Antibacterial Activity of Ginger ( Zingiber officinale ) Leaves Essential Oil Nanoemulsion against the Cariogenic Streptococcus mutans

© 2018 Nada M. Mostafa. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Nada M. Mostafa, Assistant Professor, Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt. E-mail: nadamostafa @ pharma.asu.edu.eg Antibacterial Activity of Ginger (Zingiber officinale) Leaves Essential Oil Nanoemulsion against the Cariogenic Streptococcus mutans


INTRODUCTION
Zingiber officinale Roscoe (family Zingiberaceae) is widely cultivated for its medicinal uses and as a condiment.It is used traditionally to treat the common cold, headache, muscular and rheumatic disorders (Yang et al., 2009).Numerous studies have investigated the phytochemical composition of its rhizomes, revealing zingiberene, gingerol, shogaol and their derivatives as the major components (Sivasothy et al., 2011).Reported pharmacological activities on ginger include antimicrobial, antioxidant, anti-inflammatory, hepatoprotective and antinociceptive (Abdel Azeem et al., 2013;Jeena et al., 2013;Mostafa and Singab, 2016).
Nanoemulsions are emulsions with sub-micron size that can be produced by either high-or low-energy method.The high energy method involves the application of high-pressure homogenization or micro-fluidization, while the low energy method involves continuous stirring of a mixture of oil, water, and surfactant, without any drastic condition or expensive equipment (Salama et al., 2016).The choice of the suitable surfactant is important, such as Tween-80, which is a low molecular weight non-ionic surfactant commonly used in food and pharmaceutical products (Salama et al., 2016;Nielsen et al., 2016).
The encapsulation of an essential oil in a nanoemulsion form, protects its thermolabile components, that are also sensitive to the effects of light, moisture, and air and guarantees significant decrease in their volatility, increase of their bioavailability and efficacy as a result of increasing the surface area and stability of the particles (Amaral and Bhargava, 2015;Nantarat et al., 2015).
Dental caries is an oral infection manifested by teeth enamel demineralization and destruction (Hasan et al., 2014).Its main causative agent is Streptococcus mutans, which is a colonizing and biofilm-forming bacterium with a high ability to adhere to solid surfaces (Krzyściak et al., 2014).These properties may be attributed to the bacterial ability to survive and tolerate the acids produced as a result of oral carbohydrate metabolism (Hasan et al., 2015).Dental plaque is the aggregate bacterial accumulation on the surface of teeth, where Streptococcus mutans is involved in its development and accumulation (Loesche, 1986).
The aim herein is profiling the volatile components of Zingiber officinale leaves, for the first time, from the Egyptian plant; testing the stability, anticariogenic and antiplaque effects of a formulated nanoemulsion of the ginger leaves essential oil against Streptococcus mutans, which was carried out for the first time as well.The antimicrobial and antibiofilm-forming activities were supported by transmission electron microscope imaging and an in-silico molecular docking study.

Plant material
Leaves of Zingiber officinale Roscoe, family Zingiberaceae were collected from El-Orman Botanical Garden, Cairo, Egypt.Identification of the plant was confirmed by Prof. Dr. Mohamed El Gebaly, Professor of Taxonomy, National Research Centre, Egypt.A voucher specimen was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt (PHG-P-ZO-1).

Isolation of volatile component
Fresh plant material (1 Kg) was hydrodistilled in a Clevenger-type apparatus for 4 hours.The resulting yellow oil (0.15% v/w) was then dried over anhydrous sodium sulfate and kept in separately sealed vials at −30°C until analysis.

Essential oil GC-MS analysis
The mass spectrum was recorded by using Shimadzu GC MS-QP2010 (Tokyo, Japan) equipped with Rtx-5MS fused bonded column (30 m × 0.25 mm i.d.× 0.25 µm film thickness) (Restek, USA) and a split-splitless injector.The capillary column was directly coupled to a quadrupole mass spectrometer (SSQ 7000; Thermo-Finnigan, Bremen, Germany).The initial column temperature was kept at 45°C for 2 minutes (isothermal), programmed to 300°C at a rate of 5°C/min and kept constant at 300°C for 5 minutes (isothermal).The helium carrier gas flow rate was 1.41 mL/min.The injector temperature was 250°C.The mass spectrum was recorded by applying filament emission current of 60 mA, ionization voltage of 70 eV, and 200°C ion source.A diluted sample (1% v/v) was injected with split mode with a split ratio of 1:15.

Nanoemulsion formulation
The nanoemulsion was prepared by spontaneous emulsification, composed of essential oil (1% v/v); water phase containing Tween-80 (4.5% v/v); then the water was added to complete the volume to 100% v/v.It was kept under moderate magnetic stirring for 5 min at room temperature.The surfactant percentage was determined after several trial and error experiments until reaching a visually homogenous emulsion with no phase separation.

Particle size, PDI, zeta potential and pH determinations
The particle size, polydispersity index (PDI) and charge of the prepared nanoemulsion were measured (in triplicates) by photon correlation spectroscopy (based on dynamic light scattering), using Zetasizer NanoZS 3600 (Malvern Instruments Ltd., Worcestershire, UK), after adequate aliquot dilution of the samples in distilled water (Manconi et al., 2011).The pH values of the nanoemulsion were determined (in triplicate) directly using a pH meter (Jenway pH meter, Model 3310, UK) at room temperature.

Stability study for the prepared vesicles
The prepared nanoemulsion was stored for one month at a refrigeration temperature of 2-8°C.During the storage period, the sample was regularly inspected visually for its homogeneity and consistency.The particle size, zeta potential, and polydispersity index (PDI) of the nanoemulsion were also re-measured.

Morphological analysis by transmission electron microscope (TEM)
The nanoemulsion, after appropriate dilution with distilled water, was adsorbed on a carbon-coated copper grid.Then, particles morphology and size were examined and photographed using a JEM 1200 EXII transmission electron microscope, Jeol, Japan, operated at an accelerating voltage of 60-70 KV.

Microbial strain
The nanoemulsion was tested against Streptococcus mutans ATCC 25175.

Agar diffusion method
S. mutans inoculum (density of 0.5% McFarland) was spread on the surface of Brain Heart Infusion agar (Lab M, UK), then tested via the agar diffusion method, according to CLSI methodology (2013).A negative control (containing Tween-80) was prepared as the nanoemulsion but without the essential oil.Ready discs of Clindamycin (2 µg/disc; Bioanalyse, Turkey) were used as positive control.The plates were incubated at 37°C.Each test was performed in triplicate and the results were provided as mean values in mm ± SD.

Minimum inhibitory concentration (MIC) determination
Broth microdilution method was used for the determination of minimum inhibitory concentration, according to CLSI methodology (2015), by the visual inspection of the microtiter plates (in two-fold dilutions) using a concave mirror for the absence of turbidity.

Morphological analysis by Transmission Electron Microscope (TEM)
The Streptococcus mutans suspension was photographed with and without the addition of MIC concentration of the nanoemulsion, on a carbon-coated copper grid using JEM 1200 EXII TEM, Jeol, Japan, operated at an accelerating voltage of 60-70 KV.

Molecular modeling
In-silico molecular modeling was done using Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA, USA) and applying the C-Docker protocol.The X-ray crystal structure of the C-terminal region of a Streptococcus mutans surface protein antigen (PDB ID3QE5) co-crystallized with its ligand has been downloaded from the protein data bank.The program protein preparation protocol and CHARMm force field were applied then the C-Docker binding energy of the selected docking pose was calculated by the program.

Statistical analyses
The results were measured in triplicates and reported as the mean value ± SD.The statistical analyses were made by unpaired t-test using GraphPad InStat 3 Software, Inc. La Jolla, CA, USA.The statistical significance level was set for all analyses at P < 0.05.

Essential oil analysis
Ninety compounds constituting 96.63% of the total peak area were identified in the ginger leaves oil by GC/MS analysis, as shown in Table 1, for the first time from the Egyptian chemotype.Methods for compounds identification were their experimental retention indices and mass spectra comparison with the 8 th edition of Wiley Registry of Mass Spectral Data, NIST Mass Spectral Library (December 2005), previously published literature (Ayoub et al., 2010;Singab et al., 2014;Mostafa et al., 2015;Mostafa et al., 2018) and/or authentic samples.The major components were oxygenated compounds from different classes (30.38%), of which methyl cinnamate (29.21%) represented the most abundant compound.Monoterpene hydrocarbons (23.83%) represent the second major class of compounds, rich in β-pinene (8.59%) and terpinolene (7.46%).While sesquiterpene hydrocarbons reached 20.86%, of which δ-cadinene (7.05%) was the major component.
A previous study done by Sivasothy et al. (2011) on Z. officinale var.rubrum Thielade from Malaysia identified forty-six compounds from the leaves oil.They have reported, in common with the present study, the abundance of high percentages of sesquiterpenoids (47.1%), as well as monoterpenoids (42.6%) in the leaves oil.β-caryophyllene represents the major component (31.7%) in their study, while in the present study, β-caryophyllene was detected with only 2.17%.However, our main oil component, methyl cinnamate (29.21%), was not detected at all in their corresponding study.Of the major components identified in the present study; β-pinene, δ-cadinene, and linalool were also identified in their leaves volatiles, but in much lower amounts representing only 2, 0.3 and 1.1%, respectively.Sivasothy et al. (2011) didn't detect terpinolene in their leaves oil but they rather identified it in the rhizomes oil.These variations in the leaves oil composition between the two studies may be attributed to the source, method of cultivation, regional variations and the season of plant collection (Sari et al., 2006).

Determination of particle size, distribution, zeta potential and pH
The nanoemulsion particles had a mean diameter of 151.4 nm and a polydispersity index of 0.27, which indicate a homogenous formulation.The particle size distribution is presented in Figure 1.The mean zeta potential value was −13.75 ± 3.18 mV, this negative value may be explained by the use of a hydrophilic emulsifier, which is Tween-80 with oxygen atoms in its molecule, that present a negative surface charge density (Flores et al., 2011).The mean pH value of a triplicate measurement of the formulation was 4.

