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Gold nanoparticles synthesised by flavonoid tricetin as a potential antibacterial nanomedicine to treat respiratory infections causing opportunistic bacterial pathogens
Journal Pre-proof Gold nanoparticles synthesised by flavonoid tricetin as a potential antibacterial nanomedicine to treat respiratory infections causing opportunistic bacterial pathogens Khawlah Alsamhary, Nouf Al-Enazi, Wafa A. Alshehri, Fuad Ameen PII:
S0882-4010(19)31129-5
DOI:
https://doi.org/10.1016/j.micpath.2019.103928
Reference:
YMPAT 103928
To appear in:
Microbial Pathogenesis
Received Date: 22 June 2019 Revised Date:
12 December 2019
Accepted Date: 12 December 2019
Please cite this article as: Alsamhary K, Al-Enazi N, Alshehri WA, Ameen F, Gold nanoparticles synthesised by flavonoid tricetin as a potential antibacterial nanomedicine to treat respiratory infections causing opportunistic bacterial pathogens, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/ j.micpath.2019.103928. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Gold nanoparticles synthesised by flavonoid tricetin as a potential antibacterial
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nanomedicine to treat respiratory infections causing opportunistic bacterial
3
pathogens
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Khawlah Alsamhary1,*, Nouf Al-Enazi1, Wafa A. Alshehri2, Fuad Ameen3.
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Bin Abdulaziz University, Al-kharj 11942, Saudi Arabia
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80203, Saudi Arabia
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Department of Biology, College of Science and Humanities in Al-Kharj, Prince Sattam
Department of Biological Sciences, Faculty of Science, University of Jeddah, Jeddah
Department of Botany and Microbiology, College of Science, King Saud University,
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Riyadh 11451, Saudi Arabia
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*Corresponding author. Email: [email protected]
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Abstract
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In this study, flavonoid tricetin was used as a reducing and capping agent for the
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synthesis of gold nanoparticles (AuNPs). Further, the antibacterial efficacy of the
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synthesised AuNPs was evaluated against the opportunistic bacterial pathogens that cause
27
respiratory infections. The optimum levels for the synthesis of AuNPs were found to be
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pH 8, temperature 30 °C, tricetin 125 µM and chloroauric acid 250 µM. The tricetin
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synthesised AuNPs exhibited in spherical shape with an average size of 12 nm. FT-IR
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results confirmed that the hydroxyl (OH) and carbonyl (C=O) groups of tricetin were
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mainly participated in the synthesis of AuNPs. The opportunistic bacterial pathogens
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isolated from immunocompromised patients suffering with different respiratory
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infections were identified as Staphylococcus aureus, Enterobacter xiangfangensis,
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Bacillus licheniformis, Escherichia fergusonii, Acinetobacter pittii, Pseudomonas
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aeruginosa, Aeromonas enteropelogenes and Proteus mirabilis. The antibacterial studies
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confirmed the broad-spectrum antibacterial activity of AuNPs against the tested Gram-
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positive
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biocompatibility on primary normal human dermal fibroblast (NHDF-c) cells up to 50
39
µM mL-1. Best of our knowledge, this is the first report on the synthesis of AuNPs using
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tricetin, which may be a potential antibacterial nanomedicine to treat bacterial infections.
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Keywords: Antibacterial activity; Bacteria; Gold nanoparticles; Flavonoid; Respiratory
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and
Gram-negative
infections; Tricetin
bacteria.
The
synthesised
AuNPs
showed
high
3
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Introduction
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Respiratory infections are a heavy burden in developing countries, which caused
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by microorganisms such as fungi, bacteria and viruses [1]. The opportunistic bacterial
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pathogens are mostly responsible for respiratory infections, causing a range of localized
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and invasive infections [2,3]. Nowadays, acute and chronic respiratory infections are
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frequently observed in human due to inappropriate antibacterial treatment approach [4].
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In the medical field, existing antibacterial drugs face challenging issues in the effective
52
treatment of respiratory infections due to the development of multidrug resistance (MDR)
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strains [5,6]. Thus, alternative treatment approach is expected to overcome the issues of
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MDR strains. Recently, bio-based nanomedicine has been highly attracted in modern
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medical field due to its biocompatibility and high therapeutic efficacy [7].
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Interestingly, biosynthesised metal-based nanoparticles such as silver (Ag) and
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gold nanoparticles (AuNPs) are shown excellent biomedical applications for treating
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various diseases [8–10]. In particular, AuNPs have been proven as an effective
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nanomedicine against a wide range of pathogenic microorganisms and different cancer
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cells [11]. Different kind of chemical, physical and biological methods have been
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reported for the AuNPs synthesis [12–17]. Among the biological methods, the synthesis
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of AuNPs using phytocompounds extracted from plant sources is a simple and non-toxic
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process [18,19]. Previous reports have confirmed that phytocompounds such as
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polysaccharides, flavonoids, tannins, phenolic glycosides and reducing sugars present in
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plant extracts playing an important role in the reduction and electrostatic stabilization of
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metal-based nanoparticle synthesis [19].
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Flavonoids are secondary metabolites that are extensively distributed in plants,
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which involved in the physiological functions [20]. It has been used as a natural medicine
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in human healthcare management systems for the treatment of allergy, cancer, obesity,
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oxidative stress, melanogenesis and other diseases caused by microorganisms [20–22].
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Apart from this, it has many advantages like biocompatibility and hepatoprotective
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effects [22].
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nanomedicine is greatly appreciated in modern medical field to treat different
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pathological conditions in human, including Alzheimer’s disease, cardiovascular
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disorder, cancer, diabetic, neurodegenerative disease, oxidative-nitrosative stress and
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microbial diseases, because of their high therapeutic values. Flavanoids such as myricetin
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[23], kaempferol glucoside [24], quercetin [25], proanthocyanidin [26] and genistein [27]
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have been used to synthesis AuNPs. Tricetin (5, 7-dihydroxy-2-(3, 4, 5-trihydroxyphenyl)
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chromen-4-one) is a dietary flavonoid mainly present in ginkgo nuts, tea and grains [28].
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The chemical structure of tricetin is given in Fig. 1. It has shown effective antimicrobial
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[29] and anticancer activities [30–32]. However, till date, there is no report on the
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utilization of tricetin for the metal nanoparticle synthesis.
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Recently, Sathishkumar et al. [11] underlined the flavonoid-based
Position for Fig. 1
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In this study, tricetin was used to synthesize AuNPs, which may have their
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potential in biomedical applications to treat respiratory infections causing opportunistic
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bacterial pathogens. The physicochemical property of the synthesised AuNPs was
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analyzed using ultraviolet-visible (UV-vis) spectrophotometer, transmission electron
88
microscopy (TEM), attenuated total reflection-fourier transform infrared spectroscopy
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(ATR-FTIR) and X-ray diffraction (XRD) techniques. The antibacterial efficacy of the
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synthesised AuNPs was assessed for different bacterial pathogens isolated from patients
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suffering with respiratory infections. Finally, the biocompatible nature of the synthesised
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AuNPs was confirmed by cytotoxicity studies using normal cell line.
