Synthesis and Characterization of a Lemongrass Oil Emulsion Formulation incorporating Alumina Nanoparticles for activity against Streptococcus mutans Isolated from Dental Caries

 

Kaushita Banerjee¹, Gourav Saha¹, Pitambar Sahoo¹, Sourav Chattaraj¹, Dhamodhar P²., Padma Thiagarajan¹*

¹School of Biosciences and Technology, Vellore Institute of Technology, Vellore

²Department of Biotechnology, MS Ramaiah Institite of Technology, Bengaluru, India

 

ABSTRACT:

Caries is a multifactorial infection that can be caused by Streptococcus mutans. This bacteria is responsible for the formation of oral biofilms and incorporates properties that is conducive for its survival in hostile oral environments. Long-term adverse effects like hypersensitivity reactions, supra-infections and teeth staining coupled with the risk of resistance-development limit use of chemical preparations and antibiotics for caries prevention. Hence plant-based formulations with substantial antimicrobial and anti-cariogenic properties, could serve as alternatives for caries prevention. In this study, a Cymbopogon citratus (lemongrass) oil-in-water emulsion with Tween80/Span80 was formulated and favorably characterized using particle size (252.3±1.15nm with polydispersity index of 0.111± 0.01), FTIR and AFM analyses and stability studies. Its MIC and the MIC of alumina nanoparticles against cultures and isolates of Streptococcus mutans was around 600ppm. However, with the alumina nanoparticle loaded emulsion, the MIC was observed at a much lower concentration of around 400ppm. This nanoparticle incorporated emulsion was also able to inhibit the biofilm formation, with the time kill assay showing a decrease in cell numbers within 60 to 120 minutes post incubation. Alumina incorporated lemon grass oil emulsion may thus to prevent dental caries induced by Streptococcus mutans. 

 

 

INTRODUCTION:

Despite the progress in medical sciences, prevention and control of dental caries poses a big challenge to public health and represents an issue of worldwide concern¹. It has been estimated that over 90% of the world’s population will suffer from dental caries at least one time in their lifetime². Caries formation leads to the destruction of dental tissue by organic acids produced by causative bacteria, through the fermentation of dietary carbohydrates and this leads to demineralization of the enamel layer of the tooth. It also causes the development of differently shaped and multi-colored tooth cavities.

 

Formation of dental plaque is due to acidogenic and cariogenic bacteria of Streptococcus genus, mainly Streptococcus mutans³. The symptoms associated with it are pain, inflammation of surrounding tissue and gums, development of infection, loss of teeth etc.⁴. The organism is a facultative anaerobic and a Gram-positive cocci that produces a biofilm on the rough tooth surface and reduces the plaque pH level to facilitate its attachment. The acidic condition results in demineralization of the tooth enamel. Biofilms comprise of extracellular polysaccharides that are produced in the presence of sucrose in the saliva or teeth⁵. For biofilm production, S.mutans produces three enzymes, viz., glucosyl tranferase (Gtf) B, C and D. These help in synthesizing both soluble as well as insoluble glucan chains and contribute to glucan production with a high adhesive property. One of the glucan binding proteins viz., Gbp C is responsible for cell-to-cell attachment and aggregation while Gbp A and Gbp D are responsible for integrity and rigidity of biofilm respectively⁶.

Several chemical agents like chlorhexidine⁷, fluoride⁸, triclosan and cetylpyridinium chloride have been used for caries prevention^9,10. Natural products are also employed as novel therapeutic agents¹¹ mainly to minimize adverse effects of synthetic drugs and also to provide a safer therapeutic option¹². In this context, essential oils, composed of mixtures of volatile components, and produced by aromatic plants as secondary metabolites¹³ have drawn attention due to their potent antimicrobial activities^14,15. Terpenes, along with terpenoids and also aromatic or aliphatic compounds like phenols, aldehydes etc.¹⁶ are their important constituents. Among these, monoterpenes are chiefly responsible for the potent antimicrobial activities of these oils.

 

Lemongrass oil (LGO) is a common essential oil that originates from the aerial parts of Cymbopogon citratus and well known for its ethanobotanical and medicinal properties. Cymbopogon extracts have antimicrobial, insecticidal and therapeutic properties¹⁷. LGO exhibits antioxidant as well as pro-oxidant property in eukaryotes that prevents bacterial growth and survival. Its lead component ‘citral’ (3,7-dimethyl-2,6-octadien-1-al) (75-85%,)¹⁸ is responsible for its therapeutic and antioxidant properties. The use of lemon grass oil as an emulsion¹⁹ may be more efficacious with respect to its antimicrobial potency as it facilitates gradual release of its active compounds thus producing continuous and long term effects. The stability of the emulsion is governed by the hydrophile-lipophile balance (HLB) of the oil and the surfactant used for the formulation with higher HLB’s being conducive to emulsion stability in comparison with a lower one²⁰.

