffeic acid.The presence of tannins, flavonoids, steroids, saponins, and terpenoids was confirmed by this study.Flavonoid and glycosides like orientin, isovitexin, isoorientin, vicenin 2, and vitexin have been reported from some species of Rhynchosia [22,23,27].These phytochemicals (phenolic compounds) or secondary metabolites might by responsible for the reduction of metal and hence the production of silver nanomaterials as encountered in the present study.
As mentioned, plant extracts comprised of phytochemicals like flavones, terpenoids, aldehydes, ketones, carboxylic acids, ascorbic acids, amides, and phenols, which have the potential to act as capping agent in synthesizing metal NPs.The redox process is involved in these fabrications with the virtue of primary and secondary metabolites leading to environment friendly/beneficial NP productions [28][29][30].Plant extract-derived AgNPs have also been demonstrated to exhibit antioxidant, antibacterial, cytotoxic, and antimycotic properties [31].In literature, AgNPs have been documented with antibacterial activity against Escherichia coli, with possible mechanism involving cell death due to AgNP accumulation in the cell wall [19].In another study it has been discovered that AgNPs derived from diverse saccharides have a strong broad-spectrum bactericidal activity against gram positive and gram-negative bacteria.The most notable finding of the study was that the generated AgNPs were effective against multi-resistant bacterial strains such as Staphylococcus aureus [32].
Although numerous research reports on the synthesis and characterization of AgNPs have been reported in the literature, a few findings on the green synthesis of AgNPs using R. capitata leaf extract and their biological activities have been published.Thus, the aim of this work was to highlight the biogenic approach for the biosynthesis of AgNPs from R. capitata plant leaf extract and to find out their antibacterial and antioxidant activities.

Plant sample collection
In the months of November and December, 2016, R. capitata leaves were collected from various regions of District Dir Lower Khyber Pakhtunkhwa, Pakistan.The plant was identified/authenticated by taxonomist in herbarium; Department of Botany, University of Malakand, Pakistan.After collection and authentication, the plant material (deposition number UOM-RC-10/2022) was cleaned with distilled water, dried in a shady place and then ground with a mortar and a pestle.A total of 5 g of plant materials was weighed using digital balance.

Chemicals
Silver nitrate (AgNO 3 ), deionized water, nutrient broth, and ascorbic acid were purchased from Sigma Aldrich.All chemicals utilized were of analytical grade with highest purity, used without any further processing.

Preparation of the plant extracts
R. capitata leaves were properly cleansed with deionized water and crushed into smallest possible size using a mortar and pestle.Five grams of the dried ground plant material mixed in 100 mL of deionized water was boiled for 15 min in a clean, sterile conical flask of appropriate size with continuous stirring.Using Whatman No. 1 filter paper filtration was done to produce the aqueous plant extract.

Synthesis of AgNPs using R. capitata
The conventional biogenic method reported by Evanoff and Chumanov [33] was used to produce AgNPs.R. capitata plant leaves and AgNO 3 solution were used to prepare AgNPs.AgNO 3 of 0.15 g was dissolved in 100 mL of distilled water to make AgNO 3 solution.The AgNO 3 solution was agitated at room temperature for 5 min before adding the R. capitata leaf extract.The reaction mixture was then agitated for 4 h.When R. capitata extract was added, the color of the solution immediately changed from colorless to yellowish brown.This color change confirmed AgNPs fabrication, which were then separated by centrifugation at 14,000 rpm for 35 min while maintaining a temperature of 40°C.

Physiochemical characterization of AgNPs 2.5.1 UV-visible spectroscopy
The Perkin Elmer spectrophotometer was used to get the UV-visible spectrum of the R. capitata-mediated AgNPs.
The spectrum was recorded in distilled water containing a small amount of centrifuged NPs taken in a quartz cuvette.The sample was scanned between 200 and 500 nm after an hour of solution preparation.

Scanning electron microscopy (SEM) analysis
SEM analysis was performed to visualize the morphology (particle surface size and shape) of the synthesized AgNPs using a Hitachi S-4500 SEM machine.The sample for analysis was prepared by centrifuging at 14,000 rpm for 8 min in distilled water.The pellet obtained was centrifuged after combining with deionized water.An acetone wash was done for three rounds and then sonicated for 20 min to form a homogeneous suspension.The sample was then completely dried at room temperature under ordinary light bulb.A very small quantity of the sample was placed onto the SEM grid to constitute a thin film that was subsequently coated with a sputter coater.The film on the SEM grid was then dried for 5 min under a mercury lamp.
Blotting paper was used to remove any excess solution.
Finally, the photographs were recorded following the instrument manual.

