Document Type : Original Research Article


1 Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Chemistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

2 Department of Medical Nanotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran


Objective(s): The oil-in-water (O/W) nanoemulsion is expanded in biomedical application due to their special properties. Mint and Parsley are known herbs with many health benefits. Chitosan (Ch) is a low toxic, biodegradable, biocompatible and safe polymer with the antibacterial activity which is used in production of nanomaterial. The aim of this study was to evaluate the nanoemulsion to achieve good antibacterial activity and suitable stability with low cytotoxicity.
Methods: In the presence study, the O/W nanoemulsion was prepared by Mint essential oil in the presence of Parsley aqueous extract and chitosan aqueous solution using Span 80 and Tween 80 surfactants and high-intensity homogenizer at room temperature for 30 min. The result samples were characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The stability of nanoemulsion was evaluated for 50 days at different temperatures for optimized samples. The antibacterial activity of samples was measured against an important pathogen bacterium, Escherichia coli (E. coli) using inhibition zone diameter. The rheological properties of nanoemulsion were determined by presentation of viscosity at room temperature. The cytotoxicity of samples was investigated by MTT assay for HEK239 human cell line at three different concentrations for three periods of time.
Results: Our results showed the nanoemulsion with nanometer size. The presence of chitosan was caused more particles with the smaller size. The low temperature was needed to protection of nanoemulsion stability and prevention of particle growth.
Conclusions: Our study indicated that the nanoemulsion improved its antibacterial activity with low cytotoxicity and suitable stability.



Nanotechnology has been led to a significant progress on various scientific fields and product innovation. Over the past years, microemulsions have attracted much interest as delivery systems because of unique properties such as ease of in preparation, transparency, and long-term stability [1]. The nanoemulsions have been extensively studied because of their potential applications in industrial various such as food, cosmetic, pharmacology, and agriculture in recent years [2,3]. Nanoemulsions are considered as intermediates between classical emulsions and microemulsions usually in the 20 to 500 nm size range [4-6]. The small size of nanoemulsion can be enhancing either the penetration into or absorption by cells [7]. The main categories of nanoemulsions are oil-in-water or water-in-oil, transparent or translucent, colloidal dispersions [8-10]. The components of oil-in-water emulsions can be included the lipophilic bioactive within the oil phase and an aqueous medium in presence of emulsifier. The produced droplet size depends on the component composition and the homogenization method. The thermodynamically unstable can be seen for oil-in-water conventional emulsions and nanoemulsions [11-14]. Oil-in-water nanoemulsions were stabilized by emulsifier such as Tween 20 as a non-ionic surfactant in aqueous buffer solution [15,16].

The nanoemulsions can be prepared with small particle size by high-energy or by low-energy emulsification methods and the surfactant is used to stability maintain. The high-pressure homogenizer is one of high-energy methods for the formation of nanoemulsions [17,18]. Nanomulsions have advantages than emulsion due to their small size, such as higher optical clarity, higher stability, and enhanced bioavailability of lipophilic compounds [19]. However, their Nanomulsion application is limited because of potential risks of nanostructures, such as the potential toxicity of some components (e.g. surfactants) and their ability to change the biological fate of materials and bioactive compounds, which could potentially have some adverse effects on human health [20,21].

The resistant bacteria have become a major challenge in developed countries [22,23]. The herbal oils as natural antimicrobial substances can be used as oil phase in emulsion systems to increase their efficacy and inhibit bacterial growth [24-26]. The antimicrobial activity of essential oils has been widely investigated in the last decades [27,28]. The herbal oil can be used as oil core in oil-in-water nanoemulsion [29]. The large number of volatile organic compounds is emitted from plants into the environment. These volatile compounds can be caused therapeutic and pharmacological properties [30,31].

