1. INTRODUCTION
Oral candidiasis, primarily caused by Candida albicans, is a prevalent fungal infection of the oral mucosa. Colonization rates vary from 30% to 50% in healthy individuals to 50%–65% in denture wearers, 65%–88% in long-term care patients, and 90%–95% in individuals with HIV or those undergoing corticosteroid, chemotherapy, immunosuppressive, or radiation therapy for head and neck cancers [1]. A study at Dr. Soetomo Hospital, Surabaya, reported that 47% of HIV/AIDS patients presented with oral pseudomembranous candidiasis [2]. Immunosuppression compromises host defense mechanisms, increasing susceptibility to C. albicans infections [3–5]. Prolonged glucocorticoid use, such as dexamethasone, exacerbates immunosuppression by inhibiting T-cell activation and reducing B-cell stimulation [6–8].
Current antifungal treatments, including azoles (clotrimazole, fluconazole, itraconazole, and ketoconazole) and polyenes (amphotericin B, nystatin), present challenges due to host toxicity and rising drug resistance. Surveillance data indicate an increase in antifungal resistance from 4.2% in 2008 to 7.8% in 2014, with C. albicans exhibiting resistance rates as high as 56.7% [9]. These concerns emphasize the need to develop safer and more effective antifungal alternatives, including natural compounds such as phenolics, essential oils, and botanical extracts [10]. Citrus limon essential oil, rich in limonene, α-pinene, and γ-terpinene, has demonstrated antifungal properties [11,12]. Prior studies indicate that a 0.78% concentration of C. limon essential oil gel effectively disrupts C. albicans biofilm formation [13,14] and has been evaluated for its toxicity profile and minimum inhibitory concentration (MIC) [15,16]. More recently, C. limon essential oil exhibited a MIC of 0.12% (v/v) against C. albicans ATCC 10231 in vitro [17] and an experimental oral candidiasis model reported enhanced β defensin expression following topical C. limon gel treatment [18], further supporting its antifungal and immunomodulatory potential.
This study employs a standardized oral candidiasis model in immunosuppressed Rattus norvegicus [19] to investigate the clinical, microbiological, and immunological effects of topical C. limon essential oil gel. The hypothesis is C. limon application reduces fungal colonization, inhibits hyphal formation, and modulates local immune responses in infected oral tissues.
2. MATERIALS AND METHODS
2.1. Ethical statement
The study protocol was approved by the Animal Care and Use Committee of the Health Research Ethical Clearance Commission, Faculty of Dental Medicine, Universitas Airlangga, Indonesia (Approval No.: 416/HRECC.FODM/IX/2020).
2.2. Experimental animals
Male Wistar rats (Rattus norvegicus), 3 months old and weighing 200–300 g, were used in this study. All animals were clinically healthy and acclimatized for 7 days prior to experimentation. During the experiment, rats were housed in standard polycarbonate cages (3 rats per cage) with wood shavings bedding under controlled laboratory conditions. The environment was maintained at 22°C ± 2°C with 50% ± 10% relative humidity and a 12 hours light/12 hour dark cycle. Animals were fed with a standard diet based on body weight and water ad libitum, and cages were cleaned regularly to ensure hygienic maintenance. Following inoculation with C. albicans, animals were randomly allocated into three groups (n = 10 each): disease control group (infected untreated control), positive control group (miconazole gel-treated), and experimental group (C. limon essential oil gel-treated).
The sample size was determined using Federer’s formula for experimental studies, where (t–1)(n–1) ≥ 15, with t representing the number of groups and n the number of animals per group [20]. With three groups, 10 rats per group were included, yielding a total of 30 animals. All procedures were conducted using sterile instruments and in compliance with the Guide for the Care and Use of Laboratory Animals.
2.3. Preparation of Citrus limon essential oil gel
Citrus limon from BALIJRESTO, Malang City, East Java, Indonesia, was distilled to extract its peel essential oil. 100% C. limon essential oil was added with 3% Carboxyl methylcellulose (CMC) (Bioworld, China) to obtain 0.78% C. limon essential oil gel [21].
2.4. Gas chromatography-mass spectrometry (GC-MS) of Citrus limon essential oil
Essential oil C. limon was examined with GC-MS for 60 minutes at 260oC, detector 250oC, and column 325oC. The carrier gas used was helium at a constant flow rate of 1 ml/minute. Compound identification was performed by comparing the obtained mass spectra with the NIST14.L mass spectral library (National Institute of Standards and Technology, [22]). A library match quality score was automatically generated for each peak, and only compounds with match scores ≥ 80% were considered reliably identified. Relative abundance was expressed as a percentage of peak area relative to the total ion chromatogram [23,24].
