INTRODUCTION:

Tea tree oil is a volatile essential oil. It is derived from Melaleuca alternifolia which is an Australian native plant [1].Tea tree oil has fresh camphoraceous odour. It is found to be effective as bactericide(0.002-2%) and as fungicide(0.004-0.25%) and as anti-inflammatory agent [2].Tea tree oil is composed of terpene hydrocarbons, mainly monoterpenes, sesquiterpenes and associated alcohols[1].It acts as a topical therapeutic agent[3].Tea tree oil has the ability to modulate the vasodilation and plasma extravasation associated with histamine-induced inflammation[4].

Oil of clove, also known as clove oil,is an essential oil extracted from the clove plant, Syzygium aromaticum. By an analysis using gas chromatography, it is found that the main constituents found in the clove bud oil are the phenylpropanoids eugenol, eugenyl acetate, carvacrol, thymol, cinnamaldehyde, β-caryophyllene, and 2-heptanone[5][6].

Clove buds’s standardized polyphenol-rich extracts are derived as a water soluble free flowing powder (Clovinol) which is suitable for functional food applications[7].It is an antioxidant as it acts as a very strong radical scavenger ,is antifungal as it reduces the quantity of ergosterol which is a content of fungal cell membrane and is antibacterial as it has inhibitory effect on staphylococcus spp.[8].Recent investigation has revealed that clove oil has significant anti-ulcerogenic activity (>97% inhibition of ethanol-induced stomach ulcers in Wistar rats when orally administered [7].

A worldwide increase in the incidence of fungal infections was observed for past few decades and also it has been found that mant fungus are becoming resistance to fungicides. Clinically used antifungals have many drawbacks in terms of toxicity, efficacy and cost. Essential oils have antimicrobial activity and has been frequently used recently as it inhibits the growth of various micro-organisms[9].

The purpose of this study is to examine the antifungal activity of teatree oil and clove oil.

MATERIALS AND METHODS:

Preparation of Sabouraud Dextrose Agar Plate:

The SDA media comprises of 10gm of enzymatic digest of casein,40gm of dextrose and 15gm of agar as the solidifying agent . pH of the media was adjusted to 5.6 at 25⁰ C. 65 g of the medium was suspended in one liter of purified water, heated with frequent agitation and boiled for one minute to completely dissolve the medium. Autoclaved at 121° C for 15 minutes. Cooled to 45 to 50°C and poured into petri dishes or tubes for slants.

Inoculation into the SDA plates:

For processing of specimen, in order to obtain isolated colonies, the specimen was streaked on to the medium with a sterile inoculating loop. The plates were incubated at 25 – 30°C in an inverted position (agar side up) with increased humidity. Cultures were examined weekly for fungal growth .

Preparation of fungal spore:

The filamentous fungi were grown on Sabouraud dextrose agar(SDA) slants at 28°C for 10 days. The spores were collected using sterile double distilled water and stored in refrigerator.

Agar Well diffusion method:

Antifungal activity was carried out using disc diffusion method(Murray et al.,1995).Petri plates were prepared with 20ml of sterile SDA(Hi-media, Mumbai).The test culture was swabbed on the top of the solidified media and allowed to dry for 10min.Wells were made on the media using a well borer. Different concentrations of the sample (20,40 and 60 µl per well) were loaded on the wells. Ketakonazole (10 µg/well) was used as a positive control. These plates were incubated for 48 hrs at 28°C.Zone of inhibition was recorded in millimeters (mm).

RESULTS:

Table 1 shows Antifungal activity of the sample by using disc diffusion method

Sample	Concentration (µL)	Zone of inhibition (in mm)
Sample	Concentration (µL)	*Aspergillus niger*	*Aspergillus flavus*	*Trichoderma viride*
Tea tree oil	20	10	-	-
	40	14	-	-
	60	16	-	-
Clove oil	20	11	-	8
	40	12	-	10
	60	14	11	14
Ketakonazole	10 µg	9	19	10

Antifungal activity of Teatree Oil:

Various concentration of Teatree Oil (20μL, 40μL, 60 μL) was tested for antifungal activity against Aspergillus niger (Fig No.1), Aspergillus flavus (Fig No.2), Trichoderma viride (Fig No.3).The antifungal activity was very much evident with Aspergillus niger, as the concentration of oil increased, the zone of inhibition also increased linearly but Teatree oil didn’t express any antifungal activity against Aspergillus flavus and Trichoderma viride.