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2. Materials and methods
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2.1. Chemicals
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Tricetin was procured from ExtraSynthese (Genay, France). Chloroauric acid and
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dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, USA).
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Chocolate agar, MacConkey agar, mannitol salt agar, Mueller Hinton agar (MHA),
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nutrient agar and blood agar were obtained from HiMedia Laboratories Pvt. Ltd.
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(Mumbai, India). SEEDSWAB γ (gamma) 2 was obtained from Eiken Chemical (Tokyo,
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Japan). Hydrochloric acid or sodium hydroxide was used to adjust the pH of medium and
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other reactions. All other chemicals and reagents were of analytical grade, otherwise
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mentioned in appropriate place.
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2.2. Synthesis of AuNPs using tricetin
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Initially, the synthesis of AuNPs was optimized with different range of pH (4-9),
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temperature (20-60 °C), tricetin concentration (25-150 µM) and chloroauric acid
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concentration (50-300 µM). Then, the synthesis of AuNPs was carried out under
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optimized conditions and was used for further characterization and antibacterial studies.
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AuNPs formation was confirmed by a color change from yellow to red wine/purple. The
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colloidal nanoparticles solution was centrifuged at 8,000 G for 10 min and washed with
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ultrapure water.
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2.3. Characterization of synthesised AuNPs
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The formation of AuNPs by tricetin was confirmed based on surface plasmon
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resonance (SPR) absorption band analysis using a UV-vis spectrophotometer (Shimadzu
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UV-1601) over the range of 300-800 nm. The surface morphology of AuNPs was
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determined using TEM (JEOL 1400, Japan) with a resolution limit of about 0.2 nm. For
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TEM analysis, one drop (5 µL) of AuNPs dispersion was placed on a carbon-coated
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copper (Cu) grid and the sample was allowed to dry at 40 °C. ATR-FTIR was used to
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find out the surface functional groups of tricetin participated in the formation of AuNPs.
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The spectra of freeze dried AuNPs and tricetin were recorded using ATR-FTIR
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spectrometer (Perkin Elmer, USA) from 4000 to 650 cm-1 by 32 scans at 4 cm-1
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resolution. XRD was performed to predict the crystalline structure and size of the
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synthesised AuNPs. The dried AuNPs was analyzed using XRD (Bruker AXS System,
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D8, Germany) equipped with LYNXEYE XE-T detector.
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2.4. Sample collection, isolation and identification of bacterial pathogens
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The sputum and fluid samples were collected from immunocompromised patients
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(frequently suffering from respiratory infections) diagnosed with clinical cases from King
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Fahd University Hospital, Khobar, Kingdom of Saudi Arabia using cotton swabs. Then,
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the swabs were immediately transferred to transport medium SEEDSWAB γ (gamma) 2,
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and kept at 4 °C. Further, the collected samples were plated onto chocolate and blood
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agars. After 48 h incubation at 37 °C, the colonies grown on the plates were differentiated
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by their macroscopic and microscopic appearance. The selected pathogenic bacterial
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strains were identified using classic methods based on morphological characteristics with
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biochemical assays and molecular techniques using 16S rRNA sequence. The sequences
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of the bacterial strains were compared with the database of the National Center for
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Biotechnology Information (NCBI) using the BLAST program. Finally, the sequence
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submitted to NCBI and obtained accession number for each strain.
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2.5. Antibacterial assay
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The antibacterial activity of the synthesised AuNPs was assessed against the
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selected opportunistic bacterial pathogens using agar well diffusion method. In brief, all
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of the bacterial strains were grown in nutrient broth at 37 °C in an orbital shaker. After 12
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h incubation, the exponential phase of the culture was serially diluted (optical density of
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1.0 at 590 nm) with same medium and was aseptically spread on the prepared MHA
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plates. Then, 100 µL of the AuNPs suspension (500 µg mL-1) was loaded in the MHA
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plate wells for all test organisms. As a positive control, 100 µL of gentamicin solution
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(50 mg mL-1) was loaded in the well, since it has broad spectrum antibacterial activity.
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The cultured test plates were subsequently sealed and incubated at 37 °C for 24 h.
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The zone of inhibition (mm in diameter) was calculated manually. The
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experiments were carried out in triplicate and the results were presented as the mean ±
150
SD. The growth inhibition of the tested bacterial pathogens by the synthesised AuNPs
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was categorized as follows: 10 mm (+++, strong inhibition), 2 to 10 mm (++, moderate
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inhibition), 1 mm (+, weak inhibition), and less than 1 mm (-, no inhibition), according to
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previous report [9]. Minimum inhibitory concentration (MIC) of the synthesised AuNPs
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was assessed against the selected bacterial pathogens using broth micro-dilutions
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according to “Clinical and Laboratory Safety Standards Institute (CLSI) guideline”.
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2.6. Cytotoxicity assessment
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In this study, primary normal human dermal fibroblast (NHDF-c) cell line
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obtained from PromoCell (Heidelberg, Germany) was selected to examine the
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biocompatibility of the synthesised AuNPs. In brief, NHDF cells were cultured in 96-
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multiwell culture plates with the density of 104 cells per well and was incubated in CO2
162
incubator for 24 h. Subsequently, the cells were treated with the tricetin synthesised
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AuNPs at different concentrations (0-25 µM mL-1). Then, the plates were incubated at the
164
same conditions for 24, 48 and 72 h. After incubation, the viability of the AuNPs treated
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and untreated NHDF cells was assessed using MTT Cell Proliferation Assay (ATCC®
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30-1010K), based on the conversion of MTT by reduction process to formazan crystals
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by mitochondrial reductase enzyme. In this step, the culture medium was replaced with
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fresh medium consists of 10 µL MTT (12 mM) in phosphate buffered saline (PBS). After
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4 h incubation, again the medium of culture was removed and subsequently 150 µL of
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DMSO was added. Then, the absorbance was measured at a wavelength of 590 nm in
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UV-vis spectrophotometer. Finally, the cell viability was determined by comparing the
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absorbance of samples at 590 nm with the untreated cells (considered as 100% survival).
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The experiments were performed with three replicates. Position for Table 1 and Fig. 2
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3. Results and discussion
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3.1. AuNPs synthesis
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Initially, the formation of AuNPs by tricetin was confirmed by the change of
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color from yellow to purple/red wine (Fig. 2 insert). It confirms that tricetin may act as a
9
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reducing and capping agent for the synthesis of AuNPs. Further, the significant
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parameters including pH, temperature, reducing agent concentration and gold precursor
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concentration, which influencing the nanoparticles synthesis was optimized. The
182
formation of nanoparticles were monitored using UV-vis spectrophotometer. Further, the
183
results were analyzed, according to SPR peak shift and height in UV-vis spectrum. Table
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1 demonstrates the effect of pH, temperature, tricetin concentration and chloroauric acid
185
concentration on the synthesis of AuNPs. The SPR peak of AuNPs synthesised by tricetin
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was found between 520 and 540 nm for pH 6-9, similar to the previous reports [33,34].
