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Alaa M. Shawkey1, Abeer K. Abdulall2*, Mohamed A. Rabeh 3,4, and Ashraf O. Abdellatif 5
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Department of Microbiology and immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
2 Department of Microbiology and immunology, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
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3 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
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4Department of Pharmacognosy, Faculty of Pharmacy, Nahda University, Beni-Sueif, Egypt.
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5Department of Microbiology and immunology, Faculty of Pharmacy, Karary University , Khartoum, Sudan.
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10 * Corresponding author: E-mail: [email protected]; Tele: +201111520599, Fax: +2024052968
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Abstract: Green synthesis of metal nanoparticles is an attractive research area for their divers and potential
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implications in the field of nanomedicine. In the present study, the antibacterial, antifungal, antiviral and
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larvicidal activities of greenly synthesized silver nanoparticles (SNPs) in aqueous extracts (AEs) of fruits, leaves,
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roots and seeds of Citrullus colocynthis were investigated. The C. colocynthis/SNPs had potential biological
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activities that were negligible for the plant's aqueous extracts. The greenly synthesized SNPs showed a
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significant inhibitory action against the standard strains, of Escherichia coli, Neisseria gonorrhoeae, Klebsiella
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pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus
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pyogenes; antifungal activity against Aspergillus fumigatus, Candida albicans, Geotricum candidum and
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Trichophyton mentagrophytes, with inhibition zones ranging from 15.1 ± 0.44 to 25.2 ± 0.37 mm compared to
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the AEs of C. colocynthis. TEM analysis of morphological changes in bacterial cells showed a break through
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the permeability of outer membrane resulting in the leakage of cellular materials causing a bactericidal action
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against Gram-positive S. aureus and the Gram-negative E. coli. The SNPs in leaves and roots AEs had moderate
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antiviral activity against hepatitis A virus, Herpes simplex virus type I and Herpes simplex virus type II. The
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SNPs showed a significant larvicidal activity against 3rd instars larvae of Culex pipens mosquito.
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Industrial relevance:
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Various microorganisms have evolved drug resistance to conventional chemical antimicrobial agents over
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many generations. Therefore, greenly synthesized silver nanoparticles represent an alternative way to overcome
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the drug resistance of various microorganisms. We believe that greenly synthesized SNPs of C. colocynthis can
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be developed into a broad spectrum valuable industrial pharmaceutical product.
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Keywords: green synthesis, silver nanoparticles, Citrullus colocynthis, antiviral, antimicrobial, larvicidal.
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INTRODUCTION
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The field of nanotechnology has gained momentum over the past two decades with a broad range of
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potential applications, such as increasing bioavailability of a drug, biological labeling, biosensing, antibacterial
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activity, antiviral activity, detection of genetic disorders, gene therapy and DNA sequencing (Thirumurgan et al.,
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2010).
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Advances in this field are mainly dependent on the ability to form nanoparticles of various materials,
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sizes, and shapes, and to efficiently assemble these particles into complex architectures. (David et al., 2005).
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Nanoparticles are particles with a maximum size of 100 nm. These particles have unique properties, which are
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quite different than those of larger particles (Nalwa, 2005).
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The most prominent nanoparticles for medical uses are nanosilver particles which are well recognized for
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their high anti-microbial activity. The SNPs show efficient antimicrobial property compared with other salts due
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to their extremely large surface area, which provides better contact with microorganisms (Prabhu and Poulose,
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2012)
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Silver ions are able to interact with the disulfide bonds of protein contents of microorganisms such as
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bacteria, fungi and viruses. They can change the three dimensional structure of proteins containing S-S bonds
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and block the function of the microorganism. This ability is thought to be stronger in nanosize. Though the mode
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of action of SNPs on the microbial cells is still a matter of debate, its possible mechanism of action has been
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suggested according to the morphological and structural changes in the microbial cells (Chwalibog et al., 2010;
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Prabhu and Poulose, 2012).
