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. 2022 Aug 13;18(1):190–195. doi: 10.1016/j.jtumed.2022.07.011

The effectiveness of essential oil from Citrus limon peel on Candida albicans biofilm formation: An experimental in vivo study

Desiana Radithia a,∗, Rindang Tanjungsari b, Diah Savitri Ernawati a, Adiastuti Endah Parmadiati a
PMCID: PMC9643547  PMID: 36398027

Abstract

Objectives

Candida albicans is resistant to commercial antifungal agents. Therefore, it is desirable to use material derived from natural sources as an antifungal agent. Essential oil from Citrus limon peel is able to inhibit the growth of C. albicans in vitro. The purpose of this study was to determine the most effective concentration of essential oil from C.limon peel with regards to the inhibition of C. albicans cyto-morphometric changes and biofilm formation in vivo.

Methods

Male Wistar rats weighing 200–300 g were inoculated with C. albicans for 48 h and then given a single dose of oral methylprednisolone as an immunosuppressant. Essential oil from C.limon peel, in a gel form and at three different concentrations (0.39%, 0.78% and 1.56%), was applied twice a day for 2 days. The rats were killed after 48 h and then palatal mucosa tissues were prepared and examined with a scanning electron microscope (SEM) with regards to C. albicans, cyto-morphometric changes and biofilm formation.

Results

Essential oil from C.limon peel at a concentration of 1.56% showed the strongest ability to inhibit C. albicans growth when compared to 0.78% and 0.39%. At a concentration of 1.56%, essential oil from C.limon peel disrupted cyto-morphometric changes; cells that were neither in intact nor colonised were evident, the filaments around the cells were smooth, the layer of biofilm had disappeared and there was no evidence of hyphae formation.

Conclusion

The effect of essential oil from C.limon peel on cyto-morphometric changes and biofilm formation was concentration-dependent. Essential oil from C.limon peel at a dose of 1.56% showed the strongest ability to inhibit cyto-morphometric changes and biofilm formation. These findings demonstrate that essential oil of C.limon peel is a potential antifungal candidate for the treatment of candidiasis.

Keywords: Antifungal, Biofilm, Candida albicans, Citrus limon, Essential oil

Introduction

Oral candidiasis is an opportunistic infection that affects the oral mucosa; the majority of cases are caused by Candida albicans yeast. The pathogenesis of oral candidiasis is not fully understood although several predisposing factors are known to be responsible for moving Candida from the saprophytic stage into the parasitic stage.1 Since 1995, species of Candida have become the fourth largest cause of nosocomial infections in the bloodstream with an average mortality rate of 39%. In intensive care units, Candida infection is the third most common type of nosocomial infection with an average mortality rate of 47%.2

Oral candidiasis is generally treated with antifungal agents, such as fluconazole, echinocandins or polyene. However, antifungal therapy may cause various side effects, such as high toxicity levels, recurrences or pharmacodynamic interactions; therapy may also trigger drug resistance. In addition, the main species of Candida, including C. albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata, have the ability to form biofilm layers that increase resistance to antifungal agents.3 The biofilm of C. albicans consists of biological material from specific cells of microorganisms that form a layer of an extracellular matrix that exerts a specific protective function during the growth of C. albicans, increasing resistance towards destr-uctive agents.4

Some natural ingredients have been studied to overcome this resistance to antifungal drugs. One of the natural ingredients that has been studied is essential oil derived from Citrus limon peel. Aside from this oil being readily available and inexpensive, the terpenoid content in this essential oil is particularly high. Terpenoids are known to have high antifungal properties and significant levels of coumarin, flavonoids, linalool and carotene and terpenoids have been widely studied and developed as natural antimicrobial and antioxidant agents.5

Until now, various concentrations of essential oil from C. limon peel have been investigated for their ability to inhibit the development of Candidiasis; however, these experiments have generated inconsistent results. As evidenced by DNA fraction analysis, essential oil from C. limon peel derived from n-hexane fraction solvent was proven to kill C. albicans cells.6 Other research demonstrated that a dose of 0.78% essential oil from C. limon peel was the minimum inhibitory concentration (MIC) and that a dose of 1.56% essential oil from C. limon peel was the minimum fungicidal concentration (MFC) for C. albicans in vitro.7 In our previous research, essential oil from C. limon peel at doses of 1.56% and 1.37% were both able to kill C. albicans cells through cyto-morphometric changes.8 Based on this previous data, we hypothesised that higher concentrations of essential oil from C. limon peel concentration would be directly related to the reduction in number of C. albicans. To strengthen this hypothesis, it was necessary to conduct further research in vivo to determine the most effective concentration for the inhibition of C. albicans, cyto-morphometric changes and biofilm formation in vivo.

