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Additive with a tendency to synergy interaction between Citrus aurantium essential oil and fluconazole against Aspergillus niger
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Zakład Medycyny Pracy, Katedra i Zakład Patofizjologii, Uniwersytet Medyczny w Lublinie, Polska
Paula Wróblewska-Łuczka   

Zakład Medycyny Pracy, Katedra i Zakład Patofizjologii, Uniwersytet Medyczny w Lublinie, Jaczewskiego 8b, 20-090, Lublin, Polska
Med Srod. 2022;25(1-2):8–13
Introduction and objective:
Aspergillus niger is a mould that plays an important role in agriculture and the food industry as a plant infectious agent and food contaminant. A. niger strains can release mycotoxins that are harmful to health or cause the serious human disease – aspergillosis. Treatment of fungal infections is associated with the use of azoles, but drug resistance is increasingly observed. The solution to break drug resistance may be citrus essential oils, which show a strong antimicrobial effect. The aim of the study was to combine fluconazole with the essential oil of Citrus aurantium and to evaluate the nature of the pharmacodynamic interaction between them in in vitro studies against Aspergillus niger.

Material and methods:
The experiments were carried out using the plate culture method, assessing the zones of growth inhibition of Aspergillus niger under the influence of various doses of fluconazole, orange essential oil, and combinations of the compounds. Isobolographic analysis allowed assessment of the character of interactions between the tested compounds.

The tested compounds inhibited growth of A. niger in a concentration dependent manner. The IC50 value of fluconazole against A. niger is IC50 = 2.02 ± 0.79 mg/ml and the IC50 of orange essential oil = 3.78 ± 0.48%. Isobolographic analysis showed that the combination of orange essential oil and fluconazole at a fixed 1: 1 dose ratio had an additive interaction with a tendency to synergism in plate tests for Aspergillus niger.

Essential oils are natural compounds with great therapeutic potential, and isobolographic analysis may contribute to the introduction of natural substances to the treatment of drug-resistant infections.