2.5. Induction and assessment of immunosuppressed condition
Each group of experimental animals received an intramuscular injection of 7.2 mg/kg dexamethasone (Mepro Farma Co., Jakarta, Indonesia) for 5 days to induce immunosuppression. Immunosuppression was characterized by a leukocyte count from the tail vein blood falling two standard deviations below normal, accompanied by body weight loss in the rats [19,25,26].
2.6. Induction and assessment of oral candidiasis
Inoculation was done by applying 0.2 ml of C. albicans suspension (9 x 10[8] viable cells/ml in Sabouraud Dextrose Broth, SDB; Cat. No. M033F-500G, Himedia) on the dorsal tongue and palatal mucosa after general anesthesia with intramuscular ketamine 10% (0.1–0.2 ml/kg body weight). Clinically, the tongue was observed for erythema, atrophy, and pseudomembrane formation. For microbiological confirmation, the tongue surface was swabbed and the samples serially diluted up to 10?? in SDB before plating 0.05 ml onto Sabouraud Dextrose Agar (SDA; Cat. No. M063-500G, HiMedia; supplemented with 40 mg/ml chloramphenicol). Plates were incubated anaerobically at 37°C for 48 hours, and colonies with typical C. albicans morphology were counted as Colony-forming unit (CFU). Gram staining of SDA colonies was also performed to confirm candidal hyphae, pseudohyphae, or yeast forms under oil immersion microscopy (1,000×)[19].
2.7. Treatments
The study included three groups. Treatment consisted of the topical application of either miconazole oral gel (Taisho Pharmaceutical, Indonesia) or 0.78% C. limon essential oil gel, applied to the affected tongue mucosa using a microbrush. Applications were administered twice daily at 12-hour intervals for 4 days, while the control group remained untreated. A specimen of tongue was taken to examine the macrophage and TNF-α expressions. The experimental timeline is illustrated in Figure 1.
 | Figure 1. Timeline of experiment procedures showing the schedule of immunosuppression induction, C. albicans inoculation, treatment, and clinical assessments in Rattus norvegicus. [Click here to view] |
2.8. Oral candidiasis tissue preparation
After all the groups had been treated for 4 days, the candida-affected areas of the tongue were excised and fixed in 10% neutral buffered formalin (pH 7.0) for 18–24 hour. The specimens were then rinsed in distilled water, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin blocks (melting point 56°C–58°C). Sections of 4 µm thickness were cut using a rotary microtome and mounted on poly-L-lysine–coated glass slides. The slides were incubated at 56°C–58°C to ensure proper adhesion before hematoxylin–eosin (H&E) staining and immunohistochemical staining [27].
2.9. Macrophages count analysis
A histological assessment was carried out by doing hematoxylin-eosin staining (Hematoxylin staining, C.I. 75290, Merck Germany; and Eosin staining, C.I. 45380, Merck Germany) and counting macrophages using a Nikon Eclipse E100 with 400x magnification [28] (Fig. 2).
 | Figure 2. Clinical appearance of the tongue in immunosuppressed Rattus norvegicus before and after treatment: (a) Untreated group: persistent erythema; (b) Miconazole-treated group: evenly pink coloration; (c) C. limon essential oil gel-treated: showing resolution of erythema and absence of pseudomembrane. [Click here to view] |
2.10. TNF-α expression analysis
TNF-α (1:200; monoclonal; antibody online) was used as the primary antibodies with 3,3′-diaminobenzidine tetrahydrochloride as the chromogen for visualization (Bios, USA Kit) [29].
2.11. Statistical analysis
Data were analyzed using Statistical Package for the Social Sciences software version 10.05 (SPSS Inc., Chicago, IL, USA, 1999). Analysis was performed using the Kolmogorov-Smirnov test for data distribution and The Levene test for data homogeneity. CFU counting was performed with a non-parametric test using Mann-Whitney (p < 0.05). Clinical examination and Gram staining results were performed by a non-parametric test using the McNemar test with a significance number of p < 0.05. Assessment of the difference between macrophage count and TNF-α expression between each group was performed using Kruskal–Wallis (p < 0.05). A significance threshold of α = 0.05 (two-tailed) was adopted. Exact p-values are reported, and values displayed as 0.000 by SPSS are presented as p < 0.001.
