INTRODUCTION
Nanotechnology describes nanoscale materials [1]. Nanotechnology is now widely innovated in scientific fields such as new therapies, medicine, tissue engineering, diagnostic concepts, drug delivery, and gene silencing [2].
The catalytic, electronic, magnetic, optical, and antimicrobial properties lead to their used in various fields, including chemistry, energy, and medicine [3].
The properties of nanoparticles depend on their size, morphology, and distribution. Nanoparticles such as silver, gold, zinc oxide, and platinum have medical and pharmaceutical applications.  These nanoparticles can also be used in products such as toothpaste and cosmetics [4].
Physical and chemical methods synthesize and stabilize nanoparticles [5]. These methods include solution reduction, photochemical reactions in reverse micelles, electrochemical reduction, heat evaporation, and radiation-assisting techniques. Physical and chemical methods have generally been applied successfully in synthesizing nanomaterials in large quantities over a short period [6].
However, these methods successfully produce nanoparticles; they harm human health and the environment due to hazardous and toxic chemicals [7]. An environmentally friendly and cost-effective method is used to produce nanoparticles [8]. The antimicrobial activity of silver nanoparticles is confirmed against a wide range of microorganisms. Also, recent studies showed that silver nanoparticles are antimicrobial agents. However, Sondi et al. reported the antibacterial effect of silver nanoparticles against Escherichia coli as a microbial model [9].
Generally, E.coli, Pseudomonas aeruginosa, Enterococcus, Proteus mirabilis, and Klebsiella pneumonia contribute to urinary tract infections that can form biofilms on catheters. The biofilm causes them to become resistant to antibiotics, which are a threat to health. Strategies are needed to kill the antibiotic-resistant bacteria that form a biofilm [10]. 
 Plant compounds help synthesize nanoparticles because they do not require complex processes, including intracellular synthesis, purification, and microbial cell preservation [11].
The plants include Solanum lycopersicums, Hibiscus cannabinus stem, Hibiscus cannabinus leaf, Ananas comosus, and Hibiscus cannabinus leaf have been applied for green synthesis nanoparticle Ashuk Kumar et al. synthesized AgNPs using Gloriosa superba leaf extract [12]. The red clover (Trifolium pratense L.) has a high concentration of iso-flavonoids, compounds that are widely distributed in the Leguminosae family [13].
Furthermore, it has been refined in traditional medicine, which treats coughs, asthma, eczema, and eye diseases [14]. GLC-MS analysis results determined twenty-five compositions from T. pratense leaves, flowers, and seeds [15]. The major volatile compounds were as follows: leaves (3-hexenyl acetate, 3- hexanol, and β-okimenes), flowers (acetophenone, methyl cinnamate, and 1-phenylethanol), and seed pods (β-okimenes, unknown) [16].
     For the first time, this study was conducted to synthesize novel Ag@AgCl nanocomposites using a native plant, T. pretense, as a new catalyst collected from Yasuj, Kohgiluyeh, and Boyer Ahmad Province, Iran. Also, the biological activity of this novel nanobiotic was performed using anticancer, antibiofilm, and antimicrobial activity against uropathogenic Escherichia coli isolated from urinary tract infections. Furthermore, the nanocomposites were characterized by UV-visible, XRD, FE-SEM, and EDX methods.

MATERIALS AND METHODS
Collection, identification, and extraction
Trifolium pratense flowers were collected in spring near Yasuj, Kohgiluyeh, and BoyerAhmad Province, Iran. The flowers are thoroughly washed with two liters of distilled water, stored at room temperature for two days, and then split into small pieces. To prepare the aqueous extract, 10 g T. pratense was placed in 100 ml sterile distilled water (ratio: 1:10) for 20 min at 50 °C. The extract was isolated with Whitman filter paper and kept for further processing at 4 °C.

Characteristics of silver nanoparticles
The plant extract was added to 1mM of AgNO3 solution and incubated at 60 °C. Also, the AgNO3 solution was incubated as a control. The synthesis of silver nanoparticles was confirmed using colour change to brown. To evaluate the synthesis of Ag nanoparticles, the absorbance was measured using a Schimadzu (Model No. UV 1800) spectrophotometer in the range of 300 to 800 nm at different times.
Moreover, the Fourier-transform infrared spectroscopy (FT-IR) spectra of silver nanoparticles were measured using FTIR spectrometer (Brucker, Germany) with a KBr bullet in the 4000-400 cm1 range. The crystalline silver nanoparticles were determined by X-ray diffraction (XRD) (Panalytical, Netherlands). Furthermore, the size of the nanoparticles was evaluated by the Debye-Scherrer formula [17]. Also, the energy dispersive X-ray (EDX) and scanning electron microscope (SEM) (Tescan, Czech) were performed to investigate the morphology and the chemical composition of the nanocomposites.