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Enhanced biocidal activities of Citrullus colocynthis aqueous
extracts using green nanotechnology
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Recent studies have focused on the synthesis of homogenous silver nanoparticles and evaluation of their
antimicrobial activities (Sharma et al., 2009 and Khalil et al., 2013). There are various methods for SNPs
preparation, for example; chemical precipitation, chemical vapor deposition, hydrothermal method, microwave,
and biological methods, etc. (Murthy et al., 2010; Panáček et al., 2006; Sharma et al., 2009). Biological methods
are more preferred for being eco-friendly, and don’t involve the use of toxic chemicals (Shawkey et al., 2013).
In recent years the green synthesis of nanoparticles using plant extract has emerged as a viable alternative
to traditional chemical procedures and physical methods. Gardea-Torresdey et al. (2003) firstly reported the
preparation of Silver nanoparticles by living plants. In a few years the greensynthesis of SNPs for
pharmaceutical and biological applications has become an attractive area of research ( Huang et al., 2007; Leela
and Vivekanandan, 2008; Li et al., 2007; Shankar et al., 2004; Song and Kim, 2009, Shawkey et al., 2013). The
green synthesis of nanoparticls is not only environmentally safe methodology but also, cost effective and
economic on labor, (Chandran et al., 2006).
C. colocynthis is a medicinal plant that grows widely in Egypt, Sudan and many other African counties
as well. It has been used in folk medicine in Africa for its antiinflammatory, antidiabetic, and antioxidant
activities (Gurudeeban and Ramanathan, 2010; Kumar et al., 2008). Our group demonstrated that AEs of fruits,
seeds, leaves and roots of C. colocynthis could be used to synthesize silver nanoparticles in aqueous solutions at
ambient conditions by a cheap and simple method (Shawky et al., 2013).
The aim of this study was to investigate the antibacterial, antifungal, antiviral and larvicidal activities of
the C. colocynthis/SNPs and to demonstrate their mode of action against Gram-positive S. aureus and the Gramnegative E. coli.
MATERIALS AND METHODS
Plant material:
Citrullus colocynthis fruits, seeds, leaves and roots were collected from Omdurman, Sudan (15°39′N
32°29′E), and were used to prepare the AEs.
Bacterial and fungal strains:
Nine bacterial strains; Staphylococcus aureus (RCMB 010027), Staphylococcus epidermidis (RCMB
010024), Streptococcus pyogenes (RCMB 010015), Neisseria gonorrhoeae (RCMB 010076), Proteous vulgaris
(RCMB 010085), Klebsiella pneumoniae (RCMB 0010093), Shigella flexneri (RCMB 0100542),
Pseudomonas aeruginosa (RCMB 010043) and Escherichia coli (RCMB 010056) and four fungal strains;
Aspergillus fumigatus ((RCMB 02564), Candida albicans (RCMB 05035), Geotricum candidum (RCMB 05096)
and Trichophyton mentagrophytes (RCMB 0925) were obtained from the Regional Center of Mycology and
Biotechnology Antimicrobial Unit (RCMB), Cairo, Egypt.
Cells and viral strains:
Vero cells (African green monkey kidney cell line, Hepatitis A virus (HAV), Herpes simplex Virus type I
(HSV-I) and Herpes simplex Virus type II (HSV-II) were obtained from virology center, Faculty of Medicine
(Girls), Al-Azhar University. Vero cells were maintained in MEM supplemented with 10% Fetal Bovine Serum
(FBS), 25 g/mL gentamicin and 200 mM L-glutamine
Preparation of plant aqueous extracts:
20 grams of each plant organ (fruits, leaves, seeds and roots) were thoroughly washed in distilled water,
cut into fine pieces, soaked in 200 ml distilled water for 24 hrs at 40C. The decoction obtained was then filtered
through Whatman No.1 filter paper. The same procedure was repeated twice. The aqueous extracts of two
successive extractions were pooled, concentrated under vacuum, and lyophilized.
Synthesis of silver nanoparticles using C. colocynthis AEs and their characterization by TEM analysis:
SNPs / C. colocynthis AEs were prepared as described before in our earlier study Shawkey et al., (2013).
Briefly; 2.0 mg/ml stock solutions were prepared from the extracts of the fruits, leaves, seeds and roots from C.