Park HS, Jun SC, Han KH, et al. Diversity, application, and synthetic biology of industrially important Aspergillus fungi. Adv Appl Microbiol. 2017; 100: 161–202. doi: 10.1016/bs.aambs.2017.03.001.
Parshikov IA, Woodling KA, Sutherland JB. Biotransformations of organic compounds mediated by cultures of Aspergillus niger. Appl Microbiol Biotechnol. 2015; 99(17): 6971–6986. doi: 10.1007/s00253-015-6765-0.
Abarca ML, Accensi F, Cano J, et al. Taxonomy and significance of black Aspergilli. Antonie Van Leeuwenhoek. 2004; 86(1):33–49. doi:10.1023/B:ANTO.0000024907.85688.05.
Schils R, Altdorfer A, Moerman F, et al. A rare case of invasive pulmonary aspergillosis presenting as organizing pneumonia due to Aspergillus niger in an immunocompetent host. Respir Med Case Rep. 2021; 34: 101503. doi: 10.1016/j.rmcr.2021.101503.
Workum JD, de Jong SW, Gresnigt MS, et al. Microbiological and immunological characteristics of a lethal pulmonary Aspergillus niger infection in a non-neutropenic patient. Med Mycol Case Rep. 2018; 21: 4–7. doi: 10.1016/j.mmcr.2018.03.002.
Vermeulen E, Maertens J, Meersseman P, et al. Invasive Aspergillus niger complex infections in a Belgian tertiary care hospital. Clin Microbiol Infect. 2014; 20(5): O333–5. doi: 10.1111/1469-0691.12394.
Bitar D, Lortholary O, Le Strat Y, et al. Population-based analysis of invasive fungal infections, France, 2001–2010. Emerg Infect Dis. 2014; 20(7): 1149–55. doi: 10.3201/eid2007.140087.
Okamura K, Noro R, Fujita K, et al. Acute respiratory failure due to Aspergillus niger infection with acute fibrinous and organazing pneumonia: A case report. Respir Med Case Rep. 2022; 37: 101641. doi: 10.1016/j.rmcr.2022.101641.
Watt K, Manzoni P, Cohen-Wolkowiez M, et al. Triazole use in the nursery: fluconazole, voriconazole, posaconazole, and ravuconazole. Curr Drug Metab. 2013; 14(2):193–202.
Szymańska M, Baranowski A, Płachta D. Przegląd preparatów naj-częściej stosowanych w leczeniu chorób grzybiczych. Biul Wydz Farm AMW. 2007;1:1–12.
Malhotra S, Singh S, Rana N, et al. Chemoenzymatic Synthesis, Nanotization, and Anti-Aspergillus Activity of Optically Enriched Fluconazole Analogues. Antimicrob Agents Chemother. 2017; 61(8): e00273–17. doi: 10.1128/AAC.00273-17.
Pérez-Cantero A, López-Fernández L, Guarro J, et al. Azole resistance mechanisms in Aspergillus: update and recent advances. Int J Antimicrob Agents. 2020; 55(1): 105807. doi: 10.1016/j.ijantimicag.2019.09.011.
Restuccia C, Oliveri Conti G, Zuccarello P, et al. Efficacy of different citrus essential oils to inhibit the growth and B1 aflatoxin biosynthesis of Aspergillus flavus. Environ Sci Pollut Res Int. 2019; 26(30): 31263–31272. doi: 10.1007/s11356-019-06169-9.
Wróblewska-Łuczka P. Prozdrowotne działanie olejków eterycznych cytrusów. Med Srod. 2019; 22(3–4):39–43. doi:10.26444/ms/134758.
Abd Rashed A, Rathi DG, Ahmad Nasir NAH, et al. Antifungal Properties of Essential Oils and Their Compounds for Application in Skin Fungal Infections: Conventional and Nonconventional Approaches. Molecules. 2021; 26(4): 1093. doi: 10.3390/molecules26041093.
Wróblewska-Łuczka P, Łuszczki J. Additivity interactions between fluconazole and citrus essential oils to Aspergillus fumigatus. J Pre-Clin Clin Res. 2021; 15(3): 116–120. doi: 10.26444/jpccr/140077.
Ben Hsouna A, Hamdi N, Ben Halima N, et al. Characterization of essential oil from Citrus aurantium L. flowers: antimicrobial and antioxidant activities. J Oleo Sci. 2013; 62(10): 763–72. doi: 10.5650/jos.62.763.
Oikeh EI, Ayevbuomwan M, Irabor F, et al. Evaluation of the Phenolic Content, Antioxidant and Antimicrobial Activities of Oil and Non-Oil Extracts of Citrus sinensis (L.) Osbeck Seeds. Prev Nutr Food Sci. 2020; 25(3): 280–285. doi: 10.3746/pnf.2020.25.3.280.
Suntar I, Khan H, Patel S, et al. An Overview on Citrus aurantium L.: Its Functions as Food Ingredient and Therapeutic Agent. Oxid. Med. Cell. Longev. 2018; 7864269. doi:
Malawski S, Kot N, Szefler S. Growing citrus plants in Europe in the light of historic treatises on horticulture. Agronomy Science. 2021; 76(2): 51–75.
Krzysztofik B, Dróżdż T, Sobol Z, et al. Metody zabezpieczania i utrwalania surowców oraz produktów spożywczych – studium przypadku. Kraków: Wyd. Polskie Towarzystwo Inżynierii Rolniczej; 2015.
Aloui H, Khwaldia K, Licciardello F, et al. Efficacy of the combined application of chitosan and Locust Bean Gum with different citrus essential oils to control postharvest spoilage caused by Aspergillus flavus in dates. Int J Food Microbiol. 2014; 170:21–28. doi:
Budzyńska A, Więckowska-Szakiel M, Kalemba D, et al. The optimization of methods utilized for testing the antibacterial activity of essential oils. Med Dośw Mikrobiol. 2009; 61: 281–287.
Litchfield JT Jr, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther. 1949;96:99–113.
Łuszczki JJ, Mazurkiewicz LP, Wroblewska-Luczka P, et al. Combination of phenobarbital with phenytoin and pregabalin produces synergy in the mouse tonic-clonic seizure model: An isobolographic analysis. Epilepsy Res. 2018; 145:116–122. doi: 10.1016/j.eplepsyres.2018.06.003.
