Biosynthesis of silver nanoparticles using supercritical CO 2 mediated phenolic contents extracted from Lagerstroemia speciosa leaf inhibits Klebsiella pneumoniae biofilm formation

Biosynthesis of silver nanoparticles using supercritical CO 2 mediated phenolic contents extracted from Lagerstroemia speciosa leaf inhibits Klebsiella pneumoniae biofilm formation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biosynthesis of silver nanoparticles using supercritical CO 2 mediated phenolic contents extracted from Lagerstroemia speciosa leaf inhibits Klebsiella pneumoniae biofilm formation Kiran Khandare, Shekhar Kumar, Sukesh Chander Sharma, Saswata Goswami This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4679230/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A large number of scientists are now working in order to create silver nanoparticles (AgNPs) that can be used as biomedicines against cancerous cell lines and bacteria that are resistant to drugs. In the current study, optimal supercritical fluid extract (SFE) of Lagerstroemia speciosa (LS) leaves at pressure 29.59 MPa, temperature 89.50 ºC and extraction time 53.85 min. was used to extract phenolic compounds for the synthesis of AgNPs. The synthesis was studied for 0–20 hrs. Initially the synthesis was confirmed by observing change in colour phenomenon. UV -spectroscopy confirmed the synthesis of nanoparticles (SFELS-AgNPs) demonstrated a maximum surface plasmon resonance at 430 nm. The crystallite dimension of nanoparticles was determined using XRD (13.47 nm), TEM results confirmed the diameter of the obtained silver nanoparticles between 8–20 nm. The nanoparticles possessed − 25.6 mV electric charge on the surface confirmed using zeta potential analyser. Furthermore, energy-dispersive X-ray analysis (EDAX), was used to analyze the presence of differential elements in generated materials. The developed nanoparticles were evaluated for their potential antimicrobial properties against, two gram-positive viz. Staphylococcus aureus and Bacillus cereus , and three gram-negative bacteria viz. Klebsiella pneumonia , Pseudomonas aeruginosa and Escherichia coli with different concentrations (100–400 µg/mL). The nanoparticle showed a minimum inhibitory concentration (MIC) of 64 µg/ml whereas the minimum bactericidal concentration (MBC) 128 µg/ml against K. pneumonia . They significantly inhibited K. pneumonia biofilm formation confirmed using scanning electron microscopy (SEM). The results were encouraging compared to the standards drug Chloramphenicol and other controls. The generated nanoparticles have highly effective antimicrobial properties against pathogenic bacteria. Lagerstroemia Speciosa Supercritical fluid extraction Silver Nanoparticle Klebsiella pneumoniae Biofilm Antimicrobial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Nanotechnology have been used in numerous biological applications, including biomedical such as biosensors, drug delivery systems, antibacterial, anticancer, and anti-inflammatory medicines [ 1 – 4 ]. Metal nanoparticles are the most promising nanoparticles due to their antibacterial capabilities and high surface-to-volume ratio [ 5 ]. Especially the silver nanoparticles show better chemical, physical as well as biological properties [ 6 , 7 ]. However, the synthesis of nanoparticles require both the reduction and stabilization agents [ 8 ]. Silver salt has been used as stabilization agent as numerous studies have shown that different microorganisms are quickly emerging tolerance against some antibiotics, but not against Ag ions because they need to concurrently establish host mutation processes and tolerance to protect them from Ag ions [ 9 ]. There is little information available about the bioactivity of AgNPs and how they affect human health. Therefore, developing a unique silver-based particles to manage the habitat of pathogenic bacteria on treated surfaces is the need. Synthesizing exopolysaccharides, several microorganisms have been shown to build biofilms, which protect them from harmful environmental stimuli. But the efficacy of plant based AgNPs on biofilm formation remains unchallengeable. In this study the reducing agent or polyphenols were extracted from a green source called Lagerstroemia speciosa leaf (LS) that is a plant native to tropical Asia and found in Northern Indian states [ 10 ]. It is a great source of various bioactive compounds such as antidiabetic, anti-obesity, anticancer, anti-angiogenic, and anti-microbial [ 11 – 15 ]. A large quantity of LS is the large agricultural produce and its leaf contains a significant amount of bioactive materials such as Alkaloids, Carbohydrate, Terpenoids, Tannins, Polyphenols and Flavonoids [ 16 ]. These bioactive compounds could be used as capping agent and silver salt reducing agent due to their numerous OH groups that promote their biological activities [ 17 , 18 ]. As LS is cheap and easily available plant source, an effective method to extract bioactive compounds from LS and their nanoparticle synthesis will be a beneficial technology for better utilization at commercial level. Previously reported studies conducted by Sundararajan et al., and Sai Saraswathi et al., emphasizes need of new extraction technology for the synthesis of nanoparticles from LS. Those studies revealed the significant activity of silver nanoparticles from LS leaf on Staphylococcus aureus, Proteus vulgaris, Klebsiella sp., Pseudomonas aeruginosa [ 19 ] and the photocatalytic activity of nanoparticles degrading the various dyes like methyl orange and methylene blue which suggest synthesized using LS extract [ 16 ]. But the new extraction technique is still required to synthesise the controlled synthesis of nanoparticles in terms of size, purity and environmentally friendly approach. SFE offers numerous advantages for the synthesis of nanoparticles, including high purity, precise control over particle size and morphology, environmental sustainability, and versatility. These benefits make SFE a valuable technique in various industries, from pharmaceuticals and food to cosmetics and environmental applications. The ability to produce high-quality nanoparticles with tailored properties opens up new possibilities for innovation and development in these fields. The combination of phytochemicals and their metal encapsulation, may cause synergistic effects against microorganisms and could be sources of palatable, natural substitutes. Along with that, degradation of thermolabile compounds, quality of extract are other circumstances that affects the synthesis of nanoparticles. SFE is one of the solutions for these circumstances. On other hand food and pharmaceutical industries have stated the desire to reduce the use of antibiotics and artificial chemicals as a food shield. Thus, the present work aimed to investigate the potential effect of optimal SFE extracted polyphenols and their encapsulated form on exposure to Staphylococcus aureus , Bacillus cereus , Klebsiella pneumonia , Pseudomonas aeruginosa and Escherichia coli. K. pneumoniae is one of the potentially fatal biofilm-forming bacteria that belong to the Enterobacteriaceae family and is encapsulated, nonmotile, and rod-shaped bacteria [ 20 ]. Its biofilm formation protects itself against drugs and host immunological responses, which increases its pathogenicity [ 21 ]. Certain virulence-related genes found in K. pneumoniae play important roles in the production of biofilms. Moreover, type 3 fimbriae (mrkA) genes play a significant role in K. pneumoniae biofilm development [ 22 ]. Thus, this study is specifically focused on the K. pneumonia to evaluate the effect of nanoparticles on gram-negative bacterial biofilm inhibition. According to our research survey no information available on the synthesis of nanoparticles utilising supercritical CO 2 extract of LS for the biofilm inhibition K. pneumonia so far. Although our research is the little contribution towards medicinal application of LS nanoparticles that can be used in orthopaedics, packaging, medical devices, footwear, household items as these are the fact that exists in terms of silver nanoparticles [ 23 – 26 ]. 2. Materials and methods 2.1. Chemicals and Plant sample collection Medicinal plant LS was collected from Center of Innovative and Applied Bioprocessing campus, Mohali, Punjab, India. Leaves were washed with running tap water to remove external dust and dried using fluidized bed dryer, pilot scale with (50 L) capacity at 45 0 C until they retain 8–9% water content. The moisture was analyzed using aczet MB 50, 220 V – 230 V (India) capacity moisture analyzer. The dried powder was passed through mass collider followed by sieving through 500 µm sieve to keep consistent particle size in each experiment. All analytical-grade chemicals including AgNO 3 were bought from HI-media Chemicals Ltd., Mumbai, India 2.2. Preparation of plant extract As schematically depicted in Fig. 1 , the phenolic content extraction studies were performed utilising an SFE system (SFE-Helix, Allentown, USA). In this extraction technique, liquid CO 2 was delivered into the system using a twin piston pump after being fed from a CO 2 cylinder via a syphon tube. The pump was built to withstand pressures of up to 69 MPa. An extraction vessel for supercritical CO 2 extraction was supported by this system. Through a preheater and a heating jacket placed around the vessels, the fluid was heated to its supercritical condition. To regulate the flow rate, a sturdy variable restrictor valve was employed. The restrictor was electrically warmed to prevent sample clogging. A typical experiment involved weighing and loading a 50 ml extraction jar with roughly 10 g of dried leaf material. To stop solid materials from being transferred to the tubing and clogging the system, 1.5 g of glass wool was sandwiched between the leaf sample and the ends of the extraction basket. The extraction was carried out at 300 bar pressure, 363.15 K temperature and 50 minute extraction time. The extract was collected in an dark collection vial following the initiation of supercritical fluid extraction once the necessary extraction pressure and temperature had been achieved. The extract was stored at 4°C until the synthesis of silver nanoparticles. 2.3. Determination of TPC using Folin-Ciocalteu calorimetric assay and other phytochemical scrrenings The TPC of the extract was determined using the modified Folin-Ciocalteu method [ 27 ]. The 0.5 mL extract was mixed with the Folin-Ciocalteau’s reagent (Sigma Aldrich) (0.25 mL) and 1.25 mL of 20% sodium carbonate solution. The mixture was incubated for 40 min at room temperature. The optical density of the blue-coloured samples was checked at 725 nm using a UV2700, UV–vis spectrophotometer (Shimadzu, Kyoto, Japan). The TPC were expressed as mg of gallic acid equivalents/g dry weight of the biomass (mg GAE/g DWB) using gallic acid as a calibration standard. The concentration was determined using formula as follows [ 28 ]: 𝑇𝑃𝐶 = (𝐶 x V)/𝑀 TPC = Total Phenolic Content (mg GAE/g DWB) C = concentration of gallic acid equivalent (mg/gm) V = volume of extracts used for analysis (mL) M = Weight of the extract used for analysis (mg) 2.3.1. Identification of presence of Alkaloids 2.3.1.1. Hager's Reagent Test Saturated Hager's solution (picric acid) is added in multiple drops to the 200 µL SFE-LS. A brilliant yellow precipitate indicates the presence of alkaloids. 