3. RESULTS AND DISCUSSION
3.1. Antifungal effect of bioactive compound detected in Citrus limon L. fruit peel essential oil
Gas chromatography–mass spectrometry analysis identified approximately 42 compounds in C. limon essential oil, with d-limonene being the most abundant (13.1%) (Table 1). The 10 most abundant compounds are shown in Table 1, while the complete list of 42 identified compounds and the GC-MS chromatogram are provided as supplementary material. In C. albicans, d-limonene forms stable complexes with key proteins involved in biofilm formation, including Tec1, Als3, Phospholipase B1 (Plb1), Bcr1, and Sap2. Tec1 exhibited the highest binding affinity, stabilized by an extensive van der Waals network, leading to a snug-fit conformation that may inhibit its role in biofilm development. Tec1 regulates filamentation by forming a complex with Ste12 and its inhibitors, Dig1 and Dig2, which bind to TCS motifs in the promoters of filamentation-related genes [30,31].
Table 1. The ten most abundant compounds detected in Citrus limon essential oil by gas chromatography–mass spectrometry (GC–MS). Compounds were identified by comparison with the NIST14.L mass spectral library (National Institute of Standards and Technology, [22]), with match quality scores ≥ 80% considered reliable. Relative percentage was calculated from the peak area in the total ion chromatogram.
| No | Chemical Compound | Qual | % Area |
|---|---|---|---|
| 1 | D-Limonene | 97 | 13.1% |
| 2 | Linalool | 96 | 3% |
| 3 | Citral (Z)- | 64 | 2.28% |
| 4 | 2-Cyclohexen-1-ol, 2-methyl-5-(1-methylethenyl)-, cis- | 96 | 2.25% |
| 5 | 5-Hepten-2-one, 6-methyl- | 81 | 1.9% |
| 6 | Citral | 90 | 1.9% |
| 7 | Citral (E)- | 93 | 1.85% |
| 8 | 2-Carene | 70 | 1.7% |
| 9 | D-Carvone | 97 | 1.7% |
| 10 | cis-p-Mentha-2,8-dien-1-ol | 94 | 1.45% |
Bcr1, a conserved fungal transcription factor, plays a critical role in biofilm formation. Its targets include adhesins and cell-wall proteins (ALS1, ALS3, HWP1, and RBT5), signifying its involvement in early adhesion. Secreted aspartyl proteinases serve as major virulence factors, facilitating C. albicans adhesion to tooth surfaces and degrading extracellular matrix proteins. Plb1 contributes to fungal pathogenicity and may play a role in early host invasion. The interaction of d-limonene with these proteins suggests its potential to inhibit adhesion, enzymatic activity, and biofilm formation [30,32].
These findings highlight the antifungal potential of C. limon essential oil gel in oral candidiasis, particularly in immunosuppressed R. norvegicus models. At a concentration of 0.78%, the gel demonstrated significant biofilm degradation and morphological alterations in C. albicans [13,14]. Additionally, toxicity and minimum inhibitory concentration assessments support its efficacy against C. albicans biofilms [15,16].
3.2. Reduction of Leukocyte number in Wistar rats after dexamethasone injection
Dexamethasone injection at a dosage of 7.2 mg/kg effectively induces immunosuppression in R. norvegicus [19]. As a corticosteroid, dexamethasone exhibits anti-inflammatory, antimitotic, and immunosuppressive properties, compromising host defense mechanisms and increasing susceptibility to opportunistic infections. Under these conditions, the overgrowth of commensal C. albicans facilitates its pathogenic transition, leading to oral candidiasis [33,34].
Prior to dexamethasone administration, the mean leukocyte count was 8,248.75 cells/mm³. After five consecutive days of injection, this count declined to 2,846.88 cells/mm³, representing a significant leukocyte reduction of approximately 5,402 cells/mm³. These findings confirm the immunosuppressive impact of dexamethasone in R. norvegicus, validating its efficacy as an experimental immunosuppression model [19,26].
3.3. Clinical changes on rats’ tongues post inoculation of Candida albicans
The severity of oral candidiasis in Wistar rats was assessed through clinical observation and confirmed by microbiological analysis. To evaluate the therapeutic efficacy of C. limon essential oil gel, assessments were conducted pre- and post-treatment.