Materials and Methods

Study design

This study was a laboratory experimental study that used a post-test only control group design using Wistar rats (Rattus norvegicus).

Preparation of essential oil from C.limon peel

The C. limon used in this study was obtained from the Citrus and Subtropic Fruit Research Institute (BALITJESTRO), Batu, East Java, Indonesia. The essential oil was produced using the process described by Ganesha et al.9 The essential oil from C. limon peel was extracted by steam distillation and turned into gel with 3% CMC-Na,10 producing three separ-ate concentrations of 0.39%, 0.78% and 1.56%.10

Animal preparation and C. albicans inoculation

The samples consisted of 24 male Wistar rats, each approximately 3 months of age and weighing 200–300 g. Prior to inoculation, the animals were housed in individual cages with controlled temperature (27 °C) and artificial lighting on a 12-h-on/12-h-off cycle with free access to water and standard food.

Cultures of C. albicans were obtained from swab results of oral candidiasis lesions taken from patients with stage 4 HIV/AIDS, which were then inoculated into Sabouraud broth medium and cultured at the Microbiology Laboratory, Faculty of Dentistry, Airlangga University, Surabaya. For inoculation, the Wistar rats were anesthetised intramuscularly with 10% ketamine and then inoculated with C. albicans standard Mc-Farland 1 on the palatal mucosa. Once the subjects were unconscious, they were given a single dose of oral methylprednisolone (0.05 mg/g of body weight) through a nasogastric tube. The inoculation was performed over a period of 48 h.

Essential oil from C.limon peel treatment

After 48 h of C. albicans inoculation, essential oil from C. limon peel was applied every 12 h for a period of 48 h. A cotton swab was used to apply the oil to the palatal mucosa area of each animal. The animals were divided randomly into the following four groups.

  • 1.

    A control group (four animals and no treatment),

  • 2.

    Group A (four animals receiving treatment with essential oil from C. limon at a dose of 0.39%),

  • 3.

    Group B (four animals receiving treatment with essential oil from C. limon at a dose of 0.78%),

  • 4.

    Group C (four animals receiving treatment with essential oil from C. limon peel at a dose of 1.56%).

Tissue preparation

Each animal was decapitated after the treatment period and each animal's palatal mucosa was biopsied and fixed with 2% glutaraldehyde at a temperature of 4 °C. The samples were then washed three times with phosphate buffer solution (pH 7.4), each for 5 min at 4 °C, and then treated with 1% osmic acid post-fixation solution for 1–2 h at 4 °C. Samples were re-washed three times with phosphate buffer solution (pH 7.4), each for 5 min at 4 °C and then dehydrated with a graded series of alcohol (30%, 50%, 70%, 80%, 90% and absolute) up to two times, each for 15–20 min. Alcohol dehydration at 30%–70% was carried out at 4 °C, and 80% alcohol dehydration was carried out at room temperature and was then replaced with absolute amyl acetate as a preservative while drying. The samples were then dried using a critical point drying (CPD) tool and then attached to a stub (holder) using special glue. Each sample was coated with a vacuum evaporator and a coating material made from either pure gold or carbon.

C. albicans cyto-morphometric and biofilm analysis

Palatal mucosa tissue samples were examined with an electron microscope (JEOL JSM-T100, MA, Massachusetts, USA) at magnifications of 500× and 2000×. The results of the study are presented in the form of photographs and data analysis showing the number of C. albicans colonies in the biofilms and cyto-morphometric changes in the size and shape of C. albicans cells in biofilms. The sizes and shapes were mea-sured with a comparison scale and are expressed in microns (μm).

Statistical analysis

Data were collected and normality was tested with the Shapiro–Wilk test. The Levene test was used to assess data for homogeneity. MANOVA multivariate tests, namely the Gomes Howell post-hoc test and the Bonferroni test, were used to analyse the significance of the data. The software analysis used was Statistical Product and Service Solution (SPSS) (IBM SPSS Statistic 24 for Mac, Armonk, New York, USA) All data were considered significant if p < 0.05.

Results

Biofilm analysis of C. albicans

Calculations involving C. albicans colonies on biofilms were performed using images acquired by SEM at 500× magnification. The control group presented in Figure 1A shows a solid biofilm, resembling a mesh/web of rough-looking fibres. This appearance represents mature biofilm with a solid C. albicans colony; the filamentous appearance indicated the formation of C. albicans hyphae.

Figure 1.

Figure 1

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SEM of the Candida albicans biofilm at a magnification of 500×. (A) Control, (B) Citrus limon essential oil concentration 0.39% (group A), (C) Citrus limon essential oil concentration 0.78% (group B), (D) Citrus limon essential oil concentration 1.56% (group C).