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Mosquito larvae:
Culex pipiens eggs were obtained from the Insect Research Institute, Dokki, Giza, Egypt and were kept in
dechlorinated tap water to bread the larvae .The 3 rd instars' larvae were selected for the experiments since it is the
most sensitive larval stage.
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colocynthis; 90 ml of 5.0 mM aqueous solution of silver nitrate (Sigma Aldrich, Egypt) were added to 10 ml of
stock solutions, and kept at room temperature for 24 hours. SNPs in the AE extracts were recognized by the
color change to reddish brown and by TEM analysis. For TEM analysis a drop of the silver nanoparticles
solution was placed on a formvar-coated 300 mesh Cu grids (Agar Scientific Ltd., Stansted, U.K.). After drying,
the shape and size of SNPs was analyzed using TEM JEOL model JEM-2000EX (100 keV).
Evaluation of Antimicrobial Activity of C. colocynthis /SNPs:
The antibacterial activity of SNPs/C. colocynthis AEs was evaluated by the Kirby-Bauer disc diffusion
method (Bauer et al., 1966). Briefly, sterile paper discs (6 mm) were loaded with 25 μL of C. colocynthis/SNPs
or the tested plant extracts. Several isolated colonies of each standard strain was selected from a culture of 12–18
hrs on nutrient agar (Oxoid, UK) for bacteria and 48-72 hrs on Sabouraud dextrose agar for fungi and dissolved
in sterile normal saline solution. The suspension was adjusted to match a solution of 0.5 McFarland turbidity
standards at 600 nm using spectrophotometer Shimadzu dual beam UV–visible spectrophotometer, model UV1650 (Kyoto/Japan). The surface of MHA plates (Oxoid, UK) or Sabouraud dextrose agar plates (Oxoid, UK),
were inoculated with the adjusted suspension of bacteria and fungi respectively. Impregnated discs were placed
on the surface of the inoculated medium and incubated at 37 °C for 18–24 hrs and at 30-32 °C for 48 hrs for
bacteria and fungi respectively except for Trichophyton mentagrophytes fungus which was incubated for 14
days. The diameter of the inhibition zone was measured in millimeter, and was recorded as mean ± SD of a
triplicate experiment. Ampicillins (10μg), Gentamicin (10μg) discs for bacteria, and Amphotericin B (5μg/disc)
for fungi were used as positive controls. Cultured species producing halos equal to or greater than 7 mm were
considered susceptible.
The mechanism of antibacterial activity of C. colocynthis/SNPs:
Unstained cells of S. aureus and E. coli were observed for the presence of electron-dense precipitates by
TEM. The two bacterial strains were diluted to a final concentration of 10 5 to 106 CFU/ml in milli Q water. 200
µl of the adjusted bacterial suspensions were added to 200 µl of C. colocynthis/SNPs. Control bacteria were
treated with (mili-Q water, 0.2 mg/l aqueous extracts) and simultaneously processed for imaging. Samples were
gently mixed for 15 min, 30 min, and one hour then droplets were placed on formvar-coated 300 mesh Cu grids
(Agar Scientific Ltd., Stansted, U.K.). The samples were dried in room temperature under sterile conditions and
then examined with an energy-filtering TEM operated at an accelerating voltage of 100 kV. Images were
recorded with a 4 K slow-scan charge-coupled-device camera (4000 SP; Gatan, Pleasanton, CA) (Chwalibog et
al., 2010).
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Antiviral Assay:
The antiviral activity of C. colocynthis/SNPs and the corresponding AEs of different plant organs were
tested by measuring the cytopathic effect (CPE). Briefly; Vero cells were plated in a 96-well plate (8 mm
diameter; Falcon Plastics) at a density of 10,000 cells/well in Dulbecco's Modified Eagle's Medium (DMEM)
(Sigma, Aldrich, Egypt) with 10% Fetal bovine serum (FBS) (Sigma, Aldrich, Egypt) and incubated at 37ºC in a
humidified incubator with 5% CO2. The following morning, the medium was changed and the cells were
challenged with 10-4 TCID50/mL and were simultaneously treated with two-fold serial dilutions of the C.
colocynthis/SNPs or each of the tested plant organs AEs and incubated at 37ºC for 3 to 6 days. The infection
control and untreated Vero cells control in the absence of the tested samples were included. Six wells were used
for each concentration of the tested sample. The plates were observed every 24 hours under the inverted
microscope until the virus in the control wells showed complete CPE. The monolayers were fixed with formalin
and then stained with a 0.1% crystal violet solution. The antiviral activity was assessed by the inhibition of the
cytopathic effect on the tested cell culture. Inhibition of the CPE was scored under light microscopy as negligible
(less than 25%); weak (25% to less than 50%); moderate (50% to less than 75%) and strong (more than 75%).