Antifungal activity of Clove Oil:

Various concentration of Clove oil (20μL, 40μL, 60 μL) was tested for antifungal activity against Aspergillus niger (Fig No.1), Aspergillus flavus (Fig No.2), Trichoderma viride (Fig No.3). Clove Oil exhibited antifungal activity against Aspergillus niger, Aspergillus flavus, Trichoderma viride. Zone of inhibition increased concomitantly with increase in concentration. But only 60μL of Clove oil showed zone of inhibition with Aspergillus flavus.

As compared to the standard ketakonazole ,both Teatree and Clove oil showed antifungal activity against A.niger, whereas Clove oil showed antifungal activity against T.viride and also express antifungal activity against flavus in an increased concentration. But Teatree Oil was not effective against A.flavus and T.viride.

DISCUSSION:

The antifungal activity of treetea oil is comparatively higher compared to clove oil against Aspergillus niger. Clove oil can inhibit the growth of Aspergillus flavus and Trichoderma viride but teatree oil does’nt have any inhibitory action. According to this study, the treetea oil and clove oil exhibited a higher degree of inhibition compared to that of ketakonazole standard against Aspergillus niger. Similarly, another study done by Inder Singh Rana states that clove oil is highly antifungal towards most of the fungal species and exhibited a zone of inhibition ranging from 12-22mm.The order of sensitivity in the descending array was Mucor sp., M. gypseum, F. monoliforme, T. rubrum, Aspergillus sp., F.oxysporum [9].Our study is similar to the study done by Hamini-Kadar N where the zone of inhibition of clove oil was increasing with the increasing of oil concentration(10).According to Eugenia Pinto,the fungistatic and fungicidal properties of clove oil are associated with its high eugenol content and the highest level of activity of this eugenol was observed against five different species of dermatophytes showing a MIC value of 0.16µl ml¯¹(11).In a study done by Tehmina Anjum, the antifungal activity was found using both volatile and dilution method. In volatile method, clove oil gave about 97% control on P.italicum and in dilution method, it gave about 84% mycelia growth inhibition [12]. According to a research done by K.A. Hammer, the results of Broth microdilution assay has shown that Tea tree oil MICs for all fungi ranged from 0.004% to 0.25%, and MFCs ranged from <0.03% to 8.0%. Aspergillus niger conidia showed germinated conidia to be more susceptible to Teatree oil than non-germinated conidia [13]. Francesca Mondello’s study states that Teatree oil was highly efficacious in accelerating C.albicans clearance from experimentally infected rat vagina(14).It has been found in literature that not only in fungi but also in bacteria, the clove oil have influence on biological membranes [15].Vasquez has made a study with the treatment of oropharyngeal candidiasis in AIDS patients by oral solution of Teatree oil and also showed best results [16]. Teatree oil along with butanefine is used to cure toenail onychomycosis [17]. It has also been found that Teatree oil inhibits germ-tube formation in C.albicans which causes vaginal infections(18).

CONCLUSION:

Both the oil possess antifungal activity against A. niger, A. flavus, T. viride but Clove oil was more potent as compared to Teatree Oil. In conclusion, the findings of the present study indicate that teatree oil and clove oil can be used as therapeutic option against Aspergillus niger that cause aspergillosis and otomycosis (fungal ear infection) in humans. Clove oil might be useful in the clinical manifestation of fungal sinusitis, non-invasive fungal pneumonia and aspergillosis which are caused due to Aspergillus flavus. Oral aspergillosis lesions are yellow or black in color, with a necrotic ulcerated base, classically located on the palate or posterior tongue. So this can be treated by using Clove oil.

REFERENCES:

1. C.F. Carson, K.A. Hammer, T.V. Riley;" Melaleuca alternifolia (Tea Tree) Oil: a Review of Antimicrobial and other Medicinal Properties"; Clin Microbial Rev.2006 Jan;19(1):50-62

2. Jackson Thomas, Christine F. Carson, Greg M. Peterson, Shelley F. Walton, Kate A. Hammer, Mark Naunton, Rachel C. Davey, Tim Spelman, Pascale Dettwiller, Greg Kyle, Gabrielle M. Cooper and Kavya E. Baby; Therapeutic Potential of Tea Tree Oil for Scabies; Received August 12, 2014.Accepted May 8, 2015.© The American Society of Tropical Medicine and Hygiene

3. K.A. Hammer, C.F. Carson, T.V. Riley,” Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil.” Journal of Applied Microbiology,2003;95(4):853-60.

4. Benjamin N. Roin,” Unpatentable Drugs and the Standards of Patentability.”, Texas Law Review,Vol.87,pp.503-570,February 2009.

5. 5.Chaieb K, Zmantar T, Ksouri R, Hajlaoui H, Mahdouani K, Abdelly C, et al. Antioxidant properties of the essential oil of Eugenia caryophyllata and its antifungal activity against a large number of clinical Candida species. Mycoses. 2007; 50:403–6. [PubMed]

6. Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, Mahdouani K, et al. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): A short review. Phytother Res. 2007; 21:501–6. [PubMed]

7. Issac A, Gopakumar G, Kuttan R, Maliakel B, Krishnakumar IM.Safety and anti-ulcerogenic activity of a novel polyphenol-rich extract of clove buds (Syzygium aromaticum L).Food Funct. 2015 Mar; 6(3):842-52. doi: 10.1039/c4fo00711e.

8. Namrata Dagli, Rushabh Dagli, Rasha Said Mahmoud, and Kusai Baroudi; Essential oils, their therapeutic properties, and implication in dentistry: A review; J Int Soc Prev Community Dent. 2015 Sep-Oct; 5(5): 335–340.doi: 10.4103/2231-0762.165933;PMCID: PMC4606594

9. Inder Singh Rana, Aarti Singh Rana, Ram Charan Rajak.” Evaluation of antifungal activity in essential oil of the Syzygium aromaticum (l..) By extraction, purification and analysis of its main component eugenol”. Brazilian Journal of Microbiology (2011)42:1269-1277.

10. Hamini-Kadar N, Hamdane F, Boutoutaou R, Kihal M, Henni J E.” Antifungal Activity of Clove (Syzygium aromaticum L.) Essential Oil Against Phytopathogenic Fungi of Tomato (Solanum lycopersicum L) in algeria”. Journal of Experimental Biology and Agricultural Sciences.October-2014;Volume-2(5)

11. Eugenia Pinto, Luis Vale-Silva, Carlos Cavaleiro, Ligia Salgueiro.”Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species.”Journal of Medical Microbiology (2009), 58,1454-1462.

12. Tehmina Anjum, Nosheen Akhtar.”Antifungal Activity of Essential Oils Extracted From Clove, Cumin and Cinnamon Against Blue Mold Disease on Citrus Fruit”. International Conference on Applied Life Sciences (ICALS2012). Turkey, September 10-12,2012.

13. K.A. Hammer, C.F. Carson and T.V. Riley.”In vitro activity of Melaleuca alternifolia (tea tree) oil against dermatophytes and other filamentous fungi.”Journal of Antimicrobial Chemotherapy (2002) 50,195-199.

14. Francesca Mondello, Flavia De Bernardis, Antonietta Girolamo, Giuseppe Salvatore, Antonia Cassone. ”In vitro and in vivo activity of teatree oil against azole-susceptible and -resistant human pathogenic yeasts. Journal of Antimicrobial Chemotherapy (2003)51, 1223-1229.

15. Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008). Biological effects of essential oil s-a review. Food Chem Toxicol 46,446-475.

16. Vasquez, J.A. (1999). Options for the management of mucosal candidiasis in patients with AIDS and HIV infection. Pharmocotherapy 19,76-87.

17. Syed, T.A., Qureshi,Z.A., Ali, S.M.,Ahmad,S. and Ahmad, S.A (1999). Treatment of toenail onycomycosis with 2% butenafine and 5% Melaleuca alternifolia (tea tree) oil in cream. Tropical Medicine and International Health 4,284-287.

18. Hammer, K.A., Carson, C.F. and Riley, T.V. (2000). Melaleuca alternifolia (teatree) oil inhibits germ tube formation by Candida albicans. Medical Mycology 38,355-362.

Received on 09.05.2016 Modified on 27.05.2016

Research J. Pharm. and Tech 2016; 9(8):1128-1132.

DOI: 10.5958/0974-360X.2016.00214.6