Prior to therapy, all immunosuppressed subjects exhibited oral candidiasis, characterized by atrophic mucosa and localized erythematous patches, with minimal pseudomembrane formation. After four consecutive days of treatment, clinical evaluation revealed persistent erythema in 40% (4 out of 10) of rats in the untreated group. In contrast, subjects receiving miconazole or C. limon essential oil gel demonstrated normal tongue morphology, exhibiting uniform pink coloration without signs of redness or pseudomembrane formation (Fig. 2).
McNemar’s test results showed a statistically significant difference in clinical outcomes between pre- and post-treatment assessments in the untreated group (p = 0.031), the miconazole group (p = 0.002), and the C. limon essential oil gel group (p = 0.002). Despite significant improvements in all treated groups, untreated subjects continued to exhibit clinical manifestations of oral candidiasis.
These findings suggest that C. limon essential oil gel effectively mitigates inflammation in experimental animals, potentially through the upregulation of IL-10 cytokine expression, an anti-inflammatory mediator [35]. Linalool, a key compound in C. limon essential oil, has been reported to enhance IL-10 secretion in macrophages, further supporting its therapeutic potential [36,37].
3.4. Candida albicans colony count on CFU counting
Colony-forming unit counting is a reliable method for confirming oral candidiasis. Across all sample groups, C. albicans colonies appeared white to cream, smooth, glabrous, and oval to circular with irregular edges. Representative colony morphology before and after treatment is shown in Figure 3. Consistent with clinical findings, CFU counts significantly decreased following therapy in all groups: untreated (p < 0.001), miconazole (p < 0.001), and C. limon essential oil gel (p < 0.001).
 | Figure 3. Colony morphology of C. albicans isolated from tongue swabs of immunosuppressed Rattus norvegicus before and after treatment. Colonies appeared white to cream, smooth, glabrous, and oval to circular with irregular edges. After treatment, colony numbers decreased in (a) untreated group, (b) miconazole-treated group, and (c) C. limon essential oil gel-treated group. [Click here to view] |
These findings indicate that colony formation reflects C. albicans proliferation on the tongues of Wistar rats. Reductions observed in the untreated group may partly reflect natural immune rebound after dexamethasone withdrawal or the antimicrobial properties of saliva, which act as potential confounders. However, the greater magnitude of reduction in the miconazole and C. limon groups indicates that therapeutic intervention was the primary driver of improvement. Notably, application of C. limon essential oil gel resulted in complete resolution of clinical signs of candidiasis in all subjects, comparable to the miconazole-treated group, whereas four rats in the untreated group exhibited persistent erythema.
Secondary plant metabolites, including those in C. limon, exert antifungal effects through multiple mechanisms, such as increased fungal cell wall permeability, intracellular acidification, disruption of energy production and structural component synthesis, and ultimately, genetic material damage leading to fungal cell death [38].
3.5. Hyphae formation observed on direct mycology with gram staining
Microscopic observation of hyphal formation is a crucial diagnostic test for oral candidiasis. Prior to treatment, all groups exhibited hyphae cells arranged in parallel. Figure 4 summarizes leukocyte counts, CFU quantification, clinical lesion frequency, and hyphae positivity across treatment groups before and after therapy.
 | Figure 4. Quantitative analysis before and after treatment in immunosuppressed Rattus norvegicus: (A) leukocyte counts following dexamethasone-induced immunosuppression; (B) colony-forming units of C. albicans; (C) number of samples showing clinical signs of oral candidiasis; (D) number of hyphae-positive samples on direct microscopic examination. [Click here to view] |
Following 4 days of treatment, parallel-arranged hyphae were detected in 9 out of 10 samples in the untreated group (p = 1.000), 4 out of 10 in the miconazole-treated group (p = 0.031), and 6 out of 10 in the C. limon essential oil-treated group (p = 0.125). Figure 5 illustrates Gram-stained samples showing persistent hyphae in the untreated group, whereas the treated groups predominantly exhibited budding yeast forms.
 | Figure 5. Direct Gram staining of tongue after treatment: (a) disease control group shows hyphae arranged in parallel (red arrows); (b) Miconazole-treated group shows the formation of ovoid-shaped budding yeast cells; (c) C. limon essential oil gel-treated group. Budding yeast morphology predominates in treated samples. [Click here to view] |
Although the reduction in hyphal formation was not statistically as significant in the C. limon essential oil group as in the miconazole group, a decline was nonetheless observed. The decrease may be attributed to the antifungal properties of 0.78% C. limon essential oil gel, which may require extended application to achieve hyphal inhibition comparable to standard antifungal treatments. Additionally, the d-limonene concentration in the gel was 13%, whereas higher concentrations have been reported to enhance antifungal efficacy.