The wide range of emulsions can be assembled with biomaterials. Mint is one of hundreds of different types of essential oils and the most popular [32]. Mint essential oils can be used as oil soluble part of emulsion. Recently, the antibacterial activities of Mint oil were evaluated against some Gram-positive and Gram-negative bacterial strains by agar well diffusion method [33-35]. The antioxidant properties of Mint oil were presented because of phenols one of the major groups of herbal natural compounds acting as radical scavengers that given the beneficial effect to human health [34-37]. Recently, Parsley extract was used for green synthesis of nanoparticles [38], that its aqueous solution can be used as water soluble part of emulsion. Parsley is few of the aromatic herbs with antibacterial activities against various bacterial strains [39-41]. Recently, the antioxidant capacity of Parsley as kind of herbs has attracted increasing interest compared with chemical antioxidants [42]. Chitosan is a biopolymer with biocompatibility, biodegradability, non-toxicity, and antibacterial properties [43,44]. Recently, the used of chitosan has expanded with essential oils because of the special properties of them both [45,46]. The aim of the present work was to form nanoemulsion of Mint essential oils in the presence of Parsley aqueous extract and chitosan aqueous solution and investigation of antibacterial activity against E. coli as Gram-negative bacterial model and cytotoxicity on HEK239 human cell line. This study provides important new information about nanoemulsions based on essential oils and aqueous extract.



All chemicals used were analytical grade. Ultra-pure water was used for the preparation of all reagents solutions. Chitosan (medium molecular weight) was obtained from Sigma–Aldrich. Tween 80, Span 80, and glacial acetic acid were purchased from Merck Millipore (Darmstadt, Germany). Mint essential oil and Parsley aqueous extract bought from Adonis Gol Daro (Iran).


Dynamic light scattering was reported the size and size distribution of nanoparticles (ZEN314, England). Scanning electron microscope was employed to observe the morphology and size (KYKY EM3200, China). The rheological properties were studied by investigating viscosity based on spindle speed for samples using Reologica InstrumentsAB (Lund, Sweden). The nanoemulsions were allowed to equilibrate for 72 h prior to evaluation of their rheological properties. The nanoemulsions was placed under minimum stress of 0.05 Pa and a maximum stress of 550 Pa and 600 Pa. Rheograms were produced automatically in triplicate that exerted gradually the increasing of shear stress to the specified maximum stress. The antibacterial activities were evaluated by disk diffusion method against Escherichia coli bacteria, ATCC 1399, that procured from Islamic Azad University. The cytotoxicity of samples was investigated by MTT assay for HEK239 human cell line at three different concentrations for 1, 3, and 5 days.

Nanoemulsion preparation

The nanoemulsions were prepared from a mixture of Mint essential oil and Parsley aqueous extract by homogenizing at 18000 rpm for 30 min with different percentages in presence of Tween 80 and Span 80 surfactants (Table 1).



The dynamic light scattering was determined the mean particle diameter and the particle size distribution of nanoemulsion. DLS results showed a single-peak with a narrow distribution with different size duo to the nanoemulsion components (Table 2).

Based on these results, the optimum percentage was observed for the nanoemulsion that included Mint essential oil (4% w/w), Parsley aqueous extract (94% w/w), and surfactant (2% w/w). The nanoemulsion in presence of chitosan was prepared with the optimum percentage of components by dissolving of chitosan powder (1% w/w) with 1% (v/v) acetic acid in Parsley aqueous extract. Fig. 1 was shown the DLS diagram of nanoemulsion (I) and (II) without and with chitosan in presence of two surfactants. The mean particle diameter was observed 78.1 nm in presence of Span 80 and 138 nm Tween 80 for nanoemulsion (I) and (II) respectively. Based on DLS result, the optimum nanoemulsion was nanoemulsion (I) in presence of chitosan and Span 80 surfactant.

The nanoemulsion has advantages than another oil-in-water nanoemulsions such as the smaller size compared with nanoemulsion of Sage oil with formulation containing sage oil, Tween 80 and Span 80 emulsifier, and water using probe sonicator in different times [47] and the narrow distribution compared with nanoemulsion of curcumin with formulation containing curcumin into the heated oil by homogenising [14]. The controlled size with the narrow distribution can be had potentially specific effects for different applications such as food and pharmaceutical industry in the future.