Antibacterial activity
The antimicrobial effect of the nanocomposites was measured according to the protocol of the Clinical and Laboratory Standard Institute [18]. The antibacterial effect of Ag@AgCl nanocomposites was determined against positive and negative bacteria by the agar diffusion method.
In this study, some human pathogens include p. aeruginosa (ATCC27853), K. pneumonia (ATCC 10031), Salmonella paratyphi-A serotype (ATCC 5702), Staphylococcus aureus (ATCC 29737), Shigella dysenteriae (PTCC 1188), Staphylococcus epidermis (ATCC12228), Bacillus subtilis (ATCC 6633), and Escherichia coli (ATCC 12228) were evaluated. The suspension adjusted 0.5 McFarland standard was prepared in nutrient broth at 37 °C. A 100 µl of the suspension was inoculated on Muller-Hinton agar. 
Afterward, 10 µl of the nanocomposites solution (30 mg/ml) was inserted into the well. The plates were incubated at 37 °C for 24 h. The antibacterial activity was evaluated by measuring the diameter of inhibition zones.

Determination of minimum inhibition concentration
This method calculated the minimum inhibition concentration (MIC) for the susceptible microorganisms to the nanocomposites. The MIC value was evaluated using the microdilution method. For this purpose, 95 µl of TSB medium, 5µl bacterial suspension adjusted 0.5 McFarland, and 100 µl of different concentrations of silver nanoparticles (0.03125, 0.0625, 0.025, 0.25, 0.5). (0, 1 and 2 mg/ml) were added to each well. Furthermore, 195 µl of culture medium and 5 µl of the suspension were used as controls. The microplate was then incubated at 37 °C for 24 h. Microbial growth was characterized by turbidity at the bottom of the well.
To measure lethality, after 24 h, 5μl of each clean well was inoculated on a nutrient agar medium and incubated at 37 °C for 24 h. The concentration that did not grow after 24 h killed the bacteria, and the lowest concentration was considered the minimum bactericidal concentration. 

Biofilm inhibition assay
Sixteen human pathogenic E.coli strains were isolated and collected from suspected infected Urinary tract infection (UTI) patients. According to microplate biofilm assay, the antibiofilm effect of Ag@AgCl nanocomposites was evaluated against E.coli strains. 
At first, 100 μl of the nanoparticles (2mg/ml) and 100 µl of each diluted strain were added to each well. Moreover, 200 µl of TSB and the bacteria suspensions were negative and positive control, respectively. The microplate was incubated for 24 h at 37 °C. The 1% crystal violet was then added to each well, and after 10 min, the crystal violet was washed with distilled water and allowed to dry. Finally, 200 μl of acetic acid was added to each well, and after 15 min, the optical density (OD) was measured using ELISA at 570 nm. The following formula calculated the percentage of biofilm inhibition in the samples [19].

Inhibition Percentage = 100× (C-B) - (T-B) 

In this equation, C equals the mean biofilm absorbance, the mean absorbance of the well containing the sample affected. B is the mean absorbance of the wells containing the medium (control). It was also repeated three times to ensure the test.

Evaluation of anticancer activity
The cytotoxicity activity of biosynthesized Ag@AgCl nanocomposites was investigated using Brine Shrimp Lethality Assay (BSLA).
Artemia salina eggs were grown in artificial seawater (pH 9) for 48 h. The different concentrations (0, 10, 100, 300, 500, 700, and 1000 µg) of biosynthesized Ag NPs were added to the vial, including 5 ml seawater and ten brine shrimp larvae, and incubated at 25 °C for 24h. Brine shrimp death was observed at regular intervals. Vincristine sulfate (VS) was applied as a positive control. These tests were repeated three times. The lethality percentage was recorded according to formula [20].

Lethality percentage = [(m-M)/s] ×100

m: Average number of dead larvae sampled, M: Average number of dead larvae control, s: Average number of live larvae control.