Previous studies suggest that d-limonene may disrupt C. albicans biofilm integrity by interacting with chitin bonds within the 1,3-β-glucan matrix, potentially destabilizing fungal cell structure and impairing biofilm formation [39]. Although this mechanism was not directly assessed in our study, it provides a plausible explanation for the antifungal effects observed.
3.6. Macrophages count
Given the lack of comprehensive, significant microbiological findings, we explored the immunomodulatory effects of C. limon essential oil in addressing candidiasis. Topical application of 0.78% C. limon essential oil gel led to a notable increase in macrophage count (5.1 ± 0.738) compared to the miconazole-treated (4.0 ± 0.816) and untreated groups (3.0 ± 0.816). Histological visualization of macrophage infiltration and TNF-α expression, as shown in Figure 6, corroborates quantitative findings from immunohistochemical and hematoxylin-eosin staining.
 | Figure 6. Histopathological sections of Rattus norvegicus tongue tissue after four days of treatment. Top row: Hematoxylin-eosin staining showing macrophage infiltration (black arrows) in (A) untreated, (B) miconazole-treated, and (C) C. limon gel-treated groups. Bottom row: Immunohistochemical staining showing TNF-α expression (black arrows) in (A) untreated, (B) miconazole-treated, and (C) C. limon gel-treated groups. Images captured at 400× magnification using Nikon Eclipse E100. [Click here to view] |
The significant macrophage elevation observed in the C. limon-treated group (p < 0.001) is likely due to the presence of d-limonene, a compound with established anti-inflammatory properties. Previous studies have suggested that d-limonene may enhance regulatory T cell (Treg) activity, thereby contributing to the balance between pro-inflammatory and anti-inflammatory cytokine responses. Although this mechanism was not directly assessed in our study, it provides a plausible explanation for the observed effects. Treg cells are known to facilitate monocyte differentiation into M2 macrophages (anti-inflammatory phenotype) while suppressing M1 macrophage activation (pro-inflammatory phenotype), thereby mitigating inflammatory progression. During M1 macrophage activation, monocytes concurrently polarize into M2 macrophages under IL-4 cytokine influence, leading to the secretion of anti-inflammatory cytokines, including IL-10. IL-10, predominantly secreted by macrophages, B cells, and T cells, inhibits M1 activation and suppresses pro-inflammatory mediators such as TNF-α, IL-1, and IL-6 [40,41]. These immunomodulatory effects, as suggested by previous studies, may act synergistically with the antifungal activity of d-limonene, thereby contributing to the overall therapeutic outcomes observed [39,42,43].
3.7. TNF- α expression
The topical application of 0.78% C. limon essential oil gel demonstrated significant anti-inflammatory effects, reducing TNF-α expression compared to the untreated group (p = 0.01). Although the reduction compared to the miconazole group (p = 0.06) did not reach statistical significance (p > 0.05), a decreasing trend was observed. The highest TNF-α expression was observed in the untreated group (3.5 ± 1.080), followed by the miconazole group (2.5 ± 0.707), while the C. limon essential oil gel group exhibited the lowest levels (1.6 ± 0.516). Figure 7 presents the average macrophage count (left) and TNF-α expression (right) across treatment groups, corroborating histological findings shown in Figure 6.
 | Figure 7. Bar graph showing the average number of macrophages (left) and the average expression of TNF-α (right) in the tongue tissues of Rattus norvegicus after four days of treatment. The groups included disease control group, positive control group, and C. limon L. essential oil gel-treated rats. The C. limon group showed the highest macrophage count and the lowest TNF-α expression. [Click here to view] |
The reduction in TNF-α expression in the C. limon-treated group is likely attributed to bioactive compounds that suppress TNF-α production by inhibiting TLR4 receptor signaling during C. albicans infections [15,34,36]. D-Limonene, a key component, exhibits anti-inflammatory properties by modulating lymphocyte counts and reducing pro-inflammatory cytokine production, including TNF-α and IL-6, through the inhibition of lymphocyte activation and proliferation.