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The maximum peak height of the colloidal suspension of tricetin synthesised AuNPs was
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observed at 530 for pH 8, thus pH 8 was selected for further optimization studies. The
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effect of temperature results indicates that almost equal peak height at 530 nm was
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observed between 30 °C and 50 °C. Thus, in this study, ambient temperature 30 °C was
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selected for the synthesis of AuNPs. The effect of reducing agent tricetin on the
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formation of AuNPs was assessed over the range of 25-150 µM. The result shows that the
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maximum peak height was found at 530 nm for 125 µM concentration of tricetin. Thus,
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125 µM was selected as an optimum point for the nanoparticle’s formation. Finally, the
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gold precursor concentration was optimized with the optimized levels of pH, temperature
196
and tricetin. The peak height was increased with increasing concentration of chloroauric
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acid up to 250 µM, beyond stagnant. Therefore, the optimum point of pH 8, temperature
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30 °C, tricetin 125 µM and chloroauric acid 250 µM were chosen for the synthesis of
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AuNPs. The UV-vis spectra of tricetin, chloroauric acid and the tricetin synthesised
200
AuNPs are shown in Fig. 2. The result shows that the SPR peak of the synthesised
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201
AuNPs by tricetin was found at 530 nm, which corresponds to the colloidal gold
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particles, similar to the previous report [23]. Position for Fig. 3
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3.2. Characterization of AuNPs
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Figs. 3a and b demonstrates the TEM image and selected area electron diffraction
206
(SAED) pattern of the tricetin synthesised AuNPs. The TEM image clearly shows that the
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AuNPs appeared as polydisperse with spherical shape and the average size of the
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nanoparticles was calculated as 12 nm in diameter (Fig. 3a insert), which is smaller in
209
size than the previously reported AuNPs synthesised by flavonoids such as
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dihydromyricetin (24.7 nm) [35], kaempferol glucoside (37 nm) [24], quercetin (20-45
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nm) [25], proanthocyanidin (24 nm) [26] and genistein (64.64 nm) [27]. As shown in Fig.
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3b, the SAED pattern clearly indicates that the AuNPs was found to be crystalline in
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nature.
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Fig. 3c shows the IR spectra of tricetin and the tricetin synthesised AuNPs. The
215
spectrum of tricetin shows the most important stretching vibrations for O–H, C–H, C=O,
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C=C and C–O groups at 3432 cm−1, 2899 cm−1, 1659 cm−1, 1552 cm−1, and 1028 cm−1,
217
respectively. The spectrum of the tricetin synthesised AuNPs showed the major peaks at
218
3451 cm-1, 1629 cm−1, and 1077 cm-1. These results clearly demonstrate that the
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stretching vibrations of O–H (3432 cm-1), C–O (1028 cm-1) and C=O (1659 cm-1) groups
220
of flavonoid tricetin were shifted to 3451 cm-1, 1077 cm-1 and 1629 cm−1, respectively.
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This result evidently proves that the hydroxyl and carbonyl groups of tricetin were
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mainly involved in the nanoparticles formation, similar to the previous reports [23,35].
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Recently, Sathishkumar et al. [23] found that the 5′-hydroxyl group of bioflavonoid
11
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myricetin was more suitable for the AuNPs formation. It is important to note that the
225
hydroxyl positions in the B ring of tricetin is almost similar to myricetin. Thus, 5′-
226
hydroxyl group of tricetin may be involved in the nanoparticle’s formation; however,
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further experimental and theoretical studies is essential to prove the role of hydroxyl
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groups present in tricetin for the AuNPs formation and stabilization.
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Fig. 3d demonstrates the XRD pattern of the tricetin synthesised AuNPs. The
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XRD pattern demonstrates the distinct diffractions at 38.2, 44.2, 64.6 and 77.8 degrees
231
for the (111), (200), (220), and (311) lattice planes of Au (JPCDS No.: 04-0784),
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respectively. It confirms that the synthesised AuNPs found to be well-crystalline [36–39].
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The intense diffraction peak for (111) was dominant with face centered cubic symmetry,
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which confirms the well-crystallized nanoparticles. Further, the average crystalline size
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of synthesised AuNPs was calculated as 9 nm from the primary diffraction peak,
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according to Scherrer’ equation, which is almost small to the average particle size
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observed in TEM image.
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Position for Table 2 3.3. Identification of respiratory infections causing pathogenic bacteria
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Among the 50 isolated pathogenic bacterial strains, eight morphologically
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differed strains were selected for the molecular identification using 16S rRNA sequence
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technique. Table 2 demonstrates the name and NCBI GenBank accession number of the
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identified bacterial strains. The respiratory infections causing pathogenic bacterial strains
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isolated from different immunocompromised patients were identified as Staphylococcus
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aureus (MG818959), Enterobacter xiangfangensis (MG818960), Bacillus licheniformis
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(MG818961), Escherichia fergusonii (MG818962), Acinetobacter pittii (MG818963),
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Pseudomonas aeruginosa (MG818964), Aeromonas enteropelogenes (MG818965) and
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Proteus mirabilis (MG818966). In general, these identified bacterial strains are
249
commonly known as opportunistic pathogens. Recently, Uzoamaka et al. [3] investigated
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the presence of bacteria in 954 sputum samples collected from different patients suffering
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with respiratory infections. Among these, 431 (45.2%) samples were found to be positive
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for bacteria including Klebsiella pneumoniae, E. coli, S. aureus and P. aeruginosa.
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However, in this study, K. pneumoniae and E. coli were not observed.
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Position for Figs. 4 and 5 3.4. Antibacterial activity of tricetin synthesised AuNPs
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Figs. 4 and 5 illustrate the effect of the tricetin synthesised AuNPs on the
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respiratory infections causing pathogenic bacterial strains. The result indicates that all of
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tested bacterial strains were significantly inhibited by the tricetin synthesised AuNPs at a
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concentration of 50 µM (Fig. 4). The ZOI was found to be 11.3, 14.5, 11.9, 13.6, 15.3,
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15.1, 14.3 and 16.2 mm in diameter at a 50 µg of AuNPs for S. aureus, E. xiangfangensis,
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B. licheniformis, E. fergusonii, A. pittii, P. aeruginosa, A. enteropelogenes and P.
262
mirabilis, respectively. The observed ZOI of the AuNPs was considerably higher
263
compared to tricetin at 50 µg concentrations for all the tested pathogens, as shown in Fig.
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4. It confirms that the synthesised AuNPs exhibited a strong inhibition (>10 mm; +++)
265
efficiency against all the tested bacterial strains. Notably, the antibacterial efficacy of the
266
synthesised AuNPs (50 µg) exhibited almost above 50% of the positive control drug
267
gentamicin (5 mg). MIC of the tricetin synthesised AuNPs was observed as 46.6, 31.1,
268
41.3, 38.0, 33.6, 29.9, 34.3 and 28.4 µg mL-1 for the tested bacterial strains such as S.
269
aureus, E. xiangfangensis, B. licheniformis, E. fergusonii, A. pittii, P. aeruginosa, A.
13
270
enteropelogenes and P. mirabilis, respectively (Fig. 5). These results clearly show that
271
the broad-spectrum antibacterial activity of the tricetin synthesised AuNPs was found
272
against both of the tested Gram-positive and Gram-negative pathogenic bacterial strains.
