Dr Banashree S. Sankeshwari, Dr Pranav R. Tulle, Dr Raghavendra V. Adaki, Dr Dayanand A. Huddar, Deepti S. Fulari, Dr Mokshada M. Badadare
Post graduate student,
Professor and Head,
Department of Prosthodontics
Bharati Vidyapeeth Dental College and Hospital, Sangli.
Purpose : To investigate the antifungal activity of citronella oil by incorporating in acrylic based soft liner
Materials and method : Varying concentrations of citronella oil is incorporated in acrylic based soft liner and its antifungal activity is evaluated at 1 day and 1 week and adherence of Candida albicans to the soft liner is measured at 1 day, 1 week and 15 days.
Results : Soft liner with citronella oil incorporation showed mean inhibitory zone of 10.5 mm with 400 μl, 19.8 mm with 600 μl and 33 mm with 800 μl after 1 day whereas after 1 week it showed 9.4 mm, 18 mm and 31.2 mm with 400 μl, 600 μl and 800 μl respectively.
On intergroup comparison for adherence test, after 1 day group 2 showed highest mean cell count whereas group 4 showed least mean cell count. After 7 and 15 days, group 1 showed highest mean cell count whereas group 4 showed the least mean cell count. On intragroup comparison for adherence test, the mean cell count had decreased for all the groups except the control group when measured after 1 day, 7 days and 15 days.
Conclusion : This in-vitro study shows that citronella oil can be used as antifungal agent.
Keywords : Citronella, essential oil, antifungal activity, soft liner
Citations : Sankeshwari B, Tulle P, Adaki R, Huddar D, Fulari D, Badadare M. Efficacy of citronella oil as an antifungal agent on denture soft liner. J Prosthodont Dent Mater 2020;1(1& 2): 60-64.
Denture soft liners are mainly used for therapeutic purpose in patients who are not able to tolerate denture induced stresses. The most commonly used soft liners are acrylic based or silicone based which help to absorb the masticatory forces and its distribution. Fungal growth on the surfaces of soft liners is a common phenomenon due to lack of antifungal property and increased surface roughness. The fungal growth on the surfaces of liners leads to tissue irritation and mucosal pain. The colonization and adherence of microorganisms to the surfaces leads to denture stomatitis.
The etiology of denture stomatitis though is multifactorial; the most commonly associated microorganism is Candida albicans. Treatment option includes maintenance of oral hygiene, systemic and topical application of antifungal agents.1 Systemic application has few disadvantages and to overcome these, antifungal agents are now incorporated in denture liners. Antifungal agents like ketoconazole, itraconazole, fluconazole have been used but the disadvantages include side effects of these drugs and developing resistance to these drugs.
Hence, there is a need to reduce the fungal activity without hampering the properties of soft liners with the use of natural antimicrobials. Cymbopogon nardus, popularly known as citronella, is a grass cultivated in subtropical and tropical regions of Asia, Africa, and America.2 The major chemical constituents are geraniol, citral, citronellal, and citronellol. Essential oils of Cymbopogon species are known to possess antifungal properties.3 Although, citronella provides good antifungal properties, its use in dental applications is very minimal. Therefore, the purpose of this study is to evaluate citronella as an antifungal additive to acrylic based soft liner.
MATERIALS AND METHODOLOGY
This study comprised the use of acrylic based soft liner (GC). The soft liner was processed according to the manufacturer’s instructions (2.2 gm/1.8 gm) (table 1).
Evaluation of antifungal activity:
Strains of Candida albicans were inoculated into Sabouraud Dextrose Agar in plates and incubated at 37°C. After 8 hours of incubation, the Candida albicans suspension was standardized by dilution with sterile broth. Approximately 0.5 ml of diluted Candida albicans solution was dispensed onto a sterile Sabouraud agar plate and a lawn culture was made. After the inoculum dried, a 6 mm diameter well was punched to a depth of 5 mm with a sterile punch cork borer. The punched wells were filled with soft liner with varying concentrations of citronella oil. After the wells were filled, the plates were incubated at 37°C in an incubator. The mean inhibitory zone (MIZ), in millimeters, for each test punch well was measured at 1 day and 1 week (fig 1).
