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Antimicrobial efficacy of drug blended biosynthesized colloidal gold nanoparticles from Justicia glauca against oral pathogens: A nanoantibiotic approach
Accepted Manuscript Antimicrobial efficacy of drug blended biosynthesized colloidal gold nanoparticles from Justicia glauca against oral pathogens: A nanoantibiotic approach Rayappan Emmanuel, Muthupandian Saravanan, Muhammad Ovais, Sethuramasamy Padmavathy, Zabta Khan Shinwari, Periyakaruppan Prakash PII:
S0882-4010(17)31269-X
DOI:
10.1016/j.micpath.2017.10.055
Reference:
YMPAT 2564
To appear in:
Microbial Pathogenesis
Received Date: 5 October 2017 Revised Date:
27 October 2017
Accepted Date: 27 October 2017
Please cite this article as: Emmanuel R, Saravanan M, Ovais M, Padmavathy S, Shinwari ZK, Prakash P, Antimicrobial efficacy of drug blended biosynthesized colloidal gold nanoparticles from Justicia glauca against oral pathogens: A nanoantibiotic approach, Microbial Pathogenesis (2017), doi: 10.1016/ j.micpath.2017.10.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
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Antimicrobial efficacy of drug blended biosynthesized colloidal gold nanoparticles from Justicia glauca against oral pathogens: A nanoantibiotic approach
e
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Padmavathy , Zabta Khan Shinwari , Periyakaruppan Prakash a
a
c,d
, Sethuramasamy
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Rayappan Emmanuel a, Muthupandian Saravanan *b, Muhammad Ovais
Post Graduate & Research Department of Chemistry, Thiagarajar College, Madurai-625009,
Tamilnadu, India. b
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Department of Medical Microbiology and Immunology, Institute of Biomedical Sciences, College of Health Science, Mekelle University, Mekelle-1871, Ethiopia. Department of Biotechnology, Quaid-i-Azam University, Islamabad-45320, Pakistan.
d
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National
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Center for Nanoscience and Technology of China, Beijing 100190, China. e
Department of Zoology and Microbiology, Thiagarajar College, Madurai - 625009, Tamil Nadu, India.
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Corresponding Author: *Muthupandian Saravanan,
Department of Microbiology and Immunology,
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Institute of Biomedical Sciences, College of Health Science, Mekelle University, Mekelle-1871, Ethiopia
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Tel: +251344416696, Fax: +251344416681, Email: [email protected]; [email protected]
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Graphical Abstract. Overall scheme of the study for the phytosynthesis of AuNPs, its drug
blending and potential synergistic anti-microbial activity.
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Abstract Antimicrobial resistance is a challenging task for researchers to develop new strategies. Green
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synthesis of AuNPs is an eco-friendly approach, which can be utilized in the microbistatic and microbicidal activities. The current study is focused on Justicia glauca (aqueous leaf extract) mediated AuNPs synthesis at room temperature by treating chloroaurate ions, that shows an
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antagonistic effect with Azithromycin (AZM) and Clarithromycin (CLR) antibiotics against oral pathogenic bacteria and fungi (Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus,
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Streptococcus mutans, Lactobacillus acidophilus, Escherichia coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Candida albicans). Characterization of green synthesized AuNPs
was done by using Ultraviolet-visible (UV-vis) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD) analysis and Energy Dispersive X-ray analysis (EDAX). Biosynthesized AuNPs were stable, hexagonal and
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spherical shaped with a size ~ 32.5±0.25 nm. The AuNPs and drug conjugated AuNPs showed potential antibacterial and antifungal activity against the oral pathogens. Minimum Inhibitory Concentration (MIC) values of biogenic AuNPs were observed in the range of 6.25 - 25 µg/mL
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against selected oral pathogens. Overall, we conclude that biogenic drug delivery system for
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AZM and CLR can be exploited as potential antimicrobial therapy in future, subject to detailed in-vitro and in-vivo cytotoxicity.
Key Words: Green Synthesis; Gold nanoparticles; Justicia glauca; Antimicrobial activity; Oral Pathogens.
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1. Introduction Nano-biotechnology is an exceptional approach which has influenced the synthesis of wide-
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ranging nano-biomaterials that can be used in biology and medicine. A lot of research work and manufacturing procedures were applied to synthesize gold nanoparticles (AuNPs) by different physical and chemical methods [1-4]. The bottleneck of current physical methods is the costly synthesis of nanoparticles (NPs) and low yield [5-7]. The drawback of the chemical methods
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involved toxic chemical compounds that adsorb on the surface of NPs leading to undesirable side-effects in medical applications. Moreover, the production of metal NPs is undesirable due to
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its polydispersity and instability. Foreseeing, aforementioned problems, researchers have focused to develop low cost, non-toxic and environment friendly approaches for the green synthesis of NPs with high purity, stability and low polydispersity [8]. We have unlimited biological resources in natural environment including plants, algae, fungi, yeast, bacteria and viruses. These
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organisms can be used for the synthesis of NPs through both intracellular and extracellular methods. These methods are remarkably interesting because of the synthesized NPs exhibit best
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compatibility with biomolecules [9-14].
The sensitivity of spherical NPs used for bio-detection is not feasible enough to trace the
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interaction of biomolecules [15]. Contrarily some irregular shaped AuNPs can easily adsorb biomolecules on their surface showing a good plasmon resonance and hence can be applied in detection of cancer cells [16]. Moreover, few irregular shaped AuNPs showed resemblance to certain bacteria that can penetrate the human cells. This fact suggests that the shape of NPs can be used to detect different diseases [17-19]. Previous reports are available on different applications of AuNPs i.e. immune response enhancement [20], detection and control of microorganisms [21, 22], clinical chemistry [23], cancer cell photothermolysis [24, 25], optical 4
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imaging of cancer cells by exploiting resonance scattering [26], targeted drugs delivery [25, 27], two-photon luminescence [28] and optical coherence tomography [29]. Although AgNPs alone demonstrates the highest antibacterial activity amongst metal NPs [12, 30], but antibiotic-
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conjugated AuNPs have shown the potential photo-thermal killing of protozoa and bacteria [3133]. AuNPs alone do not affect bacterial growth but conjugated AuNPs to vancomycin decreased the bacteria growth [32]. The effectiveness of several antibiotics bound by AuNPs has been
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reported that inhibit the proliferation of gram-positive and gram-negative bacteria as compared to the same quantity of antibiotics used alone [34]. It was proved that conjugated anticancer
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compound 5-fluorouracil to AuNPs showed more competence against bacteria and fungi than the compound 5-fluorouracil alone [35]. Therefore, conjugated NPs are capable of transporting antibiotics to a specific location [36, 37].