Thermal gravimetric analysis (TGA)
Melting and the recrystallization behavior of synthetic materials are be studied using the thermoanalytical approach.TGA analysis was carried out using a Diamond Series TG/DTA Perkin Elmer, USA analyzer.Al 2 O 3 was used as a reference crystal.A heating rate of 10°C at temperatures between 40 and 600°C with nitrogen flowing at a rate of 20.0 mL/min was used to quantify the sample's weight loss.The weight of the initial sample taken was 11.879 mg.
2.6 Biological activities of R. capitata extract and synthesized AgNPs

Antibacterial activities
Zone of inhibition (ZI), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) techniques were used to assess the antibacterial activity of the given plant extract and NPs against the selected bacterial strains.Strains of bacteria were spread into sterilized agar culture dishes, and 3 mm holes were formed in the media at a distance of 6 cm from each other.Amoxicillin, leaf extract, and NPs solution were placed inside the holes using a micropipette.The culture plates were incubated at 37.8°C for 24 h.Around each hole, the ZI was measured employing visual approach.To prevent contamination, the entire operation was carried out inside a laminar flow.

Determination of MIC and MBC
To calculate the MIC, a macro-broth dilution approach was used [33].Solutions of the standard (amoxicillin), AgNPs, and leaf extract (0.04-10 mg/mL) were produced in Mueller Hinton broth medium and inoculated with the selected bacterial strain (10 mL).Only one test tube containing nutrient medium was utilized as a negative control.All the test tubes were kept in an incubator overnight (37°C).The test tubes were visually inspected for turbidity, and the first tube with no changes in turbidity was chosen as the MIC.The tube that had no visible growths was incubated for further 3 days (4 days total).
The tube with no bacterial growth after 94 h was identified as MBC.

DPPH test
The percent scavenging potentials of the given plant extract and NPs were calculated using a standard technique employing the D-diphenyl-1-picrylhydrazyl (DPPH) assay [34].About 100 mL of distilled methanol and 15 mg of solid DPPH were mixed to form the DPPH stock solution.The absorbance of a 3 mL stock DPPH solution was brought to 0.75 at 515 nm (considered as control solution) in order to apply dilution to the stock solution.For the creation of free radical, the stock solution was maintained at room temperature for 24 h in a dark environment (covered with aluminum foil to avoid formation of the free radicals due to light).The extract and NPs were then dissolved in methanol at a stock concentration of 5 mg/mL, from which various dilutions ranging from 1,000 to 62.5 g/mL were made.About 3 mL of each dilution and 3 mL of the DPPH stock solution were incubated for at least 30 min to estimate the scavenging effect of the extract and NPs on DPPH.The dilution of ascorbic acid (standard) was also made the same way, and 3 mL of each dilution was incubated with 3 mL DPPH for the mentioned interval of time.The formula given below in equation (1) was used to determine the % inhibition [27]: where A shows the absorbance of oxidized DPPH and B shows the absorbance after mixing with extract/NPs.The experiments were carried out in triplicates, and the results of mean values are presented here.

2,2-Azinobis-[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) assay
The antioxidant potential of the leaf extract and the AgNPs was further evaluated using the ABTS assay in accordance with accepted and standard protocol [35][36][37].During the experiment, ascorbic acid was employed as a positive control.The given samples were diluted before being added to the ABTS stock solution (2.5 mL each).After mixing the solution was incubated for 30 min, and the absorbance at 745 nm was measured by UV-visible spectrophotometer.The scavenging potentials of the extract and NP were determined using the formula described in equation (1).

Statistical data analysis
Each experiment, including the control groups throughout the experiment, was performed in triplicates, and the findings were provided as mean ± sandard deviation.

Results and discussion
As mentioned in Section 1, different plant extracts and isolated phytochemicals have been used as reducing agents in the synthesis of metal NPs.Synthesis of NPs using plant extract has resulted in fabrication of smallest possible sized NPs with wide range of therapeutic applications.There are also reports on a variety of plantderived bioactive chemicals that have the capability of reducing metal ions into smaller sized precipitates [38].
Collectively, these compounds are termed as secondary metabolites that are frequently used as capping agents [39].These substances could increase the bioactivities of the NPs fabricated in addition to their stabilizing effects.Epigallocatechin-3-gallate, resveratrol, and fisetin are three distinct natural polyphenols that were used to make AgNPs in a reported study [40].Compared to chemically manufactured AgNPs, green-synthesized AgNPs utilizing Pycnoporus broth from white rot fungus have shown potential antibacterial efficacy against harmful bacteria [41].
Khurana et al. reported the antibacterial activities of AgNPs against P. vulgaris, S. sonnei, S. aureus, and B. megaterium.The enhancement of antibacterial action was observed with size reduction to 59 from 83 nm using plant material as bioreductant.The effectiveness of AgNPs in medical field depends on the efficiency of the technique used, size and shapes of the particles formed, the types of substances utilized as reductants, exposure period, and exposure conditions [42].

UV-visible spectroscopy
Optimization of the AgNO 3 solution to plant extract ratio is very important in the production of the AgNPs.Plant extract has excellent reducing and stabilizing powers due to the presence of organic compounds that helped to reduce Ag 1+ to Ag 0 .UV-visible spectroscopy results demonstrated the existence of Ag absorption peak at around 400 nm.Only optimum ratio results are shown in Figure 1.