Nanoemulsion stability study
The formulated nanoemulsion was kept in the refrigerator and tested for stability for a period of one month.The stability parameters are presented in Table 2.The nanoemulsion was homogenous and consistent with no apparent phase separation when inspected visually.Non-significant changes in particle size diameter, polydispersity index, and zeta potential were observed after one-month storage (P < 0.05).

Morphological analysis using transmission electron microscope (TEM)
TEM examination of the nanoemulsion showed particles of almost spherical shape and confirmed the particles nanometric size, as shown in Figure 2. The particles size observed by TEM was much smaller than that measured by the photon correlation spectroscopy due to the drying process involved in the sample preparation for TEM imaging, thus the dried oil droplets in the core would appear smaller (Ishak et al., 2017;Nantarat et al., 2015).

Inhibition zone and MIC determinations
The mean inhibition zone diameters (of three measurements) of the nanoemulsion and the clindamycin disc (2 µg/disc) are shown in Table 3.No growth inhibition was observed in the control well (Tween-80 in water at the same used concentration).MIC value of the nanoemulsion was 62.5 µL/mL that is equivalent to 0.61 µL/mL of pure essential oil.

Morphological analysis by TEM
The impact of ginger leaves nanoemulsion on the destruction of the biofilm integrity of S. mutans was illustrated by TEM, as shown in Figure 3.The control of bacterial suspension showed cells aggregation and clumping with a chainforming pattern.While, the bacterial suspension treated with the nanoemulsion (at its MIC level) showed significant cells scattering and dispersion without any apparent chain formation, suggesting reduced S. mutans glucan synthesis, which is required for bacterial adherence (Hasan et al., 2015), also the cells scattering has resulted in reduced cells interaction and impaired formation of the bacterial biofilm.All measurements are done in triplicates.

Docking study
The molecular docking was done to evaluate the possible binding mode of methyl cinnamate (the major volatile component, 29.21%) to the active site of the C-terminal region of S. mutans surface protein antigen (Ag I/II).Inspection of the active site of the bacterial protein surface antigen revealed many amino acids, such as Glu 1215, Glu 1216, Lys1299 and Thr 1244, which stabilize the complex of methyl cinnamate with the protein.The C-Docker binding energy of the selected docking pose was −8.239 kcal/ mol.The 2D-and 3D-binding modes of methyl cinnamate are shown in Figure 4, where methyl cinnamate showed a good binding affinity to the bacterial surface protein antigen through a Pi-Pi interaction between the benzene ring of the ligand and the Lys 1299 residue of the protein, besides four hydrophobic interactions of the ligand with the protein active site.This indicates a high inhibitory activity of S. mutans surface protein antigen.
The docking results of methyl cinnamate (ginger leaves oil major component) were comparable to those reported for eugenol (the major clove oil component) on S. mutans surface antigen (Ag I/II) (Adil, 2013), where the amino acid residues Glu 1215 and Glu 1216 were common in stabilizing the complex formed for either compound.Moreover, eugenol bondings with the protein active site were through hydrophobic interactions as for methyl cinnamate, in addition to H-bonding.
Eugenol had been used extensively as an antiseptic, reported for its antibacterial activity against S. mutans (Freires et al., 2015), used in dentistry as a base for fillings, and as a cement component for sealing cavities (Souza-Costa et al., 2007).On the other side, methyl cinnamate has been used in many foods, pharmaceutical industries and cosmetic products (Bathia et al., 2007).According to the results declared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA, 2009), methyl cinnamate intake is considered safe at the levels of use as a flavoring agent.Thus, further studies should be carried out to study the possible use of the ginger oil rich in methyl cinnamate or the pure compound in dentistry clinical applications.

CONCLUSION
Ginger (Zingiber officinale Roscoe) leaves volatile oil is rich in valuable phytoconstituents; the oil nanoemulsion formulation was stable, effective on Streptococcus mutans, and can be further studied for use as a gargle against dental caries and plaque formation.

CONFLICT OF INTERESTS
The author declares no conflict of interest.

Fig. 3 :
Fig. 3: Transmission electron microscope imaging of (a) Streptococcus mutans suspension alone showing biofilm and chain-forming aggregates, and (b) nanoemulsion (at MIC level) and S. mutans showing impairment of bacterial biofilm integrity.Samples magnifications were 30000 and 20000 times, respectively.

Fig. 4 :
Fig. 4: Binding of methyl cinnamate to the active C-terminal region of S. mutans surface protein antigen in [a] 3D-diagram and [b] 2D-diagram.

Table 1 :
The chemical composition of ginger (Zingiber officinale) leaves volatile oil.Compounds are listed according to elution order, RI b Kovats retention index calculated on Rtx-5MS fused bonded column, c Average of three analyses.MS, mass spectral data; RI, published retention indices; AU, co-chromatography with authentics, Tr., traces < 0.01.The major components are bold highlighted. a

Table 2 :
Stability study parameters of the prepared nanoemulsion.

Table 3 :
Antibacterial activity of ginger leaves oil nanoemulsion against S. mutans.
*Values for three determinations; NT: not tested.