Licorice and lavender plants have anti-cancer [4, 13] and antimicrobial [5, 15] properties; however, due to their low bioavailability and degradability, their use as a medicine has limitations [16, 17]. One approach to overcome these restrictions is to use nanoparticles. Nanoemotions ensure the protection of biological compounds and their controlled release [19]. This study aimed to evaluate the antiproliferative effects of nanoemulsions containing licorice extract and lavender essential oil on liver cancer cell lines (HepG2) and skin cancer cells (SK-MEL3). We also explored its antimicrobial properties on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis in vitro.

 In this experimental study, nanoemulsions containing licorice extract and lavender essential oil were generated by the spontaneous emulsion method. To make the nanoemulsion, the aqueous phase, consisting of glycerol, polyethylene glycol solvent, and water, as well as the oil phase, including lavender essential oil, licorice extract, and emulsifiers (Tween 20 & Tween 80), were each prepared separately and combined and homogenized after heating. Single-Phase and transparent nanoemulsions were obtained. The antiproliferative effect of nanoemulsion on HepG2 and SK-MEL3 cell lines was investigated using MTT colorimetric method. Four standard bacterial strains and the Minimum Inhibitory Concentration (MIC) method were used to measure the antimicrobial effect. The last batch of nanoemulsion, in which no growth was observed, was considered as MIC. To analyze the obtained data, a one-way Analysis of Variance (ANOVA) was employed in SPSS at P<0.05.

The results of the examination on HepG2 cells revealed that the IC50 level of nanoemulsion was 401 μg/mL (P<0.05). However, the concentration of 2500 licorice extracts could only kill 28% of the cells. The IC50 value for lavender essential oil was measured as 450 (P<0.05) (Figure 1).



Examining SK-MEL3 cells suggested that the IC50 nanoemulsion concentration was 82 μg/mL (P<0.05). Lavender essential oil had similar toxicity to nanoemulsions; however, licorice extract was toxic only at the concentrations of 1250 and 2500 (P<0.05) (Figure 2). 



 In the study of nanoemulsion toxicity, the desired bacterial strains were subjected to concentrations of 0.625, 1.25, 2.5, 5, and 10 mg/mL nanoemulsion (Figure 1). The collected results indicated that the nanoemulsion presented an inhibitory effect on the growth of all bacterial used strains. The highest inhibitory effect concerned Staphylococcus aureus with MIC 5 mg/mL. The MIC for the other 3 strains was equal to 10 mg. Licorice extract did not affect Staphylococcus aureus and Escherichia coli but inhibited the growth of Pseudomonas and Staphylococcus epidermidis. The lavender essential oil also inhibited the growth of all strains except Pseudomonas (Table 1).

The current study results revealed that the nanoemulsion is toxic to cancer cells. The anti-proliferative effect of licorice extract and lavender essential oil has been proven in previous studies [10, 20]. When cancer cells become malignant, they produce sustained ROS, leading to tumor growth and progression [21]. Using antioxidants, such as lavender essential oil, licorice extract, or nanoemulsions prepared from them can help improve the function of anti-cancer agents by reducing the amount of ROS.
Considering the effects of licorice and lavender, it was expected that the nanoemulsion form of licorice extract and lavender essential oil present a high potential to become a formulation with anti-proliferative properties against cancer cells due to its physical stability. The present study revealed that the mentioned nanoemulsion has such a property. Furthermore, the cytotoxicity of nanoemulsion is higher than licorice extract and equal to lavender essential oil. Given the hydrophobicity and volatility of the essential oils, the conversion of the essential oil into a stable nanoemulsion can be useful. These results were consistent with those of the study of Ziaee and associates [25].
 The antimicrobial effects of nanoemulsion highlighted that this nanoemulsion had the highest level of inhibition on the growth of Staphylococcus epidermidis; its inhibitory effect was the same on other strains. The antibacterial effect of lavender essential oil was greater than nanoemulsion in all strains; however, licorice extract provided an inhibitory effect only on Pseudomonas and Staphylococcus epidermidis.

This study was approved by the Research Ethics Committee of Kashan University of Medical Sciences (Code: IR.KAUMS.MEDNT.REC.1396.106).

The paper was extracted from the MSc. thesis of the first author, Department of Clinical Biochemistry, Faculty of medicine, Kashan University of Medical Sciences (KAUMS, Grant No: 96203). 