The enhanced expression of IL-10 observed in the C. limon-treated group may be influenced by multiple immune-regulatory mechanisms. Previous studies have reported that d-limonene can suppress inflammatory responses by modulating the nuclear factor kappa B (NF-κB) pathway, a key regulator of chemokine transcription, including ligands involved in T-helper 1 and T-helper 17 (Th17) cell migration. Through this pathway, NF-κB inhibition has been associated with reduced Th17 cell infiltration and lower pro-inflammatory cytokine production [15,28,44]. In addition, flavonoids present in C. limon have been shown to suppress TNF-α expression via antioxidant activity, which mitigates reactive oxygen species generation, enhances apoptosis, and indirectly downregulates NF-κB activity [45].
The limitations of this study include the 4-day treatment duration, which may not fully capture relapse or long-term outcomes, and the absence of a vehicle-only CMC control group, which makes it difficult to completely rule out potential contributions of the excipient. However, as CMC is widely regarded as an inert vehicle without antifungal activity, the observed therapeutic effects are most likely attributable to the bioactive compounds of C. limon essential oil. Despite these considerations, the study demonstrates notable strengths, including the use of a validated immunosuppressed R. norvegicus model and the integration of clinical, microbiological, and immunological outcome measures, thereby providing robust and comprehensive evidence for the antifungal and immunomodulatory potential of C. limon essential oil gel.
4. CONCLUSION
Based on the findings of this study, C. limon essential oil gel demonstrates significant potential as a topical therapy for oral candidiasis in immunosuppressed R. norvegicus. Its combined antifungal and anti-inflammatory properties suggest its viability as a natural alternative to conventional antifungal agents. Although its efficacy in hyphal inhibition and CFU reduction was lower than that of miconazole and the treatment duration was relatively short, the observed clinical and immunological improvements support its promise. Overall, C. limon essential oil gel offers a promising therapeutic approach for managing C. albicans infections under immunosuppressed conditions.
5. ACKNOWLEDGMENTS
The authors would like to thank the Faculty of Dental Medicine, Universitas Airlangga, for their laboratory support and research facilities. Special thanks to the animal research unit and microbiology lab staff for their valuable technical assistance throughout the study.
6. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
7. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
8. FINANCIAL SUPPORT
There is no funding to report.
9. ETHICAL APPROVALS
Ethical approval details are given in the ‘Materials and Methods’ section.
10. DATA AVAILABILITY
All data generated and analyzed are included in this research article.
11. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
12. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
REFERENCES
1. Lu SY. Oral candidosis: pathophysiology and best practice for diagnosis, classification, and successful management. J Fungi. 2021;7(7):555. CrossRef
2. Radithia D, Soebadi B, Hendarti HT, Surboyo MDC, Ayuningtyas NF, Triyono EA. Dental-related problems and oral manifestation of HIV/AIDS patients in Soetomo General Hospital Surabaya. Bali Med J. 2020;9(2):537–41. CrossRef
3. Jørgensen MR. Pathophysiological microenvironments in oral candidiasis. APMIS. 2024;132(12):956–73. CrossRef
4. Vila T, Sultan AS, Montelongo-Jauregui D, Jabra-Rizk MA. Oral candidiasis: a disease of opportunity. J Fungi. 2020;6(1):1–28. CrossRef
5. Pappas PG, Kauffman CA, Andes DR, Clancy CJ, Marr KA, Ostrosky-Zeichner L, et al. Executive summary: clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases Society of America. Clin Infect Dis. 2015;62(4):409–17. CrossRef
