Anticandidal and Antibiofilm Activity of the Essential Oils from the Leaves of Citrus limon and Citrus sinensis

Abstract

Candida albicans is an opportunistic pathogen whose virulence factors, such as polymorphism and biofilm formation, facilitate infection. Due to rising antifungal resistance, essential oils (EOs) from Citrus limon and Citrus sinensis are being investigated for their antimicrobial potential. This study analyzes the chemical composition, antifungal activity against Candida albicans in planktonic and biofilm forms, cytotoxicity on human erythrocytes, and pharmacokinetic properties of Citrus limon and Citrus sinensis essential oils. The essential oils were extracted from the leaves by hydrodistillation, and their composition was analyzed by GC-MS. Antifungal activity was assessed using broth microdilution and time-kill kinetics. Combined effects were assessed using checkerboard assays, while antibiofilm tests were evaluated using polystyrene plate assays and microscopy. Hemolytic activity tests were performed using percentage hemolysis. C. limon essential oil had limonene 40.8%, β-pinene 16.5%, and citronellal 12.3%, while C. sinensis had limonene 36.7%, β-pinene 14.6%, and eucalyptol 10.5% as its main constituents. The MIC was 0.312 – 1.25 mg/mL, with kill kinetics of up to 10 h. It showed additive effects for C. limon/fluconazole and synergistic effects for C. sinensis/fluconazole. It exhibited anti-adhesion and anti-polymorphism effects, inhibiting biofilm formation. It also exhibited low cytotoxicity, with an LC₅₀ of 14 000 µg/mL for C. limon and 13 000 µg/mL for C. sinensis. ADMET and BOILED-egg pharmacokinetic predictions indicated safety for systemic use, with caution for topical application of limonene, citronellal, and eucalyptol. Both EOs exhibited strong anti-Candida activity with minimal cytotoxicity. Its additive/synergistic effects with fluconazole highlight its potential as an alternative or adjuvant therapy against C. albicans infections.

Keywords

Candida albicans - virulence - antifungal activity - Citrus limon - Citrus sinensis - Rutaceae