2.3.1.2. Mayer's Reagent Test After combining 200 µL of SFE-LS with a few drops of diluted HCl containing Mayer's reagent (potassium mercuric iodide solution), the mixture was heated for around five minutes. The presence of alkaloids is confirmed by the yellow color of the precipitate. 2.3.2. Identification of presence of Carbohydrates 2.3.2.1. Molisch's Reagent Test 200 µL of Molisch's reagent and 200 µL of SFE-LS were mixed. Solution was boiled followed by cooling. Production of violet rings shows the presence of carbohydrates. 2.3.3. Identification of presence of Tannins 2.3.3.1. FeCl 3 Test 200 µL of FeCl3 solution is added to 100 µL of SFE-LS. The color violet indicates the presence of tannins. 2.3.4. Identification of presence of proteins and amino acids. 2.3.4.1. Ninhydrin Test After heating the SFE-LS in a 0.2% Ninhydrin solution, no violet color developed, signifying the lack of proteins and amino acids. 2.3.4.2. Million's Test After adding 100 µL of Million's reagent to 100 µL of SFE-LS, no white precipitate developed after boiling, which proves the lack of proteins and amino acids. 2.3.5. Identification of presence of Saponins 2.3.5.1. Foam Test 100 µL of SFE-LS was mixed with 100 µL of water. Lack of foam formation even after shaking suggests the absence of saponins. 2.3.6. Identification of presence of Gums & Mucilage 2.3.6.1. Swelling Test After mixing 500 µL of 100% alcohol with 100 µL of SFE-LS, the mixture was agitated for 10 minutes. Lack of gums and mucilage is indicated by the absence of swelling in the solution. 2.4. Synthesis of SFELS-AgNPs The SFE-LS in the preceding stage was employed for the environmentally friendly production of silver nanoparticles. 10 mL of SFE-LS (9.93 mg/mL) was added to 90 mL of a 1 mM aqueous silver nitrate solution. The mixture was then heated at 80°C while being constantly stirred at 400 rpm until the silver nitrate was completely reduced. The transition from yellow to dark brown was considered as a preliminary indicator of the synthesis of the SFELS-AgNPs. Centrifugation was done to separate the green-synthesised nanoparticles at 13,000 g for 10 min. from free silver. This process was carried out three times. The final silver nanoparticles produced via green synthesis, were freeze-dried and kept at 4°C until usage. 2.5. Characterization UV − vis spectroscopy (HMG Labtech SPECTRO star, U.K.) was used to check the reduction of silver nitrate with SFELS extract in the range of 220 − 700 with 1 nm resolution. The Fourier Transform Infrared Spectrophotometer (FTIR) (Agilent, Model: Carry 660 series) was used to evaluate the functional groups in the phytoconstituents that were responsible for the reduction and capping of SFELS-AgNPs utilising the KBr pellet method. The Infrared (IR) rays was kept between 4000 − 400 cm − 1 with a spectral resolution of 1 cm − 1 . The crystalline structure and phase of the SFELS-AgNPs was investigated using X-ray diffraction (XRD) study. The Cu Kα radiation (1.540 A° at 40 kV and 30 mA) were used in X-ray diffractometer (Rigaku®, SmartLab) with a scanning rate of 0.02 cm − 1 in the region of 2θ ranging between 30 and 90°. Scanning Electron Microscopy (SEM) coupled with Energy dispersive X-ray analyzer (EDX) (Hitachi S-4700) was used to investigate the external morphology, size, and atomic constituents of atoms in SFELS-AgNPs. Double-sided tape was used to secure the samples to the tubular metal stub. Gold was applied to the stub-supported samples. Ultimately, the samples coated in gold were examined under a microscope to determine their morphology. TEM analysis was performed to determine the morphology, size and shape of the silver nanoparticles. TEM measurements were done by HITACHI H-800, operating at 200 kV. The TEM grid was prepared by placing a drop of the bio-reduced diluted solution on a carbon-coated copper grid and later drying it under a lamp. Weight loss and thermal behaviour of the surface capped Ag nanoparticles was determined by using Thermogravimetric analyser in a Perkin Elmer STA 6000 (Perkin-Elmer SCIEX, Waltham, MA, USA) at a heating rate of 10°C/min. Dynamic light scattering (DLS) technique was used to determine the average hydrodynamic particle size distribution and surface charge (ζ potential) and polydispersity (PDI), with Zetasizer nano ZS (Malvern Instruments, Malvern, U.K.). The light scattering angle was set to 90° and the size measurements were performed at 25°C. Well dispersed solution was made by diluting the nanoparticles with Milli-Q water followed by ultrasonication for 10 min. 2.6. Antibacterial assay The antibacterial efficacy of the SFELS-AgNPs against gram-positive and gram-negative bacteria was evaluated by agar well diffusion assay as per Clinical and Laboratory Standard Institute (CLSI) guidelines. The Mueller Hinton Agar (HIMEDIA, M173-500G) plate, pre-inoculated with Staphylococcus aureus (MTCC 737), Bacillus cereus (MTCC 1306), Klebsiella pneumonia (MTCC109), Pseudomonas aeruginosa (MTCC 2488) and E. coli was used to check the bioactivity. Wells were created aseptically on the seeded agar plate with the help of a cork borer (HIMEDIA, 6mm diameter). 200 µl sample volume was filled into the wells and allowed to diffuse at 4°C for an hour followed by incubation at 37°C for 24 hrs. The zone of inhibition was observed and measured using calliper. 2.7. Determination of MIC and MBC MICs of SFELS-AgNPs were determined by the broth microdilution method. The primary culture of Klebsiella pneumonia was grown overnight at 37˚C with shaking at 180 rpm. The secondary culture was obtained from the overnight-grown culture. These cells were further diluted to fix the number of cells that is, 10 6 cells per well of 96-well plate. 100 µl of the diluted culture was put into every 96 well-plates and incubated with a series of 2-fold dilutions of SFELS-AgNPs ranging from 512 µg/ml to 0.5 µg/ml, and the total volume was adjusted to 200 µl. The plate was incubated for 18 hrs. at 37°C with static conditions. The lowest concentration with no visible growth was recorded as MIC. The MBC was determined using the broth microdilution method. A 100 µl aliquot of each gradient concentrated solution from the MIC activity was transferred into a nutrient agar plate and incubated at 37˚C for 48 hrs. The lowest concentration of antimicrobials that kill 99.9% of the bacteria was defined as MBC. 2.8. Single plate serial dilution spotting (SP-SDS) assay: The primary culture of Klebsiella pneumoniae was grown overnight at 37˚C with shaking at 180 rpm. The secondary culture was obtained from the overnight-grown culture. These cells were further diluted to fix 10 6 cells in 200 µl volume. The concentration of SFELS-AgNPs was kept at 64 µg/ml for all. Cells treated with SFELS-NPs along with the untreated ones were incubated at 37˚C with shaking for 0, 2, 4, 6 & 12 hrs of incubation. All the incubated cells were 10-fold serially diluted till 10 − 6 dilutions and 5 µl of each dilution were spots on the agar plate. These plates were incubated at 37˚C for 16 hrs and the images were captured. 2.9. Determination of biofilm inhibition using SEM The effect of SFELS-AgNPs on biofilm was qualitatively studied using SEM. Biofilms were formed in 6-well cell culture plates (Nunc) having coated glass coverslips. Glass coverslips were coated with poly-L-lysine (2% w/v) and further sterilized by UV radiation for 1 hr. inside the biosafety cabinet and dried before use. These coverslips were put in microtiter plate wells for biofilm initiation in the presence and absence of antimicrobial compounds. The incubation was done at static conditions with a temperature of 37°C for 24 hours. Coverslips were moved to a new 6-well plate after the incubation and washed three times with 1X PBS. PBS-washed biofilms were later fixed by formaldehyde (4% vol/vol) and glutaraldehyde (2% vol/vol) for 20 min, accompanied by dehydration with a series of ethanol solutions. Tertiary-butyl alcohol was eventually used for dehydrating the slides for 30 min and then dried in a desiccator. After the drying process, coverslips were then finally coated with gold-palladium for 135 sec at the current of 10–12 milliamperes and analyzed by scanning electron microscope (JEOL JSM-6000, NIKON Corporation) in high-vacuum mode at 10 kV. 3. Results and discussion 3.1. Quantification of phenolic compounds in SFE-LS and preliminary phytochemical screening The quantification of phenolic compounds was done using Folin-ciocalteu calorimetric assay. The SFE-LS showed 99.31 ± 2.57 mg/gm TPC of dry weight of biomass (DWB). As per the preliminary phytochemical screening done using protocol as mentioned in section 2.3.1 to 2.3.6 the contents of phytochemicals are presented in Table 1 . Table 1 Preliminary phytochemical screening of the SFE-LS S. no. Qualitative test SFE-LS 1 Alkaloids + 2 Amino acids − 3 Carbohydrate + 8 Tannins − 9 Saponins + 10 Gums & mucilage − 3.2. UV-vis study The synthesized silver nanoparticles in the colloidal solution were monitored by UV–vis spectrophotometer analysis. Figure 2 A depicts the change in colour from yellow to dark brown that confer the reduction of AgNO 3 by phenolic compounds in extract. Figure 2 B shows that the absorption spectra of silver nanoparticles formed in the reaction media has an absorbance peak at 430 nm. The increased absorbance at various time intervals (0 to 20 h) and the peak at 430 nm correspond to the surface plasmon resonance of silver nanoparticles. It is reported earlier that absorbance at around 430 nm for silver is a characteristic of these noble metal particles [ 29 ]. The λmax range found in the UV-Visible spectrum study is comparable to previous reports for AgNPs synthesized from pomegranate leaves [ 30 ]. A comparable analysis yielding essentially identical results was previously conducted in recent studies of AgNP production employing extracts from other plants, including Clerodendrum infortunatum, Azospirillum brasilense , and Allium cepa (onion) [ 31 – 33 ]. 