Currently, researchers have focused on oral health hygiene, which can improve the quality of
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life, ~ 75 bacteria and fungi strains are reported to be associated with oral diseases [38, 39]. The available chemical reagents had modified the oral microbiota which caused side effects such as; vomiting, diarrhea and tooth staining. Generally, conventional drugs can play a role in antibiotics
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therapy but there is an increasing concern of antibiotics resistance and adverse side effects like
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hypersensitivity, immune suppressive and allergic reactions [39]. Therefore, researchers are trying to make unconventional products from natural products [40]. The biomolecules present in plants can hamper the growth of oral pathogens, decrease dental plaque and diminish the symptoms of the oral diseases [41]. In the current study, we have reported biogenic AuNPs that are synthesized from leaf extract of Justicia glauca (J. glauca), one of the widespread species of Acanthaceae having ~ 600 species distributed in the tropical regions as perennial herbs [42]. A variety of water-soluble 5
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heterocyclic compounds are found in Justicia i-e lignans, alkaloids, flavonoids, steroids and terpenoids along other compounds i.e. essential oils, vitamins, fatty acids and salicylic acid [43]. Generally, lignans contain a large number of bioactive compounds that show different biological
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effects. Therefore, used for the development of new therapeutic agents with cytotoxic activity [44, 45]. Characterization of biosynthesized AuNPs was performed via UV-Vis spectroscopy; TEM, XRD and FTIR. Additionally, Minimum Inhibitory Concentration (MIC) and synergistic
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broad-spectrum antimicrobial activity of AuNPs and drug blended AuNPs has been studied
2. Experimental Procedures 2.1. Materials
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against selected oral pathogens.
All the reagents were purchased from Hi-Media Laboratories Pvt Ltd. (Mumbai, India) and
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Sigma-Aldrich Chemicals (St. Louis, USA). The collection of J. glauca leaves was done from Indian Sirumalai hills of Tamil Nadu. Selected oral pathogens (Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans, Lactobacillus acidophilus, Escherichia
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coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Candida albicans) were studied
for their antimicrobial activity. These strains were obtained from the Department of Medical
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Microbiology Laboratory, Institute of Biomedical Science, College of Health Science, Mekelle University, Ethiopia.
2.2. Leaf extract Preparation of J. glauca
Leaves of J. glauca were washed to get rid of dust particles along with other contaminations and then kept on filter paper to remove moisture content, followed by 1 hr room temperature airdrying. One gram fresh leaves were cut into tiny pieces and boiled for 15 min in 300 mL sterile
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distilled water, followed by room temperature cooling and filtering twice via Whatmann filter paper. 2.3. Biosynthesis of AuNPs via Justicia glauca leaf extract
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For the green of synthesis of AuNPs, an aqueous solution of J. glauca leaf extract and 1 mM HAuCl4.3H2O are mixed in 1:1 at room temperature. After 10 min, the leaf extract turned to pink red indicating the formation of AuNPs. The resulting solution was kept for 60 min to ensure the
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formation of AuNPs. The color changes indicate the formation of AuNPs in aqueous solution due to excitation of surface plasmon vibration in the metal nanoparticles. Later, AuNPs were
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centrifuged (5,000 rpm, 15 min), and washed three times with deionised water to remove the untreated biomolecules and impurities. The AuNPs were lyophilized at -170 ◦C to -196 ◦C under vacuum. After lyophilization, the AuNPs were stored in screw cap bottle for further characterization.
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2.4. Characterization of synthesized AuNPs
Leaf extract of J. glauca was exploited as potential reducing and capping agent for the synthesis of AuNPs. The synthesized AuNPs were analyzed by PANalyticalX’Pert PRO X-ray
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diffractometer to record the X-Ray Diffraction (XRD) spectra. UV-vis Spectroscopic analysis was done by Jasco V-560 double-beam spectrophotometer. Furthermore, determination of the
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size and morphology of synthesized AuNPs was done by TEM (JEOL JEM 2100) followed by elemental analysis via Energy Dispersive X-ray (EDX) Analyzer attached with the same instrument. Additionally, Fourier Transform Infrared (FT-IR) spectrum was detected through Shimadzu FTIR-8201PC.
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2.5. Test organism’s characterization
The oral pathogens were obtained and screened according to the method prescribed in the
followed by storage at 4 °C and sub-cultured every month. 2.6. Minimal inhibitory concentration (MIC) Determination
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manuals of Bacteriology [46]. To keep the cultures maintained, they were grown on agar slants
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Microdilution method was conducted to estimate MIC values for the antimicrobial assay. Following the established protocols, 96 well microdilution plates were utilized for the
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determination of MIC [47]. Overnight cultures of oral pathogens were sub cultured in 1 ratio 10,000 into Muller Hinton broth. After addition of 100 µL sample of bacterial cultures into 96well plates, serial dilutions of AuNPs (50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 µg/mL) were added accordingly. The microplates were incubated at 37 °C for 24 hrs to observed MICs of the tested organisms. Moreover, for the measurement of optical density (OD600) Spectrostar NANO
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Microplate Reader (BMG Labtech) was exploited. MIC value of the AuNPs was compared to Azithromycin (AZM) used as positive control. Microbial growth was demarcated by the two-fold upsurge of the OD600 in comparison of negative control (MH broth alone). The assay was
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carried out in triplicate.
