|Year : 2015 | Volume
| Issue : 5 | Page : 93-96
Susceptibility characterisation of Candida spp. to four essential oils
CC Rath, S Mohapatra
Post Graduate Department of Botany, North Orissa University, Baripada, Orissa, India
|Date of Submission||23-Jul-2013|
|Date of Acceptance||25-Aug-2014|
|Date of Web Publication||6-Feb-2015|
C C Rath
Post Graduate Department of Botany, North Orissa University, Baripada, Orissa
Source of Support: None, Conflict of Interest: None
In the present investigation, anti-Candida activity of four essential oils i.e. Black cumin (Nigella sativa), Curry leaf (Murraya koienigii), Ajwain (Trachiyspirum ammi), and Betel leaf (Piper betel) were screened against four human pathogenic species of Candida viz. Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis. The minimum inhibitory concentration (MIC) values of the oils ranged between 15.62 and 250 μl/ml while studied through tube dilution method. The oils retained their anti-Candida activities even after heat treatment (at 45ΊC, 60ΊC, 100ΊC for 1 hour) and also on autoclaving. Both Ajwain and Black Cumin leaf oils showed better anti-Candida activity against Candida albicans, resulting in an irreversible damage to the cells. The anti-Candida activity of these essential oils could be attributable to the membrane inhibition mechanism. The activity of the oils is reported to be microbicidal (Candida-cidal).
Keywords: Anti-candida activity, essential oils, minimum inhibitory concentration, minimum killing time
|How to cite this article:|
Rath C C, Mohapatra S. Susceptibility characterisation of Candida spp. to four essential oils. Indian J Med Microbiol 2015;33, Suppl S1:93-6
|How to cite this URL:|
Rath C C, Mohapatra S. Susceptibility characterisation of Candida spp. to four essential oils. Indian J Med Microbiol [serial online] 2015 [cited 2019 Aug 17];33, Suppl S1:93-6. Available from: http://www.ijmm.org/text.asp?2015/33/5/93/150903
| ~ Introduction|| |
Over the last decade fungal infections are increasing at an alarming rate , as well as a rise in the resistance of some species of fungus to different fungicidals used in medicinal practice. Candida species are currently the fourth-leading cause of hospital-acquired bloodstream infections, reaching a mortality rate of up to ~35-40% for systemic or disseminated infections. , The clinical manifestations include candidaemia, vulvovaginal infections affects women of all age groups, oropharyngeal infections and infections among persons with Human Immunodeficiency Viruses (HIV) or full blown disease of Acquired Immunodeficiency Syndrome (AIDS) patients. Candida albicans has accounted for virtually all mucosal candidiasis and responsible for about 60% of both superficial and systemic mycoses.  However, non-Albican Candida (NAC) species such as Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei which cause serious oropharyngeal candidiasis and occasionally esophageal candidiasis. ,
Because of the development of drug resistance towards the commonly used antibiotics and chemotherapeutic agents, there is a renewed interest generated among academicians and researchers towards herbal medications. For centuries, the therapeutic properties of various medicinal plants have been used to treat human diseases. It has been estimated that between 60 and 90% of the populations of developing countries use traditional and botanical medicines almost exclusively and consider them to be a normal part of primary healthcare.  Essential oils derived from plants are well known in traditional medicine and proved to have insecticidal
bactericidal, fungicidal and nematicidal effects. ,,,,, Antibacterial activity of essential oils are well cited in literature.  However, the antifungal activity of essential oils more specifically against Candida species is poorly studied. This prompted us to study the antifungal activity of selected essential oils against Candida species in this investigation.
| ~ Materials and Methods|| |
Strains of Candida [Candida albicans (MTCC-183), Candida tropicalis (MTCC-184), Candida glabrata (MTCC-3019), and Candida parapsilosis (MTCC-6510)] were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India and maintained on Sabouraud's Dextrose agar slants in the laboratory.
Hydro-steam distilled essential oils of Black Cumin (Nigella sativa, seed oil), Curry leaf (Murraya koienigii, leaf oil), Ajwain (Trachiyspirum ammi, seed oil), Betel (Piper betel, leaf oil) were procured from Southern Spice Pvt. Ltd., Madurai, India and used in the study.
