{"paper_id":"293be6d4-f171-4e62-aaa7-7374f6d8f6d0","body_text":"License and Terms: This document is copyright 2022 the Author(s); licensee Beilstein-Institut.\nThis is an open access work under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0). Please note that the reuse,\nredistribution and reproduction in particular requires that the author(s) and source are credited and that individual graphics may be subject to special legal provisions.\nThe license is subject to the Beilstein Archives terms and conditions: https://www.beilstein-archives.org/xiv/terms.\nThe definitive version of this work can be found at https://doi.org/10.3762/bxiv.2022.62.v1\nThis open access document is posted as a preprint in the Beilstein Archives at https://doi.org/10.3762/bxiv.2022.62.v1 and is\nconsidered to be an early communication for feedback before peer review. Before citing this document, please check if a final,\npeer-reviewed version has been published.\nThis document is not formatted, has not undergone copyediting or typesetting, and may contain errors, unsubstantiated scientific\nclaims or preliminary data.\nPreprint Title Assessment of the antimicrobial effect of silver nanoparticles\nsynthesized from Coriandrum sativum\nAuthors Luisa F. Barraza-Vergara, María A. Llinás-Giraldo, Alberto R. Albis\nArrieta, Ana M. Medina Buelvas and Anahí Barros Martínez\nPublication Date 13 Jul 2022\nArticle Type Full Research Paper\nORCID® iDs Luisa F. Barraza-Vergara - https://orcid.org/0000-0002-8320-3828;\nAlberto R. Albis Arrieta - https://orcid.org/0000-0003-1758-1385\n\n1 \nAssessment of the antimicrobial effect of silver \nnanoparticles synthesized from Coriandrum sativum \nLuisa F. Barraza-Vergara*1, María A. Llinás-Giraldo1, Alberto R. Albis Arrieta1, Ana M. \nMedina Buelvas2, and Anahí Barros Martínez2 \n \nAddress:  \n1Department of Chemical Engineering , Bioprocesses Laboratory, Universidad del \nAtlántico, KM 7 Puerto Colombia, Atlántico, Colombia and 2Department of \nMicrobiology, Laboratory of Immunology and Molecular Biology, Universidad Libre, Cra \n51B #135 -100, Barranquilla, Atlántico, Colombia. \n \nEmail: Luisa Fernanda Barraza Vergara; lfbarraza@mail.uniatlantico.edu.co \n* Corresponding author \n\n2 \nAbstract \nIn this study the antimicrobial effect of silver nanoparticles (AgNPs) synthesized from \nCoriandrum sativum (C. sativum) leaf extract was evaluated by varying the incubation \ntemperature from 25 °C to 40 °C, for 24 h of exposure. AgNPs were characterized \nusing Ultraviolet-Visible (UV-vis), and Transmission Electron (TEM) spectroscopy, \nevidencing the presence of nanoparticles of spherical morphology with average size s \nof 5.7, 13. 4, and 6.0 nm for nanoparticle concentrations of 1, 10 , and 100 mM, \nrespectively. The efficacy of nanoparticles as microbicidal agents was evaluated with \nthe halo inhibition method, impregnating nanoparticles in sensi -discs on agar planted \nwith ATCC standard Staphylococcus aureus  (S. aureus ), and clinically isolated  \nKlebsiella pneumoniae (K. pneumoniae ) and Pseudomonas aeruginosa  (P. \naeruginosa). AgNPs inhibited the growth of all three bacteria under all conditions. The \nclinically isolated bacteria obtained the smallest inhibitory diameters, with respect to \nthe standard bacteria. Incubation temperature had a significant effect on all bacteria, \nwith the greatest effect found with AgNPs -100mM at 25°C for P. aeruginosa and at \n35°C for S. aureus and K. pneumoniae. These results showed the potential application \nagainst pathogenic microorganisms of AgNPs synthesized from C. sativum leaf in \nhospital environments within a temperature range from 25 to 40 °C. \nKeywords \nSilver nanoparticles; antimicrobial effect; S. aureus; K. pneumoniae; P. aeruginosa; C. \nsativum; hospital environments \n \n\n3 \nIntroduction \nSilver nanoparticles (AgNPs) have emerged as novel nanomaterials for biological \napplications e.g.,  antibacterial and fungal agents.1–3 AgNPs synthesized from \nCoriander sativum (C. sativum ) are of great interest as an alternative ecofriendly \nsynthesis method. N anoparticle sizes <50 nm have been reported using C. sativum, \nwhich increases their interaction with the microorganisms of interest.4–6 However, there \nmust remain plenty of potential antimicrobial applications for Coriander Sativum-based \nAgNPs that remains unexplored. \nStaphylococcus aureus  (S. aureus ), Klebsiella pneumoniae  (K. pneumoniae ) and \nPseudomonas aeruginosa (P. aeruginosa) are multidrug-resistant bacteria of notable \nclinical importance due to their rapid spread. 7–10 Senthilkumar et al . studied the \ninhibition of these bacteria after exposing them for 24 h at 37 °C to AgNPs synthesized \nwith C. sativum  stems.11 Alsubki et al ., evaluated the antimicrobial effect, ATCC \nstandard bacteria  and 16 h of exposu re at 37 °C. Ashraf et al . investigated the \nantimicrobial potential of C. sativum  leaf-mediated AgNPs against  S. aureus.6 \nDeshpande et al. used AgNPs against S. aureus and K. pneumoniae at 37 °C.12  \nS. aureus , K. pneumoniae and P. aeruginosa  have the versatility to survive and \nreproduce over an  ample temperature range (4 to 46 °C) , which increases the \nprobability of contamination of the human-host in hospital environments. 13–15 On the \nother hand, it has been reported that clinically isolated bacteria including S. aureus and \nP. aeruginosa  are le ss susceptible to antibacterial agents than standard bacteria. \nTherefore, it is important to evaluate the antimicrobial effect of AgNPs under conditions \nthat mimics the hospital environment, i.e., against isolated bacteria and temperatures \nfrom 25 °C to 40 °C. \n\n4 \nIn the present study , environmentally friendly AgNPs were synthesized using C. \nsativum leaf, with three different concentrations of 1, 10, and 100 mM . Antimicrobial \nproperties of C. sativum  leaf-based AgNPs were studied against hospital isolated \npathogenic bacteria K. pneumoniae and P. aeruginosa, and ATCC standard S. aureus, \nduring 24 h of exposure at 25, 35, and 40 °C. \nResults and Discussion \nCharacterization of the Extract \nThe infrared spectrum of the C. sativum leaf extract (see Figure 1) shows a peak \nbetween 1568-1660 cm-1, characteristic of the functional group C -C (Alkane). Intense \nabsorption bands could also be noticed in regions of the infrared spectrum between \n3221-3508 cm -1, showing  OH groups from water, carbohydrates , and amides. \nAbsorption of 1367-1435 cm-1 is attributed to the vibrations of the C-O amide and peaks \nbetween 1028-1120 cm-1 correspond to the carboxylic acid group (COOH). In addition, \na band between 2877-2956 cm-1 is associated with the functional group C-H (Alkane). \n5,16,17  \n \nFigure 1: Fourier-transform infrared spectroscopy spectrum of coriander leaf extract \n\n\n5 \nCharacterization of Silver Nanoparticles: \nThe  color change from colorless to brown  when synthesizing the AgNPs  \ndemonstrates the reducing properties of coriander attributed to secondary metabolites \nsuch as flavonoids that may reduce silver ions to nanoparticles counterpart. 1,4,5 \nUV-vis Characterization: \nUV-vis spectra of silver nanoparticles suspended in NaOH with a pH ~ 10 are shown \nin Figure 2.  