Fig 1: Mean inhibitory zone after 1 week (800 μl)
Specimen preparation for adherence test:
Specimens of dimensions (25 mm diameter x 2 mm thickness) (fig 2) were prepared by adding citronella oil in different concentrations to soft liner and were processed by conventional compression molding technique.
Fig 2 :Mold for specimen fabrication
A total of 12 samples were made and randomly divided into 4 groups based on different concentrations of citronella oil.
Group 1- Control
Group 2- 400 µl
Group 3- 600 µl
Group 4- 800 µl
Total sample size- 3 samples per group
All the specimens were prepared to a uniform size with smooth surfaces by placing polyester film over them. The prepared specimens were allowed to polymerize for 30 minutes at room temperature.
Adherence test :
The standard strain of Candida albicans was inoculated into 25 ml of Sabouraud Dextrose broth in a sterile petri dish and incubated at 37°C for 8 hours. After 30 minutes of setting, the disks were retrieved from the mold, placed in Sabouraud dextrose broth, and incubated at 37°C. At the end of 1 day, 7 days and 15 days storage in the broth, the disks were washed twice with sterile broth for 1 minute. The washed disks were dried, and the adherent yeast cells were fixed in methanol and stained with Crystal violet for 1 minute. Subsequently, the disks were washed with distilled water for 30 seconds and examined under light microscopy. Adherent yeast cells were quantified at 10 different fields on the disks and reported as yeast cells per mm2 (fig 3 and 4).
The mean inhibitory zone after 24 hours was 33 mm for 800 μl and least was 10.5 mm for 400 μl (table 2).
On intergroup comparison for adherence test, after 1 day group 2 showed highest mean cell count (509 cells per mm2) whereas group 4 showed least mean cell count (69 cells per mm2) (table 4).
After 15 days, group 1 showed highest mean cell count (699.33 cells per mm2) whereas group 4 showed the least mean cell count (15.33 cells per mm2) (table 6).
On intragroup comparison of group 1, the highest mean cell count was seen after 7 days (807.33 cells per mm2) whereas with groups 2, 3 and 4, the mean cell count was highest after 1 day (509, 148 and 69 cells per mm2 respectively) and gradually reduced till 15th day (231.66, 105 and 15.33 cells per mm2 respectively).
Soft liners do not possess significant antifungal activity by themselves. The antimicrobial effect of citronella originates from the natural biological activity of its chemical constituent, such as citronellal. Citronella essential oil is thought to disrupt cell membranes and degrade other organic structures.4 Hence, when essential oil was incorporated in acrylic liner, there was an increase in the zone of inhibition as the concentration of oil was increased. There was a decrease in mean inhibitory zone for all the groups after 1 week as compared to that after 1 day.
On intragroup comparison for adherence test, the mean cell count per mm2 had decreased for all the groups except the control group when measured after 1 day, 7 days and 15 days respectively.
On intergroup comparison for adherence test, after 1 day- group 2 showed highest cell count as compared to other groups. After 7 days and 15 days- control group showed highest cell count followed by group 2, 3 and 4 respectively. Group 4 showed the least cell count after 1, 7 and 15 days indicating that it had the highest antifungal activity followed by group 3 and group 2 respectively.
This study concludes that as the concentration of citronella oil increases, there is an increase in the mean inhibitory zone (MIZ). However, after a period of 1 week the MIZ is seen to decrease. Similarly, as the concentration of citronella oil increases, the adherence of Candida albicans cells decreases when measured after 1, 7 and 15 days.
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2. De Toledo L, Ramos M, Spósito L, Castilho E, Pavan F, Lopes É et al. Essential oil of Cymbopogon nardus (L.) Rendle: a strategy to combat fungal infections caused by Candida species. Int J Mol Sci 2016;17(8):1252.
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