Determination of minimum inhibitory concentrations
Minimum Inhibitory Concentration (MIC) of the oils was determined by two-fold tube dilution method of Das et al., 2012  in Sabouraud's Dextrose Broth supplemented with Tween-20 (0.75%) to facilitate miscibility of the oils.
Determination of minimum killing time of the oils
Similarly, an experiment was designed to determine the Minimum Killing Time (MKT) of the oils against the test pathogens.  Briefly, 1 ml of SDB with T-20 (0.75%), at the MIC level of the oils was prepared and inoculated with 0.1ml of freshly grown test organisms and incubated at 4°C, room temperature (28±2°C) and 37°C separately. One loopful of the sample from the above test tubes were subcultured onto SDA palates at 0, 5, 10, 15, 30, 45, 60, 120, 180, 240, 300, 360, 420, 480 min. intervals and incubated overnight. Two sets of tubes were incubated for each test organism at a specific temperature from which subculturing was carried out alternatively (to minimise the time lapse during subculture). The activity was observed after overnight incubation of the plates at 28 ± 2°C. No growth on the streaking line was considered to be the time required by the oil to kill the organism.
Determination of effect of temperature and pressure on anti-Candida activity of the oils
The effect of temperature and pressure was studied by heating the oils at different temperatures i.e. 45°C, 60°C, 100°C (for 1 hr) in a water bath and autoclaving at 121°C for 20 min and the activity was studied at MIC levels of the oils. ,
| ~ Results|| |
Minimum inhibitory concentration
Minimum Inhibitory Concentration (MIC) of oils ranged between 15.62 and 250 μl/ml [Table 1]. Lowest MIC value of 15.62 μl/ml was reported in case of Nigella sativa oil against Candida albicans and Candida tropicalis. Murraya koenigii showed MIC value 125 μl/ml against Candida albicans and Candida glabrata. Whereas, the same oil showed higher MIC values of 250 μl/ml against C. tropicalis and C. parapsilosis.
|Table 1: Minimum inhibitory cincentration of the oils against test pathogens|
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Determination of fungicidal/fungistatic nature of the oils
An attempt was made to study the fungicidal/fungistatic nature of the oils by subculturing one loopful of the sample from the MIC dilution tubes onto SDA plates. It was observed that all the oils showed the fungicidal (Candida-cidal) nature, as no growth was observed on the SDA plates after the incubation period.
Minimum killing time
During this study an attempt was made to study the time required to kill the pathogens at a particular temperature at MIC level of the oils. A variation was observed in MKT of the oils when tested at three different temperatures [Table 2]. Immediate killing of Piper betel oil was observed against C. albicans at all the three temperatures studied. However, similar effect of Piper betel oil and Trachiyspirum ammi oil was also observed against C. glabrata at 37°C and C. albicans at 4°C, respectively.
Effect of temperature and pressure on anti-candida activity of the oils
This experiment was designed to study the effect of temperature and pressure on the anti-Candida activity of the oils. Oils were heated in a boiling water bath (45, 60, 100°C for 1 hr and autoclaved at 121°C and 15lb pressure for 20 min) and studied for their anti-Candida activities by Disc Diffusion Method loading MIC values of the oils onto the discs. Surprisingly, a significant increase in the activity of the oils was reported due to heating in case of Trachispirum ammi, Nigella sativa and Piper betel that showed complete inhibition of the test pathogens (pathogens showing poor growth at the centre of the Petri dish More Details and no growth around the discs), on SDA plates.