3.3. FTIR and XRD studies Figure 3 show the FT-IR spectra of SFELS-AgNPs that confers the presence of functional groups in Ag nanoparticles. FT-IR spectrum showed the major peak positions at 3334 cm − 1 , 2165 cm − 1 , 2007 cm -1 , 1727 cm -1 , 1625 cm − 1 , 1461 cm -1 , 1349 cm − 1 , 1041 cm − 1 , 1041 cm − 1 , 524 cm − 1 and 455 cm − 1 . The peak at 3334 cm -1 intimates OH group in phenolic compounds and carbohydrates [ 34 ]. The stretching vibrations of the aliphatic hydrocarbon chains (CH band) observed at 2,925 cm -1 , either from the lipids' methyl or methylene groups in the SFE extract. The C = O stretch of aromatic alkenes in the phenolic content showed the band at 1625 cm -1 . The band at 1,461 cm -1 is of CH 2 and CH 3 of aliphatic chains. The band at 1349 cm -1 is attributed to C = C. The stretching vibration of C–O–C is responsible for the prominent peak at 1,041 cm -1 . The described FT-IR data suggested that the SFE extracts consist of bioactive compounds with functional groups like OH and COOH. These functional groups serve as stabilising agents in addition to being in charge of the AgNO 3 reduction [ 35 , 36 ]. XRD pattern revealed distinct peaks at 2 θ values, which can be attributed to 111, 200, 220 and 311 crystalline planes of silver NPs at 38.02°, 44.16°, 64.34° and 77.26° as shown in Fig. 4 . These peaks are associated with the face-centred cubic lattice. Bragg’s reflections of face centre cubic (fcc) structure of metallic silver respectively similar to Joint Committee on Powder Diffraction Standards (JCPDS) file no: ICDD-PDF2, Release 2007, PA, USA, 2007, revealing that synthesized nanoparticles are of pure crystalline silver. The crystalline size of the SFELS-AgNPs formed were calculated using Debye–Scherrer equation (Eq. 1) which was around 13.48 nm, were good in agreement with TEM results. D= (kλ /β cos θ) ………………………. (1) Where, D is size of particle, k is Scherrer’s constant, λ is the X-ray wavelength, β is full width at half maximum (FWHM) 3.4. FESEM, HRTEM and EDAX analysis The morphology of SFELS-AgNPs was analyzed through FESEM image (Fig. 5 A). The majority of particles were spherical in shape and there were a few oval AgNPs as well. Biosynthesized nanoparticles had been spread thoroughly in the solution. The size of some selected biosynthesized nanoparticles was around 50 nm according to FESEM images. These results strongly confirmed that SFE-LS extract acts as a reducing and capping agent for the synthesis silver nanoparticles. Figure 5 B demonstrated energy dispersive spectrum of the SFELS-AgNPs, which has silver as a ingredient element. Silver nanoparticles generally shows the peak at 3 keV, due to its surface plasmon resonance. The fig also showed the presence of C, O due to the reaction with mixed phenolic groups. This is one of the advantages of synthesis of silver nanoparticles using plant extract. Results of EDAX in this research are conservative with the silver nanoparticles synthesized using Rheum palmatum and Colius aromaticus extract [ 37 , 38 ]. The morphology and size of the synthesized silver nanoparticles were determined by TEM images and they are shown in Fig. 6 A and B. The particles formed were spherical in shape. It was determined that the generated nanospheres have a large surface area. The size of the formed nanoparticles ranged from 8 to 20 nm. The average particle size is 14 nm which is share exactly similar results by Saraswathi et al., 2017 [ 16 ]. 3.5. DLS and zeta potential of SFELS-AgNps Using the DLS technique, the surface zeta potential of synthesized SFELS-AgNPs in aqueous colloidal solution was measured. The negative zeta potential was determined to be − 25.6 mV in this investigation, with a zeta deviation of 4.63 mV. Figure 7 B. The zeta potential values give us the information regarding the surface charge of nanoparticles which appeared to be negative here. The high negative value indicated that synthesized silver nanoparticles did not agglomerate. Additionally, it gives us an idea about the stability of nanoparticles and the zeta potential value obtained for SFELS-AgNPs lays within the stable range, signifying the nanoparticle are stable in aqueous solution [ 39 ]. The zeta potential range of ± 30 mV is thought to be the most stable for silver nanoparticles [ 40 ]. Figure 7 A demonstrates that the average particle size of SFELS-AgNps is around 116 nm with the polydispersity index of 0.244. 3.6. Weight loss and thermal behaviour Thermogravimetric analysis of SFELS-AgNPs was carried out to examine the influence of temperature on nanomaterial. Figure 8 shows that the nanoparticles lost its first weight at 102.56 ºC. which may be due to the moisture in material. Temperature between 210.96 ºC to 348.18 ºC almost lost 30% of its weight. Nanoparticles almost started degrading after 348.18 ºC, which suggest the threshold of thermal sensitivity of the material. 3.6 Antibacterial activity The growth inhibition by SFELS-AgNPs against Staphylococcus aureus , Bacillus cereus , Klebsiella pneumonia , Pseudomonas aeruginosa and Escherichia coli was studied using well diffusion method. To describe the expansion of the clean zone, three different concentration 100 µg/mL, 200 µg/mL and 400 µg/mL of silver capped SFE-LS extract and SFE-LS extract itself were used to check the concentration dependent growth inhibition. Results were compared with standard antibacterial drug Chloramphenicol and Gentamycin. SFELS extract showed low clear zone but the SFELS-AgNPs showed significant antibacterial activity (Fig. 9 ). The zone of inhibition at every sample was counted using a pair of callipers. The results are presented in Table 2 . Table 2 Bactericidal activity (Zone of inhibition in mm) of SFE-LS-NPs against pathogenic bacteria Sr. No. Name of samples Bacterial Zone of inhibition (mm) Gram positive Gram negative B. cereus (MTCC1306) S. aureus (MTCC737) K. pneumoniae (MTCC109) P. aeruginosa (MTCC2488) E. coli 1 100 SFELS-AgNps 12 15 20 12 ND 2 200 SFELS-AgNps 13 17 22 13 10 3 400 SFELS-AgNps 18 16 24 14 12 4 Chloramphenicol/ Gentamycin 20 24 22 20 14 5 100 SFE-LS ND ND ND ND ND 6 200 SFE-LS ND ND ND ND ND 7 400 SFE-LS ND ND ND ND ND 8 Water ND ND ND ND ND ND: Not determined The bacterial cell membrane damages due to electrostatic interference of AgNPs which form pores on the surface, that alters the structure of the bacteria and ultimately leads to cell death. As a result, the zone of inhibition increased with concentration [ 41 ]. The AgNPs in prokaryotic bacteria interact with the bacterial cell membrane, bind to the mesosomal cell organelle, impair mesosome function, and enhance the production of reactive oxygen species (ROS). Silver nanoparticles bind with thiol groups in proteins, which inactivates both DNA replication and protein synthesis [ 42 ]. Simultaneously, oxygen combines with silver and reacts with the sulfhydryl (-S-H) groups on the bacterial cell wall to eliminate the -H atoms. This prevents the sulphur atoms from forming an R-S-S-R bond and kills the bacterial cells by preventing respiration [ 43 ]. The silver nanoparticles are responsible for the disruption of membrane integrity in bacteria and even limits the growth of bacteria by mitigating oxygen, sulphur and nitrogen in essential biological molecules. Despite numerous papers describing the antibacterial action of AgNPs produced by plant materials, the plant extract often exhibited little to no inhibition while the synthesized SFELS-AgNPs displayed minimal to moderate and good to exceptional activity [ 44 ]. The results of this study intimated that SFELS-AgNPs are more effective on K. pneumoniae compared to other bacteria. 3.7 Evaluation of MIC and MBC The effectiveness of SFELS-AgNPs was further confirmed by determining the MIC and MBC against the K. pneumonia based on the agar well diffusion assay. The SFELS-AgNPs showed a MIC of 64 µg/ml whereas the MBC was determined as 128 µg/ml. The experiment was repeated three times and each concentration was done in triplicates. Our results showed that SFELS-AgNPs are more successful in suppressing the growth of bacteria, confirming the theory that the antibacterial activity is strongly influenced by the modification of the nanoparticles-bacteria interface [ 45 ]. Several studies reported that the major reason of AgNPs stop the growth of microorganisms by interfering with microbial DNA replication, contact death, and the generation of reactive oxygen species [ 46 , 47 ]. 3.8 Effect of SFELS-AgNPS on K. pneumonia biofilm formation The MIC (64 µg/ml) was used to evaluate the effect of SFELS-AgNPS on bacterial cells. The biofilm formation was assessed in control (Untreated) and treated strains of K. pneumonia as shown in Fig. 10 , that revealed cells in the untreated biofilm are congregated and intact. After the treatment, biofilm breaks and cells dispersed and the breakages of cells (red arrow) are also visible. Numerous studies showed the herbal nano formulations has potential to reduce biofilm [ 48 ]. The suppression of biofilm formation may be the result of SFELS-AgNPs penetrating the bacterial cell wall, suppressing the synthesis of exo-polysaccharide layers, and interfering with cell adhesion and communication [ 49 ]. Mousavi et al., recently studied biofilm reduction in K. pneumonia strains using biologically synthesized silver nanoparticles their results are comparable with this study as they have obtained significant reduction at 128 µg/mL of nanoparticles [ 50 ]. Additionally, prior research indicated that these NPs might be effective antibacterial agents against a variety of bacterial infections, such as K. pneumoniae [ 51 ]. Although several processes have been hypothesized, the precise mechanism by which AgNPs exert their antibacterial effect remains unknown. The capacity of these NPs to attach the bacterial cell wall and their subsequent entry into the cell, which causes structural alterations in the cellular membrane and causes cell lysis and content leaking, is one of the hypothesized mechanisms [ 52 ]. Biofilm-forming microorganisms can cause a wide range of illnesses. In fact, a report from the National Institutes of Health and the Centers for Disease Control states that 65–80% of infections are the cause of these microbes [ 53 ]. Therefore, use of biofilm inhibitors is one of the effective ways to reduce the diseases caused by these microorganisms. Numerous studies have reported the effect of AgNPs as biofilm inhibitors against multi drug resistant K. pneumoniae [ 51 ]. We discussed how AgNPs may be used to target K pneumoniae biofilm development in the current investigation. The current findings demonstrated that at 64 µg/ml concentration of SFELS-AgNPs, Klebsiella pneumoniae strain was failed to generate biofilms. 3.9. SP-SDS Procedure Bacterial dilution spotting (10-fold) assay was performed to quantify the growth and sensitivity of K. pneumonia upon treatment with SFELS-AgNPs using MIC. A fixed SFELS-AgNPs concentration (64 µg/ml) was used with varying treatment times (0, 2, 4, 6 & 12 hrs). The sensitivity of K. pneumonia could be correlated with the percentage of cells that survive and form a colony. As evident in the result, with increasing incubation time, the viability decreases at 10 − 4, 10 − 5, & 10 − 6 dilution after 2 hrs treatment; 10 − 3, 10 − 4, 10 − 5, & 10 − 6 dilution after 4 hrs treatment and 10 − 2, 10 − 3, 10 − 4, 10 − 5, & 10 − 6 dilution after 6 hrs treatment and hence there is no or less bacterial colony visible at the spot (Fig. 11 ). At 12 hours of treatment, complete growth inhibition can be seen at all the dilutions. These results suggests that synthesized nanoparticles are capable of complete inhibition of the k. pneumoniae on 12 hrs of treatment. 4. Conclusion In the current research, methodology was developed for the synthesis of silver nanoparticles using supercritical fluid extracted polyphenols from LS leaves, as reducing agent. The extract supports the synthesis of nanoparticles at room temperature with a quick kinetics and without the use of any hazardous chemicals, therefore the results are quite encouraging. Research using UV-vis spectroscopy reveals that LS is a plant that is abundant in bioactive compounds like polyphenols that can be used as reducing agents and may also be found in other plants. These compounds when encapsulated with stabilisers for silver nanoparticles, the same chemical mechanisms gave them significant antibacterial properties. According to the results SFELS-AgNPs showed highest antibacterial activity against K. pneumoniae with MIC 64 µg/ml and MBC 128 µg/ml. Furthermore, it significantly inhibited the K. pneumoniae biofilm formation. The development of biofilm plays a crucial part in bacterial pathogenicity, shielding the bacteria from antibiotics. In our investigation, plant-mediated synthesized AgNPs shown initial biofilm formation inhibitory action. Therefore, LS-mediated AgNPs may be a novel approach to treating infections linked to bacterial biofilms. According to TEM, the diameter of the silver nanoparticles is between 8 and 20 nm. Interestingly, XRD results revealed that the population of silver nanoparticles is made up of a variety of face-centered cubic forms. This technique revealed the presence of the unusual hexagonal crystal structure 4H for silver nanoparticles, providing a novel way to investigate the antibacterial capabilities and optical response of this nanomaterial. Moreover, SFELS-AgNPs could be added to formulations to create antibacterial goods like hand washes, sanitizers, and creams to reduce the spread of harmful bacteria in the environment, public spaces, hospitals, etc. To address issues with the usage of nanomaterials, especially their pharmacokinetics and pharmacodynamics properties, more research is necessary. As per the safety concerns, clinically testing of nanoparticles is also required. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment Authors would like to acknowledge Center of Innovative and Applied Bioprocessing for research support and Council of Scientific and Industrial Research. They would also like to acknowledge Panjab University, Chandigarh. Author Contributions Kiran Khandare: Investigation, Experimental analysis, Writing-original draft, Manuscript editing and revision; Shekhar Kumar: Experimental analysis, Manuscript editing and revision; Sukesh Chander Sharma: Supervision, Manuscript review, and editing; Saswata Goswami: Supervision, Conceptualization, Manuscript review, and editing, Resources, Funding acquisition Funding No funding is obtained for this work Data Availability The corresponding author can provide the data that were utilized to support the study's conclusions upon request. Compliance with Ethical Standards Research Involving Humans and Animals Statement-None. Informed Consent- None. References Tan, P., Li, H. S., Wang, J., & Gopinath, S. C. B. (2021). Silver nanoparticle in biosensor and bioimaging: Clinical perspectives. Biotechnol Appl Biochem , 68 (6), 1236–1242. Prasher, P., Sharma, M., Mudila, H., Gupta, G., Sharma, A. K., Kumar, D., Bakshi, H. A., Negi, P., Kapoor, D. N., Chellappan, D. K., et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4679230","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322760205,"identity":"8e78ca7a-61c7-41d7-8d42-6bb5517830e0","order_by":0,"name":"Kiran Khandare","email":"","orcid":"","institution":"Center of Innovative and Applied Bioprocessing","correspondingAuthor":false,"prefix":"","firstName":"Kiran","middleName":"","lastName":"Khandare","suffix":""},{"id":322760208,"identity":"53e74c7d-3d82-48c9-a31b-7448acefc1e0","order_by":1,"name":"Shekhar Kumar","email":"","orcid":"","institution":"Center of Innovative and Applied Bioprocessing","correspondingAuthor":false,"prefix":"","firstName":"Shekhar","middleName":"","lastName":"Kumar","suffix":""},{"id":322760210,"identity":"0e45c7f1-a3f0-4410-838b-3245a461c123","order_by":2,"name":"Sukesh Chander Sharma","email":"","orcid":"","institution":"Departement of biochemistry, Panjab University, India","correspondingAuthor":false,"prefix":"","firstName":"Sukesh","middleName":"Chander","lastName":"Sharma","suffix":""},{"id":322760212,"identity":"92900a66-86ca-47b0-a46b-443a5bbf1938","order_by":3,"name":"Saswata Goswami","email":"data:image/png;base64,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","orcid":"","institution":"Center of Innovative and Applied Bioprocessing","correspondingAuthor":true,"prefix":"","firstName":"Saswata","middleName":"","lastName":"Goswami","suffix":""}],"badges":[],"createdAt":"2024-07-03 09:28:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4679230/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4679230/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61349610,"identity":"6b8ac57f-21ca-41e4-aaee-07b450716bf7","added_by":"auto","created_at":"2024-07-29 18:32:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55182,"visible":true,"origin":"","legend":"\u003cp\u003eFlow sheet diagram of supercritical fluid extraction (SFE) apparatus C\u003csub\u003e1\u003c/sub\u003e: CO\u003csub\u003e2\u003c/sub\u003e cylinder; H\u003csub\u003e1\u003c/sub\u003e: Condenser; H\u003csub\u003e2\u003c/sub\u003e: Electric heater; V\u003csub\u003e1\u003c/sub\u003e: CO\u003csub\u003e2\u003c/sub\u003e recycle unit; V\u003csub\u003e2\u003c/sub\u003e: Extraction cell; V3: Micro metering valve/collecting vessel; P\u003csub\u003e1\u003c/sub\u003e: CO\u003csub\u003e2\u003c/sub\u003e pump; P\u003csub\u003e2\u003c/sub\u003e: Backpressure regulator; P\u003csub\u003e3\u003c/sub\u003e: Backpressure regulator.\u003c/p\u003e","description":"","filename":"Fig1SFEFlowsheetdiagram.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/fb548c76b6287ce3b877ee9e.png"},{"id":61349612,"identity":"9996f58b-1d91-48e8-8c5c-d5f65c96edb2","added_by":"auto","created_at":"2024-07-29 18:32:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56620,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Colour change of extract from pale yellow to dark brown after incubation at 80 Cº with AgNO\u003csub\u003e3\u003c/sub\u003e (B) UV spectra of the SFELS-AgNPs.\u003c/p\u003e","description":"","filename":"Fig2UV2.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/40720a41df9a4836080e4d8d.png"},{"id":61348724,"identity":"60d66c2a-410e-4e51-8105-e9967486e5de","added_by":"auto","created_at":"2024-07-29 18:24:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28508,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR analysis of SFELS-AgNPs\u003c/p\u003e","description":"","filename":"Fig3FTIR.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/a2c242a5b15ece2cb959cd62.png"},{"id":61348726,"identity":"1878fbca-4200-411f-9307-5dd6bac116d5","added_by":"auto","created_at":"2024-07-29 18:24:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74381,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction profile of SFE-LSNPs\u003c/p\u003e","description":"","filename":"Fig4XraydiffractionprofileofSFELSAgNPs.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/c92af53ea0a77c2666c644aa.png"},{"id":61350467,"identity":"d2e26aca-5db7-42bf-9e53-f1d10823a51f","added_by":"auto","created_at":"2024-07-29 18:40:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":387143,"visible":true,"origin":"","legend":"\u003cp\u003eElectron microscopy study:(A) Scanning Electron Microscopy (B) EDX Spectra of SFELS-AgNPs\u003c/p\u003e","description":"","filename":"Fig5Electronmicroscopystudy.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/6dd213eb3b7ad991fe166460.png"},{"id":61348736,"identity":"ab2f5203-ae8c-4224-8a51-f1530ab90679","added_by":"auto","created_at":"2024-07-29 18:24:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":511014,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission Electron Microscopy image of SFELS-AgNPs\u003c/p\u003e","description":"","filename":"Fig6TEM.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/ea64d485e104942f947072da.png"},{"id":61350468,"identity":"b5507291-0cf0-484c-a08e-1b05f3c883bc","added_by":"auto","created_at":"2024-07-29 18:40:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":172670,"visible":true,"origin":"","legend":"\u003cp\u003eSFELSAg-NPs\u003csub\u003e \u003c/sub\u003esize measurement (A) size and PDI analysis (B) zeta potential\u003c/p\u003e","description":"","filename":"Fig7SFELSAgNPssizemeasurementAsizeandPDIanalysisBzetapotential.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/adaa7ab69b849d5a31b63691.png"},{"id":61348735,"identity":"c9ab5a33-fc3f-4e45-b055-15dfc41d97fd","added_by":"auto","created_at":"2024-07-29 18:24:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":53048,"visible":true,"origin":"","legend":"\u003cp\u003eThermogravimetric analysis of SFELS-AgNPs\u003c/p\u003e","description":"","filename":"Fig8ThermogravimetricanalysisofSFELSAgNPs.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/24c07cc78a79e6d211b5d3f7.png"},{"id":61348734,"identity":"ee1efb0b-2d2e-4c9c-a78e-e30fc0e764a9","added_by":"auto","created_at":"2024-07-29 18:24:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":906689,"visible":true,"origin":"","legend":"\u003cp\u003eBactericidal activity of SFELS-AgNO\u003csub\u003e3 \u003c/sub\u003e(a-e) Chloramphenicol or Gentamycin as positive control, water as negative control and 100-400 \u003cem\u003eµg/mL \u003c/em\u003eSFE-LS on \u003cem\u003eP. aeruginosa, S. aureus, K. pneumoniae, E. coli and B. cereus \u003c/em\u003erespectively; (a՛-e՛)\u003csub\u003e \u003c/sub\u003e100-400 \u003cem\u003eµg/mL \u003c/em\u003eSFELS-AgNPs on \u003cem\u003eP. aeruginosa, S. aureus, K. pneumoniae, E. coli and B. cereus \u003c/em\u003erespectively\u003c/p\u003e","description":"","filename":"Fig9Antimicrobialresults.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/6f17d6f84953435a3e9f52d6.png"},{"id":61348731,"identity":"697aeab7-12f1-43cd-9207-7c4c06be3bcb","added_by":"auto","created_at":"2024-07-29 18:24:08","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":382707,"visible":true,"origin":"","legend":"\u003cp\u003eScanning Electron Micrograph (SEM) image of untreated and treated \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e (MTCC 109) biofilm with SFELS-AgNPs.\u003c/p\u003e","description":"","filename":"Fig10BiofilmSEM.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/317efd990f8e0e191fae48b5.png"},{"id":61349613,"identity":"379ec0de-9636-4946-adb6-fb909a291a31","added_by":"auto","created_at":"2024-07-29 18:32:08","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":154851,"visible":true,"origin":"","legend":"\u003cp\u003eA bacterial dilution spotting (10-fold) assay was performed to measure the toxicity against \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e (MTCC 109) upon treatment with SFE-LS-NPS.\u003c/p\u003e","description":"","filename":"Fig11Bacterialdilutionassay.png","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/b343ec8e6f15cd7cee9e4e40.png"},{"id":66285500,"identity":"b12cfd0a-0960-4d82-9495-a894e480914c","added_by":"auto","created_at":"2024-10-09 17:04:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4442892,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4679230/v1/6e8ba635-e418-4328-8bc8-f3f97b32534b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biosynthesis of silver nanoparticles using supercritical CO 2 mediated phenolic contents extracted from Lagerstroemia speciosa leaf inhibits Klebsiella pneumoniae biofilm formation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanotechnology have been used in numerous biological applications, including biomedical such as biosensors, drug delivery systems, antibacterial, anticancer, and anti-inflammatory medicines [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Metal nanoparticles are the most promising nanoparticles due to their antibacterial capabilities and high surface-to-volume ratio [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Especially the silver nanoparticles show better chemical, physical as well as biological properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the synthesis of nanoparticles require both the reduction and stabilization agents [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Silver salt has been used as stabilization agent as numerous studies have shown that different microorganisms are quickly emerging tolerance against some antibiotics, but not against Ag ions because they need to concurrently establish host mutation processes and tolerance to protect them from Ag ions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. There is little information available about the bioactivity of AgNPs and how they affect human health. Therefore, developing a unique silver-based particles to manage the habitat of pathogenic bacteria on treated surfaces is the need. Synthesizing exopolysaccharides, several microorganisms have been shown to build biofilms, which protect them from harmful environmental stimuli. But the efficacy of plant based AgNPs on biofilm formation remains unchallengeable.