Conceptualization and validation methodology of data analysis: Mohammad Esmaeil Shahaboddin, Mohammad Hossein Aarabi; Research, analysis, and sources: Ali Nazari-Alam, Mohammad Shayestehpour, Majid Nejati, Hamidreza Gilasi, and Afshin Salehi; Drafting: Zohreh Karimi Taheri.

The authors declared no conflicts of interest.

We would like to thank the esteemed the Vice Chancellor for Research and Technology of Kashan University of Medical Sciences.
 

Reference

1. Bent S. Herbal medicine in the United States: review of efficacy, safety, and regulation. J Gen Intern Med. 2008; 23(6):854-9. [DOI:10.1007/s11606-008-0632-y] [PMID] [PMCID]
2. Hosseinzadeh H, Nassiri-Asl M. Pharmacological effects of Glycyrrhiza spp. and its bioactive constituents: update and review. Phytother Res. 2015; 29(12):1868-86. [DOI:10.1002/ptr.5487] [PMID]
3. Nomura T, Fukai T, Akiyama T. Chemistry of phenolic compounds of licorice (Glycyrrhiza species) and their estrogenic and cytotoxic activities. Pure Appl Chem. 2002; 74(7):1199-206. [DOI:10.1351/pac200274071199]
4. Basar N, Oridupa OA, Ritchie KJ, Nahar L, Osman NMM, Stafford A, et al. Comparative cytotoxicity of Glycyrrhiza glabra roots from different geographical origins against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cells. Phytother Res. 2015; 29(6):944-8. [DOI:10.1002/ptr.5329] [PMID]
5. Nitalikar MM, Munde KC, Dhore BV, Shikalgar SN. Studies of antibacterial activities of Glycyrrhiza glabra root extract. Int J Pharm Tech Res. 2010; 2(1):899-901.
6. Ghadiri MK, Gorji A. Lavender for medicine: A brief review of clinical effects. Avicenna. 2002; 1:23-7. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.116.6864&rep=rep1&type=pdf#page=24
7. Shiina Y, Funabashi N, Lee K, Toyoda T, Sekine T, Honjo S, et al. Relaxation effects of lavender aromatherapy improve coronary flow velocity reserve in healthy men evaluated by transthoracic Doppler echocardiography. Int J Cardiol. 2008; 129(2):193-7. [DOI:10.1016/j.ijcard.2007.06.064] [PMID]
8. Shahnazi M, Nikjoo R, Yavarikia P, Mohammad-Alizadeh-Charandabi S. Inhaled lavender effect on anxiety and pain caused from intrauterine device insertion. J Caring Sci. 2012; 1(4):255. [doi: 10.5681/jcs.2012.035]
9. SILVA GL, Luft C, Lunardelli A, Amaral RH, MELO DA, Donadio MV, et al. Antioxidant, analgesic and anti-inflammatory effects of lavender essential oil. An Acad Bras Cienc. 2015; 87(2 S):1397-408. [DOI:10.1590/0001-3765201520150056] [PMID]
10. Kozics K, Srancikova A, Sedlackova E, Horvathova E, Melusova M, Melus V, et al. Antioxidant potential of essential oil from Lavandula angustifolia in in vitro and ex vivo cultured liver cells. Neoplasma. 2017; 64(4):485-93. [DOI:10.4149/neo_2017_401] [PMID]
11. Prusinowska R, Śmigielski KB. Composition, biological properties and therapeutic effects of lavender (Lavandula angustifolia L). A review. Herba Pol. 2014; 60(2):56-66. [DOI:10.2478/hepo-2014-0010]
12. Imre S, Eşianu S, Miklos A, Tiuca I, Dicher I, Tero-Vescan A, et al. Qualitative assay of essential oils of Lavender and Peppermint in commercial products through spectral and chromatographic methods. Farmacia. 2016; 64(6):857-62. https://farmaciajournal.com/wp-content/uploads/2016-06-art-09-Imre_857-862.pdf
13. Dalilan S, Rezaei-Tavirani M, Nabiuni M, Heidari-Keshel S, Azodi MZ, Zali H. Aqueous extract of Lavender angustifolia inhibits lymphocytes proliferation of Hodgkin’s lymphoma patients. Iran J Cancer Prev. 2013; 6(4):201-8. [PMCID]
14. Roller S, Ernest N, Buckle J. The antimicrobial activity of high-necrodane and other lavender oils on methicillin-sensitive and-resistant Staphylococcus aureus (MSSA and MRSA). J Altern Complement Med. 2009; 15(3):275-9. [DOI:10.1089/acm.2008.0268] [PMID]