6. Kraaij MD, Van Der Kooij SW, Reinders MEJ, Koekkoek K, Rabelink TJ, Van Kooten C, et al. Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages. Mol Immunol. 2011;49(3):549–57. CrossRef
7. Xiao JL, Xu GC, De Hoog S, Qiao JJ, Fang H, Li YL. Oral prevalence of candida species in patients undergoing systemic glucocorticoid therapy and the antifungal sensitivity of the isolates. Infect Drug Resist. 2020;13:2601–7. CrossRef
8. Couvaras L, Trijau S, Delamotte G, Pradel V, Pham T, Lafforgue P. Epidemiological description of long-term oral glucocorticoid use: results from the French health insurance database. Revue De Med Interne. 2018;39(10):777–81. CrossRef
9. Bona E, Cantamessa S, Pavan M, Novello G, Massa N, Rocchetti A, et al. Sensitivity of Candida albicans to essential oils: are they an alternative to antifungal agents?. J Appl Microbiol. 2016;121(6):1530–45. CrossRef
10. Perlin DS, Rautemaa-Richardson R, Alastruey-Izquierdo A. The global problem of antifungal resistance: prevalence, mechanisms, and management. Lancet Infect Dis. 2017;17(12):e383–392. CrossRef
11. Voo SS, Grimes HD, Lange BM. Assessing the biosynthetic capabilities of secretory glands in Citrus peel. Plant Physiol. 2012;159(1):81–94. CrossRef
12. Negri M, Salci TP, Shinobu-Mesquita CS, Capoci IRG, Svidzinski TIE, Kioshima ES. Early state research on antifungal natural products. Molecules. 2014;19(3):2925–56. CrossRef
13. 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(1):190–5. CrossRef
14. Prabajati R, Hernawan I, Hendarti HT. Effects of Citrus limon essential oil (Citrus limon L.) on cytomorphometric changes of Candida albicans. Dental J. 2017;50(1):43. CrossRef
15. Tjahyono N, Parmadianti, AE, Mahdani FY. Cytotoxicity test of Citrus limon peel essential oil in human gingival fibroblast. Oral Med Dental J. 2017;9(1):53–60. CrossRef
16. Hernawan I, Radithia D, Hadi P, Ernawati DS. Fungal inhibitory effect of Citrus Limon peel essential oil on Candida albicans. Dental J. 2015;48(2):84–8. CrossRef
17. Gharzouli M, Aouf A, Mahmoud E, Ali H, Alsulami T, Badr AN, et al. Antifungal effect of Algerian essential oil nanoemulsions to control Penicillium digitatum and Penicillium expansum in Thomson Navel oranges (Citrus sinensis L. Osbeck). Front Plant Sci. 2024;15:1–8. CrossRef
18. Femilian A. Expression of ß defensin in an experimental model of oral candidiasis treated with Citrus limon L. In AIP Conf Proc, AIP Publishing, Melville, NY, 2025. 3312(1). CrossRef
19. Al Abrori UNH, Ayuningtyas NF, Ernawati DS, Hendarti HT, Surboyo MDC, Radithia D. Oral candidiasis in immunosuppressed wistar rats (Rattus norvegicus) post dexamethasone injection at 7.2 mg/kg and 16 mg/kg doses. Braz Dent Sci. 2020;23(4):1–7. CrossRef
20. Rukmana A, Supardi LA, Sjatha F, Nurfadilah M. Responses of humoral and cellular immune mediators in BALB/c Mice to LipX (PE11) as seed tuberculosis vaccine candidates. Genes. 2022;13(11) :1954. CrossRef
21. Ganesha R, Hernawan I, Hendarti HT, Radithia D, Hadi P, Ayuningtyas NF, et al. Expression of FGF-2 and fibronectin in Citrus limon fruit peel malang essential oil gel treated traumatic ulcer in diabetic wistar rats. Res J Pharm and Tech. 2019;12(7):3350–4. CrossRef
22. National Institute of Standards and Technology. NIST Mass Spectral Library, Release 2014, version 1.7. Available from: https://www.nist.gov/system/files/documents/srd/NIST1aVer22Man.pdf
23. Hotmian E, Suoth E, Fatimawali F, Tallei T. Analisis GC-MS (gas chromatography - mass spectrometry) ekstrak metanol dari umbi rumput teki (Cyperus rotundus L.). Pharmacon. 2021;10(2):849. CrossRef
24. Ranjan Maji S, Roy C, Sinha SK. Gas chromatography–mass spectrometry (GC-MS): a comprehensive review of synergistic combinations and their applications in the past two decades. J Anal Sci Appl Biotechnol. 2023;5(2):72–85. Available from: https://revues.imist.ma/index.php/JASAB/article/view/40209
25. Vosooghi S, Mahmoudabady M, Neamati A, Aghababa H. Preventive effects of hydroalcoholic extract of saffron on hematological parameters of experimental asthmatic rats. Avicenna J Phytomed. 2013;3(3):279–87.