273
Furthermore, the result indicates that Gram-negative bacterial strains were highly
274
susceptible for AuNPs, compared to the Gram-positive bacterial strains. Previously few
275
reports mentioned that the biosynthesised AuNPs were more effective against Gram-
276
negative bacterial strains, compared to Gram-positive bacteria, since it has thicker layer
277
of peptidoglycan, which protect the attack of the AuNPs [40–42]. Interestingly, the
278
present study confirms that the tricetin synthesised AuNPs showed broad-spectrum
279
activity against both kind of bacterial groups. Previously, Pistelli et al. [29] confirmed the
280
antibacterial activity of tricetin. In addition, similar kind of flavonoids like isopomiferin
281
inhibited methionyl-tRNA synthetase (MetRS) enzyme activity and showed broad
282
spectrum antibacterial activity [43]. Therefore, the observed broad-spectrum activity
283
might be due to the bioflavonoid tricetin used for the AuNPs synthesis, since it has
284
antibacterial activity. Apart from this, the smaller size of the nanoparticles also may offer
285
larger surface-to-volume ratio and also highly effective contact with the surface of
286
bacteria, which enhancing the antimicrobial efficiency of the tricetin synthesised AuNPs
287
[44]. Thus, in this study, capping agent tricetin may effectively attack all of the tested
288
bacterial strains along with nano Au conductivity. These results clearly indicate that the
289
tricetin synthesised AuNPs may agreeable to bring as a therapeutic drug for the bacterial
290
infections in respiratory tract. Position for Fig. 6
291 292
3.5. Cytotoxicity assessment
14
293
As shown in Fig. 6, the synthesised AuNPs exhibits very less cytotoxicity (6%)
294
up to 50 µg mL-1. In the case of 75 µg mL-1 and 100 µg mL-1 concentrations of AuNPs,
295
83% and 54% of cell viability was observed. It explains that the tricetin synthesised
296
AuNPs may have dose-dependent cytotoxicity on the NHDF cells. This result confirms
297
that the synthesised AuNPs was biocompatible up to 50 µg mL-1. According to the
298
antibacterial activity results (Fig. 5), the MIC of AuNPs was found to be within 50 µg
299
mL-1 for all of the tested respiratory infections causing pathogenic bacterial strains. Thus,
300
the tricetin synthesised AuNPs might be an efficient candidate to treat respiratory
301
infections causing pathogenic bacterial strains.
302
4. Conclusions
303
In this study, AuNPs were successfully synthesised using flavonoid tricetin and
304
further used to treat respiratory infections causing bacterial pathogens. The synthesised
305
nanoparticles were observed to be spherical in shape with the size of 12 nm in diameter.
306
The identified respiratory infections causing pathogenic bacterial strains were S. aureus,
307
E. xiangfangensis, B. licheniformis, E. fergusonii, A. pittii, P. aeruginosa, A.
308
enteropelogenes and P. mirabilis. The tricetin synthesised AuNPs demonstrated broad
309
spectrum antibacterial activity against both of the tested Gram-negative and Gram-
310
positive bacterial strains. The cytotoxicity result confirms that the synthesised AuNPs is
311
more biocompatible up to 50 µg mL-1 for therapeutic applications. Finally, this study
312
suggests that tricetin synthesised AuNPs might be an effective drug to treat respiratory
313
infections causing pathogenic bacterial strains without any adverse side effects on host.
314
Acknowledgment
15
315
This project was supported by the Deanship of Scientific Research at Prince
316
Sattam Bin Abdulaziz University under the research project 2019/01/10137.
317
Conflict of interests
318
The authors declare that have no competing interests in this investigation.
319
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459 460 461 462
Figure legends
463
Figure 1. Chemical structure of flavonoid tricetin
464
Figure 2. Uv-vis spectrum of the tricetin synthesised AuNPs under optimized conditions
465
(pH 8, temperature 30 °C, tricetin concentration 125 µM and chloroauric acid
466
250 µM). Insert: corresponding photograph of (i) chloroauric acid, (ii) tricetin
467
and (iii) the tricetin synthesised AuNPs
468 469 470 471 472 473 474
Figure 3. (a) TEM image (insert: histogram of nanoparticles), (b) SAED pattern, (c) FTIR spectrum and (d) XRD pattern of the synthesised AuNPs Figure 4. Antibacterial activity: ZOI of AuNPs against tested opportunistic bacterial pathogens Figure 5. Antibacterial activity: MIC of AuNPs against tested opportunistic bacterial pathogens Figure 6. Cytotoxicity of AuNPs against primary normal human dermal fibroblast cells
Table 1. Effect of pH, temperature, tricetin and chloroauric acid levels on surface plasmon resonance (SPR) of AuNPs formation Optimization experiments Effect of pH 4 5 6 7 8 9 Effect of temperature 20 °C 30 °C 40 °C 50 °C 60 °C Effect of tricetin 25 µM 50 µM 75 µM 100 µM 125 µM 150 µM Effect of chloroauric acid 50 µM 100 µM 150 µM 200 µM 250 µM 300 µM
SPR of AuNPs in UV-vis spectrum Peak intensity Wavelength (nm) 0.37 0.43 0.53 0.58 0.58 0.50
545 541 538 534 530 522
0.43 0.58 0.60 0.61 0.55
538 530 530 530 530
0.18 0.38 0.46 0.61 0.61 0.18
530 530 530 530 530 535
0.13 0.61 0.69 0.79 0.83 0.84
525 530 530 530 530 535
Experimental conditions for AuNPs synthesis optimization studies. (i) pH optimization: temperature 30 °C, tricetin concentration 100 µM and chloroauric acid 100 µM; (ii) Temperature optimization: pH 8 and tricetin concentration 100 µM and chloroauric acid 100 µM; and (iii) Tricetin concentration optimization: pH 8, Temperature 30 °C, and chloroauric acid concentration 100 µM; and (iv) Chloroauric acid concentration optimization: pH 8, Temperature 30 °C, and tricetin concentration 125 µM. Results expressed with the mean value of triplicate experiments.
Table 2. Opportunistic bacterial pathogens isolated from immunocompromised patients with respiratory infections S. No 1 2 3 4 5 6 7 8
Organism name Staphylococcus aureus Enterobacter xiangfangensis Bacillus licheniformis Escherichia fergusonii Acinetobacter pittii Pseudomonas aeruginosa Aeromonas enteropelogenes Proteus mirabilis
GenBank accession number MG818959 MG818960 MG818961 MG818962 MG818963 MG818964 MG818965 MG818966
Identity (%) 99 99 99 99 99 99 99 100
Fig. 1
i
Fig. 2
ii
iii
a
b
c
d
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Highlights AuNPs were successfully synthesized using flavonoid tricetin. AuNPs are polydispersed and spherical in shape with the size of 12 nm. AuNPs exhibited broad-spectrum antibacterial activity. AuNPs showed high biocompatibility on NHDF-c cells up to 50 µM mL-1.