\u003c/p\u003e \u003cp\u003eIn this study the reducing agent or polyphenols were extracted from a green source called \u003cem\u003eLagerstroemia speciosa\u003c/em\u003e leaf (LS) that is a plant native to tropical Asia and found in Northern Indian states [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is a great source of various bioactive compounds such as antidiabetic, anti-obesity, anticancer, anti-angiogenic, and anti-microbial [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A large quantity of LS is the large agricultural produce and its leaf contains a significant amount of bioactive materials such as Alkaloids, Carbohydrate, Terpenoids, Tannins, Polyphenols and Flavonoids [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These bioactive compounds could be used as capping agent and silver salt reducing agent due to their numerous OH groups that promote their biological activities [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As LS is cheap and easily available plant source, an effective method to extract bioactive compounds from LS and their nanoparticle synthesis will be a beneficial technology for better utilization at commercial level. Previously reported studies conducted by Sundararajan et al., and Sai Saraswathi et al., emphasizes need of new extraction technology for the synthesis of nanoparticles from LS. Those studies revealed the significant activity of silver nanoparticles from LS leaf on \u003cem\u003eStaphylococcus aureus, Proteus vulgaris, Klebsiella sp., Pseudomonas aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and the photocatalytic activity of nanoparticles degrading the various dyes like methyl orange and methylene blue which suggest synthesized using LS extract [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. But the new extraction technique is still required to synthesise the controlled synthesis of nanoparticles in terms of size, purity and environmentally friendly approach. SFE offers numerous advantages for the synthesis of nanoparticles, including high purity, precise control over particle size and morphology, environmental sustainability, and versatility. These benefits make SFE a valuable technique in various industries, from pharmaceuticals and food to cosmetics and environmental applications. The ability to produce high-quality nanoparticles with tailored properties opens up new possibilities for innovation and development in these fields.\u003c/p\u003e \u003cp\u003eThe combination of phytochemicals and their metal encapsulation, may cause synergistic effects against microorganisms and could be sources of palatable, natural substitutes. Along with that, degradation of thermolabile compounds, quality of extract are other circumstances that affects the synthesis of nanoparticles. SFE is one of the solutions for these circumstances. On other hand food and pharmaceutical industries have stated the desire to reduce the use of antibiotics and artificial chemicals as a food shield. Thus, the present work aimed to investigate the potential effect of optimal SFE extracted polyphenols and their encapsulated form on exposure to \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eEscherichia coli.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e is one of the potentially fatal biofilm-forming bacteria that belong to the Enterobacteriaceae family and is encapsulated, nonmotile, and rod-shaped bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Its biofilm formation protects itself against drugs and host immunological responses, which increases its pathogenicity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Certain virulence-related genes found in \u003cem\u003eK. pneumoniae\u003c/em\u003e play important roles in the production of biofilms. Moreover, type 3 fimbriae (mrkA) genes play a significant role in \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilm development [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, this study is specifically focused on the \u003cem\u003eK. pneumonia\u003c/em\u003e to evaluate the effect of nanoparticles on gram-negative bacterial biofilm inhibition. According to our research survey no information available on the synthesis of nanoparticles utilising supercritical CO\u003csub\u003e2\u003c/sub\u003e extract of LS for the biofilm inhibition \u003cem\u003eK. pneumonia\u003c/em\u003e so far. Although our research is the little contribution towards medicinal application of LS nanoparticles that can be used in orthopaedics, packaging, medical devices, footwear, household items as these are the fact that exists in terms of silver nanoparticles [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1. Chemicals and Plant sample collection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMedicinal plant LS was collected from Center of Innovative and Applied Bioprocessing campus, Mohali, Punjab, India. Leaves were washed with running tap water to remove external dust and dried using fluidized bed dryer, pilot scale with (50 L) capacity at 45 \u003csup\u003e0\u003c/sup\u003eC until they retain 8\u0026ndash;9% water content. The moisture was analyzed using aczet MB 50, 220 V \u0026ndash; 230 V (India) capacity moisture analyzer. The dried powder was passed through mass collider followed by sieving through 500 \u0026micro;m sieve to keep consistent particle size in each experiment. All analytical-grade chemicals including AgNO\u003csub\u003e3\u003c/sub\u003e were bought from HI-media Chemicals Ltd., Mumbai, India\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2. Preparation of plant extract\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs schematically depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the phenolic content extraction studies were performed utilising an SFE system (SFE-Helix, Allentown, USA). In this extraction technique, liquid CO\u003csub\u003e2\u003c/sub\u003e was delivered into the system using a twin piston pump after being fed from a CO\u003csub\u003e2\u003c/sub\u003e cylinder via a syphon tube. The pump was built to withstand pressures of up to 69 MPa. An extraction vessel for supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction was supported by this system. Through a preheater and a heating jacket placed around the vessels, the fluid was heated to its supercritical condition. To regulate the flow rate, a sturdy variable restrictor valve was employed. The restrictor was electrically warmed to prevent sample clogging.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA typical experiment involved weighing and loading a 50 ml extraction jar with roughly 10 g of dried leaf material. To stop solid materials from being transferred to the tubing and clogging the system, 1.5 g of glass wool was sandwiched between the leaf sample and the ends of the extraction basket. The extraction was carried out at 300 bar pressure, 363.15 K temperature and 50 minute extraction time. The extract was collected in an dark collection vial following the initiation of supercritical fluid extraction once the necessary extraction pressure and temperature had been achieved. The extract was stored at 4\u0026deg;C until the synthesis of silver nanoparticles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of TPC using Folin-Ciocalteu calorimetric assay and other phytochemical scrrenings\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe TPC of the extract was determined using the modified Folin-Ciocalteu method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The 0.5 mL extract was mixed with the Folin-Ciocalteau\u0026rsquo;s reagent (Sigma Aldrich) (0.25 mL) and 1.25 mL of 20% sodium carbonate solution. The mixture was incubated for 40 min at room temperature. The optical density of the blue-coloured samples was checked at 725 nm using a UV2700, UV\u0026ndash;vis spectrophotometer (Shimadzu, Kyoto, Japan). The TPC were expressed as mg of gallic acid equivalents/g dry weight of the biomass (mg GAE/g DWB) using gallic acid as a calibration standard. The concentration was determined using formula as follows [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003e\u0026#119879;\u0026#119875;\u0026#119862; = (\u0026#119862; x V)/\u0026#119872;\u003c/p\u003e \u003cp\u003eTPC\u0026thinsp;=\u0026thinsp;Total Phenolic Content (mg GAE/g DWB)\u003c/p\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;concentration of gallic acid equivalent (mg/gm)\u003c/p\u003e \u003cp\u003eV\u0026thinsp;=\u0026thinsp;volume of extracts used for analysis (mL)\u003c/p\u003e \u003cp\u003eM\u0026thinsp;=\u0026thinsp;Weight of the extract used for analysis (mg)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Identification of presence of Alkaloids\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section4\"\u003e \u003ch2\u003e2.3.1.1. Hager's Reagent Test\u003c/h2\u003e \u003cp\u003eSaturated Hager's solution (picric acid) is added in multiple drops to the 200 \u0026micro;L SFE-LS. A brilliant yellow precipitate indicates the presence of alkaloids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section4\"\u003e \u003ch2\u003e2.3.1.2. Mayer's Reagent Test\u003c/h2\u003e \u003cp\u003eAfter combining 200 \u0026micro;L of SFE-LS with a few drops of diluted HCl containing Mayer's reagent (potassium mercuric iodide solution), the mixture was heated for around five minutes. The presence of alkaloids is confirmed by the yellow color of the precipitate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Identification of presence of Carbohydrates\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e2.3.2.1. Molisch's Reagent Test\u003c/h2\u003e \u003cp\u003e200 \u0026micro;L of Molisch's reagent and 200 \u0026micro;L of SFE-LS were mixed. Solution was boiled followed by cooling. Production of violet rings shows the presence of carbohydrates.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Identification of presence of Tannins\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.3.3.1. FeCl\u003csub\u003e3\u003c/sub\u003e Test\u003c/h2\u003e \u003cp\u003e200 \u0026micro;L of FeCl3 solution is added to 100 \u0026micro;L of SFE-LS. The color violet indicates the presence of tannins.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. Identification of presence of proteins and amino acids.\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003ch2\u003e2.3.4.1. Ninhydrin Test\u003c/h2\u003e \u003cp\u003eAfter heating the SFE-LS in a 0.2% Ninhydrin solution, no violet color developed, signifying the lack of proteins and amino acids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section4\"\u003e \u003ch2\u003e2.3.4.2. Million's Test\u003c/h2\u003e \u003cp\u003eAfter adding 100 \u0026micro;L of Million's reagent to 100 \u0026micro;L of SFE-LS, no white precipitate developed after boiling, which proves the lack of proteins and amino acids.