Group A presented with a solid biofilm, dense and separate colonies, resembling a mesh web of rough-looking fibres. Figure 1B shows that there was slight damage to the mature biofilm but confirms the formation of C. albicans hyphae.

Group B presented with a rather dense biofilm with many colonies separated by a thin filamentous formation. Fig-ure 1C illustrates degradation of the C. albicans biofilm with reduced density and compaction of C. albicans colonies.

In group C, there were only a few separated C. albicans colonies with less filamentous formations; furthermore, the C. albicans colonies appeared to be thinner than those of group A and group B. Figure 1D illustrates the occurrence of biofilm degradation along with C. albicans cells undergoing necrosis; there was no new hyphae formation (see Figure 1D).

The C. albicans colony counts for the biofilms care given in Table 1. Group C had the lowest number of colonies when compared to the control group (p = 0.000), group A (p = 0.001) and group B (p = 0.021).

Table 1.

Colony counts and cell sizes of Candida albicans in biofilm.

Groups Colony count Cell size (μm)
Control 83.33 ± 12.45a 2.28 ± 0.25830a
Group A 35.67 ± 6.38a 2.12 ± 0.19a
Group B 15.67 ± 1.86a 1.66 ± 0.13a
Group C 11.83 ± 1.17a 1.17 ± 0.15a
p-value 0.000a
0.001a
0.021a 		0.000a
0.001a
0.360b
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Group A, treatment with Citrus limon essential oil 0.39%; group B, treatment with Citrus limon essential oil 0.78%; group C, treatment with Citrus limon essential oil 1.56%.

Data is presented as mean ± standard deviation. The same superscript letter shows significant differences in each group.

a

Significant differences using the Gomes–Howell test at p < 0.05.

b

Significant differences using the Gomes–Howell test p > 0.00.

Cyto-morphometric analysis of C. albicans

The cyto-morphometry of C. albicans formation was examined by SEM at 2000× magnification. Cyto-morpho-metric analyses in the control group revealed round cells that were dense and tightly colonised with a dense and rough filamentous appearance at the base of the colonies. The C. albicans in the biofilm was in good condition with a high number of round and intact cells, dense and compact colonies and hyphae formation (see Figure 2A).

Figure 2.

Figure 2

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The size of Candida albicans cells, as determined by SEM at a magnification of 2000×. (A) Control, (B) Citrus limon essential oil concentration 0.39% (group A), (C) Citrus limon essential gel concentration 0.78% (group B), (D) Citrus limon essential oil concentration 1.56% (group C).

Cyto-morphometric analysis in group A revealed round cells and intact colonies that had started to separate but maintained a filamentous appearance around the base. Figure 2B shows that the C. albicans were still in good condition with round, intact cells but also show that damage had begun to appear in the C. albicans biofilm, marked by separate colonies.

The cyto-morphometric appearance in group B presented with flat, oval-shaped cells; the colonies were separated and there was a clear lack of structure. The filaments were less formed and were smooth in appearance. Figure 2C shows damage in the biofilm and that the damage incurred by C. albicans was characterised by cyto-morphometric changes in cell shape.

In group C, the cyto-morphometric of C. albicans revealed cells that were neither intact nor colonised with fewer filaments around them; the cells also appeared smooth. Figure 2C shows damage to the C. albicans and the biofilm marked by changes in cell shape, a disappearance of the biofilm layer and no new cell or hyphae formation.

A comparison of C. albicans cell sizes is given in Table 1. The fewest cyto-morphometric changes in C. albicans were found in group C (1.17 ± 0.15); the cyto-morphometric changes in the control group were significantly more extensive than both group B and group C (both p = 0.000).

Discussion

Research on the effects of essential oil from C. limon peel has never been carried out on animals. The concentration of essential oil from C. limon peel used in this study was based on previous research stating that the minimum inhibitory concentration on C. albicans biofilm was (0.78%) and the fungicidal activity against C. albicans was 1.56%.8 The essential oil from C. limon peel used in this study was provided in a gel form to facilitate application because this material immediately melts when it comes into contact with the mucosa where it forms a layer that functions as a covering agent. In addition, the absorption rate of the gel by the mucosa is better than the absorption rates of other semi-solid forms.11

This study provides evidence that essential oil from C. limon peel not only induces cyto-morphometric changes but also affects biofilm formation. Essential oil from C. limon peel contains citral, a substance that is known to inhibit the S-phase in the cell cycle and reduce the DNA replication of C. albicans. Citral also inhibits the synthesis of ergosterol, the largest lipid component in the cell membranes of C. albicans. If ergosterol synthesis is inhibited, there will be an imbalance in membrane permeability. Cytoplasmic protein will experience denaturation, and the respiratory function and proliferation of C. albicans cells will then be disrupted. Disruption to cellular function prevents C. albicans from proliferating and inducing cyto-morphometric changes.12,13 In this study, this effect increased as the concentration of C. limon peel increased. Essential oil from C. limon peel at a dose of 1.56% provided the maximum effect in terms of the disruption of cyto-morphometric changes, presenting cells that lacked shape, structure and colonisation. Essential oil from C. limon peel at a dose of 1.56% also resulted in fewer filaments; in addition, the filaments were smooth in appearance, the biofilm layer had disappeared, and no new hyphae formation was observed.