SEM analysis of AgNPs
High-density AgNPs formed by R. capitata extract are shown in Figure 2 from SEM analysis.The white, isolated spots seen in the SEM picture are AgNPs.It has been noted that AgNPs are consistently spherical.The particles' dimensions fell within the nanometric range.Rhynchosia capitata leaves based nanoparticles synthesis  5 The capping agent in the form of extract scattered in the given picture, implies that the NPs had been stabilized because they were not in contact even after they had aggregated.At some places the aggregates have formed the bigger AgNPs.

TGA
TGA, a thermoanalytical technique, was used to assess the stability and behavior of materials.Figure 3 shows the results of the TGA, which reveals how much weight loss has been encountered by AgNPs as the temperature increases.The given sample was heated to a temperature of 40-195°C to decompose the AgNPs; at around 40°C, the sample starts to decompose and its size steadily decreases until 80°C.The elimination of moisture contents from the NPs caused the sample's weight to decrease when the temperature was raised from 40 to 80°C.The capping agent being organically decomposed rapidly with the increase in temperature and resulted in abrupt mass loss from 40 to 80°C.The weight loss continued till 195°C and very little amount was left over as evident from a parallel line above the x-axis zero line.Our TGA analysis results and the reported results in the literature coincide quite closely [43,44].

Antioxidant activities of R. capitata leaf aqueous extract and formulated AgNPs
The extract and AgNPs' free radical scavenging capacities and IC 50 values, as measured by DPPH and ABTS tests, are displayed in Table 1.Antioxidant activity of AgNPs was stronger and more promising than in aqueous extract.Additionally, the observed antioxidant activity was concentration dependent.Ascorbic acid served as positive control in this study.

Antibacterial activities of extract and AgNPs
The extract and fabricated NPs were evaluated for antibacterial activity against Salmonella typhi, E. coli, S. aureus, and Streptococcus pneumoniae using the agar well diffusion, MIC, and MBC methods.Amoxicillin antibiotic was used as a standard.

Determination of ZI
Zones of inhibition of AgNPs and leaf extract against various bacterial strains are shown in Table 3.The inhibitory zone of the plant leaf extract was smaller when compared to the synthesized AgNPs.In comparison to leaf extract, AgNPs exhibited an 18 mm ZI against E. coli, whereas the latter had an 11 mm ZI. S. aureus was inhibited by the NPs with ZI of 16 mm, whereas the ZI of the leaf extract was close to 13 mm.AgNPs had a ZI of 19 mm, whereas the extract had a ZI of 10 mm.Similarly, S. pneumoniae created a ZI of 19 mm, while the extract ZI was 9 mm.Amoxicillin was used as a positive control in the experiment, and inhibited the strains more potently.demonstrated that the values of MIC and MBC are high for AgNPs as compared to plant extract, whereas comparable to those of the standard.The greatest activity against S. typhi was shown by both the aqueous leaf extract and the AgNPs, with MIC values of 135 and 80 μg/mL and MBC values of 255 and 95 μg/mL, respectively.In another investigation, biosynthesized AgNPs have exhibited strong, broad-spectrum bactericidal efficacy against both gramnegative and gram-positive bacteria.The study's striking finding was that the generated AgNPs were effective against microorganisms with multiple resistance traits, including S. aureus [32].In another literature findings AgNPs have shown exceptional antibacterial activity toward the studied bacteria as evaluated by MIC and MBC [45].

Conclusion
In the current research, AgNPs were synthesized via biological method which would limit the amount of toxic waste that could be added to the environment.The synthesized AgNPs with a size of 2-60 nm, showed an absorption peak at 400 nm as depicted by UV-visible spectroscopy confirmed the synthesis of AgNPs.The TGA revealed an abrupt mass loss from 40 to 80°C.SEM results confirmed the production of round and spherical AgNPs with different sizes.According to the results of the DPPH and ABTS tests, the aqueous leaf extract and the AgNPs both demonstrated a significant degree of antioxidant potential.ZI, MIC, and MBC values for AgNPs were comparable with the standard rather than for extract.The synthesized AgNPs also had greater antioxidant activity in comparison to the extract.Therefore, it can be concluded that AgNPs synthesized through R. capitata leaf extract mediation should be used as potential antibacterial and antioxidant agents in therapeutic applications.The study is a green and environment friendly approach; therefore, it is urged that biological resources such as plants should be utilized in the synthesis of AgNPs as different plant materials contain different phytochemicals with differential quantities and thus have varied potentials in reducing the metal ions to get the smallest sized particles with improved properties.Further research work is necessary to test the potential bioactivities of the plantsbased AgNPs prepared in this study.
Table 2 lists the IC 50 values and ABTS scavenging potentials of AgNPs and R. capitata leaf extract, respectively.

Table 4
depicts the MIC and MBC values in μg/mL for synthesized AgNPs, R. capitata leaf extract, and standard antibiotic (amoxicillin).The findings listed in the table

Table 1 :
DPPH scavenging potentials, IC 50 values of AgNPs and leaf extract of R. capitata at different concentrations

Table 2 :
Percent ABTS scavenging potentials, IC 50 values of AgNPs and leaf extract of R. capitata and AgNPs