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2.7. Analysis of antimicrobial activity
Well diffusion assay was adopted with some alteration in the previously reported procedure Schillinger and Lücke [48]. The sample concentrations were measured in µg/mL. Muller Hinton Agar medium (pH of 7.2 ± 0.2) was poured in plates at 7 mm depth and solidified by refrigeration. Tested organisms were grown overnight and the bacterial suspension was adjusted to McFarland Standard 0.5. Standard inoculum concentration was maintained in the plates to make sure a confluent growth of organisms. A well cutter was used to make 6 mm wells in agar 8
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plates poured with 100 µL (100 µg/mL) of AuNPs sample in each well. Incubation of the plates was done at 37 °C for 24 hrs and subsequently examined for clear inhibition zone surrounding by the wells. The assay was repeated thrice for the tested pathogens and AuNPs alone were
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maintained as control [49]. Following the recommended protocol, the synergistic antimicrobial activity of AuNPs with Azithromycin (AZM) and Clarithromycin (CLR) antibiotics was checked in 1:1 ratio (50 µg:50 µg) with a final concentration of 100 µg/mL against the entire set of oral
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pathogens.
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3. Results and discussion
Green synthesis of metallic nanoparticles via plant extracts have many advantages over other biosynthesis routes i-e from fungi, bacteria etc [50]. The phytochemicals present in plant extract act as both reducing and capping agents, resulting in the synthesis of highly stable and
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biocompatible metallic nanoparticles. In the leaf extract of Justicia glauca there are many versatile essential phytochemicals which are responsible for the reduction of [AuCl4]- present in gold chloride solution to Au0. Moreover, along with other low and high molecular weight
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proteins these phytochemicals stabilize the synthesized AuNPs by capping. The diverse set of phytochemicals includes iridods, diterpenoids, lignans, docosanoic acid, salicylic acid and many
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other essential oils, vitamins and fatty acid [42]. The proposed mechanism of AuNPs synthesis via leaf extract of J. glauca is shown in Figure 1.
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Figure 1. Proposed mechanism of AuNPs biosynthesis via Justicia glauca leaf extract.
3.1. Surface Plasmon resonance
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SPR peaks attained in UV–vis spectroscopy is one of the versatile techniques to confirm the formation of metal NPs. SPR was generated due to the coherence of electrons on the surface of
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AuNPs. As shown in Figure 2, the UV-vis spectra of biogenic colloidal AuNP shifted from 588, to 562, to 542 nm over time 10, 30 and 60 minutes of reaction respectively. As the time increases (10-60 min), the number of AuNPs increases and the size of AuNPs decreases which was confirmed by UV-Vis spectra. The conjugation length and intensities decrease from 588 to 542 nm which indicates the decrease in size. The shift to the blue or red in the λmax of the SPR peak could be related to obtaining gold nanoparticles at various shapes, sizes or extract dependencies
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of formed AuNPs [51, 52]. As depicted form spectra, the peak intensity gradually increases as a function of reaction time and the colour change was first noticed within 10 min of reaction form
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pale yellow to pink red [53, 54].
Figure 2. UV-Vis spectra of Justicia glauca leaf mediated green synthesized AuNPs recorded at
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various time intervals.
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3.2. Crystal structure study
The XRD pattern correlated with JCPDS data No. 04-0784 confirms the crystalline nature of AuNPs by showing sharp diffraction peak at 38.2°. Due to the capping of AuNPs with plant phytochemicals, the suggest strong X-ray diffraction peaks 38.2° in the crystalline phase can correspond to the (111) reflection of the metallic gold with fcc structure. Moreover, comparatively weak diffraction peaks at 44.3°, 64.6° and 78° are corresponding with (200), (220) and (311) reflections respectively. Further, the unassigned peak appearing at 25.6° is due to the 11
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crystallization of bioorganic phases which occurs on the surface of the nanoparticles. For specific particle size recognition, the broadening of peaks in the XRD patterns is a key indicator. Thus,
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from the XRD pattern, highly crystalline nature of biogenic AuNPs is confirmed (Figure 3).
Figure 3. X-ray diffraction patterns of Justicia glauca leaf extract mediated biosynthesized
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AuNPs, showing highly crystalline nature.
3.3. Size and Morphological analysis of AuNPs
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The TEM images confirmed that green synthesized AuNPs are hexagonal, some are spherical and nanoprism shaped at different magnifications (Figure 4 a, b, c). EDX analysis of AuNPs has been done to recognise the composition of gold. As shown in Figure 4 d a sharp signal at 2 ev confirms AuNPs formation. The selected area (electron) diffraction (SAED) pattern shown that bright circular rings correspond to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of fcc gold
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planes. It obviously pinpoints that the entire AuNPs are crystalline with the same lattice
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orientation (Figure 5).
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Figure 4. TEM images of AuNPs at different magnifications (a–c). The corresponding EDX
profile of AuNPs (d).
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Figure 5. Selected area (electron) diffraction (SAED) pattern of biogenic AuNPs, showing the diffraction planes. 3.4. FT-IR spectral analysis of AuNPs
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FTIR spectra of the J. glauca leaf extract powder (a) and its synthesized AuNPs (b) is shown in Figure 6. The J. glauca leaf extract spectrum exhibits significant absorption bands at 3371 cm−1 (O-H stretching), 2925 cm−1 and 2850 cm−1 (C–H– stretching), 1649 cm−1 (C=O stretching),
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1603 cm−1 and 1514 cm−1 (C=C stretching), 1400 cm−1 (C–H bending), 1153 cm−1 (C–O stretching) and small peaks in the region of 500–1000 cm−1 (aromatic C–H out of plan bending)
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(54-55). The AuNPs showed significant peaks at 3435 cm−1 (O-H stretching), 2925 and 2850 cm−1 (C–H– stretching), 1767 cm−1 (OH - in aromatic stretching), 1599 (C-C aromatic stretching), 1425 (C-H group), 500–1000 cm−1 region (C–H out of plane bending) which are of characteristics of phenolic compounds. The band pattern of the functional groups attributed to the adsorption of polyphenols and flavonoids, which may be responsible for the capping and stabilization of biogenic AuNPs.