While inhibiting cyto-morphometric changes, essential oil from C. limon peel was also able to inhibit and degrade biofilm formation. Limonene was responsible for this effect as it can damage the structure of the extracellular matrix of C. albicans biofilm by damaging the chitin bond in the 1,3-β-glucan composition and inhibit chitinase synthesis in the formation of the extracellular matrix, thus disrupting the C. albicans cyto-morphometric changes that lead to hyphae formation.14 A significant level of chitin is formed during the cyto-morphometric changes that lead to the formation of hyphae. When adhesion occurs and initiates hyphae morphogenesis, C. albicans requires a supportive environment for cyto-morphometric changes into pathogens. Single yeast develop when the pH is less than 6, and hyphae form when the pH is more than 7.. During hyphae formation, C. albicans undergo adaption by increasing the uptake of amino acids, amines, polyamine and glucose. These molecules then break the substrate intracellularly with the help of Dur1,2 urea amidolase and releases the product (ammonia) through the ammonia transport outward (Ato) protein. This activity creates alkalinisation to support the cyto-morphometric changes that lead to hyphae formation. Limonene undergoes oxygenation metabolism that is dominated by three elements: perillic acid, dihydro-perillic acid and limonene-1,2-diol. These elements can neutralise the pH of the mucosa. Therefore, this does not support the adhesion of C. albicans. This activity is inhibited by limonene metabolite elements by neutralising alkalinisation of pH and decreasing Dur1,2 urea amidolase activity through oxygenase activity. If there is no supporting extracellular environment, chitin synthesis is inhibited and cyto-morphometric changes of the hyphae fails.15,16 The inhibition and degradation of biofilm formation was induced by essential oil from C. limon peel at a dose of 1.56%, as evidenced by the formation of fewer colonies with thin filamentous formations. This finding was confirmed by the observation of biofilm degradation by C. albicans cells undergoing necrosis and no new hyphae formation.

This study provided evidence relating to the essential oils derived from C. limon peel. C. limon inhibits C. albicans and is known to inhibit C. glabrata, Candida krusei, C. orthopsilosis, C. parapsilosis and C. tropicalis.17, 18, 19

Although, we demonstrated that essential oil from C. limon peel has an effect on C. albicans biofilm formation and degradation, there were some limitations to this study that need to be considered. First, this study was preliminary; we still need to prove the effectiveness of C. limon peel on C. albicans before it can be compared with the currently available anti-fungal drugs. Second, no specific active ingredient has been identified in the essential oil from C. limon peel that causes the degradation of C. albicans biofilm. However, our findings can be used as a basis for developing further research on the antifungal potential of essential oil from C. limon peel and its phytocomponents against C. albicans.

This study confirmed that the effectiveness of essential oil from C. limon peel on cyto-morphometric changes and biofilm formation is influenced by its concentration. Essential oil from C. limon peel at a dose of 1.56% showed the strongest ability to inhibit cyto-morphometric changes and biofilm formation. This finding highlights the potential of essential oil from C. limon peel as an antifungal candidate for the treatment of candidiasis. The integration of essential oil from C. limon peel into other drug delivery systems can be adapted to the mechanisms of candidiasis treatment as req-uired.

Conclusion

The effect of essential oil from C. limon peel on cyto-morphometric changes and biofilm formation is influenced by its concentration. Essential oil from C. limon peel at a dose of 1.56% showed the strongest ability to inhibit cyto-morphometric changes and biofilm formation. Based on our findings, essential oil of C. limon can eradicate C. albicans biofilm and has potential as an antifungal agent for the treatment of oral candidiasis.

Source of funding

This research did not receive any specific grant from any public, commercial or not-for-profit agencies.

Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

This study was approved and supervised by the Research Ethical Clearance Commission, Universitas Airlangga Faculty of Dental Medicine (No. 063/HRECC.FODM/V/2017).

Authors contributions

DR: conceptualisation, methodology, writing and review and editing; RT: investigation, data curation, formal analysis and writing (original draft); DSE: conceptualisation and supervision; AEP: conceptualisation and supervision. All authors author has critically reviewed and approved the final draft and is responsible for the content and similarity index of the manuscript.

Footnotes

Peer review under responsibility of Taibah University.

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