RESULTS AND DISCUSSION
Characterization of nanoparticles
The formation of extracellular was observed with a change in color from yellowish to deep brown due to the excitation of the localized surface plasmon vibrations of the nanoparticles. 
The changing color from yellowish to brown confirmed the synthesis of the nanoparticles.  
One of the properties of metal nanoparticles is their optical properties, which change [21]. The color change indicated the biosynthesis of silver nanoparticles using an aqueous extract of T. pretense (Fig. 1).
Free electrons in silver nanoparticles are excited by visible light absorption and transmitted to higher energy levels. The unstable electrons return to their original energy levels and emit a photon simultaneously [22].
Fig. 1 shows a maximum wavelength of about (450 nm), similar to other researchers [23]. The optimal conditions for preparing these nanoparticles were 1mM of silver nitrate and 5 ml of aqueous extract at 65 °C for 24 h. The effect of contact time (0, 2, 8, 12, 24h) on the formation of nanoparticles was investigated under constant conditions.
As exhibited in Fig. 1, with the increased time, an increasing trend in absorbance was exhibited.

FTIR 
Comparing the spectral pattern of silver nanoparticles and the extract showed that nanoparticles contain compounds in the extract. They are usually formed as a layer around the nanoparticles and can play a role in the stability of nanoparticles. The results showed peaks in regions 3404 cm-1 determined the OH bond in phenols and alcohols. Moreover, the absorption band indicated in region 2925 cm-1 corresponds to CH stretching vibration in alkyls (methylene group), and 1607 cm-1 corresponds to NH bonds in first amines (Fig. 2). Furthermore, the band at 1404 cm-1 corresponding OH stretching indicates that phenols. The absorption band determined in region 1073 cm-1 corresponds to the CO stretching in the first alcohols. Phenolic compounds present in the plant are among the major contributors to reducing silver ions and the synthesis of nanoparticles.

SEM analysis
The surface morphology and structure of the synthesized silver nanoparticles were characterized using SEM. Fig. 3 exhibited that the silver nanoparticle was almost spherical, ranging between 19 and 46 nm and having an average size of 34.42 nm. The results of the size distribution analysis of silver nanoparticles are shown in (Fig. 3).

EDX
The EDX result confirmed the presence of silver as a major element. Moreover, the present O, C, and N were determined in the EDX spectrum (Fig. 3). The presence of biomaterial is one of the advantages of nanoparticles synthesized using plant extracts compared to chemical methods. 
Similar results also reported the formation of silver nanoparticles performed using Artemisia nilagirica leaf and Artocarpus heterophyllus seed extract [24].

XRD
X-ray diffraction is used to study the structure of crystalline materials. The spectral region results can obtain information on the structure, material, and quantities of the elements [25]. X-ray diffraction analysis was used to investigate and study the synthesized silver nanoparticles (Fig. 3).
 The average crystal size was calculated by calculating the width of peaks formed in the samples using the Debay-Scherer formula [26].

D= (0.9λ/β cosϴ)

Where β is the width of the peaks at half the maximum height, λ is the X-ray wavelength equal to 54.1 nm. Moreover, ϴ is the angle between the reflected beam and the radiation, and D is the crystal size. As shown in the figure, clear peaks in the areas 2ϴ = 27.82, 32.25, 38.07, 44.25, 46.25, 64, 57, and 77.3 are the reason for the successful synthesis of nanoparticles. The presence of sharp peaks in the patterns indicates a high degree of crystallinity for the nanoparticles. The average size of the synthesized crystal was estimated at 22.25 nm, utterly consistent with SEM results.