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5. Identification of presence of Saponins\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section4\"\u003e \u003ch2\u003e2.3.5.1. Foam Test\u003c/h2\u003e \u003cp\u003e100 \u0026micro;L of SFE-LS was mixed with 100 \u0026micro;L of water. Lack of foam formation even after shaking suggests the absence of saponins.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.3.6. Identification of presence of Gums \u0026amp; Mucilage\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section4\"\u003e \u003ch2\u003e2.3.6.1. Swelling Test\u003c/h2\u003e \u003cp\u003eAfter mixing 500 \u0026micro;L of 100% alcohol with 100 \u0026micro;L of SFE-LS, the mixture was agitated for 10 minutes. Lack of gums and mucilage is indicated by the absence of swelling in the solution.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Synthesis of SFELS-AgNPs\u003c/h2\u003e \u003cp\u003eThe SFE-LS in the preceding stage was employed for the environmentally friendly production of silver nanoparticles. 10 mL of SFE-LS (9.93 mg/mL) was added to 90 mL of a 1 mM aqueous silver nitrate solution. The mixture was then heated at 80\u0026deg;C while being constantly stirred at 400 rpm until the silver nitrate was completely reduced. The transition from yellow to dark brown was considered as a preliminary indicator of the synthesis of the SFELS-AgNPs. Centrifugation was done to separate the green-synthesised nanoparticles at 13,000 g for 10 min. from free silver. This process was carried out three times. The final silver nanoparticles produced via green synthesis, were freeze-dried and kept at 4\u0026deg;C until usage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.5. Characterization\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eUV\u0026thinsp;\u0026minus;\u0026thinsp;vis spectroscopy (HMG Labtech SPECTRO star, U.K.) was used to check the reduction of silver nitrate with SFELS extract in the range of 220\u0026thinsp;\u0026minus;\u0026thinsp;700 with 1 nm resolution.\u003c/p\u003e \u003cp\u003eThe Fourier Transform Infrared Spectrophotometer (FTIR) (Agilent, Model: Carry 660 series) was used to evaluate the functional groups in the phytoconstituents that were responsible for the reduction and capping of SFELS-AgNPs utilising the KBr pellet method. The Infrared (IR) rays was kept between 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a spectral resolution of 1 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe crystalline structure and phase of the SFELS-AgNPs was investigated using X-ray diffraction (XRD) study. The Cu Kα radiation (1.540 A\u0026deg; at 40 kV and 30 mA) were used in X-ray diffractometer (Rigaku\u0026reg;, SmartLab) with a scanning rate of 0.02 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the region of 2θ ranging between 30 and 90\u0026deg;.\u003c/p\u003e \u003cp\u003eScanning Electron Microscopy (SEM) coupled with Energy dispersive X-ray analyzer (EDX) (Hitachi S-4700) was used to investigate the external morphology, size, and atomic constituents of atoms in SFELS-AgNPs. Double-sided tape was used to secure the samples to the tubular metal stub. Gold was applied to the stub-supported samples. Ultimately, the samples coated in gold were examined under a microscope to determine their morphology.\u003c/p\u003e \u003cp\u003eTEM analysis was performed to determine the morphology, size and shape of the silver nanoparticles. TEM measurements were done by HITACHI H-800, operating at 200 kV. The TEM grid was prepared by placing a drop of the bio-reduced diluted solution on a carbon-coated copper grid and later drying it under a lamp.\u003c/p\u003e \u003cp\u003eWeight loss and thermal behaviour of the surface capped Ag nanoparticles was determined by using Thermogravimetric analyser in a Perkin Elmer STA 6000 (Perkin-Elmer SCIEX, Waltham, MA, USA) at a heating rate of 10\u0026deg;C/min.\u003c/p\u003e \u003cp\u003eDynamic light scattering (DLS) technique was used to determine the average hydrodynamic particle size distribution and surface charge (ζ potential) and polydispersity (PDI), with Zetasizer nano ZS (Malvern Instruments, Malvern, U.K.). The light scattering angle was set to 90\u0026deg; and the size measurements were performed at 25\u0026deg;C. Well dispersed solution was made by diluting the nanoparticles with Milli-Q water followed by ultrasonication for 10 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Antibacterial assay\u003c/h2\u003e \u003cp\u003eThe antibacterial efficacy of the SFELS-AgNPs against gram-positive and gram-negative bacteria was evaluated by agar well diffusion assay as per Clinical and Laboratory Standard Institute (CLSI) guidelines. The Mueller Hinton Agar (HIMEDIA, M173-500G) plate, pre-inoculated with \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MTCC 737), \u003cem\u003eBacillus cereus\u003c/em\u003e (MTCC 1306), \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e (MTCC109), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (MTCC 2488) and \u003cem\u003eE. coli\u003c/em\u003e was used to check the bioactivity. Wells were created aseptically on the seeded agar plate with the help of a cork borer (HIMEDIA, 6mm diameter). 200 \u0026micro;l sample volume was filled into the wells and allowed to diffuse at 4\u0026deg;C for an hour followed by incubation at 37\u0026deg;C for 24 hrs. The zone of inhibition was observed and measured using calliper.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Determination of MIC and MBC\u003c/h2\u003e \u003cp\u003eMICs of SFELS-AgNPs were determined by the broth microdilution method. The primary culture of \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e was grown overnight at 37˚C with shaking at 180 rpm. The secondary culture was obtained from the overnight-grown culture. These cells were further diluted to fix the number of cells that is, 10\u003csup\u003e6\u003c/sup\u003e cells per well of 96-well plate. 100 \u0026micro;l of the diluted culture was put into every 96 well-plates and incubated with a series of 2-fold dilutions of SFELS-AgNPs ranging from 512 \u0026micro;g/ml to 0.5 \u0026micro;g/ml, and the total volume was adjusted to 200 \u0026micro;l. The plate was incubated for 18 hrs. at 37\u0026deg;C with static conditions. The lowest concentration with no visible growth was recorded as MIC.\u003c/p\u003e \u003cp\u003eThe MBC was determined using the broth microdilution method. A 100 \u0026micro;l aliquot of each gradient concentrated solution from the MIC activity was transferred into a nutrient agar plate and incubated at 37˚C for 48 hrs. The lowest concentration of antimicrobials that kill 99.9% of the bacteria was defined as MBC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Single plate serial dilution spotting (SP-SDS) assay:\u003c/h2\u003e \u003cp\u003eThe primary culture of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e was grown overnight at 37˚C with shaking at 180 rpm. The secondary culture was obtained from the overnight-grown culture. These cells were further diluted to fix 10\u003csup\u003e6\u003c/sup\u003e cells in 200 \u0026micro;l volume. The concentration of SFELS-AgNPs was kept at 64 \u0026micro;g/ml for all. Cells treated with SFELS-NPs along with the untreated ones were incubated at 37˚C with shaking for 0, 2, 4, 6 \u0026amp; 12 hrs of incubation. All the incubated cells were 10-fold serially diluted till 10\u0026thinsp;\u0026minus;\u0026thinsp;6 dilutions and 5 \u0026micro;l of each dilution were spots on the agar plate. These plates were incubated at 37˚C for 16 hrs and the images were captured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Determination of biofilm inhibition using SEM\u003c/h2\u003e \u003cp\u003eThe effect of SFELS-AgNPs on biofilm was qualitatively studied using SEM. Biofilms were formed in 6-well cell culture plates (Nunc) having coated glass coverslips. Glass coverslips were coated with poly-L-lysine (2% w/v) and further sterilized by UV radiation for 1 hr. inside the biosafety cabinet and dried before use. These coverslips were put in microtiter plate wells for biofilm initiation in the presence and absence of antimicrobial compounds. The incubation was done at static conditions with a temperature of 37\u0026deg;C for 24 hours. Coverslips were moved to a new 6-well plate after the incubation and washed three times with 1X PBS. PBS-washed biofilms were later fixed by formaldehyde (4% vol/vol) and glutaraldehyde (2% vol/vol) for 20 min, accompanied by dehydration with a series of ethanol solutions. Tertiary-butyl alcohol was eventually used for dehydrating the slides for 30 min and then dried in a desiccator. After the drying process, coverslips were then finally coated with gold-palladium for 135 sec at the current of 10\u0026ndash;12 milliamperes and analyzed by scanning electron microscope (JEOL JSM-6000, NIKON Corporation) in high-vacuum mode at 10 kV.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Quantification of phenolic compounds in SFE-LS and preliminary phytochemical screening\u003c/h2\u003e \u003cp\u003eThe quantification of phenolic compounds was done using Folin-ciocalteu calorimetric assay. The SFE-LS showed 99.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57 mg/gm TPC of dry weight of biomass (DWB). As per the preliminary phytochemical screening done using protocol as mentioned in section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.3.1\u003c/span\u003e to \u003cspan refid=\"Sec18\" class=\"InternalRef\"\u003e2.3.6\u003c/span\u003e the contents of phytochemicals are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePreliminary phytochemical screening of the SFE-LS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQualitative test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSFE-LS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAlkaloids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmino acids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarbohydrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTannins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSaponins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGums \u0026amp; mucilage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.2. UV-vis study\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe synthesized silver nanoparticles in the colloidal solution were monitored by UV\u0026ndash;vis spectrophotometer analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA depicts the change in colour from yellow to dark brown that confer the reduction of AgNO\u003csub\u003e3\u003c/sub\u003e by phenolic compounds in extract. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows that the absorption spectra of silver nanoparticles formed in the reaction media has an absorbance peak at 430 nm. The increased absorbance at various time intervals (0 to 20 h) and the peak at 430 nm correspond to the surface plasmon resonance of silver nanoparticles. It is reported earlier that absorbance at around 430 nm for silver is a characteristic of these noble metal particles [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The λmax range found in the UV-Visible spectrum study is comparable to previous reports for AgNPs synthesized from pomegranate leaves [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A comparable analysis yielding essentially identical results was previously conducted in recent studies of AgNP production employing extracts from other plants, including \u003cem\u003eClerodendrum infortunatum, Azospirillum brasilense\u003c/em\u003e, and \u003cem\u003eAllium cepa\u003c/em\u003e (onion) [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.3. FTIR and XRD studies\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e show the FT-IR spectra of SFELS-AgNPs that confers the presence of functional groups in Ag nanoparticles. FT-IR spectrum showed the major peak positions at 3334 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2165 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2007 cm\u003csup\u003e-1\u003c/sup\u003e, 1727 cm\u003csup\u003e-1\u003c/sup\u003e, 1625 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1461 cm\u003csup\u003e-1\u003c/sup\u003e, 1349 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1041 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1041 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 524 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 455 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The peak at 3334 cm\u003csup\u003e-1\u003c/sup\u003e intimates OH group in phenolic compounds and carbohydrates [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The stretching vibrations of the aliphatic hydrocarbon chains (CH band) observed at 2,925 cm\u003csup\u003e-1\u003c/sup\u003e, either from the lipids' methyl or methylene groups in the SFE extract. The C\u0026thinsp;=\u0026thinsp;O stretch of aromatic alkenes in the phenolic content showed the band at 1625 cm\u003csup\u003e-1\u003c/sup\u003e. The band at 1,461 cm\u003csup\u003e-1\u003c/sup\u003e is of CH\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e3\u003c/sub\u003e of aliphatic chains. The band at 1349 cm\u003csup\u003e-1\u003c/sup\u003e is attributed to C\u0026thinsp;=\u0026thinsp;C. The stretching vibration of C\u0026ndash;O\u0026ndash;C is responsible for the prominent peak at 1,041 cm\u003csup\u003e-1\u003c/sup\u003e. The described FT-IR data suggested that the SFE extracts consist of bioactive compounds with functional groups like OH and COOH. These functional groups serve as stabilising agents in addition to being in charge of the AgNO\u003csub\u003e3\u003c/sub\u003e reduction [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eXRD pattern revealed distinct peaks at 2\u003cem\u003eθ\u003c/em\u003e values, which can be attributed to 111, 200, 220 and 311 crystalline planes of silver NPs at 38.02\u0026deg;, 44.16\u0026deg;, 64.34\u0026deg; and 77.26\u0026deg; as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. These peaks are associated with the face-centred cubic lattice. Bragg\u0026rsquo;s reflections of face centre cubic (fcc) structure of metallic silver respectively similar to Joint Committee on Powder Diffraction Standards (JCPDS) file no: ICDD-PDF2, Release 2007, PA, USA, 2007, revealing that synthesized nanoparticles are of pure crystalline silver. The crystalline size of the SFELS-AgNPs formed were calculated using Debye\u0026ndash;Scherrer equation (Eq.\u0026nbsp;1) which was around 13.48 nm, were good in agreement with TEM results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eD= (kλ /β cos θ) \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;. (1)\u003c/p\u003e \u003cp\u003eWhere, D is size of particle, k is Scherrer\u0026rsquo;s constant, λ is the X-ray wavelength, β is full width at half maximum (FWHM)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.4. FESEM, HRTEM and EDAX analysis\u003c/h2\u003e \u003cp\u003eThe morphology of SFELS-AgNPs was analyzed through FESEM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The majority of particles were spherical in shape and there were a few oval AgNPs as well. Biosynthesized nanoparticles had been spread thoroughly in the solution. The size of some selected biosynthesized nanoparticles was around 50 nm according to FESEM images. These results strongly confirmed that SFE-LS extract acts as a reducing and capping agent for the synthesis silver nanoparticles. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB demonstrated energy dispersive spectrum of the SFELS-AgNPs, which has silver as a ingredient element. Silver nanoparticles generally shows the peak at 3 keV, due to its surface plasmon resonance. The fig also showed the presence of C, O due to the reaction with mixed phenolic groups. This is one of the advantages of synthesis of silver nanoparticles using plant extract. Results of EDAX in this research are conservative with the silver nanoparticles synthesized using \u003cem\u003eRheum palmatum\u003c/em\u003e and \u003cem\u003eColius aromaticus\u003c/em\u003e extract [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe morphology and size of the synthesized silver nanoparticles were determined by TEM images and they are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B. The particles formed were spherical in shape. It was determined that the generated nanospheres have a large surface area. The size of the formed nanoparticles ranged from 8 to 20 nm. The average particle size is 14 nm which is share exactly similar results by Saraswathi et al., 2017 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.5. DLS and zeta potential of SFELS-AgNps\u003c/h2\u003e \u003cp\u003eUsing the DLS technique, the surface zeta potential of synthesized SFELS-AgNPs in aqueous colloidal solution was measured. The negative zeta potential was determined to be \u0026minus;\u0026thinsp;25.6 mV in this investigation, with a zeta deviation of 4.63 mV. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. The zeta potential values give us the information regarding the surface charge of nanoparticles which appeared to be negative here. The high negative value indicated that synthesized silver nanoparticles did not agglomerate. Additionally, it gives us an idea about the stability of nanoparticles and the zeta potential value obtained for SFELS-AgNPs lays within the stable range, signifying the nanoparticle are stable in aqueous solution [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The zeta potential range of \u0026plusmn;\u0026thinsp;30 mV is thought to be the most stable for silver nanoparticles [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA demonstrates that the average particle size of SFELS-AgNps is around 116 nm with the polydispersity index of 0.244.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Weight loss and thermal behaviour\u003c/h2\u003e \u003cp\u003eThermogravimetric analysis of SFELS-AgNPs was carried out to examine the influence of temperature on nanomaterial. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows that the nanoparticles lost its first weight at 102.56 \u0026ordm;C. which may be due to the moisture in material. Temperature between 210.96 \u0026ordm;C to 348.18 \u0026ordm;C almost lost 30% of its weight. Nanoparticles almost started degrading after 348.18 \u0026ordm;C, which suggest the threshold of thermal sensitivity of the material.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Antibacterial activity\u003c/h2\u003e \u003cp\u003eThe growth inhibition by SFELS-AgNPs against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e was studied using well diffusion method. To describe the expansion of the clean zone, three different concentration 100 \u0026micro;g/mL, 200 \u0026micro;g/mL and 400 \u0026micro;g/mL of silver capped SFE-LS extract and SFE-LS extract itself were used to check the concentration dependent growth inhibition. Results were compared with standard antibacterial drug Chloramphenicol and Gentamycin. SFELS extract showed low clear zone but the SFELS-AgNPs showed significant antibacterial activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The zone of inhibition at every sample was counted using a pair of callipers. The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBactericidal activity (Zone of inhibition in mm) of SFE-LS-NPs against pathogenic bacteria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSr.\u003c/p\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eName of samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eBacterial Zone of inhibition (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eGram positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eGram negative\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e (MTCC1306)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e (MTCC737)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e (MTCC109)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e (MTCC2488)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 SFELS-AgNps\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 SFELS-AgNps\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400 SFELS-AgNps\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChloramphenicol/ Gentamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 SFE-LS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 SFE-LS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400 SFE-LS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eND: Not determined\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe bacterial cell membrane damages due to electrostatic interference of AgNPs which form pores on the surface, that alters the structure of the bacteria and ultimately leads to cell death. As a result, the zone of inhibition increased with concentration [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The AgNPs in prokaryotic bacteria interact with the bacterial cell membrane, bind to the mesosomal cell organelle, impair mesosome function, and enhance the production of reactive oxygen species (ROS). Silver nanoparticles bind with thiol groups in proteins, which inactivates both DNA replication and protein synthesis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Simultaneously, oxygen combines with silver and reacts with the sulfhydryl (-S-H) groups on the bacterial cell wall to eliminate the -H atoms. This prevents the sulphur atoms from forming an R-S-S-R bond and kills the bacterial cells by preventing respiration [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The silver nanoparticles are responsible for the disruption of membrane integrity in bacteria and even limits the growth of bacteria by mitigating oxygen, sulphur and nitrogen in essential biological molecules. Despite numerous papers describing the antibacterial action of AgNPs produced by plant materials, the plant extract often exhibited little to no inhibition while the synthesized SFELS-AgNPs displayed minimal to moderate and good to exceptional activity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The results of this study intimated that SFELS-AgNPs are more effective on \u003cem\u003eK. pneumoniae\u003c/em\u003e compared to other bacteria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.7 Evaluation of MIC and MBC\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe effectiveness of SFELS-AgNPs was further confirmed by determining the MIC and MBC against the \u003cem\u003eK. pneumonia\u003c/em\u003e based on the agar well diffusion assay. The SFELS-AgNPs showed a MIC of 64 \u0026micro;g/ml whereas the MBC was determined as 128 \u0026micro;g/ml. The experiment was repeated three times and each concentration was done in triplicates. Our results showed that SFELS-AgNPs are more successful in suppressing the growth of bacteria, confirming the theory that the antibacterial activity is strongly influenced by the modification of the nanoparticles-bacteria interface [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Several studies reported