26. Saettini F, Bonanomi S, Orlandi S, Biondi A, Balduzzi A, Coliva T. Isolated leukopenia in children and adolescents referred to a pediatric hematology clinic. J Pediatric Neonatal Individualized Med. 2023;12(1):e120108. CrossRef
27Kim Suvarna S, Christopher Layton JDB. Bancroft’s theory and practice of histological techniques. London, United Kingdom: Churchill Livingstone; 2012. CrossRef
28. Surboyo MDC, Mahdani FY, Ernawati DS, Hadi P, Hendarti HT, Parmadiati AE, et al. Number of macrophages and transforming growth factor β expression in Citrus limon L. Tlekung peel oil-treated traumatic ulcers in diabetic rats. Trop J Pharm Res. 2019;18(7):1427–33. CrossRef
29. Lee JH, Lee TK, Kim IH, Lee JC, Won MH, Park JH, et al. Changes in histopathology and tumor necrosis factor-α levels in the hearts of rats following asphyxial cardiac arrest. Clin Exp Emerg Med. 2017;4(3):160–7. CrossRef
30. 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(3):100285. CrossRef
31. Sahni N, Yi S, Daniels KJ, Srikantha T, Pujol C, Soll DR. Genes selectively up-regulated by pheromone in white cells are involved in biofilm formation in Candida albicans. PLoS Pathog. 2009;5(10):e1000601. CrossRef
32. Zhao Z, Zhou Y, Wang R, Xie F, Zhai Z. Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli. 3 Biotech. 2020;10(12):538. CrossRef
33. Whirledge S, Defranco DB. Glucocorticoid signaling in health and disease: insights from tissue-specific GR knockout mice. Endocrinology. 2018;159(1):46–64. CrossRef
34. Searle T, Ali FR, Al-Niaimi F. Perioral dermatitis: diagnosis, proposed etiologies, and management. J Cosmet Dermatol. 2021;20(12):3839–4848. CrossRef
35. Mahdani FY, Parmadiati AE, Ernawati DS, Husain H, Ekaperdana SAP, Rachmaningayu U, et al. Citrus limon peel essential oil-induced type IV hypersensitivity reaction. J Exp Pharmacol. 2020;12:213–20. CrossRef
36. Ramalho T, Filgueiras L, Pacheco De Oliveira M, Lima A, Bezerra-Santos C, Jancar S, et al. Gamma-terpinene modulation of LPS-stimulated macrophages is dependent on the PGE2/IL-10 axis. Planta Med. 2016;82(15):1341–5. CrossRef
37. Plastina P, Apriantini A, Meijerink J, Witkamp R, Gabriele B, Fazio A. In Vitro anti-inflammatory and radical scavenging properties of chinotto (Citrus myrtifolia Raf.) essential oils. Nutrients. 2018;10(6) :783. CrossRef
38. Ammad F, Moumen O, Gasem A, Othmane S, Hisashi KN, Zebib B, et al. The potency of lemon (Citrus limon L.) essential oil to control some fungal diseases of grapevine wood. C R Biol. 2018;341(2):97–101. CrossRef
39. Yoo Y, Choi HT. Antifungal chitinase against human pathogenic yeasts from Coprinellus congregatus. J Microbiol. 2014;52(5):441–3. CrossRef
40. Husain H, Hendarti HT, Hadi P, Radithia D. Jumlah Sel Makrofag dan Ekspresi IFN-? Pada Uji Hipersensitivitas Tipe IV Setelah Aplikasi Topikal Essential Oil Kulit Buah Citrus Limon L. Repository Unair. 2019;2019:1–70. Available from: http://repository.unair.ac.id/id/eprint/80290
41. Larjava H. Oral wound healing cell biology and clinical management(1st ed). 1st ed. West Sussex: Wiley Blackwell; 2012. CrossRef
42. 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(1):1–24. CrossRef
43. Yoon WJ, Lee NH, Hyun CG. Limonene suppresses lipopolysaccharide-induced production of nitric oxide, prostaglandin E2, and pro-inflammatory cytokines in RAW 264.7 macrophages. J Oleo Sci. 2010;59(8):415–21. CrossRef
44. Ben Hsouna A, Ben Halima N, Smaoui, S, Hamdi N. Citrus lemon essential oil: chemical composition, antioxidant and antimicrobial activities with its preservative effect against Listeria monocytogenes inoculated in minced beef meat. Lipids Health Dis. 2017;16(1):1–1. CrossRef
45. Sharma N, Dobhal M, Joshi Y, Chahar M. Flavonoids: a versatile source of anticancer drugs. Pharmacogn Rev. 2011;5(9):1–2. CrossRef