AgNPs were labelled as AgNPs-1mM, AgNPs-10mM, and AgNPs-100mM \nfor AgNPs obtained using AgNO3 solutions at concentrations of 1, 10, and 100 mM, \nrespectively. Maximum absorption peaks, determined by UV -vis spectroscopy, \nappeared at 421 nm, 423  nm and 430 nm  for the AgNPs -1mM, AgNPs-10mM and \nAgNPs-100mM samples, respectively. These values confirm ed the presence of \nAgNPs.18 \n  \nFigure 2: UV-vis spectra of  C. sativum leaf-based AgNPs-1mM, AgNPs-10mM, and \nAgNPs-100mM. \n\n\n6 \nTEM Characterization: \nTEM analysis wa s performed on samples of reduced AgNPs with C. sativum to \nestablish the presence, size, and morphology of AgNPs suspended in aqueous \nmedium. Figures 3a, 3b, and 3c shows TEM images of AgNPs synthesized, which \ndemonstrate a predominant spherical morphology for each concentration. The images \nshow a good dispersion of AgNPs in the aqueous  medium without agglomerations.  \nThe average nanoparticles diameter found was 5.7 nm , 13.4 nm and 6.0 nm for \nAgNPs-1mM, AgNPs-10mM, and AgNPs -100mM, respectively. The obtained \nnanoparticles sizes is between the typical sizes previously reported , i.e., 8 to 75 nm.5 \nThe nanoparticles obtained by this method of synthesis are relatively small, which \nmakes them have a greater contact surface area, thus increasing their reactivity and \ntheir antimicrobial activity. \n \nFigure 3: TEM characterization of (a) AgNPs-1mM, (b) AgNPs-10mM and (c) AgNPs-\n100mM with scale bar 50 nm.  \nAntimicrobial effect of AgNPs against bacteria  \nThe antimicrobial effect of the AgNPs was determined against three multidrug-\nresistance bacteria according to the World Health Organization (WHO) with priority 1 \n(S. aureus) and priority 2 (K. pneumoniae and P. aeruginosa).7 The average inhibition \n(a)                                           (b)                                         (c) \n \n\n\n7 \nhalo for each condition is shown in Table 1. The positive control of the standard \nantibiotic Ciprofloxacin was used, which had an inhibitory effect on the three bacteria. \nTable 1: Main effects of inhibition halo formed by AgNPs against bacteria. \nTemperature \nSilver nitrate \nconcentration (mM) \nAverage inhibition halo (mm) \nS. aureus \nK. \npneumoniae \nP. \naeruginosa \n25 °C \n1 - a 6.3±0.2 8.0±0.1 \n10 - a 6.7±0.4 8.3±0.4 \n100 - a 6.8±0.4 9.0±0.1 \n35 °C \n1 8.1±1 7.6±0.5 7.7±0.6 \n10 10.1±1 8.2±1 8.4±0.6 \n100 12.1±0.9 8.6±0.5 8.7±0.5 \n40 °C \n1 7.2±0.4 7.2±0.4 6.5±0.1 \n10 8.2±0.4 8.4±0.2 7.5±0.1 \n100 9.5±0.7 8.5±0.1 8.0±0.1 \naThe bacteria did not grow in this condition. \nSynthesized C. sativum leaf-based AgNPs induced growth inhibition in all three \nbacteria studied. The largest average diameter of inhibition was obtained with the \nATCC standard bacteria S. aureus , which may be du e to the higher susceptibility \ncompared to clinically isolated bacteria  K. pneumoniae  and P. aeruginosa . \nFurthermore, gram -positive bacteria have an absence of the outer membrane \ncharacteristic of gram -negative bacteria, which makes them more resistant to \nantimicrobials. However, gram-positive bacteria have a thicker peptidoglycan layer that \nprotects it from penetration by silver ions.19,20 \n\n8 \nThe halo is indicative of bacterial growth inhibition o n a Mueller Hinton agar plate \ninoculated with each bacterium. Incubation temperature and AgNO3 concentration for \nnanoparticle synthesis seem to have a significant effect on the average diameters of \nthe inhibition halos (ANOVA P-Value <0.05). However, the combination of both factors \n(temperature and concentration)  only had a significant effect on S. aureus , which \nmeans that the impact of the concentration of AgNPs on the diameter of the inhibition \nzone depends on the temperature used. The maximum diameter indicated in S. aureus \nand K. pneumoniae was 12.1 and 8.6 mm, respectively, both obtained at 35  °C and \nAgNPs-100mM. In contrast, P. aeruginosa showed the largest halo of 9  mm at 25 °C \nand AgNPs-100mM.  \nThe increase in the concentration of the synthesized C. sativum leaf-based AgNPs has \na greater antimicrobial efficacy against the three bacteria studied. This result is \nattributed to the increased antimicrobial properties of silver at the nanoscale. Due to \nAgNPs have a high surface area, the chemical reactivit y of the surface is increased, \nwhich enhances interaction with bacteria. 21 Although the mechanism of action of \nAgNPs in bacteria is not completely clear, it have been  suggested that AgNPs can \ndenature the outer membrane or peptidoglycan layer of bacteria.22 This action induces \nthe depletion of intracellular ATP due to the electrostatic attraction between the positive \ncharge of the nanoparticles and the negative charge of the cell wall of the \nmicroorganisms.22 The increased antimicrobial activity of the AgNPs obtained using a \nconcentration of 100 mM is probably a result of a higher concentration of AgNPs and \nnot of the change of the dimensions of the nanoparticles . It should be noted that the \nonly component that is exerting inhibition against growth is AgNPs, since the controls \nthat were performed  (see Figure 4), do not inhibit the growth of these bacteria. \nCiprofloxacin was also used as standard and positive control to show antimicrobial \n\n9 \nactivity, where inhibition halos of 2.8 cm, 3.2 cm and 2.2 cm were obtained for \nStaphylococcus Aureus , Klebsiella pneumoniae and Pseudomonas aeruginosa , \nrespectively. Figures 5, 6 and 7 represent the inhibition halos formed by AgNPs-1mM, \nAgNPs-10mM, and AgNPs-100mM, at 35°C against Staphylococcus Aureus , \nKlebsiella pneumoniae and Pseudomonas aeruginosa, respectively. \n \nFigure 4: Controls: Ciprofloxacin (+), Coriandrum sativum (-1) sodium hydroxide (-2) \nagainst bacteria: (a) Staphylococcus Aureus, (b) Klebsiella pneumoniae and (c) \nPseudomonas aeruginosa. \n \nFigure 5: Inhibition halo formed by (a) AgNPs-1mM, (b) AgNPs-10mM, and (c) AgNPs-\n100mM, at 35°C against S. aureus. \n\n\n10 \n \nFigure 6: Inhibition halo formed by (a) AgNPs-1mM, (b) AgNPs-10mM, and (c) \nAgNPs-100mM, at 35°C against K. pneumoniae. \n \nFigure 7: Inhibition halo formed by (a) AgNPs-1mM, (b) AgNPs-10mM, and (c) \nAgNPs-100mM, at 35°C against P. aeruginosa. \nConclusion  \nSynthesized C. sativum leaf-based AgNPs proved to be antimicrobial agents against \nbacteria of clinical interest and multidrug -resistant S. aureus, K. pneumoniae and P. \naeruginosa. The increase in the concentration of AgNO3 in the synthesis of AgNPs was \ndirectly correlated with the inhibition halo. Clinically isolated bacteria K. pneumoniae \nand P. aeruginosa were more resistant to AgNPs, with lower inhibition halos compared \nto standard S. aureus bacterium. Furthermore, temperature had a significant effect on \nthe inhibition of all three bacte ria, implying that C. sativum leaf-based AgNPs appear \n\n\n11 \nto be viable for use as an antimicrobial against clinically isolated pathogenic bacteria \nin hospital environments with a temperature range from 25 °C to 40 °C. \nExperimental  \nPreparation of Coriander Leaf Extract \n200 g of coriander leaves gathered from a local market, were used and