Fig. 2 was shown the SEM images for nanoemulsion (I) and (II) without and with chitosan in presence of Span 80 and Tween 80 with spherical morphology. The particle size was estimated for nanoemulsion (I) without and with chitosan about 70 nm (with broad distribution) and 60 nm (with narrow distribution) respectively. The particle size was estimated for nanoemulsion (II) without and with chitosan about 75 nm (with broad distribution) and 50 nm (with narrow distribution) respectively. According to the results, the presence of chitosan was caused more particles due to suitable nucleation and the smaller size because of the effect of capping agent. The advantages of the nanoemulsion include the spherical morphology with narrow distribution and more nanoparticles compared with previous report from researchers [47].

Rheological properties

The viscosity was investigated at different shear rates for nanoemulsions (I) and (II) without and with chitosan (Table 3). According to the result, the viscosity of sample was as following: nanoemulsions (I) with chitosan > nanoemulsions (II) with chitosan > nanoemulsions (I) > nanoemulsions (II) in the same spindle speed. The higher viscosity was observed for nanoemulsions with chitosan inParsley aqueous extract and Span 80 surfactant because of their nature and properties. The rheological properties are according to studies of Wulff-Pérez and coworkers in 2013 [49]. The flow ability of the nanoemulsion is determined by measuring spindle speed and viscosity, which indicated an important role in samples for food and pharmaceutical applications in future.

Antibacterial activity

The inhibition zone diameter was investigated for determination of sample antibacterial activity against Escherichia coli as Gram-negative bacterial model. The E. coli cells were cultivated on Mueller-Hinton Agar and incubated at 37 ºC for 1 day. The inhibition zone diameter was 1 cm for nanoemulsions (I) and (II) and 1.5 cm for these nanoemulsions with chitosan. Based on the result, the presence of chitosan was increased antibacterial activity in nanoemulsions. The antibacterial activity is according to previous reports from researchers [28, 47]. The antibacterial activity of nanoemulsion is related to essential oils and the presence of chitosan is caused the increase of antibacterial activity. The nanoemulsion can keep essential oils from easily evaporate and decompose during preparation, owing to direct exposure to heat, pressure and light [51].

MTT assay

The cytotoxicity of samples was investigated by MTT assay for HEK239 human cell line at three different concentrations 0.25, 0.5, and 1% (v/v) for three periods of time (1, 3 and 5 days). Based on the result, concentration and time were two effective factors on cytotoxicity of nanoemulsions. The higher cytotoxicity was observed because of the increase of concentration and time and the cell viability was shown more than 50% for all samples. The MTT assay advantages of the nanoemulsion include acceptable toxicity in longer time compared with previous report from researchers [50].


The stability was performed for the optimum sample that’s mean nanoemulsion (I) with chitosan by SEM analysis into the different temperatures (-20, -4, 15, and 38 Co) after 50 days. Based on the results, the particle size didn’t change at -20, -4, and 15 °C, but the particle agglomerated at 38 °C (Fig. 4). Therefore, the low temperature was needed for protection of stable nanoemulsion to prevent growth and agglomeration of particle. The stability advantages of the nanoemulsion are according to studies of Mann and coworkers in 2015 and the increase of treatment caused to the increase of average size [48]. Nanoemulsion stability was reported for the first time in low temperature.


The present study demonstrated the nanoemulsion with Mint essential oil and Parsley aqueous extract in the presence of chitosan can be good candidate for antibacterial activity against Escherichia coli with low cytotoxicity for HEK239 human cell line. The ph ysical stability of nanoemulsion was good and suitable for 50 days at the temperatures of -20, -4, and 15 Co with little evidence of phase separation or particle growth. The optimum sample was included Mint essential oil (4% w/w), Parsley aqueous extract (94% w/w), and Span 80 surfactant (2% w/w) in presence of chitosan. Based on our results, it can be proposed that the presence of chitosan can be controlled the size, nucleation, and antibacterial activity in the nanoemulsion. The prepared nanoemulsion can be a cost-effective way with good potential to different applications in variety research fields of food and pharmaceutical in future.