Authors statement This manuscript has not been published and is not under consideration for publication elsewhere. Authors have no conflicts of interest to disclose. Declaration of interests ☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Sincerely, Dr. Fuad Ameen, Assistance Professor, Dept. of Botany & Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
S0882-4010(19)31129-5
DOI:
https://doi.org/10.1016/j.micpath.2019.103928
Reference:
YMPAT 103928
To appear in:
Microbial Pathogenesis
Received Date: 22 June 2019 Revised Date:
12 December 2019
Accepted Date: 12 December 2019
Please cite this article as: Alsamhary K, Al-Enazi N, Alshehri WA, Ameen F, Gold nanoparticles synthesised by flavonoid tricetin as a potential antibacterial nanomedicine to treat respiratory infections causing opportunistic bacterial pathogens, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/ j.micpath.2019.103928. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Gold nanoparticles synthesised by flavonoid tricetin as a potential antibacterial
2
nanomedicine to treat respiratory infections causing opportunistic bacterial
3
pathogens
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Khawlah Alsamhary1,*, Nouf Al-Enazi1, Wafa A. Alshehri2, Fuad Ameen3.
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1
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Bin Abdulaziz University, Al-kharj 11942, Saudi Arabia
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2
8
80203, Saudi Arabia
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3
Department of Biology, College of Science and Humanities in Al-Kharj, Prince Sattam
Department of Biological Sciences, Faculty of Science, University of Jeddah, Jeddah
Department of Botany and Microbiology, College of Science, King Saud University,
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Riyadh 11451, Saudi Arabia
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*Corresponding author. Email: [email protected]
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23
Abstract
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In this study, flavonoid tricetin was used as a reducing and capping agent for the
25
synthesis of gold nanoparticles (AuNPs). Further, the antibacterial efficacy of the
26
synthesised AuNPs was evaluated against the opportunistic bacterial pathogens that cause
27
respiratory infections. The optimum levels for the synthesis of AuNPs were found to be
28
pH 8, temperature 30 °C, tricetin 125 µM and chloroauric acid 250 µM. The tricetin
29
synthesised AuNPs exhibited in spherical shape with an average size of 12 nm. FT-IR
30
results confirmed that the hydroxyl (OH) and carbonyl (C=O) groups of tricetin were
31
mainly participated in the synthesis of AuNPs. The opportunistic bacterial pathogens
32
isolated from immunocompromised patients suffering with different respiratory
33
infections were identified as Staphylococcus aureus, Enterobacter xiangfangensis,
34
Bacillus licheniformis, Escherichia fergusonii, Acinetobacter pittii, Pseudomonas
35
aeruginosa, Aeromonas enteropelogenes and Proteus mirabilis. The antibacterial studies
36
confirmed the broad-spectrum antibacterial activity of AuNPs against the tested Gram-
37
positive
38
biocompatibility on primary normal human dermal fibroblast (NHDF-c) cells up to 50
39
µM mL-1. Best of our knowledge, this is the first report on the synthesis of AuNPs using
40
tricetin, which may be a potential antibacterial nanomedicine to treat bacterial infections.
41
Keywords: Antibacterial activity; Bacteria; Gold nanoparticles; Flavonoid; Respiratory
42 43 44
and
Gram-negative
infections; Tricetin
bacteria.
The
synthesised
AuNPs
showed
high
3
45
Introduction
46
Respiratory infections are a heavy burden in developing countries, which caused
47
by microorganisms such as fungi, bacteria and viruses [1]. The opportunistic bacterial
48
pathogens are mostly responsible for respiratory infections, causing a range of localized
49
and invasive infections [2,3]. Nowadays, acute and chronic respiratory infections are
50
frequently observed in human due to inappropriate antibacterial treatment approach [4].
51
In the medical field, existing antibacterial drugs face challenging issues in the effective
52
treatment of respiratory infections due to the development of multidrug resistance (MDR)
53
strains [5,6]. Thus, alternative treatment approach is expected to overcome the issues of
54
MDR strains. Recently, bio-based nanomedicine has been highly attracted in modern
55
medical field due to its biocompatibility and high therapeutic efficacy [7].
56
Interestingly, biosynthesised metal-based nanoparticles such as silver (Ag) and
57
gold nanoparticles (AuNPs) are shown excellent biomedical applications for treating
58
various diseases [8–10]. In particular, AuNPs have been proven as an effective
59
nanomedicine against a wide range of pathogenic microorganisms and different cancer
60
cells [11]. Different kind of chemical, physical and biological methods have been
61
reported for the AuNPs synthesis [12–17]. Among the biological methods, the synthesis
62
of AuNPs using phytocompounds extracted from plant sources is a simple and non-toxic
63
process [18,19]. Previous reports have confirmed that phytocompounds such as
64
polysaccharides, flavonoids, tannins, phenolic glycosides and reducing sugars present in
65
plant extracts playing an important role in the reduction and electrostatic stabilization of
66
metal-based nanoparticle synthesis [19].
4
67
Flavonoids are secondary metabolites that are extensively distributed in plants,
68
which involved in the physiological functions [20]. It has been used as a natural medicine
69
in human healthcare management systems for the treatment of allergy, cancer, obesity,
70
oxidative stress, melanogenesis and other diseases caused by microorganisms [20–22].
71
Apart from this, it has many advantages like biocompatibility and hepatoprotective
72
effects [22].
73
nanomedicine is greatly appreciated in modern medical field to treat different
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pathological conditions in human, including Alzheimer’s disease, cardiovascular
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disorder, cancer, diabetic, neurodegenerative disease, oxidative-nitrosative stress and
76
microbial diseases, because of their high therapeutic values. Flavanoids such as myricetin
77
[23], kaempferol glucoside [24], quercetin [25], proanthocyanidin [26] and genistein [27]
78
have been used to synthesis AuNPs. Tricetin (5, 7-dihydroxy-2-(3, 4, 5-trihydroxyphenyl)
79
chromen-4-one) is a dietary flavonoid mainly present in ginkgo nuts, tea and grains [28].
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The chemical structure of tricetin is given in Fig. 1. It has shown effective antimicrobial
81
[29] and anticancer activities [30–32]. However, till date, there is no report on the
82
utilization of tricetin for the metal nanoparticle synthesis.
83
Recently, Sathishkumar et al. [11] underlined the flavonoid-based
Position for Fig. 1
84
In this study, tricetin was used to synthesize AuNPs, which may have their
85
potential in biomedical applications to treat respiratory infections causing opportunistic
86
bacterial pathogens. The physicochemical property of the synthesised AuNPs was
87
analyzed using ultraviolet-visible (UV-vis) spectrophotometer, transmission electron
88
microscopy (TEM), attenuated total reflection-fourier transform infrared spectroscopy
89
(ATR-FTIR) and X-ray diffraction (XRD) techniques. The antibacterial efficacy of the
5
90
synthesised AuNPs was assessed for different bacterial pathogens isolated from patients
91
suffering with respiratory infections. Finally, the biocompatible nature of the synthesised
92
AuNPs was confirmed by cytotoxicity studies using normal cell line.
93
2. Materials and methods
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2.1. Chemicals
95
Tricetin was procured from ExtraSynthese (Genay, France). Chloroauric acid and
96
dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, USA).
97
Chocolate agar, MacConkey agar, mannitol salt agar, Mueller Hinton agar (MHA),
98
nutrient agar and blood agar were obtained from HiMedia Laboratories Pvt. Ltd.