 

Metal oxide nanoparticles like those of zinc oxide, alumina and silver oxide also possess good antimicrobial activity and may be helpful in synergistically preventing dental plaque formation by S. mutans, along with essential oils. Among metal oxides, aluminum oxide has a wide range of activities²¹.

 

In present study, a stable lemon grass oil emulsion (LGE) has been formulated with the Tween 80 – Span 80 surfactants and subjected to physiochemical characterization in terms of emulsion size and zeta potential. Its FTIR spectral analysis was also carried out along with atomic force microscopic analysis. Relevant stability tests have been carried out. Its Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) values have been determined against MTCC culture and clinical isolates of Streptococcus mutans. Thereafter, alumina nanoparticles have been incorporated into it and tested for its activity against the bacteria. Experiments for inhibition of biofilm formation have also been performed along with time kill assay. To the best of our knowledge, this is a first novel report on an LGE incorporated alumina nanoparticle formulation, that is active against Streptococcus mutans.

 

MATERIAL AND METHODS:

Chemicals, bacterial strains and growth conditions:

LGO, Tween 80/Span 80 surfactants and alumina nanoparticles were purchased directly from Falcon and SRL, India Pvt. Ltd. respectively. Media were from Hi media. Milli Q water was used for all studies. S.mutans (strain 890) was obtained from MTCC (Chandigarh, India) and clinical isolates were collected from M.S Ramaiah Dental College and Hospital, Bengaluru. For preservation of the organism stocks, Brain Heart Infusion (BHI) agar slants were used and stored at -4°C. All bacterial cultures were made into BHI broth. For the experiments, different stock culture aliquots were inoculated separately into BHI broth.

 

Synthesis of Lemongrass oil emulsion (LGE) and its characterization studies:

43.92ml of Tween 80 and 56.07ml of Span 80 was mixed to get a surfactant mixture with an HLB value of 8.0. This along with 5ml aliquots of the oil in a series of conical flasks, were all heated to 40ºC. The surfactant mixture was then added in increments of 0.5ml (from 0.5 to 5ml) to each of the conical flasks containing the oil, and the total volumes in the flasks were made up to 50ml with water maintained at 40ºC. Emulsions wherein there were no visually observed phase separations were subjected to sonication (Vibra Cell, Sonics and Materials Inc., USA) for 5 minutes (pulse rate of 2/min, duty cycle 16.66%). After 24 hours at 37ºC, their particle sizes were measured (in triplicate, 1:100 dilution, SZ-100 particle size analyzer, Horiba Scientific accompanied by Windows (Z type) version 2.00 software]. Performance of one way ANOVA and Tukey test using Origin software (version 9.0) revealed significant differences (if any) between the particle sizes of the formulations so that the emulsion with the lowest particle size could be selected. This emulsion was labeled as LGE and its zeta potential was measured with the above instrument after a 1:100 dilution.

 

FTIR spectra (4000 to 500 cm^-1, Bruker Optics, Germany, ATR technique) of LGO and LGE were recorded using and its topology was studied using AFM analysis (Nanosurf Easy Scan2). A thin smear of LGE, prepared and dried for 24 hours was used.

 

For stability studies, 5g of LGE was maintained in a closed beaker at 37°C for 90 days and observed every 7 days visually and any signs of its cracking, creaming, oil-droplet formation and (/or) phase separation was noted if any. In another experiment, 5g of LGE was maintained at -18°C in a closed beaker for a time interval of 48 hours. After this interval, the beaker was brought back to 37°C and similar observations as stated above were recorded. It was then taken back to -18°C for another 48 hours. This test procedure was repeated for three consecutive cycles. LGE was subjected to centrifugation at 10,000rpm, using REMI Microprocessor Research Centrifuge (PR-24), for 30 minutes and destabilization phenomenon, if any were noted.

 

MIC and MBC studies of LGE, alumina nanoparticles and their emulsion NIE against cultures of S. mutans:

200-1000ppm of LGE as well as alumina nanoparticles were used separately for determining their MIC and MBC against clinical isolates and MTCC culture of S. mutans by micro broth-dilution method with BHI as per CLSI guidelines. 180µl of each concentration of LGE and alumina nanoparticles were pipetted into different wells of a 96-well microtitre plate along with 20µl of bacterial culture to give a final concentration of 1×10⁵ CFU/ml. After incubation for 24 hours at 37°C, the absorbance was measured at 595nm with an ELISA reader (BioRad 6.0). Thereafter, the concentration equal to MIC value of the nanoparticles was dissolved in required volume of distilled water, sonicated, and used for the formulation of the nanoparticle incorporated emulsion (NIE) by using the same procedure for synthesis as described for LGE. MIC and MBC were determined for NIE against all the cultures also as per  standard CLSI guidelines.