prepared \naccording to the procedure described in the literature.4 In a typical synthesis, 100 g of \nleaves washed with distilled water were used and their size was reduced by cru shing \nin a mortar and pestle. Then it was poured into a beaker with 250 mL of distilled water \nand heated on a plate with continuous stirring until boiling (~300 rpm and 10 minutes). \n100 g of additional leaves were then added with continued stirring and hea ting. \nSubsequently, the leaves were removed, and the concentrated extract was heated until \nits volume was reduced by evaporation to  50 mL. The final extract was filtered, \ndeposited in glass vials, and cooled to room temperature. \nSynthesis of AgNPs \nA solution of AgNO3 purchased from Merck Millipore (Emsure, ACS reagent) was used \nas a precursor. A stock solution of 100 mM AgNO3 was prepared, and dilutions of 10, \nand 1 mM were made .23 1.5 mL of coriander extract was combined with 25 m L of \nAgNO3 solution. Subsequently, the temperature of the solution was increased to 55 °C \nand nanoparticles were collected by centrifugation at 7500 rpm for 20 min. The AgNPs \nwere suspended in distilled water adding 0.1 M NaOH (purchased from Merck \nMillipore) drops until reaching a pH of 10. The suspensions were brought to ultrasound \n\n12 \nfor 30 minutes, allowed to settle  for a period of one week, to remove any precipitate \nthat may had formed  \nCharacterization     \nTo carry out the characterization of the coriander leaf ex tract, an IRAffinity model \nFourier transform infrared spectroscope (FTIR) was used, with a resolution of 4 cm -1 \nand a wavelength range between 4000 and 400 cm -1. The characterization of the \nAgNPs was carried out through the ultraviolet visible spectroscope  (UV-Vis) model \nEvolution 60S to determine the wavelength between 400 -450 nm using samples \ndiluted at 1% v/v. TEM analysis was performed using the high resolution (0.1 nm) FEI \nTecnai G2 F20 S-TWIN HR(S)TEM equipment. \nAssessment of the Antimicrobial Effect of AgNPs \nTo evaluate the antimicrobial activity of AgNPs, gram-negative bacteria strains K. \npneumoniae and P. aeruginosa  were isolated from local hospital samples and t he \ngram-positive bacteria S. aureus was purchased to Thermo Scientific (ATCC 25923). \nTo determine the effectiveness of AgNPs as a microbicidal agent, the disk diffusion or \ninhibition halo method was used.24  Each inoculum of bacteria was suspended in saline \nand turbidity was adjusted to McFarland's 0.5 standards (~1.5x108 CFU/ml).25 Petri \ndishes were prepared with 15 m L of Mueller -Hinton Agar and the entire plate was \ninoculated homogeneously with a sterile cotton swab. Circular filter paper discs of 6 \nmm of diameter were soaked with 10 µl of AgNPs dispersion of 1 mM, 10 mM and 100 \nmM were put on the freshly seeded agar and incubated at 25 °C, 35 °C, and 40 °C for \n24 hours. After the incubation period, the diameter of the halo around the filter paper \nwas determined. Negative controls were NaOH  and coriander extract and positive \ncontrol Ciprofloxacin Sensi-disc (Becton, Dickinson, and Company). \n\n13 \nStatistical Analysis \nA multifactorial analysis of variance ANOVA was carried out to determine the statistical \nsignificance of each factor (concentration of silver nitrate solution and incubation \ntemperature) and the interactions between them on the growth of the inhibition halo. \nReferences \n(1)  Sathishkumar, P.; Preethi, J.; Vijayan, R.; Mohd Yusoff, A. R.; Amee n, F.; \nSuresh, S.; Balagurunathan, R.; Palvannan, T. 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