99
(Mumbai, India). SEEDSWAB γ (gamma) 2 was obtained from Eiken Chemical (Tokyo,
100
Japan). Hydrochloric acid or sodium hydroxide was used to adjust the pH of medium and
101
other reactions. All other chemicals and reagents were of analytical grade, otherwise
102
mentioned in appropriate place.
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2.2. Synthesis of AuNPs using tricetin
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Initially, the synthesis of AuNPs was optimized with different range of pH (4-9),
105
temperature (20-60 °C), tricetin concentration (25-150 µM) and chloroauric acid
106
concentration (50-300 µM). Then, the synthesis of AuNPs was carried out under
107
optimized conditions and was used for further characterization and antibacterial studies.
108
AuNPs formation was confirmed by a color change from yellow to red wine/purple. The
109
colloidal nanoparticles solution was centrifuged at 8,000 G for 10 min and washed with
110
ultrapure water.
111
6
112
2.3. Characterization of synthesised AuNPs
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The formation of AuNPs by tricetin was confirmed based on surface plasmon
114
resonance (SPR) absorption band analysis using a UV-vis spectrophotometer (Shimadzu
115
UV-1601) over the range of 300-800 nm. The surface morphology of AuNPs was
116
determined using TEM (JEOL 1400, Japan) with a resolution limit of about 0.2 nm. For
117
TEM analysis, one drop (5 µL) of AuNPs dispersion was placed on a carbon-coated
118
copper (Cu) grid and the sample was allowed to dry at 40 °C. ATR-FTIR was used to
119
find out the surface functional groups of tricetin participated in the formation of AuNPs.
120
The spectra of freeze dried AuNPs and tricetin were recorded using ATR-FTIR
121
spectrometer (Perkin Elmer, USA) from 4000 to 650 cm-1 by 32 scans at 4 cm-1
122
resolution. XRD was performed to predict the crystalline structure and size of the
123
synthesised AuNPs. The dried AuNPs was analyzed using XRD (Bruker AXS System,
124
D8, Germany) equipped with LYNXEYE XE-T detector.
125
2.4. Sample collection, isolation and identification of bacterial pathogens
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The sputum and fluid samples were collected from immunocompromised patients
127
(frequently suffering from respiratory infections) diagnosed with clinical cases from King
128
Fahd University Hospital, Khobar, Kingdom of Saudi Arabia using cotton swabs. Then,
129
the swabs were immediately transferred to transport medium SEEDSWAB γ (gamma) 2,
130
and kept at 4 °C. Further, the collected samples were plated onto chocolate and blood
131
agars. After 48 h incubation at 37 °C, the colonies grown on the plates were differentiated
132
by their macroscopic and microscopic appearance. The selected pathogenic bacterial
133
strains were identified using classic methods based on morphological characteristics with
134
biochemical assays and molecular techniques using 16S rRNA sequence. The sequences
7
135
of the bacterial strains were compared with the database of the National Center for
136
Biotechnology Information (NCBI) using the BLAST program. Finally, the sequence
137
submitted to NCBI and obtained accession number for each strain.
138
2.5. Antibacterial assay
139
The antibacterial activity of the synthesised AuNPs was assessed against the
140
selected opportunistic bacterial pathogens using agar well diffusion method. In brief, all
141
of the bacterial strains were grown in nutrient broth at 37 °C in an orbital shaker. After 12
142
h incubation, the exponential phase of the culture was serially diluted (optical density of
143
1.0 at 590 nm) with same medium and was aseptically spread on the prepared MHA
144
plates. Then, 100 µL of the AuNPs suspension (500 µg mL-1) was loaded in the MHA
145
plate wells for all test organisms. As a positive control, 100 µL of gentamicin solution
146
(50 mg mL-1) was loaded in the well, since it has broad spectrum antibacterial activity.
147
The cultured test plates were subsequently sealed and incubated at 37 °C for 24 h.
148
The zone of inhibition (mm in diameter) was calculated manually. The
149
experiments were carried out in triplicate and the results were presented as the mean ±
150
SD. The growth inhibition of the tested bacterial pathogens by the synthesised AuNPs
151
was categorized as follows: 10 mm (+++, strong inhibition), 2 to 10 mm (++, moderate
152
inhibition), 1 mm (+, weak inhibition), and less than 1 mm (-, no inhibition), according to
153
previous report [9]. Minimum inhibitory concentration (MIC) of the synthesised AuNPs
154
was assessed against the selected bacterial pathogens using broth micro-dilutions
155
according to “Clinical and Laboratory Safety Standards Institute (CLSI) guideline”.
156
8
157
2.6. Cytotoxicity assessment
158
In this study, primary normal human dermal fibroblast (NHDF-c) cell line
159
obtained from PromoCell (Heidelberg, Germany) was selected to examine the
160
biocompatibility of the synthesised AuNPs. In brief, NHDF cells were cultured in 96-
161
multiwell culture plates with the density of 104 cells per well and was incubated in CO2
162
incubator for 24 h. Subsequently, the cells were treated with the tricetin synthesised
163
AuNPs at different concentrations (0-25 µM mL-1). Then, the plates were incubated at the
164
same conditions for 24, 48 and 72 h. After incubation, the viability of the AuNPs treated
165
and untreated NHDF cells was assessed using MTT Cell Proliferation Assay (ATCC®
166
30-1010K), based on the conversion of MTT by reduction process to formazan crystals
167
by mitochondrial reductase enzyme. In this step, the culture medium was replaced with
168
fresh medium consists of 10 µL MTT (12 mM) in phosphate buffered saline (PBS). After
169
4 h incubation, again the medium of culture was removed and subsequently 150 µL of
170
DMSO was added. Then, the absorbance was measured at a wavelength of 590 nm in
171
UV-vis spectrophotometer. Finally, the cell viability was determined by comparing the
172
absorbance of samples at 590 nm with the untreated cells (considered as 100% survival).
173
The experiments were performed with three replicates. Position for Table 1 and Fig. 2
174 175
3. Results and discussion
176
3.1. AuNPs synthesis
177
Initially, the formation of AuNPs by tricetin was confirmed by the change of
178
color from yellow to purple/red wine (Fig. 2 insert). It confirms that tricetin may act as a
9
179
reducing and capping agent for the synthesis of AuNPs. Further, the significant
180
parameters including pH, temperature, reducing agent concentration and gold precursor
181
concentration, which influencing the nanoparticles synthesis was optimized. The
182
formation of nanoparticles were monitored using UV-vis spectrophotometer. Further, the
183
results were analyzed, according to SPR peak shift and height in UV-vis spectrum. Table
184
1 demonstrates the effect of pH, temperature, tricetin concentration and chloroauric acid
185
concentration on the synthesis of AuNPs. The SPR peak of AuNPs synthesised by tricetin
186
was found between 520 and 540 nm for pH 6-9, similar to the previous reports [33,34].