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Figure 6. FT-IR spectra of Justicia glauca leaf powder and its biosynthesized AuNPs. 3.5. Screening of MIC for the green synthesized AuNPs
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The antimicrobial activities of green synthesized AuNPs were carried out among selected strains
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comprised both pathogens and opportunistic pathogens of dental caries and periodontal diseases. The MIC of Au nanoparticles was determined by treating the strains with different concentrations (50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 µg/mL) of Au nanoparticles. MIC of AuNPs was checked by broth micro-dilution method. The optical density of bacterial suspension was measured at 600 nm wavelength. The green synthesized AuNPs showed significant activity with MIC ranged 25-6.2 µg/mL for bacterial and 12.5 µg/mL for fungal pathogens respectively (Figure 7 and Table 1). MIC against S. mutans, L. acidophilus and E. coli showed 25 µg/mL
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whereas against M. luteus, B. subtilis, S. aureus, S. cerevisiae and C. albicans was found to be
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12.5 µg/mL. MIC in case of AuNPs against P. aeruginosa was exhibited 6.25 µg/mL.
Figure 7. MICs of AuNPs against tested bacterial and fungal strains. For the MIC determination
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Serial dilutions of biogenic AuNPs were exposed to bacterial strains. ML-Micrococcus luteus, BABacillus subtilis, SA - Staphylococcus aureus, SM- Streptococcus mutans, LA-. Lactobacillus
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acidophilus, EC- Escherichia coli, PA- Pseudomonas aeruginosa, SC- Saccharomyces cerevisiae, CACandida albican PC- Positive Control, NC- Negative Control. Black Arrows denote the MICs for
this specific experiment.
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Table 1. Minimum inhibitory concentrations (µg/mL ± SD) of gold nanoparticles of Justicia
MIC (µg/mL ± SD)
Micrococcus luteus
12.5 ± 0.2
Bacillus subtilis
12.5 ± 0.0
Staphylococcus aureus
12.5 ± 0.3
Streptococcus mutans
25 ± 0.2
Escherichia coli Pseudomonas aeruginosa
25 ± 0.0 25 ± 0.4
6.25 ± 0.1 12.5 ± 0.0
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Saccharomyces cerevisiae
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Lactobacillus acidophilus
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Tested Microorganisms
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glauca against oral pathogens.
Candida albicans
12.5 ± 0.3
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3.6. Screening for Antimicrobial Activity of drug loaded AuNPs Synergistic activity of biogenic AuNPs with antibiotics was conducted on selected oral
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pathogens as shown in Figure 8. The AuNPs (100 µg) and drug conjugated AuNPs (50 µg drug + 50 µg AuNPs) exhibit broad-spectrum antimicrobial activities against the tested pathogens. Our AuNPs showed antimicrobial activity by making a zone of inhibition ranged 14-17 mm against tested pathogens except for B. subtilis, which exhibit less response of 9 mm zone. The AuNPs exhibits maximum antifungal efficacy against S. cerevisiae and C. albicans, by making 19 and 17 mm zones respectively. Figure 9 shows that 100 µg of antibiotics (AZM and CLR) has
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moderate activity against the selected pathogens, when it was compared to drug blended AuNPs
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(1: 1 ratio).
Figure 8. Broad spectrum antimicrobial efficacy of drug blended green synthesized AuNPs
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against the selected oral pathogens 1). Micrococcus luteus, 2). Bacillus subtilis, 3). Staphylococcus aureus, 4). Streptococcus mutans, 5) Lactobacillus acidophilus, 6). Escherichia coli, 7). Pseudomonas aeruginosa, 8). Saccharomyces cerevisiae, 9). Candida albicans. (In each image: AuNPs - gold nanoparticles, AZM - Azithromycin, CLR- Clarithromycin, AZM +
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AuNPs- Azithromycin conjugated- gold nanoparticles, CLR + AuNPs - Clarithromycin
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conjugated- gold nanoparticles).
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Figure 9. Evaluation of broad spectrum antimicrobial activity of AuNPs, Drug alone and drug
blended AuNPs against dental caries and periodontal disease causing oral pathogenic
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microorganisms.
In comparison to biogenic AuNPs and drugs alone the AuNPs blended antibiotics (50 µg each)
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were found to be highly effective against the potential oral pathogens. The proposed reason for highly effective activity of drug conjugated AuNPs can be attributed to AuNPs drug carrier potential. As shown in Figure 10 the hydrophobic nature of bacterial cell membrane made of phospholipids and glycoproteins may facilitate the transfer of drug loaded AuNPs (hydrophilic) across the membrane [55-60]. The proposed mechanism of bacterial growth inhibition is the interference of NPs with growth-signaling pathway inside the cell. Moreover, via modulating tyrosine phosphorylation of growth essential peptides substrate, bacterial cell growth is inhibited 19
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[33]. The lack of aggregation in NPs depict high stability due to the coating of plant phytochemical. There is great potential in the proposed anti-microbial activity of biogenic NPs
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synthesized via J. glauca.
4. Conclusion
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Figure 10. The plausible mechanism for biogenic AuNPs killing bacterial cells.
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Green synthesis of AuNPs from Justicia glauca extract is a novel approach towards the wellbeing of humankind. The broad-spectrum antimicrobial efficacy increased by coating NPs with antibiotics against selected oral pathogens suggest that synthesized AuNPs can be used as a potential tool to combat oral pathogens. With the recent trends in ongoing research biogenic AuNPs can be explored in diverse biomedical applications such as drug delivery. Moreover, the plant-based NPs synthesis may have commercial viability to be developed as future nanomedicine. 20
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Acknowledgements The support of Thiagarajar College, Madurai, Tamilnadu, India, SAIF (Sophisticated Analytical Instrument Facility) IIT, Chennai, India, and Department of Biotechnology, Quaid-i-Azam
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University, Islamabad, Pakistan is highly acknowledged for the provision of research facilities.
for providing facilities for anti-microbial studies. Conflict of interest
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The authors declare no conflict of interest.
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Department of Microbiology laboratory, Mekelle University, Ethiopia is highly acknowledged
Author’s contribution
MS, RE, PP for conceiving the idea. RE, SP, MO for performing the experimental. MO, MS, RE,
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nanoparticles: green approach. Environ Sci Pollut Res Int. (2017). doi: 10.1007/s11356-017-
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Highlights
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Eco-friendly approach for the biosynthesis of gold nanoparticles (AuNPs) form a novel plant Justicia glauca has been reported. The phytosynthesized AuNPs were blended with Azithromycin (AZM) and Clarithromycin (CLR) antibiotics. The biogenic AuNPs shows an antagonistic effect with antibiotics against oral pathogenic bacteria and fungi.