CPE was measured in three independent experiments, and each experiment was performed in triplicates,
(Dargan, 1998; Vijayan et al., 2004).
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Collection of C. colocynthis
(fruits,seeds,leaves and roots)
Preparation Of C. colocynthis Aqueous Extracts (AEs)
Synthesis of SNPs in C. colocynthis AEs
Characterization of SNPs/C. colocynthis AEs by TEM analysis
Evaluation of the biological activities of SNPs/C. colocynthis AEs
Evaluation of the
antibacterial activity
Evaluation of
Evaluation of the
Evaluation of the
the antifungal activity
antiviral activity
larvicidal activity
Investigation of the mechanism of action of SNPs/ C. colocynthis
AE on Staphylococcus aureus and E. coli by TEM analysis
Figure 1: Experiments flowchart
RESULTS
Characterization of SNPs/C. colocynthis:
A color change of the AEs to reddish brown was the early sign for the formation of SNPs, due to the
excitation of surface plasmon vibrations in SNPs. Further confirmation of SNPs synthesis by TEM analysis
showed formation of homogenous SNPs in the range of 7.398 nm to 19.267 nm in different AEs solutions (Fig.
2).
Figure 2. TEM pictures Showing formation of homogenous SNPs in the range of 7.398 nm to 19.267 nm in
different AEs solutions of different parts of C. colocynthis after treatment with AgNO3.
Aqueous solution of AgNO3 was added to C. colocynthis AEs and kept at room temperature for 24 hours. The
shape and size of SNPs were examined by TEM, (A). SNPs formed in Fruits AE, (B) SNPs formed in Seeds AE,
(C) SNPs formed in leaves AE and (D) SNPs formed in Roots AE.
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Determination of larvicidal activity on mosquito larvae of Culex pipiens:
Larvicidal tests were performed according to the World Health Organization method (WHO, 1996). The
larvae colonies were maintained continuously at 28 ± 1 °C, at photoperiod of 16:8 hrs (light: dark) and
65% ± 5% relative humidity. Concentrations ranging from 0.5 to 3.0 mg/mL were tested to determine the 50 %
lethal dose (LD50) for AEs and their SNPs. Each concentration has been replicated four times comprising 100
larvae. Glass beakers were left at room temperature and mortality was recorded after 24 h, 48h and 72h of
exposure. Mortality percentage was always corrected according to Abbott's formula when the mortality exceeded
10% , among larvae that were treated with control (AgNO3) (Abbott, 1925).
The experimental work is summarized in a flowchart presented in fig (1).
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Antibacterial and antifungal activities C. colocynthis/SNPs against a wide range of bacteria and fungi
Greenly synthesized SNPs from different parts of C. colocynthis showed significant inhibition to the
tested standard strains with inhibition zones ranging from 15.1 ± 0.44 to 25.2 ± 0.37 mm (Table 1) compared to
the fruits, leaves, roots and seeds AEs of C. colocynthis, all of which produced little or no antimicrobial
activity. These results indicate that the C. colocynthis/SNPs exhibited significant antibacterial and antifungal
activities against a wide range of tested strains.