187
The maximum peak height of the colloidal suspension of tricetin synthesised AuNPs was
188
observed at 530 for pH 8, thus pH 8 was selected for further optimization studies. The
189
effect of temperature results indicates that almost equal peak height at 530 nm was
190
observed between 30 °C and 50 °C. Thus, in this study, ambient temperature 30 °C was
191
selected for the synthesis of AuNPs. The effect of reducing agent tricetin on the
192
formation of AuNPs was assessed over the range of 25-150 µM. The result shows that the
193
maximum peak height was found at 530 nm for 125 µM concentration of tricetin. Thus,
194
125 µM was selected as an optimum point for the nanoparticle’s formation. Finally, the
195
gold precursor concentration was optimized with the optimized levels of pH, temperature
196
and tricetin. The peak height was increased with increasing concentration of chloroauric
197
acid up to 250 µM, beyond stagnant. Therefore, the optimum point of pH 8, temperature
198
30 °C, tricetin 125 µM and chloroauric acid 250 µM were chosen for the synthesis of
199
AuNPs. The UV-vis spectra of tricetin, chloroauric acid and the tricetin synthesised
200
AuNPs are shown in Fig. 2. The result shows that the SPR peak of the synthesised
10
201
AuNPs by tricetin was found at 530 nm, which corresponds to the colloidal gold
202
particles, similar to the previous report [23]. Position for Fig. 3
203 204
3.2. Characterization of AuNPs
205
Figs. 3a and b demonstrates the TEM image and selected area electron diffraction
206
(SAED) pattern of the tricetin synthesised AuNPs. The TEM image clearly shows that the
207
AuNPs appeared as polydisperse with spherical shape and the average size of the
208
nanoparticles was calculated as 12 nm in diameter (Fig. 3a insert), which is smaller in
209
size than the previously reported AuNPs synthesised by flavonoids such as
210
dihydromyricetin (24.7 nm) [35], kaempferol glucoside (37 nm) [24], quercetin (20-45
211
nm) [25], proanthocyanidin (24 nm) [26] and genistein (64.64 nm) [27]. As shown in Fig.
212
3b, the SAED pattern clearly indicates that the AuNPs was found to be crystalline in
213
nature.
214
Fig. 3c shows the IR spectra of tricetin and the tricetin synthesised AuNPs. The
215
spectrum of tricetin shows the most important stretching vibrations for O–H, C–H, C=O,
216
C=C and C–O groups at 3432 cm−1, 2899 cm−1, 1659 cm−1, 1552 cm−1, and 1028 cm−1,
217
respectively. The spectrum of the tricetin synthesised AuNPs showed the major peaks at
218
3451 cm-1, 1629 cm−1, and 1077 cm-1. These results clearly demonstrate that the
219
stretching vibrations of O–H (3432 cm-1), C–O (1028 cm-1) and C=O (1659 cm-1) groups
220
of flavonoid tricetin were shifted to 3451 cm-1, 1077 cm-1 and 1629 cm−1, respectively.
221
This result evidently proves that the hydroxyl and carbonyl groups of tricetin were
222
mainly involved in the nanoparticles formation, similar to the previous reports [23,35].
223
Recently, Sathishkumar et al. [23] found that the 5′-hydroxyl group of bioflavonoid
11
224
myricetin was more suitable for the AuNPs formation. It is important to note that the
225
hydroxyl positions in the B ring of tricetin is almost similar to myricetin. Thus, 5′-
226
hydroxyl group of tricetin may be involved in the nanoparticle’s formation; however,
227
further experimental and theoretical studies is essential to prove the role of hydroxyl
228
groups present in tricetin for the AuNPs formation and stabilization.
229
Fig. 3d demonstrates the XRD pattern of the tricetin synthesised AuNPs. The
230
XRD pattern demonstrates the distinct diffractions at 38.2, 44.2, 64.6 and 77.8 degrees
231
for the (111), (200), (220), and (311) lattice planes of Au (JPCDS No.: 04-0784),
232
respectively. It confirms that the synthesised AuNPs found to be well-crystalline [36–39].
233
The intense diffraction peak for (111) was dominant with face centered cubic symmetry,
234
which confirms the well-crystallized nanoparticles. Further, the average crystalline size
235
of synthesised AuNPs was calculated as 9 nm from the primary diffraction peak,
236
according to Scherrer’ equation, which is almost small to the average particle size
237
observed in TEM image.
238 239
Position for Table 2 3.3. Identification of respiratory infections causing pathogenic bacteria
240
Among the 50 isolated pathogenic bacterial strains, eight morphologically
241
differed strains were selected for the molecular identification using 16S rRNA sequence
242
technique. Table 2 demonstrates the name and NCBI GenBank accession number of the
243
identified bacterial strains. The respiratory infections causing pathogenic bacterial strains
244
isolated from different immunocompromised patients were identified as Staphylococcus
245
aureus (MG818959), Enterobacter xiangfangensis (MG818960), Bacillus licheniformis
246
(MG818961), Escherichia fergusonii (MG818962), Acinetobacter pittii (MG818963),
12
247
Pseudomonas aeruginosa (MG818964), Aeromonas enteropelogenes (MG818965) and
248
Proteus mirabilis (MG818966). In general, these identified bacterial strains are
249
commonly known as opportunistic pathogens. Recently, Uzoamaka et al. [3] investigated
250
the presence of bacteria in 954 sputum samples collected from different patients suffering
251
with respiratory infections. Among these, 431 (45.2%) samples were found to be positive
252
for bacteria including Klebsiella pneumoniae, E. coli, S. aureus and P. aeruginosa.
253
However, in this study, K. pneumoniae and E. coli were not observed.
254 255
Position for Figs. 4 and 5 3.4. Antibacterial activity of tricetin synthesised AuNPs
256
Figs. 4 and 5 illustrate the effect of the tricetin synthesised AuNPs on the
257
respiratory infections causing pathogenic bacterial strains. The result indicates that all of
258
tested bacterial strains were significantly inhibited by the tricetin synthesised AuNPs at a
259
concentration of 50 µM (Fig. 4). The ZOI was found to be 11.3, 14.5, 11.9, 13.6, 15.3,
260
15.1, 14.3 and 16.2 mm in diameter at a 50 µg of AuNPs for S. aureus, E. xiangfangensis,
261
B. licheniformis, E. fergusonii, A. pittii, P. aeruginosa, A. enteropelogenes and P.
262
mirabilis, respectively. The observed ZOI of the AuNPs was considerably higher
263
compared to tricetin at 50 µg concentrations for all the tested pathogens, as shown in Fig.
264
4. It confirms that the synthesised AuNPs exhibited a strong inhibition (>10 mm; +++)
265
efficiency against all the tested bacterial strains. Notably, the antibacterial efficacy of the
266
synthesised AuNPs (50 µg) exhibited almost above 50% of the positive control drug
267
gentamicin (5 mg). MIC of the tricetin synthesised AuNPs was observed as 46.6, 31.1,
268
41.3, 38.0, 33.6, 29.9, 34.3 and 28.4 µg mL-1 for the tested bacterial strains such as S.
269
aureus, E. xiangfangensis, B. licheniformis, E. fergusonii, A. pittii, P. aeruginosa, A.
13
270
enteropelogenes and P. mirabilis, respectively (Fig. 5). These results clearly show that
271
the broad-spectrum antibacterial activity of the tricetin synthesised AuNPs was found
272
against both of the tested Gram-positive and Gram-negative pathogenic bacterial strains.