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DOI:
10.1016/j.micpath.2017.10.055
Reference:
YMPAT 2564
To appear in:
Microbial Pathogenesis
Received Date: 5 October 2017 Revised Date:
27 October 2017
Accepted Date: 27 October 2017
Please cite this article as: Emmanuel R, Saravanan M, Ovais M, Padmavathy S, Shinwari ZK, Prakash P, Antimicrobial efficacy of drug blended biosynthesized colloidal gold nanoparticles from Justicia glauca against oral pathogens: A nanoantibiotic approach, Microbial Pathogenesis (2017), doi: 10.1016/ j.micpath.2017.10.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
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Antimicrobial efficacy of drug blended biosynthesized colloidal gold nanoparticles from Justicia glauca against oral pathogens: A nanoantibiotic approach
e
c
Padmavathy , Zabta Khan Shinwari , Periyakaruppan Prakash a
a
c,d
, Sethuramasamy
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Rayappan Emmanuel a, Muthupandian Saravanan *b, Muhammad Ovais
Post Graduate & Research Department of Chemistry, Thiagarajar College, Madurai-625009,
Tamilnadu, India. b
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Department of Medical Microbiology and Immunology, Institute of Biomedical Sciences, College of Health Science, Mekelle University, Mekelle-1871, Ethiopia. Department of Biotechnology, Quaid-i-Azam University, Islamabad-45320, Pakistan.
d
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National
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Center for Nanoscience and Technology of China, Beijing 100190, China. e
Department of Zoology and Microbiology, Thiagarajar College, Madurai - 625009, Tamil Nadu, India.
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Corresponding Author: *Muthupandian Saravanan,
Department of Microbiology and Immunology,
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Institute of Biomedical Sciences, College of Health Science, Mekelle University, Mekelle-1871, Ethiopia
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Tel: +251344416696, Fax: +251344416681, Email: [email protected]; [email protected]
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Graphical Abstract. Overall scheme of the study for the phytosynthesis of AuNPs, its drug
blending and potential synergistic anti-microbial activity.
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Abstract Antimicrobial resistance is a challenging task for researchers to develop new strategies. Green
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synthesis of AuNPs is an eco-friendly approach, which can be utilized in the microbistatic and microbicidal activities. The current study is focused on Justicia glauca (aqueous leaf extract) mediated AuNPs synthesis at room temperature by treating chloroaurate ions, that shows an
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antagonistic effect with Azithromycin (AZM) and Clarithromycin (CLR) antibiotics against oral pathogenic bacteria and fungi (Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus,
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Streptococcus mutans, Lactobacillus acidophilus, Escherichia coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Candida albicans). Characterization of green synthesized AuNPs
was done by using Ultraviolet-visible (UV-vis) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD) analysis and Energy Dispersive X-ray analysis (EDAX). Biosynthesized AuNPs were stable, hexagonal and
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spherical shaped with a size ~ 32.5±0.25 nm. The AuNPs and drug conjugated AuNPs showed potential antibacterial and antifungal activity against the oral pathogens. Minimum Inhibitory Concentration (MIC) values of biogenic AuNPs were observed in the range of 6.25 - 25 µg/mL
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against selected oral pathogens. Overall, we conclude that biogenic drug delivery system for
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AZM and CLR can be exploited as potential antimicrobial therapy in future, subject to detailed in-vitro and in-vivo cytotoxicity.
Key Words: Green Synthesis; Gold nanoparticles; Justicia glauca; Antimicrobial activity; Oral Pathogens.
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1. Introduction Nano-biotechnology is an exceptional approach which has influenced the synthesis of wide-
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ranging nano-biomaterials that can be used in biology and medicine. A lot of research work and manufacturing procedures were applied to synthesize gold nanoparticles (AuNPs) by different physical and chemical methods [1-4]. The bottleneck of current physical methods is the costly synthesis of nanoparticles (NPs) and low yield [5-7]. The drawback of the chemical methods
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involved toxic chemical compounds that adsorb on the surface of NPs leading to undesirable side-effects in medical applications. Moreover, the production of metal NPs is undesirable due to
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its polydispersity and instability. Foreseeing, aforementioned problems, researchers have focused to develop low cost, non-toxic and environment friendly approaches for the green synthesis of NPs with high purity, stability and low polydispersity [8]. We have unlimited biological resources in natural environment including plants, algae, fungi, yeast, bacteria and viruses. These
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organisms can be used for the synthesis of NPs through both intracellular and extracellular methods. These methods are remarkably interesting because of the synthesized NPs exhibit best
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compatibility with biomolecules [9-14].
The sensitivity of spherical NPs used for bio-detection is not feasible enough to trace the
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interaction of biomolecules [15]. Contrarily some irregular shaped AuNPs can easily adsorb biomolecules on their surface showing a good plasmon resonance and hence can be applied in detection of cancer cells [16]. Moreover, few irregular shaped AuNPs showed resemblance to certain bacteria that can penetrate the human cells. This fact suggests that the shape of NPs can be used to detect different diseases [17-19]. Previous reports are available on different applications of AuNPs i.e. immune response enhancement [20], detection and control of microorganisms [21, 22], clinical chemistry [23], cancer cell photothermolysis [24, 25], optical 4
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imaging of cancer cells by exploiting resonance scattering [26], targeted drugs delivery [25, 27], two-photon luminescence [28] and optical coherence tomography [29]. Although AgNPs alone demonstrates the highest antibacterial activity amongst metal NPs [12, 30], but antibiotic-
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conjugated AuNPs have shown the potential photo-thermal killing of protozoa and bacteria [3133]. AuNPs alone do not affect bacterial growth but conjugated AuNPs to vancomycin decreased the bacteria growth [32]. The effectiveness of several antibiotics bound by AuNPs has been
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reported that inhibit the proliferation of gram-positive and gram-negative bacteria as compared to the same quantity of antibiotics used alone [34]. It was proved that conjugated anticancer
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compound 5-fluorouracil to AuNPs showed more competence against bacteria and fungi than the compound 5-fluorouracil alone [35]. Therefore, conjugated NPs are capable of transporting antibiotics to a specific location [36, 37].