The authors declare that there are no conflicts of interest regarding the publication of this manuscript.



1. Kreilgaard M. Influence of microemulsions on cutaneous drug delivery. Advanced Drug Delivery Reviews. 2002;54:S77-S98.

2. Izquierdo P, Esquena J, Tadros TF, Dederen JC, Feng J, Garcia-Celma MJ, et al. Phase Behavior and Nano-emulsion Formation by the Phase Inversion Temperature Method. Langmuir. 2004;20(16):6594-8.

3. Guglielmini G. Nanostructured novel carrier for topical application. Clinics in Dermatology. 2008;26(4):341-6.

4. Anton N, Benoit J-P, Saulnier P. Design and production of nanoparticles formulated from nano-emulsion templates—A review. Journal of Controlled Release. 2008;128(3):185-99.

5. Tadros T, Izquierdo P, Esquena J, Solans C. Formation and stability of nano-emulsions. Advances in Colloid and Interface Science. 2004;108-109:303-18.

6. Ee SL, Duan X, Liew J, Nguyen QD. Droplet size and stability of nano-emulsions produced by the temperature phase inversion method. Chemical Engineering Journal. 2008;140(1-3):626-31.

7. Lin C-C, Lin H-Y, Chen H-C, Yu M-W, Lee M-H. Stability and characterisation of phospholipid-based curcumin-encapsulated microemulsions. Food Chemistry. 2009;116(4):923-8.

8. Porras M, Solans C, González C, Martínez A, Guinart A, Gutiérrez JM. Studies of formation of W/O nano-emulsions. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2004;249(1-3):115-8.

9. Sajjadi S. Erratum to “Formation of fine emulsions by emulsification at high viscosity or low interfacial tension: A comparative study” [Colloids Surf. A: Physicochem. Eng. Aspects 299 (2007) 73–78]. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2007;303(3):258.

10. Al-Edresi S, Baie S. Formulation and stability of whitening VCO-in-water nano-cream. International Journal of Pharmaceutics. 2009;373(1-2):174-8.

11. Solans C, Izquierdo P, Nolla J, Azemar N, Garciacelma M. Nano-emulsions. Current Opinion in Colloid & Interface Science. 2005;10(3-4):102-10.

12. Acosta E. Bioavailability of nanoparticles in nutrient and nutraceutical delivery. Current Opinion in Colloid & Interface Science. 2009;14(1):3-15.

13. McClements DJ, Rao J. Food-Grade Nanoemulsions: Formulation, Fabrication, Properties, Performance, Biological Fate, and Potential Toxicity. Critical Reviews in Food Science and Nutrition. 2011;51(4):285-330.

14. Ahmed K, Li Y, McClements DJ, Xiao H. Nanoemulsion- and emulsion-based delivery systems for curcumin: Encapsulation and release properties. Food Chemistry. 2012;132(2):799-807.

15. Sajjadi S, Zerfa M, Brooks BW. Phase inversion in p-xylene/water emulsions with the non-ionic surfactant pair sorbitan monolaurate/polyoxyethylene sorbitan monolaurate (Span 20/Tween 20). Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2003;218(1-3):241-54.

16. Qian C, Decker EA, Xiao H, McClements DJ. Nanoemulsion delivery systems: Influence of carrier oil on β-carotene bioaccessibility. Food Chemistry. 2012;135(3):1440-7.

17. Izquierdo P, Feng J, Esquena J, Tadros TF, Dederen JC, Garcia MJ, et al. The influence of surfactant mixing ratio on nano-emulsion formation by the pit method. Journal of Colloid and Interface Science. 2005;285(1):388-94.

18. Solè I, Maestro A, González C, Solans C, Gutiérrez JM. Optimization of Nano-emulsion Preparation by Low-Energy Methods in an Ionic Surfactant System. Langmuir. 2006;22(20):8326-32.