Publication History

Received: 17 August 2025

Accepted after revision: 03 February 2026

Article published online:
02 March 2026

• References

• 1 Reinhardt LC, Nascente PS, Ribeiro JS, Guimarães VBS, Etges A, Lund RG. Sensitivity to antifungals by Candida spp samples isolated from cases of chronic atrophic candidiasis (CAC). Braz J Biol 2019; 80: 266-272
• 2 Xiao M, Chen SC, Kong F, Xu XL, Yan L, Kong HS, Fan X, Hou X, Cheng JW, Zhou ML, Li Y, Yu SY, Huang JJ, Zhang G, Yang Y, Zhang JJ, Duan SM, Kang W, Wang H, Xu YC. Distribution and antifungal susceptibility of Candida species causing candidemia in China: An update from the CHIF-NET study. J Infect Dis 2020; 221: 139-147
• 3 Kadosh D. Regulatory mechanisms controlling morphology and pathogenesis in Candida albicans . Curr Opin Microbiol 2019; 52: 27-34
• 4 Abdulghani M, Iram R, Chidrawar P, Bhosle K, Kazi R, Patil R, Kharat K, Zore G. Proteomic profile of Candida albicans biofilm. J Proteomics 2022; 265: 104661
• 5 Vitiello A, Ferrara F, Boccellino M, Ponzo A, Cimmino C, Comberiati E, Zovi A, Clemente S, Sabbatucci M. Antifungal drug resistance: An emergent health threat. Biomedicines 2023; 11: 1063
• 6 Carmo A, Rocha M, Pereirinha P, Tomé R, Costa E. Antifungals: From pharmacokinetics to clinical practice. Antibiotics 2023; 12: 884
• 7 Sánchez-Villacreses EM, Tapia JC, Cáceres-Valdiviezo MJ, Morey-León G, Fernández-Cadena JC, Andrade-Molina DM. Basal expression of ERG11, MDR1 and CDR1 genes in clinical isolates of Candida albicans from Ecuador: insights into azole resistance surveillance. All Life 2025; 18: 2551757
• 8 Hon KLE, Chan VP, Leung AK, Leung KKY, Hui WF. Invasive fungal infections in critically ill children: Epidemiology, risk factors and antifungal drugs. Drugs Context 2024; 13: 2023-2029
• 9 Iqbal M, Parveen R, Parveen A, Parveen B, Aref IM. Establishing the botanical identity of plant drugs based on their active ingredients under diverse growth conditions. J Environ Biol 2018; 39: 123-136
• 10 Klimek-Szczykutowicz M, Szopa A, Ekiert H. Citrus limon (Lemon) phenomenon–A review of the chemistry, pharmacological properties, applications in the modern pharmaceutical, food, and cosmetics industries, and biotechnological studies. Plants 2020; 9: 119
• 11 Favela-Hernández JMJ, González-Santiago O, Ramírez-Cabrera MA, Esquivel-Ferriño PC, Camacho-Corona MDR. Chemistry and pharmacology of Citrus sinensis . Molecules 2016; 21: 247
• 12 Dosoky NS, Setzer WN. Biological activities and safety of Citrus spp. essential oils. Int J Mol Sci 2022; 23: 5895
• 13 Manzur M, Luciardi MC, Blázquez MA, Alberto MR, Cartagena E, Arena ME. Citrus sinensis essential oils an innovative antioxidant and antipathogenic dual strategy in food preservation against Spoliage bacteria. Antioxidants 2023; 12: 246
• 14 Hamdan M, Jaradat N, Al-Maharik N, Ismail S, Qadi M. Chemical composition, cytotoxic effects and antimicrobial activity of combined essential oils from Citrus meyeri, Citrus paradise, and Citrus sinensis leaves. Ind Crops Prod 2024; 210: 118096
• 15 Roos VC, Denardi LB, Teixeira CA, Machado VS, Verdi CM, Dos Santos ID, Wagner R, Sagrillo MR, Pacheco PS, Gindri AL, Santos RCV. Chemical composition, antifungal activity, antibiofilm and citotoxicity of the essential oil of Citrus deliciosa tenore. Nat Prod Res 2024; 38: 4059-4064
• 16 Chaudhary PK, Saini D, Srivastava AK, Prasad R. Assessment of antifungal and anti-biofilm potential of essential oil active constituents alone and in combinatorial mode of limonene and linalool against Candida albicans and Candida tropicalis . Nat Prod Res 2025; 39: 1-6
• 17 Nidhi P, Rolta R, Kumar V, Dev K, Sourirajan A. Synergistic potential of Citrus aurantium L. essential oil with antibiotics against Candida albicans . J Ethnopharmacol 2020; 262: 113135
• 18 Ambrosio CMS, Diaz-Arenas GL, Agudelo LPA, Stashenko E, Contreras-Castillo CJ, da Gloria EM. Chemical composition and antibacterial and antioxidant activity of a Citrus essential oil and its fractions. Molecules 2021; 26: 2888
• 19 Fokas R, Giormezis N, Vantarakis A. Synergistic approaches to foodborne pathogen control: A narrative review of essential oils and bacteriophages. Foods 2025; 14: 1508
• 20 Thakre A, Zore G, Kodgire S, Kazi R, Mulange S, Patil R, Shelar A, Santhakumari B, Kulkarni M, Kharat K, Karuppayil SM. Limonene inhibits Candida albicans growth by inducing apoptosis. Med Mycol 2018; 56: 565-578
• 21 Lu H, Shrivastava M, Whiteway M, Jiang Y. Candida albicans targets that potentially synergize with fluconazole. Crit Rev Microbiol 2021; 47: 323-337
• 22 Sobel JD. Resistance to fluconazole of Candida albicans in vaginal isolates: A 10-year study in a clinical referral center. Antimicrob Agents Chemother 2023; 67: e0018123