15. Hossain S, Heo H, De Silva B, Wimalasena S, Pathirana H, Heo G-J. Antibacterial activity of essential oil from lavender (Lavandula angustifolia) against pet turtle-borne pathogenic bacteria. Lab Anim Res. 2017; 33(3):195-201. [DOI:10.5625/lar.2017.33.3.195] [PMID] [PMCID]
16. Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils-a review. Food Chem Toxicol. 2008; 46(2):446-75. [DOI:10.1016/j.fct.2007.09.106] [PMID]
17. Raut JS, Karuppayil SM. A status review on the medicinal properties of essential oils. Ind Crops Prod. 2014; 62:250-64. [DOI:10.1016/j.indcrop.2014.05.055]
18. Thakkar PJ, Madan P, Lin S. Transdermal delivery of diclofenac using water-in-oil microemulsion: Formulation and mechanistic approach of drug skin permeation. Pharm Dev Technol. 2014; 19(3):373-84. [DOI:10.3109/10837450.2013.788658] [PMID]
19. El Asbahani A, Miladi K, Badri W, Sala M, Addi EA, Casabianca H, et al. Essential oils: From extraction to encapsulation. Int J Pharm. 2015; 483(1-2):220-43. [DOI:10.1016/j.ijpharm.2014.12.069] [PMID]
20. Khazraei-Moradian S, Ganjalikhani-Hakemi M, Andalib A, Yazdani R, Arasteh J, Kardar GA. The effect of licorice protein fractions on proliferation and apoptosis of gastrointestinal cancer cell lines. Nutr Cancer. 2017; 69(2):330-9. [DOI:10.1080/01635581.2017.1263347] [PMID]
21. Fu Y, Chen J, Li Y-J, Zheng Y-F, Li P. Antioxidant and anti-inflammatory activities of six flavonoids separated from licorice. Food Chem. 2013; 141(2):1063-71. [DOI:10.1016/j.foodchem.2013.03.089] [PMID]
22. Vlaisavljević S, Šibul F, Sinka I, Zupko I, Ocsovszki I, Jovanović-Šanta S. Chemical composition, antioxidant and anticancer activity of licorice from Fruska Gora locality. Ind Crops Prod. 2018; 112:217-24. [DOI:10.1016/j.indcrop.2017.11.050]
23. Mehdinezhad Doghikolayi S, Mohammadi M. [The antioxidant and cytotoxic effects of lavandulla anguostifouliya on the 8305C cell line (Persian)]. New Cell Mol Biotechnol J. 2018; 8(32):91-8. https://ncmbjpiau.ir/browse.php?a_id=1146&sid=1&slc_lang=en
24. Gezici S. Promising anticancer activity of lavender (Lavandula angustifolia Mill.) essential oil through induction of both apoptosis and necrosis. Ann Phytomed. 2018; 7(2):38-45. [DOI:10.21276/ap.2018.7.2.5]
25. Ziaei Hezarjaribi H, Nadeali N, Saeedi M, Soosaraei M, Jorjani ON, Momeni Z, et al. [The effect of lavender essential oil and nanoemulsion on Trichomonas vaginalis in vitro (Persian)]. Feyz. 2017; 21(4):326-34. http://feyz.kaums.ac.ir/article-1-3154-en.html
26. Giovannini D, Gismondi A, Basso A, Canuti L, Braglia R, Canini A, et al. Lavandula angustifolia mll. Essential oil exerts antibacterial and anti-inflammatory effect in macrophage mediated immune response to staphylococcus aureus. Immunol Invest. 2016; 45(1):11-28. [DOI:10.3109/08820139.2015.1085392] [PMID]
27. Masoomi VO, Tajik H, Moradi M, Forough M, Shahabi N. [Antimicrobial effects of Zataria multiflora boiss. essential oil nanoemulsion against Escherichia coli O157: H7 (Persian)]. Stud Med Sci. 2016; 27(7):608-17. http://umj.umsu.ac.ir/browse.php?a_code=A-10-2520-1&sid=1&slc_lang=en
28. Sabbioni C, Ferranti A, Bugamelli F, Forti GC, Raggi MA. Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products. Phytochem Anal. 2006; 17(1):25-31. [DOI:10.1002/pca.877] [PMID] [PMCID]
29. Bradley PR. British herbal compendium: A handbook of scientific information on widely used plant drugs. United Kingdom: British Herbal Medicine Association; 1992. https://books.google.com/books/about/British_Herbal_Compendium.html?id=lrUFBAAACAAJ