Table 1: Antibacterial and antifungal activities of SNPs described as minimum zone of inhibition
Tested microorganism
Fungi
Aspergillus fumigatus
(RCMB 02564)
Candida albicans
(RCMB 05035)
Geotricum candidum
(RCMB 05096)
Trichophyton mentagrophytes
(RCMB 0925)
Gram positive bacteria
Staphylococcus aureus
(RCMB 010027)
Staphylococcus epidermidis
(RCMB 010024)
Streptococcus pyogenes
(RCMB 010015)
Gram negative bacteria
Neisseria gonorrhoeae
(RCMB 010076)
Proteous vulgaris
(RCMB 010085)
Klebsiella pneumoniae
(RCMB 0010093)
Shigella flexneri
(RCMB 0100542)
Escherichia coli
(RCMB 010056)
Pseudomonas aeruginosa
(RCMB 010043)
Extracts SNPs
Leaves
Roots
SNPs
SNPs
16.6 ± 0.58
17.7± 0.37
Fruits
SNPs
18.2± 0.25
Standard
Amphotericin B
15.3 ± 0.44
15.6 ± 0.44
16.7± 0.44
17.7± 0.37
21.9± 0.12
15.9 ± 0.27
16.4 ± 0.44
17.4± 0.44
19.2± 0.44
26.4± 0.20
15.1 ± 0.44
15.6 ± 0.37
16.6± 0.25
18.9± 0.25
25.4± 0.16
20.6 ± 0.44
21.2 ± 0.25
20.6± 0.44
22.2 ± 0.44
Ampicillin
22.9 ± 0.14
23.7 ± 0.58
23.3 ± 0.37
24.0 ± 0.37
25.2 ± 0.37
25.4 ± 0.18
20.2 ± 0.19
22.9 ± 0.25
23.8 ± 0.37
24.2 ± 0.37
26.4 ± 0.34
16.4 ± 0.25
15.8 ± 0.44
15.8± 0.44
16.6± 0.44
Gentamycin
19.9± 0.18
17.8 ± 0.37
17.6 ± 0.58
18.3 ± 0.25
19.1± 0.44
23.4± 0.3
19.1 ± 0.44
22.4 ± 0.58
24.1 ± 0.58
24.2 ± 0.28
26.3± 0.15
15.4 ± 0.58
14.6 ± 0.44
16.8 ± 0.44
17.3± 0.28
24.8± 0.24
19.5 ± 0.25
20.3 ± 0.19
19.9 ± 0.19
21.2 ± 0.58
25.3± 0.18
13.2 ± 0.37
13.3 ± 0.37
12.7 ± 0.58
13.2 ± 0.58
17.3± 0.12
23.7± 0.10
The test was done using the diffusion agar technique, Well diameter: 6.0 mm, (25 µl was tested). Data are
expressed in the form of mean ± SD
RCMB: Regional Center for Mycology and Biotechnology Antimicrobial unit test organisms
C. colocynthis/SNPs distorts Staphylococcus aureus and Escherichia coli cells
We sought to determine the mechanism of action of SNPs on bacterial cells. To test that, we used S.
aureus and E. coli as representatives for Gram positive and Gram negative bacteria respectively. The
morphological changes in bacterial cells upon treatment with C. colocynthis/SNPs were measured using TEM
analysis.
The control S. aureus and E coli cells retained their normal morphology and seemed to be normal (Fig. 3
A1 & A2). In contrast, S. aureus and E.coli cells treated with the C. colocynthis/SNPs were severely distorted
and became disrupted (Fig. 3 B1 to F1 and Fig. 3 B2 to F2). Interestingly, the SNPs aggregated and were
localized non-specifically on the cell wall, and were seen within the cell wall or the cytoplasm. TEM analysis
showed that the cell wall was damaged at few spots. Particularly in case of S. aureus the cytoplasmic membrane
became separated from cell wall demonstrating a clear bactericidal effect especially on the Gram positive
bacteria.
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Seeds
SNPs
15.1 ± 0.58
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C. colocynthis /SNPs antiviral activity
By testing the SNPs for antiviral activities against Herpes simplex (HSV-1, HSV-2) and Hepatitis A
viruses (HAV), all synthesized SNPs showed moderate to weak antiviral activities against tested viruses
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Figure 3. TEM pictures showing the morphological changes in Staphylococcus aureus and Escherichia coli
cell structure after interaction with SNPs. The external morphology of unstained Staphylococcus aureus cells
was observed by TEM; (A1) Untreated bacteria, (B1, C1, D1, E1 and F1) Staphylococcus aureus treated with
SNPs. The external morphology of unstained E.coli cells observed by TEM; (A2) control bacteria. (B2, C2, D2,
E2 and F2) E. coli treated with SNPs. Electron-dense particles were found around damaged cells and SNPs
aggregated and located non-specifically on the cell wall, also, SNPs were seen within the cell wall or the cell of
bacteria. The cells became severely distorted and disrupted (arrows).