Currently, researchers have focused on oral health hygiene, which can improve the quality of
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life, ~ 75 bacteria and fungi strains are reported to be associated with oral diseases [38, 39]. The available chemical reagents had modified the oral microbiota which caused side effects such as; vomiting, diarrhea and tooth staining. Generally, conventional drugs can play a role in antibiotics
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therapy but there is an increasing concern of antibiotics resistance and adverse side effects like
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hypersensitivity, immune suppressive and allergic reactions [39]. Therefore, researchers are trying to make unconventional products from natural products [40]. The biomolecules present in plants can hamper the growth of oral pathogens, decrease dental plaque and diminish the symptoms of the oral diseases [41]. In the current study, we have reported biogenic AuNPs that are synthesized from leaf extract of Justicia glauca (J. glauca), one of the widespread species of Acanthaceae having ~ 600 species distributed in the tropical regions as perennial herbs [42]. A variety of water-soluble 5
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heterocyclic compounds are found in Justicia i-e lignans, alkaloids, flavonoids, steroids and terpenoids along other compounds i.e. essential oils, vitamins, fatty acids and salicylic acid [43]. Generally, lignans contain a large number of bioactive compounds that show different biological
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effects. Therefore, used for the development of new therapeutic agents with cytotoxic activity [44, 45]. Characterization of biosynthesized AuNPs was performed via UV-Vis spectroscopy; TEM, XRD and FTIR. Additionally, Minimum Inhibitory Concentration (MIC) and synergistic
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broad-spectrum antimicrobial activity of AuNPs and drug blended AuNPs has been studied
2. Experimental Procedures 2.1. Materials
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against selected oral pathogens.
All the reagents were purchased from Hi-Media Laboratories Pvt Ltd. (Mumbai, India) and
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Sigma-Aldrich Chemicals (St. Louis, USA). The collection of J. glauca leaves was done from Indian Sirumalai hills of Tamil Nadu. Selected oral pathogens (Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans, Lactobacillus acidophilus, Escherichia
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coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Candida albicans) were studied
for their antimicrobial activity. These strains were obtained from the Department of Medical
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Microbiology Laboratory, Institute of Biomedical Science, College of Health Science, Mekelle University, Ethiopia.
2.2. Leaf extract Preparation of J. glauca
Leaves of J. glauca were washed to get rid of dust particles along with other contaminations and then kept on filter paper to remove moisture content, followed by 1 hr room temperature airdrying. One gram fresh leaves were cut into tiny pieces and boiled for 15 min in 300 mL sterile
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distilled water, followed by room temperature cooling and filtering twice via Whatmann filter paper. 2.3. Biosynthesis of AuNPs via Justicia glauca leaf extract
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For the green of synthesis of AuNPs, an aqueous solution of J. glauca leaf extract and 1 mM HAuCl4.3H2O are mixed in 1:1 at room temperature. After 10 min, the leaf extract turned to pink red indicating the formation of AuNPs. The resulting solution was kept for 60 min to ensure the
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formation of AuNPs. The color changes indicate the formation of AuNPs in aqueous solution due to excitation of surface plasmon vibration in the metal nanoparticles. Later, AuNPs were
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centrifuged (5,000 rpm, 15 min), and washed three times with deionised water to remove the untreated biomolecules and impurities. The AuNPs were lyophilized at -170 ◦C to -196 ◦C under vacuum. After lyophilization, the AuNPs were stored in screw cap bottle for further characterization.
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2.4. Characterization of synthesized AuNPs
Leaf extract of J. glauca was exploited as potential reducing and capping agent for the synthesis of AuNPs. The synthesized AuNPs were analyzed by PANalyticalX’Pert PRO X-ray
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diffractometer to record the X-Ray Diffraction (XRD) spectra. UV-vis Spectroscopic analysis was done by Jasco V-560 double-beam spectrophotometer. Furthermore, determination of the
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size and morphology of synthesized AuNPs was done by TEM (JEOL JEM 2100) followed by elemental analysis via Energy Dispersive X-ray (EDX) Analyzer attached with the same instrument. Additionally, Fourier Transform Infrared (FT-IR) spectrum was detected through Shimadzu FTIR-8201PC.
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2.5. Test organism’s characterization
The oral pathogens were obtained and screened according to the method prescribed in the
followed by storage at 4 °C and sub-cultured every month. 2.6. Minimal inhibitory concentration (MIC) Determination
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manuals of Bacteriology [46]. To keep the cultures maintained, they were grown on agar slants
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Microdilution method was conducted to estimate MIC values for the antimicrobial assay. Following the established protocols, 96 well microdilution plates were utilized for the
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determination of MIC [47]. Overnight cultures of oral pathogens were sub cultured in 1 ratio 10,000 into Muller Hinton broth. After addition of 100 µL sample of bacterial cultures into 96well plates, serial dilutions of AuNPs (50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 µg/mL) were added accordingly. The microplates were incubated at 37 °C for 24 hrs to observed MICs of the tested organisms. Moreover, for the measurement of optical density (OD600) Spectrostar NANO
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Microplate Reader (BMG Labtech) was exploited. MIC value of the AuNPs was compared to Azithromycin (AZM) used as positive control. Microbial growth was demarcated by the two-fold upsurge of the OD600 in comparison of negative control (MH broth alone). The assay was
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carried out in triplicate.