Antimicrobial activity
The antimicrobial effect of the silver nanoparticles, the extract, and the composite of extract and silver nanoparticles were measured by the present or absent zone of the inhibition, and the results are shown in Table1.
Yazdani et al. reported that silver quantum dots synthesized by Teucrium polium L. had antimicrobial activity [27]. 
Silver nanoparticles synthesized by Eugenia roxburghii DC. were evaluated against bacterial biofilms. Their results showed that the silver nanoparticles inhibited biofilms [28].
The results showed that the nanoparticles had an antimicrobial effect. In contrast, the extract shows no antimicrobial activity.
The nanoparticles showed a zone of inhibition against B. subtilis, S. epidermidis, and P. aeruginosa (9 mm). Moreover, S.paratyphi A serotype exhibited the inhibition zone (10 mm), and the zone of S. aureus was 7 mm. The composite of extract and silver nanoparticles has no antimicrobial effect except for two strains: B. subtilis (9 mm) and S. epidermidis (7 mm).
Allafchian et al. reported the antibacterial effect of silver nanoparticles against Gram-positive (S. aureus and B. cereus) and Gram-negative (S. typhimurium and E.coli) bacteria using the agar well diffusion. These nanoparticles were synthesized by Phlomis cancellata Bunge leaf extract. Their results proved to be almost parallel with our findings [6].
Furthermore, in the study by Hashoosh et al., silver nanoparticles synthesized by Aloe vera plant and Aloe vera extract had different antibacterial effects against Gram-negative bacteria (E.coli) and Gram-positive bacteria (S.aureus). The results showed that the aqueous extract of Aloe vera had no inhibitory effect. In contrast, silver nanoparticles had an inhibitory effect against E.coli and S.aureus [4]. According to a recent study, Ag/AgCl nanoparticles synthesized from Staphylococcus pasteuri ZAR1 exhibited antibacterial effect on 66 pathogenic strains [29].
Nazar Ul Islam et al. reported synthesized silver nanoparticles using Prunus armeniaca. They investigated the antimicrobial properties of the nanoparticles and the extract against S. aureus, E.coli, and P. aeruginosa with the diameter of inhibition zones 18, 10.2, and 11.2 mm, respectively. However, the plant extract alone did not have antimicrobial activity [30]. In another study by S. Yallappa et al., copper nanoparticles synthesized using an aqueous extract of Terminalia arjuna tree bark showed good antimicrobial activity against S. aureus, E.coli, S. typhi, and P. aeruginosa [31].

Biofilm Inhibition
The antibacterial effect of the silver nanoparticles was evaluated against sixteen E.coli strains isolated from patients with urinary tract infections. The results show that the MIC and MBC of silver nanoparticles range from 1.00 to 0.0312 and 2.00 to 0.0312, respectively (Table 2). 
The biofilm formation ability was measured for each assay as a control. The percentage of biofilm inhibition of the silver nanoparticles was between 21.12-97.10%. The nanoparticles exhibited high ability antibiofilm against E. coli 579, E. coli 4701, E. coli 228, E. coli 726, E. coli 4828, E. coli 885, and E. coli 3059. Furthermore, the lowest ability antibiofilm was against E. coli 5149.
Biofilm inhibitory activity of nanoparticles synthesized from Cordia. Dichotoma was evaluated. The results showed that silver nanoparticles (100 μg/ml) inhibited S.aureus and E.coli biofilm (92% and 95%) after 12h [32].
Morones-Amirez et al. reported that AgNPs inhibited biofilm activity by approximately 20% [33].
The other report evaluated the antibiofilm effect of biosynthesized Ag/AgCl composite against pathogenic bacterial strains. Their results showed that the composite inhibited biofilm in the 5-100% range [34].

Cytotoxicity
The anticancer activity of the silver nanoparticles and the percentage of dead larvae were determined at different concentrations (μg/ml).
The previous study determined that LC50 (0.5-1 mg/ml) was weak in the toxicity against A. salina. Moreover, The LC50 between 0 and 0.1 mg/ml exhibited high toxicity and 0.1-0.5 mg/ml moderate cytotoxicity. LC50 above 1 mg/ml indicated a lack of toxicity against A. salina [35].
Our study showed that the silver nanoparticles with LC50 1.3 μg/ml had high cytotoxicity and anticancer properties. Furthermore, the LC50 of vincristine sulfate was 0.751 μg/ml as a positive control. Based on these results, T. pratense L.  extract showed high cytotoxicity (Fig. 5).
A similar research reported copper nanoparticles synthesized by Prunus mahaleb L. have cytotoxic and anticancer effects, according to the results of BSLA [36].  

CONCLUSION
For the first time in the present study, silver nanoparticles were synthesized using the T. pratense. The antimicrobial and antibiofilm activity of the nanoparticles was determined. Additionally, the formation of silver nanoparticles was confirmed using UV-Vis, XRD, SEM, EDX, and FTIR. However, this study reports a novel, rapid, economical, and environmentally friendly procedure for producing silver nanoparticles. 
Based on the results, the nanoparticles have anticancer, antimicrobial, and antibiofilm activity against UTI.  Silver nanoparticles may play a role in neutralizing cell adhesives, thus preventing biofilm formation. Bacterial biofilm is highly resistant to antibiotics. Therefore, silver nanoparticles may play a major role in biofilm formation on urinary catheters.

ACKNOWLEDGEMENT
We are grateful to University of Kashan for supporting this work.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.