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compared to negative controls (AgNO3 and AEs). SNPs from leaves and roots showed moderate activity against
all tested virus whereas all SNPs showed moderate activity against HSV-1 (Table 2).
Table 2: Antiviral activity of Citrullus colocynthis fruits, leaves, seeds and roots SNPs against Hepatitis A virus
and Herpes simplex type I & II:
Sample
HAV
HSV-II
weak
Moderate
Fruits SNPs
Moderate
Moderate
Leaves SNPs
Moderate
Moderate
Seeds SNPs
Moderate
Moderate
Roots SNPs
HAV: Hepatitis A virus; HSV-I: Herps simplex type I virus; HSV-II: Herps simplex type II virus
weak
Moderate
weak
Moderate
C. colocynthis /SNPs larvicidal activity
Testing SNPs for their activity against C. pipiens larva, AEs did not show any activity before 48 hours,
whereas SNPs showed larvicidal activity just after 24 hours incubation. It was observed that AEs were not lethal
to the larvae. The leaves AEs showed the lowest LD50 of 2.7 mg/mL after 72 hours exposure; On the other hand,
seeds/SNPs extract showed LD50 of 0.5 mg/mL after 24 hours exposure. The comparison of LD50 of different
AEs versus their corresponding SNPs showed a significant improvement in larvicidal activity after SNPs
synthesis (Fig. 4). The average larval mortality data of each AE and its corresponding SNPs were subjected to
the chi-square test formula to analyze whether the larvicidal activity increased significantly or not, after SNPs
synthesis.
Results with p<0.05 were considered to be statistically significant. We found the chi-square values as follow:
Seeds x2 = 37.7*, Leaves x2 = 12*, Roots x2 = 22.8* , Fruits
x2 = 15.3*
* Significant at P<0.05 level.
All the calculated chi-square values were highly significant at p < 0.05 level, indicating that the larvicidal
activity of AEs increased significantly after SNPs synthesis process.
Seeds AE
20
Seeds SNPs
18
Leaves AE
16
LD 50 in mg/mL
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Antiviral effect on
HSV-I
Leaves SNPs
14
Roots AE
12
Roots SNPs
10
Fruits AE
8
Fruits SNPs
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4
2
0
After 24 hrs
After 48 hrs
After 72 hrs
Contact time
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Figure 4. SNPs show lower LD50 than AEs counterparts; The 50 % lethal dose of SNPs was determined
and then compared with the LD50 for the AEs. Glass beakers containing the treated and the untreated larva
were left at room temperature and mortality was recorded after 24 h, 48h and 72h of exposure. Mortality
percentage was always corrected according to Abbott's formula when the mortality exceeded 10%, among larvae
that were treated with control (AgNO3).
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Various microorganisms have evolved drug resistance to conventional chemical antimicrobial agents over
many generations. Therefore, an alternative way to overcome the drug resistance of various microorganisms is
needed desperately (Kim et al., 2007).
Although that Ag ions and Ag-based compounds have strong antimicrobial effects (Furno et al., 2004),
Ag ions or salts has only limited usefulness as an antimicrobial agent for several reasons, including the
interfering effects of salts and the antimicrobial mechanism and the continuous release of enough concentration
of Ag ion from the metal form. In contrast, these limitations can be overcome by the use of SNPs (Kim et al.,
2007). To use SNPs in various fields against microorganisms, it is essential to prepare the SNPs with cost
effective method. We were able be prepare SNPs by an easy, reproducible, eco-friendly and cost effectively
method using the aqueous extracts from different parts of C. colocynthis, and these SNPs were homogeneous
(Shawkey et al., 2013).
In the present study, the greenly synthesized C. colocynthis /SNPs as antimicrobial agents have come up
as a promising candidate offering a practical solution for the problem.