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2.7. Analysis of antimicrobial activity
Well diffusion assay was adopted with some alteration in the previously reported procedure Schillinger and Lücke [48]. The sample concentrations were measured in µg/mL. Muller Hinton Agar medium (pH of 7.2 ± 0.2) was poured in plates at 7 mm depth and solidified by refrigeration. Tested organisms were grown overnight and the bacterial suspension was adjusted to McFarland Standard 0.5. Standard inoculum concentration was maintained in the plates to make sure a confluent growth of organisms. A well cutter was used to make 6 mm wells in agar 8
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plates poured with 100 µL (100 µg/mL) of AuNPs sample in each well. Incubation of the plates was done at 37 °C for 24 hrs and subsequently examined for clear inhibition zone surrounding by the wells. The assay was repeated thrice for the tested pathogens and AuNPs alone were
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maintained as control [49]. Following the recommended protocol, the synergistic antimicrobial activity of AuNPs with Azithromycin (AZM) and Clarithromycin (CLR) antibiotics was checked in 1:1 ratio (50 µg:50 µg) with a final concentration of 100 µg/mL against the entire set of oral
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3. Results and discussion
Green synthesis of metallic nanoparticles via plant extracts have many advantages over other biosynthesis routes i-e from fungi, bacteria etc [50]. The phytochemicals present in plant extract act as both reducing and capping agents, resulting in the synthesis of highly stable and
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biocompatible metallic nanoparticles. In the leaf extract of Justicia glauca there are many versatile essential phytochemicals which are responsible for the reduction of [AuCl4]- present in gold chloride solution to Au0. Moreover, along with other low and high molecular weight
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proteins these phytochemicals stabilize the synthesized AuNPs by capping. The diverse set of phytochemicals includes iridods, diterpenoids, lignans, docosanoic acid, salicylic acid and many
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other essential oils, vitamins and fatty acid [42]. The proposed mechanism of AuNPs synthesis via leaf extract of J. glauca is shown in Figure 1.
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Figure 1. Proposed mechanism of AuNPs biosynthesis via Justicia glauca leaf extract.
3.1. Surface Plasmon resonance
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SPR peaks attained in UV–vis spectroscopy is one of the versatile techniques to confirm the formation of metal NPs. SPR was generated due to the coherence of electrons on the surface of
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AuNPs. As shown in Figure 2, the UV-vis spectra of biogenic colloidal AuNP shifted from 588, to 562, to 542 nm over time 10, 30 and 60 minutes of reaction respectively. As the time increases (10-60 min), the number of AuNPs increases and the size of AuNPs decreases which was confirmed by UV-Vis spectra. The conjugation length and intensities decrease from 588 to 542 nm which indicates the decrease in size. The shift to the blue or red in the λmax of the SPR peak could be related to obtaining gold nanoparticles at various shapes, sizes or extract dependencies
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of formed AuNPs [51, 52]. As depicted form spectra, the peak intensity gradually increases as a function of reaction time and the colour change was first noticed within 10 min of reaction form
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pale yellow to pink red [53, 54].
Figure 2. UV-Vis spectra of Justicia glauca leaf mediated green synthesized AuNPs recorded at
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various time intervals.
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3.2. Crystal structure study
The XRD pattern correlated with JCPDS data No. 04-0784 confirms the crystalline nature of AuNPs by showing sharp diffraction peak at 38.2°. Due to the capping of AuNPs with plant phytochemicals, the suggest strong X-ray diffraction peaks 38.2° in the crystalline phase can correspond to the (111) reflection of the metallic gold with fcc structure. Moreover, comparatively weak diffraction peaks at 44.3°, 64.6° and 78° are corresponding with (200), (220) and (311) reflections respectively. Further, the unassigned peak appearing at 25.6° is due to the 11
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crystallization of bioorganic phases which occurs on the surface of the nanoparticles. For specific particle size recognition, the broadening of peaks in the XRD patterns is a key indicator. Thus,
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from the XRD pattern, highly crystalline nature of biogenic AuNPs is confirmed (Figure 3).
Figure 3. X-ray diffraction patterns of Justicia glauca leaf extract mediated biosynthesized
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AuNPs, showing highly crystalline nature.
3.3. Size and Morphological analysis of AuNPs
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The TEM images confirmed that green synthesized AuNPs are hexagonal, some are spherical and nanoprism shaped at different magnifications (Figure 4 a, b, c). EDX analysis of AuNPs has been done to recognise the composition of gold. As shown in Figure 4 d a sharp signal at 2 ev confirms AuNPs formation. The selected area (electron) diffraction (SAED) pattern shown that bright circular rings correspond to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of fcc gold
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planes. It obviously pinpoints that the entire AuNPs are crystalline with the same lattice
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orientation (Figure 5).
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Figure 4. TEM images of AuNPs at different magnifications (a–c). The corresponding EDX
profile of AuNPs (d).
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Figure 5. Selected area (electron) diffraction (SAED) pattern of biogenic AuNPs, showing the diffraction planes. 3.4. FT-IR spectral analysis of AuNPs
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FTIR spectra of the J. glauca leaf extract powder (a) and its synthesized AuNPs (b) is shown in Figure 6. The J. glauca leaf extract spectrum exhibits significant absorption bands at 3371 cm−1 (O-H stretching), 2925 cm−1 and 2850 cm−1 (C–H– stretching), 1649 cm−1 (C=O stretching),
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1603 cm−1 and 1514 cm−1 (C=C stretching), 1400 cm−1 (C–H bending), 1153 cm−1 (C–O stretching) and small peaks in the region of 500–1000 cm−1 (aromatic C–H out of plan bending)
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(54-55). The AuNPs showed significant peaks at 3435 cm−1 (O-H stretching), 2925 and 2850 cm−1 (C–H– stretching), 1767 cm−1 (OH - in aromatic stretching), 1599 (C-C aromatic stretching), 1425 (C-H group), 500–1000 cm−1 region (C–H out of plane bending) which are of characteristics of phenolic compounds. The band pattern of the functional groups attributed to the adsorption of polyphenols and flavonoids, which may be responsible for the capping and stabilization of biogenic AuNPs.