19. McClements DJ, Xiao H. Potential biological fate of ingested nanoemulsions: influence of particle characteristics. Food Funct. 2012;3(3):202-20.

20. McClements DJ. Edible lipid nanoparticles: Digestion, absorption, and potential toxicity. Progress in Lipid Research. 2013;52(4):409-23.

21. Pinheiro AC, Coimbra MA, Vicente AA. In vitro behaviour of curcumin nanoemulsions stabilized by biopolymer emulsifiers – Effect of interfacial composition. Food Hydrocolloids. 2016;52:460-7.

22. Hemaiswarya S, Kruthiventi AK, Doble M. Synergism between natural products and antibiotics against infectious diseases. Phytomedicine. 2008;15(8):639-52.

23. Murbach Teles Andrade BF, Nunes Barbosa L, da Silva Probst I, Fernandes Júnior A. Antimicrobial activity of essential oils. Journal of Essential Oil Research. 2013;26(1):34-40.

24. Alizadeh Behbahani B, Tabatabaei Yazdi F, Vasiee A, Mortazavi SA. Oliveria decumbens essential oil: Chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection. Microbial Pathogenesis. 2018;114:449-52.

25. Huang W, Wang J, Song H, Zhang Q, Liu G. Effect of Antimicrobial Packaging Film Coated with Ajowan and Cinnamon Essential Oil on Shelf Life of Fresh Chilled Pork. Lecture Notes in Electrical Engineering: Springer Singapore; 2018. p. 787-97.

26. Salvia-Trujillo L, Rojas-Graü A, Soliva-Fortuny R, Martín-Belloso O. Physicochemical characterization and antimicrobial activity of food-grade emulsions and nanoemulsions incorporating essential oils. Food Hydrocolloids. 2015;43:547-56.

27. Seow YX, Yeo CR, Chung HL, Yuk H-G. Plant Essential Oils as Active Antimicrobial Agents. Critical Reviews in Food Science and Nutrition. 2013;54(5):625-44.

28. Moghimi R, Ghaderi L, Rafati H, Aliahmadi A, McClements DJ. Superior antibacterial activity of nanoemulsion of Thymus daenensis essential oil against E. coli. Food Chemistry. 2016;194:410-5.

29. Zorzi GK, Carvalho ELS, von Poser GL, Teixeira HF. On the use of nanotechnology-based strategies for association of complex matrices from plant extracts. Revista Brasileira de Farmacognosia. 2015;25(4):426-36.

30. Kesselmeier J, Staudt M. Biogenic Volatile Organic Compounds (VOC): An Overview on Emission, Physiology and Ecology. J Atmos Chem. 1999;33:23-88.

31. Tholl D, Boland W, Hansel A, Loreto F, Röse USR, Schnitzler J-P. Practical approaches to plant volatile analysis. The Plant Journal. 2006;45(4):540-60.

32. Verma RS, Rahman L, Verma RK, Chauhan A, Yadav AK, Singh A. Essential oil composition of menthol mint (Mentha arvensis) and peppermint (Mentha piperita) cultivars at different stages of plant growth from Kumaon Region of western Himalaya. J Med Aromat Plants. 2010;1:13-18.

33. Donsì F, Ferrari G. Essential oil nanoemulsions as antimicrobial agents in food. Journal of Biotechnology. 2016;233:106-20.

34. Sandasi M, Leonard CM, Van Vuuren SF, Viljoen AM. Peppermint (Mentha piperita) inhibits microbial biofilms in vitro. South African Journal of Botany. 2011;77(1):80-5.

35. Singh R, Shushni MAM, Belkheir A. Antibacterial and antioxidant activities of Mentha piperita L. Arabian Journal of Chemistry. 2015;8(3):322-8.

36. Biswas NN, Saha S, Ali MK. Antioxidant, antimicrobial, cytotoxic and analgesic activities of ethanolic extract of Mentha arvensis L. Asian Pacific Journal of Tropical Biomedicine. 2014;4(10):792-7.