We investigated the morphological changes in S. aureus and E. coli cells upon treatment with C.
colocynthis /SNPs by TEM analysis of unstained bacteria. Our results clearly demonstrated that SNPs inhibit the
tested strains compared to the AEs, and this inhibition can be interpreted depending on previous studies
suggested that nanometer-sized silvers possess different properties, which might come from morphological,
structural and physiological changes (Nel et al., 2006). A similar effect was described when E. coli bacteria were
treated with highly reactive metal oxide nanoparticles (Stoimenov et al., 2002). A bacterial membrane with this
morphology exhibits a significant increase in permeability, leaving the bacterial cells incapable of properly
regulating transport through the plasma membrane and, finally, causing cell death.
In general, the SNPs attached to the cell wall affect its membrane integrity, leading to leakage of
metabolites, essential enzymes, nucleic acids, and peptides. Furthermore, nanoparticles may enter the microbial
cells and inactivate the essential enzymes, deplete the intracellular ATP (Lok et al., 2006) and interfere with the
cell membranes permeability leading to the cell death (Li et al., 2010). Owing to their small size, SNPs impair
the sulfur and phosphorus containing essential macromolecules such as proteins and DNA (Wei et al., 2009)
,Thus, antimicrobial action of SNPs appears to be a consequence of adherence to and penetration inside the cell
wall of the target microorganism.
On the other hand the interaction of SNPs with viruses is a largely unexplored field. The viral capsid
proteins bind to the host cellular surface specific receptors. This attachment can induce the viral envelope protein
to undergo changes that results in the fusion of viral and cellular membranes and may lead to an infection. For
this reason there is a high interest studying possible mechanisms of binding SNPs to the viral capsid and inhibit
the later fusion.
Recently, it has been suggested that nanoparticles bind with a viral envelope glycoprotein and inhibit the
virus by binding to the disulfide bond regions of the CD4 binding domain within the gp120 glycoprotein, as
demonstrated in vitro (Elechiguerra et al., 2005).
Metal nanoparticles, especially those produced with silver or gold, have proven to exhibit a good antiviral
activity against a broad-spectrum of viruses and reduce viral infectivity of cultured cells (Galdiero et al., 2011).
Beside the direct interaction with viral surface glycoproteins, metal nanoparticles may gain access into the cell
and exert their antiviral activity through interactions with the viral genome (DNA or RNA). Further mechanism
of action is suggested to be the interaction of metal nanoparticles with intracellular components of a virally
infected cell and host cellular factors that are needed to allow viral replication.
Previous studies on SNPs biological activities were done on mosquitoes biological control field, a significant
larvicidal activity of greenly synthesized SNPs was reported against Aedes aegypti (Salunkhe et al., 2011) and
Aedes albopictus (Sareen et al., 2012). The resulted larvicidal activity may be attributed to the interaction
between silver ions and molecules of an extracellular lipoprotein matrix increases the permeability of the plasma
membrane of microbial cells and eventually causes their death, and/or due to the denaturation of the sulfurcontaining proteins or phosphorous containing compound like DNA that, leads to the denaturation of organelles
and enzymes and thus reduces the cellular membrane permeability and education in ATP synthesis which finally
causes the loss of the cellular function and cell death (Sap-Iam et al., 2010; Sondi and Salopek-Sondi, 2004).
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DISCUSSION
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Based on the present study, the action model of SNPs may be described as SNPs making a break through
the permeability of outer membrane firstly, resulting in the leakage of cellular materials. Secondly, SNPs enter
the inner membrane and inactivate respiratory chain dehydrogenases, thus inhibiting respiration and growth of
cells. Simultaneously, SNPs could affect some proteins and phosphate lipids and induce collapse of membrane,
resulting in cell decomposition and death eventually. Taking into account the mobility of SNPs into cells and
their fate in a bioprocess or even in the environment, the risk aspects for the application in larger scales and in
the environment should be strengthened in future study.
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CONCLUSION
This study proved a potent and swift bactericidal action of the C. colocynthis/SNPs on both Grampositive and Gram-negative bacteria and should be explored further for antimicrobial applications in various
fields such as medical devices and antimicrobial systems.
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