 

Time kill assay of NIE:

Each bacterial sample, diluted with fresh BHI broth (final concentration of 1.5×10⁵ CFU/ml) was incubated with MIC concentration of NIE for 0, 30, 60, 120, 240, 480 and 720 minutes at 37°C. 50μl of each of the above bacterial samples were then spread on BHI agar medium, incubated at 37°C for 24 hours. CFU was arrived at as follows: (number of colonies x dilution factor)/Volume of sample on plate. The time kill effect was arrived at by plotting the log₁₀ values of CFU versus specific time points.

 

Studies on inhibition of biofilm formation of S. mutans:

20μl aliquots, taken from 24 hour old broth cultures of isolates and MTCC 890 strain were inoculated into wells of 96 microtiter plate with 180μl of NIE and incubated for 48 hours at 37°C. Wells were then washed out with sterile distilled water, and 100μl of methanol was added. The plate was incubated for 15 minutes for proper adhesion of biofilm to well surface. Methanol was removed by air drying and staining was done with 20μl (0.1% w/v) of crystal violet solution. Thereafter, bound dye was removed by adding 20μl of 30% (v/v) glacial acetic acid into the wells. Absorbances were then recorded at 595 nm using ELISA reader (BioRad 6.0). The assay was conducted separately for the MIC concentration of each of LGE, nanoparticle suspension and NIE. The biofilm inhibition (%)was thereafter calculated for each test strain.

 

RESULTS AND DISCUSSION:

LGO is known for its versatile medicinal properties and acts as an antiseptic, astringent etc. However, the use of the concentrated oil is a matter of concern due to its dosage toxicity. The effects also vary in cells, organs and tissues depending on the conditions used²². Therefore it is essential to have an effective and safe delivery system for LGO. In this context, LGO-based emulsion formulation reported in this study may be useful. The surfactants used (Tween 80/ Span 80 combination) for blending the oil with water are sorbitan monooleate and its poly-ethoxylated derivative.

 

Formulation and characterization of LGE:

Formulations with oil:surfactant ratios of 1:0.5, 1:0.6 and 1:0.7 were visually stable. Further after ultrasonication, since there were low significant differences (p<0.05) in the particle sizes of these three formulations as determined by one way ANOVA and Tukey tests, the formulation with the middle ratio of 1:0.6 was selected and labeled as LGE for further studies. The final composition of this emulsion was 5ml oil, 3ml surfactant mixture (43.92ml of Tween80 and 56.07ml of Span80) and 48ml of water in a total volume of 50ml. 

 

The mean particle size and polydispersity index (PDI) of LGE were found to be 252.3±1.15nm (Fig.1) and 0.111± 0.01 respectively. Particle size is vital in determining the pharmacokinetics, distribution of actives in the tissues as well as their clearance. LGE with a size of around 252nm would serve well in this regard. Also there is a very low tendency for such sub micron sized particles to aggregate and destabilize the emulsion as is observed with micro and higher sized particles. Further, PDI is an indication of the constituent particle size distribution in any system and varies from 0 (monodisperse) to 1.0 (highly polydisperse) with monodispersed systems showing enhanced stability²³. This is again due to their lower propensity for aggregation and system destabilization. The value of PDI in case of LGE (0.111±0.01), is below the value of 0.2 that is commonly deemed to be acceptable for emulsion systems with regard to drug delivery and also stability²⁴.

 

Figure 1  Particle size-distribution of LGE

 

FTIR analysis (Fig. 2) was aimed at functional group analysis of the components of LGO and LGE. The C-H (2854.55 cm^-1 to 2996.52 cm^-1), C=C (1631.78 cm^-1 to 1672.28 cm^-1) and =C-H bonds (441.41cm^-1 to 983.70cm^-1) present in the oil’s constituents reveal the FTIR bands at their respective wave numbers. The highly intense broad peak at 3321.42 cm^-1 is of the -OH of Durenol, β- linalool, Germacrene-D-4-ol, Geranyl linalool. The alcoholic C-O bonds present in the constituents also show sharp peaks at 1120.64 cm^-1 and 1193.94 cm^-1. Sharp and intense peaks at 2960.73, 2920.23, 2856.58 2927.94 and 2854.65cm^-1 correspond to stretching vibrations due to –C-H bonds in almost all constituents of the oil. Similarly, bending vibrations of =C-H bonds, that are characteristic of several compounds present in the oil, result in formation of sharp peaks at 983.70, 840.96, 746.45, 594.08 and 449.11cm^-1.