• 23 Vitale RG. Role of antifungal combinations in difficult to treat Candida infections. J Fungi 2021; 7: 731
• 24 Ahmedi S, Khan I, Ghanem S, Manzoor N. Limonene synergistically augments fluconazole susceptibility in clinical Candida isolates from cleft lip and palate patients. Natl J Maxillofac Surg 2024; 15: 47-54
• 25 Soulaimani B, Varoni E, Iriti M, Mezrioui NE, Hassani L, Abbad A. Synergistic anticandidal effects of six essential oils in combination with fluconazole or amphotericin B against four clinically isolated Candida strains. Antibiotics 2021; 10: 1049
• 26 Soulaimani B, Nafis A, Kasrati A, Rochdi A, Mezrioui NE, Abbad A, Hassani L. Chemical composition, antimicrobial activity and synergistic potential of essential oil from endemic Lavandula maroccana (Mill.). S Afr J Bot 2019; 125: 202-206
• 27 Nafis A, Saad FE, Khalloufi FE, Kasrati A, Abbad A, Mezrioui N, Oudra B, Vasconcelos V, Hassani L. New insight into antimicrobial activities of Linaria ventricosa essential oil and its synergetic effect with conventional antibiotics. Arch Microbiol 2021; 203: 4361-4366
• 28 Elhidar N, Nafis A, Kasrati A, Goehler A, Bohnert JA, Abbad A, Hassani L, Mezrioui NE. Chemical composition, antimicrobial activities and synergistic effects of essential oil from Senecio anteuphorbium, a Moroccan endemic plant. Ind Crops Prod 2019; 130: 310-315
• 29 Talapko J, Juzbašić M, Matijević T, Pustijanac E, Bekić S, Kotris I, Škrlec I. Candida albicans – The virulence factors and clinical manifestations of infection. J Fungi 2021; 7: 79
• 30 Kovács R, Majoros L. Antifungal lock therapy: An eternal promise or an effective alternative therapeutic approach?. Lett Appl Microbiol 2022; 74: 851-862
• 31 Atriwal T, Azeem K, Husain FM, Hussain A, Khan MN, Alajmi MF, Abid M. Mechanistic understanding of Candida albicans biofilm formation and approaches for its inhibition. Front Microbiol 2021; 12: 46-78
• 32 Ahmedi S, Pant P, Raj N, Manzoor N. Limonene inhibits virulence associated traits in Candida albicans: In-vitro and in-silico studies. Phytomedicine Plus 2022; 2: 100285
• 33 Radithia D, Tanjungsari R, Ernawati DS, Parmadiati AE. The effectiveness of essential oil from Citrus limon peel on Candida albicans biofilm formation: An experimental in vivo study. J Taibah Univ Med Sci 2023; 18: 190-195
• 34 Cavalcante CSP, Falcão CB, Fontenelle RO, Andreu D, Rádis-Baptista G. Anti-fungal activity of Ctn [15–34], the C-terminal peptide fragment of crotalicidin, a rattlesnake venom gland cathelicidin. J Antibiot (Tokyo) 2017; 70: 231-237
• 35 Sánchez-Martínez JD, Bueno M, Alvarez-Rivera G, Tudela J, Ibañez E, Cifuentes A. In vitro neuroprotective potential of terpenes from industrial orange juice by-products. Food Funct 2021; 12: 302-314
• 36 Niu Z, Xiao X, Wu W, Cai Q, Jiang Y, Jin W, Wang M, Yang G, Kong L, Jin X, Yang G, Chen H. PharmaBench: Enhancing ADMET benchmarks with large language models. Scientific Data 2024; 11: 985
• 37 Adams RP. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. 5th online ed. Gruver, TX, USA: Texensis Publishing 2017; 5: 46-52
• 38 Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard–Third Edition. CLSI document M27-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2008: 3
• 39 Fontenelle ROS, Morais SM, Brito EHS, Kerntopf MR, Brilhante RSN, Cordeiro RA, Tomé AR, Queiroz MGR, Nascimento NRF, Sidrim JJC, Rocha MFG. Chemical composition, toxicological aspects and antifungal activity of essential oil from Lippia sidoides Cham. J Antimicrob Chemother 2007; 59: 934-940
• 40 Kwolek-Mirek M, Zadrag-Tecza R. Comparison of methods used for assessing the viability and vitality of yeast cells. FEMS Yeast Res 2014; 14: 1068-1079
• 41 Johnson MD, Macdougall C, Ostrosky-Zeichner L, Perfect JH. Combination antifungal therapy. Antimicrob Agents Chemother 2004; 48: 693-715
• 42 Pierce CG, Uppuluri P, Tristan AR, Wormley jr. FL, Mowat E. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 2008; 3: 1494-1500
• 43 Fu Y, Wang W, Zeng Q, Wang T, Qian W. Antibiofilm efficacy of luteolin against single and dual species of Candida albicans and Enterococcus faecalis . Front Microbiol 2021; 12: 715156
• 44 Hajlaoui H, Arraouadi S, Noumi E, Aouadi K, Adnan M, Khan MA, Kadri A, Snoussi M. Antimicrobial, antioxidant, anti-acetylcholinesterase, antidiabetic, and pharmacokinetic properties of Carum carvi L. and Coriandrum sativum L. essential oils alone and in combination. Molecules 2021; 26: 3625
• 45 Rauf A, Khan H, Khan M, Abusharha A, Serdaroğlu G, Daglia M. In silico, SwissADME, and DFT studies of newly synthesized oxindole derivatives followed by antioxidant studies. J Chem 2023; 2023: 5553913