37. Benabdallah A, Rahmoune C, Boumendjel M, Aissi O, Messaoud C. Total phenolic content and antioxidant activity of six wild Mentha species (Lamiaceae) from northeast of Algeria. Asian Pacific Journal of Tropical Biomedicine. 2016;6(9):760-6.

38. Hajiashrafi S, Motakef-Kazemi N. Green synthesis of zinc oxide nanoparticles using parsley extract. Nanomed Res J. 2018;3(1):44-50.

39. Semeniuc CA, Pop CR, Rotar AM. Antibacterial activity and interactions of plant essential oil combinations against Gram-positive and Gram-negative bacteria. Journal of Food and Drug Analysis. 2017;25(2):403-8.

40. Karimi F, Rezaei M, Shariatifar N, Sayadi M, Mohammadpourfard I, Malekabad E, Jafari H. Running head: Antimicrobial activity of Parsley antimicrobial activity of essential oil of Parsley (PetroselinumCrispum) against FoodPathogenic bacteria. World Appl Sci J. 2014;31:1147–1150.

41. Khalil AF, Elkatry HO, El Mehairy HF. Protective effect of peppermint and parsley leaves oils against hepatotoxicity on experimental rats. Annals of Agricultural Sciences. 2015;60(2):353-9.

42. Vranješ M, Popović BM, Štajner D, Ivetić V, Mandić A, Vranješ D. Effects of bearberry, parsley and corn silk extracts on diuresis, electrolytes composition, antioxidant capacity and histopathological features in mice kidneys. Journal of Functional Foods. 2016;21:272-82.

43. Silalahi J, Situmorang P, Patilaya P, Ce Silalahi Y. ANTIBACTERIAL ACTIVITY OF CHITOSAN AND HYDROLYZED COCONUT OIL AND THEIR COMBINATION AGAINST BACILLUS CEREUS AND ESCHERICHIA COLI. Asian Journal of Pharmaceutical and Clinical Research. 2016;9(5):69.

44. Goy RC, Morais STB, Assis OBG. Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on E. coli and S. aureus growth. Revista Brasileira de Farmacognosia. 2016;26(1):122-7.

45. Peng Y, Yin L, Li Y. Combined effects of lemon essential oil and surfactants on physical and structural properties of chitosan films. International Journal of Food Science & Technology. 2012;48(1):44-50.

46. Li Y, Wu C, Wu T, Wang L, Chen S, Ding T, et al. Preparation and characterization of citrus essential oils loaded in chitosan microcapsules by using different emulsifiers. Journal of Food Engineering. 2018;217:108-14.

47. Moghimi R, Aliahmadi A, McClements DJ, Rafati H. Investigations of the effectiveness of nanoemulsions from sage oil as antibacterial agents on some food borne pathogens. LWT - Food Science and Technology. 2016;71:69-76.

48. Sari TP, Mann B, Kumar R, Singh RRB, Sharma R, Bhardwaj M, et al. Preparation and characterization of nanoemulsion encapsulating curcumin. Food Hydrocolloids. 2015;43:540-6.

49. Wulff-Pérez M, Martín-Rodríguez A, Gálvez-Ruiz MJ, de Vicente J. The effect of polymeric surfactants on the rheological properties of nanoemulsions. Colloid and Polymer Science. 2012;291(3):709-16.

50. Ahmad M,1 Sahabjada, Akhtar J, Hussain A, Badaruddeen, Arshad Md, Mishra A. Development of a new rutin nanoemulsion and its application on prostate carcinoma PC3 cell line. EXCLI J. 2017;16:810–823.

51. Marei G, Rabea E, Badawy M. Ultrasonic Emulsification and Characterizations of Bio-based Nanoemulsion Formulations Containing Citral with Their Antimicrobial Activity. Egyptian Academic Journal of Biological Sciences, F Toxicology & Pest Control. 2017;9(3):169-82.