 

Figure 2 FTIR spectra of LGO and LGE

 

AFM analysis was carried out to study the surface topology and characteristics of the emulsion. The surface scans generated reveal the fairly smooth surface of LGE (Fig.3).

 

Emulsion systems tend to destabilize due to different mechanisms operating in them like creaming, flocculation, coalescence and Ostwald ripening²⁵. Such mechanisms may occur when they are stored at ambient temperatures where in they are intended for use, over a period of time or when subjected to certain temperature extremes and visually observed as creaming, cracking, oil droplet formation, and phase separations. Reports on such accelerated stability tests reveal that the selection of methodologies, temperatures, time periods and test cycles depend on the intended temperatures of storage and usage of the respective products that have been formulated in the studies and the procedures are tailor made accordingly^26,27,28.

    

    

Figure 3 Atomic Force Microscopic analysis of LGE

In this context, LGE was stored at an intended usage temperature of 37ºC for a period of 90 days for monitoring its shelf life stability and no destabilizing mechanisms were found to be operating as observed visually. The colour and texture also remained unchanged. Similarly, it was also deemed relevant to study stability of LGE if it is stored and transported at -18ºC for 48 hours and brought back to 37ºC for usage. During this process and also during centrifugation, no destabilization was observed visually

 

MIC and MBC studies of LGE, alumina nanoparticles and their emulsion (NIE) against cultures of S. mutans:

MIC of LGE and alumina nanoparticles were recorded around 600ppm whereas the MIC of NIE was lower at around 400ppm. LGE and nanoparticles thus appear to act synergistically. MBC was determined to be approximately 800ppm (Fig.4a and b). If the MBC is upto 4 times of MIC, a compound can be considered to exhibit antibacterial activity against the organism being studied. Nearly similar values for MIC and MBC were obtained with 0.2%w/v solution of chlorhexidine gluconate (positive control), which shows strong antimicrobial activity against dental pathogens²⁹.

 

 

Figure 4  (a) MIC against clinical isolates (b) MIC against MTCC 890 (LGE-Lemongrass oil emulsion, NP-Al₂O₃ nanoparticles, NIE- Nanoparticle incorporated LGE,CHX - chlorhexidine gluconate solution-0.2% w/v)

 

Time kill assay of NIE:

A quick decrease in the log₁₀ value of CFU/ml, within 60 to 120 minutes of incubation was observed with 400ppm (MIC value) of NIE showing that the growth of S. mutans is inhibited by the NIE (Fig.5). This is in contrast to the control plate (without NIE) wherein organisms grew gradually and steadily gradual over a period of time. The growth stabilised eventually.

 

 

Figure 5 Time kill assay of NIE against MTCC890 and representative five clinical isolates (average values) of S mutans

 

Studies on inhibition of biofilm formation of S. mutans:

NIE inhibited the biofilm formation considerably (Fig.6). Inhibition percentage with nanoparticle suspension was higher than with LGE. NIE showed such inhibitory action due to synergistic antimicrobial actions of both LGE and alumina nanoparticles. The mechanism of action may involve disruption of the bacterial cell membrane and this can occur by several factors that include induction of oxidative stress, release of metal ions or penetration of cell membrane followed by DNA interactions^9,30.

 

Figure 6   Inhibitionof biofilm of five clinical isolates (average values) of S.mutans with LGE (CIE), alumina Nanoparticles (CINP) and NIE (CINIE), and MTCC890 strain with LGE (ME), alumina nanoparticles and (MNP) and NIE (MNIE)

 

CONCLUSION:

MIC’s of LGE and nanoparticle suspension against clinical isolates of S. mutans and MTCC culture was 600 ppm whereas MBC was twice the value. However, NIE, that was formulated by combining LGE with alumina nanopaticles showed an MIC of around 400ppm with MBC being twice this value. Further NIE not only had a higher antibacterial potency as compared to LGE and alumina nanoparticles separately, but also inhibited biofilm formation by the organisms to a greater extent. The activity was also comparable to a commercial chlorhexidine gluconate (gold standard) preparation. Due to this potent antibacterial activity of NIE against cariogenic strains of S. mutans, it can be further explored for potential use in mouthwash formulations to prevent dental caries. It can hence serve as a safer alternative natural medication for this condition.  

 

ACKNOWLEDGEMENT:

The authors acknowledge the facilities rendered by Vellore Institute of Technology, Vellore, India for this study.   

 

CONFLICT OF INTEREST:

Nil.

 

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Accepted on 12.11.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(5):2291-2296.