273
Furthermore, the result indicates that Gram-negative bacterial strains were highly
274
susceptible for AuNPs, compared to the Gram-positive bacterial strains. Previously few
275
reports mentioned that the biosynthesised AuNPs were more effective against Gram-
276
negative bacterial strains, compared to Gram-positive bacteria, since it has thicker layer
277
of peptidoglycan, which protect the attack of the AuNPs [40–42]. Interestingly, the
278
present study confirms that the tricetin synthesised AuNPs showed broad-spectrum
279
activity against both kind of bacterial groups. Previously, Pistelli et al. [29] confirmed the
280
antibacterial activity of tricetin. In addition, similar kind of flavonoids like isopomiferin
281
inhibited methionyl-tRNA synthetase (MetRS) enzyme activity and showed broad
282
spectrum antibacterial activity [43]. Therefore, the observed broad-spectrum activity
283
might be due to the bioflavonoid tricetin used for the AuNPs synthesis, since it has
284
antibacterial activity. Apart from this, the smaller size of the nanoparticles also may offer
285
larger surface-to-volume ratio and also highly effective contact with the surface of
286
bacteria, which enhancing the antimicrobial efficiency of the tricetin synthesised AuNPs
287
[44]. Thus, in this study, capping agent tricetin may effectively attack all of the tested
288
bacterial strains along with nano Au conductivity. These results clearly indicate that the
289
tricetin synthesised AuNPs may agreeable to bring as a therapeutic drug for the bacterial
290
infections in respiratory tract. Position for Fig. 6
291 292
3.5. Cytotoxicity assessment
14
293
As shown in Fig. 6, the synthesised AuNPs exhibits very less cytotoxicity (6%)
294
up to 50 µg mL-1. In the case of 75 µg mL-1 and 100 µg mL-1 concentrations of AuNPs,
295
83% and 54% of cell viability was observed. It explains that the tricetin synthesised
296
AuNPs may have dose-dependent cytotoxicity on the NHDF cells. This result confirms
297
that the synthesised AuNPs was biocompatible up to 50 µg mL-1. According to the
298
antibacterial activity results (Fig. 5), the MIC of AuNPs was found to be within 50 µg
299
mL-1 for all of the tested respiratory infections causing pathogenic bacterial strains. Thus,
300
the tricetin synthesised AuNPs might be an efficient candidate to treat respiratory
301
infections causing pathogenic bacterial strains.
302
4. Conclusions
303
In this study, AuNPs were successfully synthesised using flavonoid tricetin and
304
further used to treat respiratory infections causing bacterial pathogens. The synthesised
305
nanoparticles were observed to be spherical in shape with the size of 12 nm in diameter.
306
The identified respiratory infections causing pathogenic bacterial strains were S. aureus,
307
E. xiangfangensis, B. licheniformis, E. fergusonii, A. pittii, P. aeruginosa, A.
308
enteropelogenes and P. mirabilis. The tricetin synthesised AuNPs demonstrated broad
309
spectrum antibacterial activity against both of the tested Gram-negative and Gram-
310
positive bacterial strains. The cytotoxicity result confirms that the synthesised AuNPs is
311
more biocompatible up to 50 µg mL-1 for therapeutic applications. Finally, this study
312
suggests that tricetin synthesised AuNPs might be an effective drug to treat respiratory
313
infections causing pathogenic bacterial strains without any adverse side effects on host.
314
Acknowledgment
15
315
This project was supported by the Deanship of Scientific Research at Prince
316
Sattam Bin Abdulaziz University under the research project 2019/01/10137.
317
Conflict of interests
318
The authors declare that have no competing interests in this investigation.
319
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Figure legends
463
Figure 1. Chemical structure of flavonoid tricetin
464
Figure 2. Uv-vis spectrum of the tricetin synthesised AuNPs under optimized conditions
465
(pH 8, temperature 30 °C, tricetin concentration 125 µM and chloroauric acid
466
250 µM). Insert: corresponding photograph of (i) chloroauric acid, (ii) tricetin
467
and (iii) the tricetin synthesised AuNPs
468 469 470 471 472 473 474
Figure 3. (a) TEM image (insert: histogram of nanoparticles), (b) SAED pattern, (c) FTIR spectrum and (d) XRD pattern of the synthesised AuNPs Figure 4. Antibacterial activity: ZOI of AuNPs against tested opportunistic bacterial pathogens Figure 5. Antibacterial activity: MIC of AuNPs against tested opportunistic bacterial pathogens Figure 6. Cytotoxicity of AuNPs against primary normal human dermal fibroblast cells
Table 1. Effect of pH, temperature, tricetin and chloroauric acid levels on surface plasmon resonance (SPR) of AuNPs formation Optimization experiments Effect of pH 4 5 6 7 8 9 Effect of temperature 20 °C 30 °C 40 °C 50 °C 60 °C Effect of tricetin 25 µM 50 µM 75 µM 100 µM 125 µM 150 µM Effect of chloroauric acid 50 µM 100 µM 150 µM 200 µM 250 µM 300 µM
SPR of AuNPs in UV-vis spectrum Peak intensity Wavelength (nm) 0.37 0.43 0.53 0.58 0.58 0.50
545 541 538 534 530 522
0.43 0.58 0.60 0.61 0.55
538 530 530 530 530
0.18 0.38 0.46 0.61 0.61 0.18
530 530 530 530 530 535
0.13 0.61 0.69 0.79 0.83 0.84
525 530 530 530 530 535
Experimental conditions for AuNPs synthesis optimization studies. (i) pH optimization: temperature 30 °C, tricetin concentration 100 µM and chloroauric acid 100 µM; (ii) Temperature optimization: pH 8 and tricetin concentration 100 µM and chloroauric acid 100 µM; and (iii) Tricetin concentration optimization: pH 8, Temperature 30 °C, and chloroauric acid concentration 100 µM; and (iv) Chloroauric acid concentration optimization: pH 8, Temperature 30 °C, and tricetin concentration 125 µM. Results expressed with the mean value of triplicate experiments.
Table 2. Opportunistic bacterial pathogens isolated from immunocompromised patients with respiratory infections S. No 1 2 3 4 5 6 7 8
Organism name Staphylococcus aureus Enterobacter xiangfangensis Bacillus licheniformis Escherichia fergusonii Acinetobacter pittii Pseudomonas aeruginosa Aeromonas enteropelogenes Proteus mirabilis
GenBank accession number MG818959 MG818960 MG818961 MG818962 MG818963 MG818964 MG818965 MG818966
Identity (%) 99 99 99 99 99 99 99 100
Fig. 1
i
Fig. 2
ii
iii
a
b
c
d
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Highlights AuNPs were successfully synthesized using flavonoid tricetin. AuNPs are polydispersed and spherical in shape with the size of 12 nm. AuNPs exhibited broad-spectrum antibacterial activity. AuNPs showed high biocompatibility on NHDF-c cells up to 50 µM mL-1.
Authors statement This manuscript has not been published and is not under consideration for publication elsewhere. Authors have no conflicts of interest to disclose. Declaration of interests ☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Sincerely, Dr. Fuad Ameen, Assistance Professor, Dept. of Botany & Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia