M. fragrans is commonly known as nutmeg. S. aromaticum is called as clove. In Siddha medicine, these are used as one of the ingredients in medicines that are used to treat vaginal diseases. The aim of the study was to evaluate the anticandidal activity of oils of seed and leaf of M.fragrans and clove against C. tropicalis ATCC 13803, C. krusei ATCC 6258, C. albicans ATCC 90028, C. glabrata ATCC 90030, C. parapsilosis ATCC 22019, C. albicans 3 clinical isolates. Oils were distilled using the Clevenger apparatus. Anticandidal activity of oils was screened using disc diffusion method. Minimum Inhibitory Concentration (MIC) of the oils was determined in two different method of two fold micro-broth dilution. Minimum Bactericidal Concentration (MBC) was determined wells with no turbidity in the micro-broth MIC method (BHI) were sub cultured onto a blood agar plate. All three oils showed activity against all tested Candida sp ZOI from 8.3 ± 0.5 - 30.0 ± 0.0mm. MIC of three oils are similar for all tested Candida sp in both methods. The extremely low MIC (0.0045µg/mL) of the oil of S. aromaticum for all the tested Candida strains is note worthy. However, all the tested oils were active against Candida with MICs ranging from 0.0045 –2.5 µg/mL. MBC was the same or differed by only one dilution as the MIC for tested Candida sp. suggesting that the oils are fungicidal. Three oils have ability to inhibit Candida sp with low MIC
Keywords: Anticandidal activity; essential oils; Myristica fragrans; Syzygium aromaticum
Essential oils are one of the secondary metabolites of aromatic plants and are present in buds, flowers, leaves, seeds, fruits, roots, bark and wood. They are stored in secretary cells, cavities and canals of plants and are soluble in lipids and organic solvents which have a lower density than water. Chemically, essential oils are a combination of benzene derivatives, terpenes, various hydrocarbons and straight chain compounds [1, 4]. Water distillation (hydrodistillation) is one of the techniques used for the distillation of essential oils from aromatic plants. Hydrodistillation is the simplest method for obtaining essential oils. Essential oils are highly volatile at room temperature and have been shown to possess antimicrobial activity [2]. In recent years, many essential oils have been used for the preparation of facial creams, head oils, lotions, herbal soaps and toothpaste. Monoterpenes are present in all essential oils and have been shown to possess antibacterial activity. M. fragrans seed, leaf and S. aromaticum are aromatic plants. M. fragrans belongs to the family Myristicaeae is commonly known as nutmeg in English, Sadikka in Sinhala and Sathikkai in Tamil [11]. In Siddha medicine (a type of traditional medicine), the seeds and leaves are used as one of the ingredients in medicines that are used to treat skin diseases, respiratory diseases and arthritic conditions [3]. S. aromaticum (synonym: Eugenia carophyllata) is called as clove in English, karambu-neti in Sinhala and illavangappu or kirambu in Tamil. It belongs to the Family Myrtaceae. In Siddha medicine, along with other ingredients, it is used in the treatment of respiratory, gastro intestinal, urinary and skin diseases. Antimicrobial activity of S. aromaticum oil has been previously evaluated against S. aureus, E. coli and Bacillus cereus, Streptococcus mutans, Lactobacillus acidophilus, Saccharomyces cerevisiae [5] and Aspergillus sp and Dermatophytes [6]. Antimicrobial activity of the essential oils of M. fragrans seed has been reported against Shigella dysenteriae [8], E. coli and P. aeruginosa [7] In Siddha medicine, M. fragrans leaf, seeds and S. aromaticum are used to prepare different medicines for the treatment of vaginal discharge [9]. The aim of the study is to evaluate the anticandidal activity of oils of these plants against selected Candidal sp.
M. fragrans seed and leaf and S. aromaticum flower buds were collected from Kandy. The specimens of plants were sent and obtained voucher specimen numbers (M. fragrans- S.M/04, S. aromaticum – S.M/03) from the National herbarium of Royal Botanical Garden, Peradeniya. These were pounded as coarse powder and transferred to a flask. 100g of M. fragrans seed, leaf and flower buds of S. aromaticum were distilled separately using Clevenger apparatus for 8 hours and 6mL, 2mL and 14.5mL oils were obtained respectively. Oils were stored at 4 C until used for the anticandidal assay.
Anticandidal activity of oils was screened using disc diffusion method against selected Candidal sp. Three replicates were carried out for the entire procedure [10].
Whatman’sNo.1 filter paper was used to prepare the discs. Using a borer 6mm diameter discs were cut out from the filter paper and placed in universal bottles and autoclaved.
A broth suspension with turbidity equivalent to 0.5 McFarland standards was prepared from a pure culture of each of the test organisms. A Sabouraud dextrose agar plate was inoculated with 1 mL of the broth suspension and the plate rotated to allow even spreading of the inoculum. After removal of the excess fluid, the plate was allowed to dry at 37°C for 15 min. Sterile blank discs (6mm) placed on the seeded plate were impregnated with 5μL of the oil and left on the bench for 30 min. for absorption of the oil. The plates were incubated at 37°C for 24 h. After incubation, the diameters of the zones of inhibition were measured.
This method was carried out for 8Candida species (Table 1).
Minimum inhibitory concentration of the oils was determined using two micro-broth dilution methods.
i. Using Brain heart infusion (BHI) broth Under aseptic conditions, U bottomed 96 well microtiter (Corning Life Sciences, Canada) used for this assay. The first column of the microtiter plate was filled with 180 μL BHI, 10 μL of oil and 10 μL of 1/50 diluted Tween 80.Other wells of the microtiter plate were filled with 100 μL of BHI. Two fold serial dilutions were made by subsequent well in the next column of the same row so that each well had 100 μL of broth in serially descending concentrations. A broth suspension with turbidity equivalent to 0.5 McFarland standards was prepared from a pure culture of each of the test organisms. One mL of each suspension was transferred to 9 mL of sterile distilled water. Finally, 10 μL of this bacterial suspension was added to each well. The last two columns of each row contained only BHI and Tween 80 respectively which served as controls. The plates were incubated at 37°C for 24 h. After 24 h incubation the turbidity in the well was observed visually. The lowest concentration of the fraction at which no turbidity observed was noted as
The first column of the microtiter plate was filled with 180 μL SGB, 10 μL of oil and 10 μL of 1/50 diluted Tween 80. Other wells of microtitre plates were filled with 100 μL of SGB. Two fold serial dilution was made by starting transferring 100 μL material from first column to the subsequent wells in the next column of the same row and so that each well has 100 μL of material in serially descending concentrations. Finally, 10 μL of bacterial suspensions were added to each well. Last two columns served as the control column for SGB and SGB with Tween80 respectively. The plates were incubated at 37°C for 24 h. After 24 h incubation the colour change to pink was observed in plate. The lowest concentration of oil at which no colour change occurred was recorded as the MIC of the oil for the organisms in the row (Figure 2).
Wells with no turbidity in the micro-broth MIC method (BHI) were sub cultured onto a blood agar plate. The MBC was the lowest concentration at which no growth was detected after incubation at 37 C for 24 hours.
Table 2 shows that the screening results for anticandidal activity of essential oils using disc diffusion method. All three oils showed activity against all tested Candida sp.
Table 3 shows that the MIC of oils (S. aromaticum, M.
fragrans seed and leaf are similar for all tested Candida sp in both methods. Both methods gave results which were identical or varied by one dilution as shown in table 3.
The extremely low MIC (0.0045μg/mL) of the oil of S.
aromaticum for all the tested Candida strains is noteworthy. However, all the tested oils were active against Candida with MICs ranging from 0.0045 – 2.5 μg/mL.
Table 4 shows that the MBC was the same or differed by only one dilution as the MIC for tested Candida sp.
suggesting that the oils are fungicidal. MIC and MBC of S. aromaticum and M. fragrans leaf and seed oils for all Candida sp. ranges 0.004–2.5 μg/mL. S. aromaticum oil showed very low MIC and MBC for all tested organisms. In Siddha medicine, M.fragrans leaf, seed and S. aromaticum are used to prepare different medicines for the treatment of vaginal discharge [9]. Vaginal Candidiasis due to C. albicans may lead to vaginal discharge. The current study showed that three oils showed activity against all tested Candida sp.
The MIC of oils of S. aromaticum, M. fragrans seed and leaf oils for all tested Candida sp ranged from 0.004 – 0.31μg/mL. S. aromaticum oil was found to have an extremely low MIC of 0.004 μg/mL for all tested Candida sp.
Fluconazole is the drug to treat the Candidal infection. Previously, the Subcommittee for Antifungal Testing of the Clinical and Laboratory Standards Institute (CLSI) mentioned MIC interpretive breakpoints for fluconazole on Candida sp was 8μg/mL. An organism-drug combination is essential to determine the MIC [12]. The current study shows that the tested oils have lower MIC than Fluconazole.
Three oils have ability to inhibit Candida sp with low MIC. Further study on this widely used medicinal plant oils is indicated to explore the possibility of finding novel antifungal compounds.
Financial assistance by Ministry of Higher Education, (HETC Scholarship) is kindly acknowledged.