| ~ Discussion|| |
Four different essential oils were tested for their antimicrobial properties against Candida species. During the investigation we observed lower MIC values of test essential oils against the Candida species is indicative of their high degree of effectiveness against these pathogens. Observance of lower MIC value of essential oils against both bacteria and fungi is being reported in literature. ,, Rath et al., (1999a)  reported a higher MIC values 62.5 and 500 μl/ml of turmeric leaf and rhizome essential oils, respectively, against C. albicans in contrast to the findings observed in this investigation. While studying the microbicidal/microbiostatic nature of these oils, the oils were reported to be microbicidal (Candida-cidal). Microbicidal nature of different essential oils is well documented in literature. ,,
During the determination of the MKT of the oils it was observed that Piper betel killed C. albicans immediately at all three test temperatures. Similar results were also observed for Trachispirum ami and Piper betel oil against C. albicans and C. glabrata when tested at 4°C and 37°C, respectively. Killing of the Candida species immediately by these oils indicates that the oils cause an irreversible damage to the structure of the test organisms when they come in contact with the oil mixture. Immediate killing of pathogens and irreversible damage to cellular structure by essential oils is well recorded in literature. ,,,,,, Since the oils showed activity against Candida spp. at both lower temperatures 4°C and at 37°C, it implies that the activities of these oils are energy independent. Energy-independent bactericidal activities of lemongrass, palmarosa and Eucalyptus oil against E. coli and other bacteria is also recorded.  Rath  et al., (2001) reported similar observations while studying the antifungal activity of fractionally distilled and neat turmeric leaf oil. They reported that neat oil killed C. albicans, Cryptococcus neoformans, Trichophyton rubrum and Microsporum gypsum within 1 minute of treatment whereas, 1 hr and 2 hr fractionally distilled oil took a longer time to kill the same pathogens in comparison to neat oil. In agreement to our observations here, Rath  et al., (1999a) reported immediate killing of C. albicans and C. neoformans by turmeric leaf oil when it comes in contact with the oil, causing an irreversible damage to the cells. They also reported that turmeric rhizome oil kills C. albicans and C. neoformans when treated for a longer time (15 min) in comparison to turmeric leaf oil.
Anti-Candida activity of the oils was not lost even on heating the oils at 45, 60, 100°C for 1 hr and autoclaved at 121°C and 15 lb pressure for 20 min. Similar properties of various essential oils have been reported by researchers while studying their antimicrobial activities ,,, including Candida species. On heat treatment, a significant increase in anti-Candida activity of Trachispirum ami, Nigella sativa and Piper betel oils was recorded during the investigation. This could be attributable to the change in charge of the compounds present in these essential oils and increase in their mobility. Further, it suggests that the anti-Candida components that are present in these oils are heat stable and withstand a temperature of 121°C and 15-lb pressure, indicating their thermostable and barostable nature. Gupta et al.,(2004)  reported the persistence of antibacterial activity of carrot (Daucus carota) and celery (Apium graveolens) seed essential oils against both Gram positive and Gram negative pathogens after heat treatment (100ΊC) and autoclaving, indicating the presence of heat stable components in these essential oils as reported in our investigation. Similar results are also reported in literature  while studying the antibacterial activity of lime (Citrus limonum) and juniper (Juniperus communis) oils against 32 strains of methicillin resistant S. aureus. Das et al., (2009)  reported the antibacterial activity of essential oils of three Ocimum spp. and their cocktail mixture at high temperature and pressure concluding the presence of heat stable and baro-stable components in essential oils, as reported here in our studies.
Susceptibility of these pathogens to the test essential oils may be due to inhibition of cell membrane synthesis, specifically by extracting the sterols from the membrane or inhibiting steroid synthesis. Senhaji et al., (2007)  observed the antibacterial activity of essential oil from Cinnamum zeylanicum against E. coli 0157:H7 is through outer membrane disintegration and increasing the permeability to ATP through cytoplasmic membrane. Similarly, Rath et al., (2005)  also reported the anti-Staphylococcal activity of Juniper and Lime essential oils against methicillin-resistant S. aureus (MRSA) through inhibition of cell membrane synthesis. Further, it is to add that the essential oils are rich in terpenes (monoterpenes, oxygenated monoterpenes and sesquiterpenes). However, the mode of action of terpenic constituents (essential oils) on microorganisms is not fully understood. But, in view of their hydrophobicity, it is considered that they are involved in mechanism such as permeability of cytoplasmic membrane, coagulation of cell contents and disruption of the proton motive force.  Therefore, the anti-Candida activity of these essential oils through membrane inhibition could be attributable to the hydrophobicity of essential oils that enables them to make partitions in the membrane, rendering permeability due to extraction of steroid molecules present on the membrane and leading to leakage of cell contents resulting in death of the cells.
In conclusion it can be told that in this investigation we have established the anti-Candida activity of these essential oils against human pathogens is suggestive of their use in pharmaceuticals and cosmetic industries for production of drugs and aroma products. However, further scientific research is essential to investigate the side effects of these oils before consideration of their use.
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[Table 1], [Table 2]