that the major reason of AgNPs stop the growth of microorganisms by interfering with microbial DNA replication, contact death, and the generation of reactive oxygen species [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Effect of SFELS-AgNPS on \u003cem\u003eK. pneumonia\u003c/em\u003e biofilm formation\u003c/h2\u003e \u003cp\u003eThe MIC (64 \u0026micro;g/ml) was used to evaluate the effect of SFELS-AgNPS on bacterial cells. The biofilm formation was assessed in control (Untreated) and treated strains of \u003cem\u003eK. pneumonia\u003c/em\u003e as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, that revealed cells in the untreated biofilm are congregated and intact. After the treatment, biofilm breaks and cells dispersed and the breakages of cells (red arrow) are also visible. Numerous studies showed the herbal nano formulations has potential to reduce biofilm [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The suppression of biofilm formation may be the result of SFELS-AgNPs penetrating the bacterial cell wall, suppressing the synthesis of exo-polysaccharide layers, and interfering with cell adhesion and communication [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Mousavi et al., recently studied biofilm reduction in \u003cem\u003eK. pneumonia\u003c/em\u003e strains using biologically synthesized silver nanoparticles their results are comparable with this study as they have obtained significant reduction at 128 \u0026micro;g/mL of nanoparticles [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Additionally, prior research indicated that these NPs might be effective antibacterial agents against a variety of bacterial infections, such as \u003cem\u003eK. pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough several processes have been hypothesized, the precise mechanism by which AgNPs exert their antibacterial effect remains unknown. The capacity of these NPs to attach the bacterial cell wall and their subsequent entry into the cell, which causes structural alterations in the cellular membrane and causes cell lysis and content leaking, is one of the hypothesized mechanisms [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Biofilm-forming microorganisms can cause a wide range of illnesses. In fact, a report from the National Institutes of Health and the Centers for Disease Control states that 65\u0026ndash;80% of infections are the cause of these microbes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, use of biofilm inhibitors is one of the effective ways to reduce the diseases caused by these microorganisms. Numerous studies have reported the effect of AgNPs as biofilm inhibitors against multi drug resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. We discussed how AgNPs may be used to target \u003cem\u003eK pneumoniae\u003c/em\u003e biofilm development in the current investigation. The current findings demonstrated that at 64 \u0026micro;g/ml concentration of SFELS-AgNPs, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strain was failed to generate biofilms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e3.9. SP-SDS Procedure\u003c/h2\u003e \u003cp\u003eBacterial dilution spotting (10-fold) assay was performed to quantify the growth and sensitivity of \u003cem\u003eK. pneumonia\u003c/em\u003e upon treatment with SFELS-AgNPs using MIC. A fixed SFELS-AgNPs concentration (64 \u0026micro;g/ml) was used with varying treatment times (0, 2, 4, 6 \u0026amp; 12 hrs). The sensitivity of \u003cem\u003eK. pneumonia\u003c/em\u003e could be correlated with the percentage of cells that survive and form a colony. As evident in the result, with increasing incubation time, the viability decreases at 10\u0026thinsp;\u0026minus;\u0026thinsp;4, 10\u0026thinsp;\u0026minus;\u0026thinsp;5, \u0026amp; 10\u0026thinsp;\u0026minus;\u0026thinsp;6 dilution after 2 hrs treatment; 10\u0026thinsp;\u0026minus;\u0026thinsp;3, 10\u0026thinsp;\u0026minus;\u0026thinsp;4, 10\u0026thinsp;\u0026minus;\u0026thinsp;5, \u0026amp; 10\u0026thinsp;\u0026minus;\u0026thinsp;6 dilution after 4 hrs treatment and 10\u0026thinsp;\u0026minus;\u0026thinsp;2, 10\u0026thinsp;\u0026minus;\u0026thinsp;3, 10\u0026thinsp;\u0026minus;\u0026thinsp;4, 10\u0026thinsp;\u0026minus;\u0026thinsp;5, \u0026amp; 10\u0026thinsp;\u0026minus;\u0026thinsp;6 dilution after 6 hrs treatment and hence there is no or less bacterial colony visible at the spot (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). At 12 hours of treatment, complete growth inhibition can be seen at all the dilutions. These results suggests that synthesized nanoparticles are capable of complete inhibition of the \u003cem\u003ek. pneumoniae\u003c/em\u003e on 12 hrs of treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn the current research, methodology was developed for the synthesis of silver nanoparticles using supercritical fluid extracted polyphenols from LS leaves, as reducing agent. The extract supports the synthesis of nanoparticles at room temperature with a quick kinetics and without the use of any hazardous chemicals, therefore the results are quite encouraging. Research using UV-vis spectroscopy reveals that LS is a plant that is abundant in bioactive compounds like polyphenols that can be used as reducing agents and may also be found in other plants. These compounds when encapsulated with stabilisers for silver nanoparticles, the same chemical mechanisms gave them significant antibacterial properties. According to the results SFELS-AgNPs showed highest antibacterial activity against \u003cem\u003eK. pneumoniae\u003c/em\u003e with MIC 64 \u0026micro;g/ml and MBC 128 \u0026micro;g/ml. Furthermore, it significantly inhibited the \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilm formation. The development of biofilm plays a crucial part in bacterial pathogenicity, shielding the bacteria from antibiotics. In our investigation, plant-mediated synthesized AgNPs shown initial biofilm formation inhibitory action. Therefore, LS-mediated AgNPs may be a novel approach to treating infections linked to bacterial biofilms. According to TEM, the diameter of the silver nanoparticles is between 8 and 20 nm. Interestingly, XRD results revealed that the population of silver nanoparticles is made up of a variety of face-centered cubic forms. This technique revealed the presence of the unusual hexagonal crystal structure 4H for silver nanoparticles, providing a novel way to investigate the antibacterial capabilities and optical response of this nanomaterial. Moreover, SFELS-AgNPs could be added to formulations to create antibacterial goods like hand washes, sanitizers, and creams to reduce the spread of harmful bacteria in the environment, public spaces, hospitals, etc. To address issues with the usage of nanomaterials, especially their pharmacokinetics and pharmacodynamics properties, more research is necessary. As per the safety concerns, clinically testing of nanoparticles is also required.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to acknowledge Center of Innovative and Applied Bioprocessing for research support and Council of Scientific and Industrial Research. They would also like to acknowledge Panjab University, Chandigarh.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKiran Khandare: Investigation, Experimental analysis, Writing-original draft, Manuscript editing and revision; Shekhar Kumar: Experimental analysis, Manuscript editing and revision; Sukesh Chander Sharma: Supervision, Manuscript review, and editing; Saswata Goswami: Supervision, Conceptualization, Manuscript review, and editing, Resources, Funding acquisition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding is obtained for this work\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author can provide the data that were utilized to support the study\u0026apos;s conclusions upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch Involving Humans and Animals Statement-None.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed Consent- None. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTan, P., Li, H. 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Bacterial biofilm and associated infections. \u003cem\u003eJournal of the chinese medical association\u003c/em\u003e, \u003cem\u003e81\u003c/em\u003e(1), 7\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lagerstroemia Speciosa, Supercritical fluid extraction, Silver Nanoparticle, Klebsiella pneumoniae, Biofilm, Antimicrobial","lastPublishedDoi":"10.21203/rs.3.rs-4679230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4679230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA large number of scientists are now working in order to create silver nanoparticles (AgNPs) that can be used as biomedicines against cancerous cell lines and bacteria that are resistant to drugs. In the current study, optimal supercritical fluid extract (SFE) of \u003cem\u003eLagerstroemia speciosa\u003c/em\u003e (LS) leaves at pressure 29.59 MPa, temperature 89.50 \u0026ordm;C and extraction time 53.85 min. was used to extract phenolic compounds for the synthesis of AgNPs. The synthesis was studied for 0\u0026ndash;20 hrs. Initially the synthesis was confirmed by observing change in colour phenomenon. UV -spectroscopy confirmed the synthesis of nanoparticles (SFELS-AgNPs) demonstrated a maximum surface plasmon resonance at 430 nm. The crystallite dimension of nanoparticles was determined using XRD (13.47 nm), TEM results confirmed the diameter of the obtained silver nanoparticles between 8\u0026ndash;20 nm. The nanoparticles possessed \u0026minus;\u0026thinsp;25.6 mV electric charge on the surface confirmed using zeta potential analyser. Furthermore, energy-dispersive X-ray analysis (EDAX), was used to analyze the presence of differential elements in generated materials. The developed nanoparticles were evaluated for their potential antimicrobial properties against, two gram-positive viz. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eBacillus cereus\u003c/em\u003e, and three gram-negative bacteria viz. \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e with different concentrations (100\u0026ndash;400 \u0026micro;g/mL). The nanoparticle showed a minimum inhibitory concentration (MIC) of 64 \u0026micro;g/ml whereas the minimum bactericidal concentration (MBC) 128 \u0026micro;g/ml against \u003cem\u003eK. pneumonia\u003c/em\u003e. They significantly inhibited \u003cem\u003eK. pneumonia\u003c/em\u003e biofilm formation confirmed using scanning electron microscopy (SEM). The results were encouraging compared to the standards drug Chloramphenicol and other controls. The generated nanoparticles have highly effective antimicrobial properties against pathogenic bacteria.\u003c/p\u003e","manuscriptTitle":"Biosynthesis of silver nanoparticles using supercritical CO 2 mediated phenolic contents extracted from Lagerstroemia speciosa leaf inhibits Klebsiella pneumoniae biofilm formation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 18:24:03","doi":"10.21203/rs.3.rs-4679230/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ccd5d8b-22be-45df-81a2-1d4e06fc0d33","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-09T16:56:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 18:24:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4679230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4679230","identity":"rs-4679230","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}