Łuszczki JJ. Isobolographic analysis of interaction between drugs with nonparallel dose-response relationship curves: a practical application. Naunyn Schmiedebergs Arch Pharmacol. 2007; 375:105–114.
Grabarska A, Wróblewska-Łuczka P, Kukula-Koch W, et al. Palmatine, a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica, Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin. Molecules. 2021; 26: 6253.
Wróblewska-Łuczka P, Grabarska A, Florek-Łuszczki M, et al. Synergy, Additivity, and Antagonism between Cisplatin and Selected Coumarins in Human Melanoma Cells. Int. J. Mol. Sci. 2021; 22: 537.
Wróblewska-Łuczka P. Isobolographic in vitro interactions of fluconazole with citrus essential oils against Cladosporium cladosporioides. J Pre-Clin Clin Res. 2021; 15(1): 15–19. doi: 10.26444/jpccr/132014.
Elefanti A, Mouton JW, Verweij PE, et al. Amphotericin B- and voriconazole-echinocandin combinations against Aspergillus spp.: Effect of serum on inhibitory and fungicidal interactions. Antimicrob Agents Chemother. 2013; 57(10):4656–4663. doi: 10.1128/AAC.00597-13.
Ferro BE, Meletiadis J, Wattenberg M, et al. Clofazimine prevents the regrowth of Mycobacterium abscessus and Mycobacterium avium type strains exposed to amikacin and clarithromycin. Antimicrob Agents Chemother. 2015; 60(2):1097–1105. doi: 10.1128/AAC.02615-15.
Riaz T, Abbasi MA, Rehman A, et al. Enzyme inhibitory, antifungal, antibacterial and hemolytic potential of various fractions of Colebrookia oppositifolia. Pak J Pharm Sci. 2017; 30(1):105–112.
Łukowska-Chojnacka E, Mierzejewska J, Milner-Krawczyk M, et al. Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity. Bioorg Med Chem. 2016; 24(22):6058–6065. doi: 10.1016/j.bmc.2016.09.066.
Kauthale S, Tekale S, Damale M, et al. Synthesis, antioxidant, antifungal, molecular docking and ADMET studies of some thiazolyl hydrazones. Bioorg Med Chem Lett. 2017; 27(16):3891–3896. doi: 10.1016/j.bmcl.2017.06.043.
Kumar S, Lim SM, Ramasamy K, et al. Bis-pyrimidine acetamides: design, synthesis and biological evaluation. Chem Cent J. 2017; 11(1):80.
Sun N, Li D, Zhang Y, et al. Repurposing an inhibitor of ribosomal biogenesis with broad anti-fungal activity. Sci Rep. 2017; 7(1):17014. doi: 10.1038/s41598-017-17147-x.
Wu J, Ni T, Chai X, et al. Molecular docking, design, synthesis and antifungal activity study of novel triazole derivatives. Eur J Med Chem. 2018; 143:1840–1846. doi: 10.1016/j.ejmech.2017.10.081.
Łuszczki JJ, Gustaw-Rothenberg K, Chmielewski J, et al. Perspektywy zastosowania leków ziołowych w związku z postępującym zanieczyszczeniem środowiska. Med Srod. 2019; 22(1–2):5–8. doi:10.26444/ms/117884.
Cisneros-Zevallos L. The power of plants: how fruit and vegetables work as source of nutraceuticals and supplements. Int J Food Sci Nutr. 2021; 72(5): 660–664. doi: 10.1080/09637486.2020.1852194.
Sachdeva V, Roy A, Bharadvaja N. Current Prospects of Nutraceuticals: A Review. Curr Pharm Biotechnol. 2020; 21(10): 884–896. doi: 10.2174/1389201021666200130113441.
Adaszyńska M, Swarcewicz M, Markowska-Szczupak A. Porównanie składu chemicznego i aktywności przeciwdrobnoustrojowej olejku eterycznego otrzymanego z różnych krajowych odmian lawendy wąskolistnej (Lavandula angustifolia L.). Post Fitoter. 2013; 2:90–96.
Tariq S, Wani S, Rasool W, et al. A comprehensive review of the antibacterial, antifungal and antiviral potential of essential oils and their chemical constituents against drug-resistant microbial pathogens. Microb Pathog. 2019; 134: 103580. doi: 10.1016/j.micpath.2019.103580.
Mahato N, Sharma K, Koteswararao R, et al. Citrus essential oils: extraction, authentication and application in food preservation. Crit Rev Food Sci Nutr. 2019; 59: 611–625.
Phi NT, van Hung P, Chi PT, et al. Impact of growth locations and genotypes on antioxidant and antimicrobial activities of citrus essential oils in Vietnam. J Essent Oil-Bear Plants. 2015; 18: 1421– 1432.
Sheikh M, Mehnaz S, Sadiq MB. Prevalence of fungi in fresh tomatoes and their control by chitosan and sweet orange (Citrus sinensis) peel essential oil coating. J Sci Food Agric. 2021; 101(15): 6248–6257. doi: 10.1002/jsfa.11291.
Sharma N, Tripathi A. Effects of Citrus sinensis (L.) Osbeck epicarp essential oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem. Microbiol Res. 2008; 163(3): 337–44. doi: 10.1016/j.mic-res.2006.06.009.
Velázquez-Nunez MJ, Avila-Sosa R, Palou E, et al. Antifungal activity of orange (Citrus sinensis var. Valencia) peel essential oil applied by direct addition or vapor contact. Food Control. 2013; 31: 1– 4.
Gniewosz M, Kraśniewska K, Kosakowska O, et al. Chemical compounds and antimicrobial activity of petitgrain (Citrus aurantium L. var. amara) essential oil. Herba Polonica. 2017; 63(4): 18–25.
Nidhi P, Rolta R, Kumar V, et al. Synergistic potential of Citrus aurantium L. essential oil with antibiotics against Candida albicans. J Ethnopharmacol. 2020; 262: 113135. doi: 10.1016/j.jep.2020.113135.