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Figure 6. FT-IR spectra of Justicia glauca leaf powder and its biosynthesized AuNPs. 3.5. Screening of MIC for the green synthesized AuNPs
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The antimicrobial activities of green synthesized AuNPs were carried out among selected strains
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comprised both pathogens and opportunistic pathogens of dental caries and periodontal diseases. The MIC of Au nanoparticles was determined by treating the strains with different concentrations (50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 µg/mL) of Au nanoparticles. MIC of AuNPs was checked by broth micro-dilution method. The optical density of bacterial suspension was measured at 600 nm wavelength. The green synthesized AuNPs showed significant activity with MIC ranged 25-6.2 µg/mL for bacterial and 12.5 µg/mL for fungal pathogens respectively (Figure 7 and Table 1). MIC against S. mutans, L. acidophilus and E. coli showed 25 µg/mL
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whereas against M. luteus, B. subtilis, S. aureus, S. cerevisiae and C. albicans was found to be
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12.5 µg/mL. MIC in case of AuNPs against P. aeruginosa was exhibited 6.25 µg/mL.
Figure 7. MICs of AuNPs against tested bacterial and fungal strains. For the MIC determination
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Serial dilutions of biogenic AuNPs were exposed to bacterial strains. ML-Micrococcus luteus, BABacillus subtilis, SA - Staphylococcus aureus, SM- Streptococcus mutans, LA-. Lactobacillus
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acidophilus, EC- Escherichia coli, PA- Pseudomonas aeruginosa, SC- Saccharomyces cerevisiae, CACandida albican PC- Positive Control, NC- Negative Control. Black Arrows denote the MICs for
this specific experiment.
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Table 1. Minimum inhibitory concentrations (µg/mL ± SD) of gold nanoparticles of Justicia
MIC (µg/mL ± SD)
Micrococcus luteus
12.5 ± 0.2
Bacillus subtilis
12.5 ± 0.0
Staphylococcus aureus
12.5 ± 0.3
Streptococcus mutans
25 ± 0.2
Escherichia coli Pseudomonas aeruginosa
25 ± 0.0 25 ± 0.4
6.25 ± 0.1 12.5 ± 0.0
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Saccharomyces cerevisiae
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Lactobacillus acidophilus
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Tested Microorganisms
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glauca against oral pathogens.
Candida albicans
12.5 ± 0.3
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3.6. Screening for Antimicrobial Activity of drug loaded AuNPs Synergistic activity of biogenic AuNPs with antibiotics was conducted on selected oral
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pathogens as shown in Figure 8. The AuNPs (100 µg) and drug conjugated AuNPs (50 µg drug + 50 µg AuNPs) exhibit broad-spectrum antimicrobial activities against the tested pathogens. Our AuNPs showed antimicrobial activity by making a zone of inhibition ranged 14-17 mm against tested pathogens except for B. subtilis, which exhibit less response of 9 mm zone. The AuNPs exhibits maximum antifungal efficacy against S. cerevisiae and C. albicans, by making 19 and 17 mm zones respectively. Figure 9 shows that 100 µg of antibiotics (AZM and CLR) has
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moderate activity against the selected pathogens, when it was compared to drug blended AuNPs
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(1: 1 ratio).
Figure 8. Broad spectrum antimicrobial efficacy of drug blended green synthesized AuNPs
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against the selected oral pathogens 1). Micrococcus luteus, 2). Bacillus subtilis, 3). Staphylococcus aureus, 4). Streptococcus mutans, 5) Lactobacillus acidophilus, 6). Escherichia coli, 7). Pseudomonas aeruginosa, 8). Saccharomyces cerevisiae, 9). Candida albicans. (In each image: AuNPs - gold nanoparticles, AZM - Azithromycin, CLR- Clarithromycin, AZM +
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AuNPs- Azithromycin conjugated- gold nanoparticles, CLR + AuNPs - Clarithromycin
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conjugated- gold nanoparticles).
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Figure 9. Evaluation of broad spectrum antimicrobial activity of AuNPs, Drug alone and drug
blended AuNPs against dental caries and periodontal disease causing oral pathogenic
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microorganisms.
In comparison to biogenic AuNPs and drugs alone the AuNPs blended antibiotics (50 µg each)
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were found to be highly effective against the potential oral pathogens. The proposed reason for highly effective activity of drug conjugated AuNPs can be attributed to AuNPs drug carrier potential. As shown in Figure 10 the hydrophobic nature of bacterial cell membrane made of phospholipids and glycoproteins may facilitate the transfer of drug loaded AuNPs (hydrophilic) across the membrane [55-60]. The proposed mechanism of bacterial growth inhibition is the interference of NPs with growth-signaling pathway inside the cell. Moreover, via modulating tyrosine phosphorylation of growth essential peptides substrate, bacterial cell growth is inhibited 19
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[33]. The lack of aggregation in NPs depict high stability due to the coating of plant phytochemical. There is great potential in the proposed anti-microbial activity of biogenic NPs
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synthesized via J. glauca.
4. Conclusion
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Figure 10. The plausible mechanism for biogenic AuNPs killing bacterial cells.
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Green synthesis of AuNPs from Justicia glauca extract is a novel approach towards the wellbeing of humankind. The broad-spectrum antimicrobial efficacy increased by coating NPs with antibiotics against selected oral pathogens suggest that synthesized AuNPs can be used as a potential tool to combat oral pathogens. With the recent trends in ongoing research biogenic AuNPs can be explored in diverse biomedical applications such as drug delivery. Moreover, the plant-based NPs synthesis may have commercial viability to be developed as future nanomedicine. 20
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Acknowledgements The support of Thiagarajar College, Madurai, Tamilnadu, India, SAIF (Sophisticated Analytical Instrument Facility) IIT, Chennai, India, and Department of Biotechnology, Quaid-i-Azam
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University, Islamabad, Pakistan is highly acknowledged for the provision of research facilities.
for providing facilities for anti-microbial studies. Conflict of interest
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The authors declare no conflict of interest.
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Department of Microbiology laboratory, Mekelle University, Ethiopia is highly acknowledged
Author’s contribution
MS, RE, PP for conceiving the idea. RE, SP, MO for performing the experimental. MO, MS, RE,
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Highlights
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Eco-friendly approach for the biosynthesis of gold nanoparticles (AuNPs) form a novel plant Justicia glauca has been reported. The phytosynthesized AuNPs were blended with Azithromycin (AZM) and Clarithromycin (CLR) antibiotics. The biogenic AuNPs shows an antagonistic effect with antibiotics against oral pathogenic bacteria and fungi.
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