1,2Laboratory of Microbiology,Department of Botany, University of Rajasthan, Jaipur
Received: 17May 2017 Revised and Accepted: 31 Aug 2017
Objective: The study was designed to determined antidermatophytic activityof Thymus vulgaris essential oil against fungi causing superficial skin infections in human and animal.
Methods: Minimum inhibitory concentration(MIC) of essential oilwas screened against selected pathogenic fungi namely Trichophytonrubrum(MTCC 296),T. mentagrophytes(MTCC 7687), Microsporum gypseum (MTCC 4524),M.fulvum(MTCC2837), T.soudanenseandT. interdigitale throughsemi solid agar antifungal susceptibility testing method(SAAS). Minimum fungicidal concentration(MFC) was also determined by modified semi solid agar antifungal susceptibility method.T.soudanense and T. interdigitalis were isolatedthrough TO. KA. VA hair bating technique from animal habitats soil of Jaipur district.
Results: T. vulgaris essential oil exhibited excellent antideramtophytic activity against all selected dermatophytes. Minimum inhibitory concentration was ranged from 0.020±0.000μl/ml to 0.1±0.033 μl/ml. MFC were found little higher than MIC(0.02±0.000μl/ml to.2±0.000μl/ml). M. gypseum was found to be most susceptible fungus as compared to other test fungi.
Conclusion: T. vulgaris was found to be most effective fungicidal agent against human pathogenic fungi.Present findings provide for a rationale basis of a possible utilization of this oil in fields requiring safe and cheap compounds with antiseptic and preservative properties, such as cosmetic, pharmaceutical and food industries.
Keywords:Thymus vulgaris, Essential oil, Dermatophytes, Trichophyton, Microsporum
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Infection caused by fungi in man and animal are more common in tropical and subtropical countries due to prevailing moisture, over population, poor hygienic living conditionsand temperature regimes [1-5]. Skin infection due to dermatophytes has become a severe health problem affecting children, adolescent and adults. They produce keratinase which degrade the keratin and thus, invade the superficial skin tissue, nails and hair. These infectionsare generally cutaneous and restricted to the non-living, cornified layers of the skin. However, in constantly reoccurring conditions, the dermatophytes invade deeper tissues and simultaneously caused infections with other organisms. In general, the dermatophytes lack the ability to invade deeper tissues or organs of the host. The dermatophytic infections are generally referredas ringworm infections due to their ring like appearance. Recently, there has been an increase in the incidence of fungal infections in developing countries. This may be the result of frequent use of antibiotics, environmental factors, resistant strains and various conditions, like organ transplantation, lymphomas, leukemia and human immunodeficiency virus. However, clinical values of these agents have been limited due to having high toxicity and emergence of drug resistance in their antifungal activities. These factors emphasize the urgent need for the development of new effective treatment alternatives.
The reduced adverse effects, decreased drug interactions, improved cure rate andcost effectivemedicine preparation from available topical agents are present day needs. In recent year there has been a gradual revival of interest in the use of medicinal plants and their products for the treatment dermatophytoses, and other fungal infections where a topical therapy is required.Spices and herbs are a major part of the daily food in India comprise the most important products used for flavoring foods and play a major role as the topical or systemic treatment of a wide range of diseases including infectious diseases.Beside their importance for general well-being, they are frequently used astraditional medicine . In recent years, a lots of studies has been conducted onthe antifungal effects of plant-derived essential oils on fungi causing superficial infections[10-14].These finding promoted us to explore more spice essential oil for the treatment of dermatophytic infections.
Thymus vulgarisL. belonging to family Lamiaceaeis a pleasant smelling perennialshrub, cultivating all over the world.Thyme is widely used for seasoning fish, poultry farms, vegetables, soups, for flavoring liqueurs, herbal tea preparations.T. vulgarisessential oil andextract has also been used in the treatment of gum diseases, rheumatism, sore throat, tonsillitis, and arthritis[15-16].The essential oil of T. vulgaris is a known antiseptic, antiviral, antimicrobial agents[17-20]. Lots of work on antifungal activity of T.vulgaris essential oil against plant pathogenic, food born fungi, saprophytic fungi andCandida albicans has been reported[21-24]but very few reports are available on the management of human pathogenic fungi usingThymus oil despite its proven biological actions.
In present investigation a most important spice of European countries, T. vulgarisL.was explore against dermatophytic fungi. The high antidermatophytic activity suggests thatthymus essential oilis a potential new antifungal agent for a natural product.
Plant essential oil
Essential oil of Thymus vulgaris(red thyme) was procured from sigma Aldrich (São Paulo, SP, Brazil)company. Oil was clear light yellowcolour with pleasant odor. Oil was stored in refrigerator.
Chemical and reagents
Sabouraud dextrose agarand Brain heart infusion agar media were purchased from Sigma Aldrich chemical company.
Microorganism for in vitro studies
T. vulgaris essential oil was evaluated fortheir antifungal properties against selected dermatophytes and keratinophilic fungi.For antidermatophytic studies four dermatophytes were procured from the Imtech Chandigarh. These are T. mentagrophytes(MTCC 7687), M. gypseum(MTCC 4524)and Microsporum fulvum(MTCC2837), T.rubrum (MTCC 296).Trichophytonsoudanense and T. interdigitale were isolated from soil sample collected from animal habitats of Jaipur district. These fungi were isolated through TO. KA. VA hair baiting technique of Vanbreuseghem. Identification was based on macroscopic and microscopic examination of the culture isolates. The macroscopic examination of fungi was characterized by duration of growth, surface morphology and pigment production on the reverse. Strains were identified by their morphological and physiological characteristics according to the procedure described by Conant et al.,and Forbes et al.,. These selected fungi are maintained on Sabouraud’s dextrose agar media and Potato dextrose agar media. Cultures were stored at 4 °C and subcultured once in the month.
Determination of MIC and MFC by semisolid agar antifungal susceptibility method (SAAS)
Semisolid agar antifungal susceptibility testing methodof Provine and Hadley  was applied for end point determination. For this method Brain heart agar medium was used.
Sterile swab dipped into sterile tween 80 was used to pick the pure colony of yeast. This was then suspended in 3-4 ml of sterile normal saline and vortexed. The turbidity of the homogenous suspension was adjusted to ~0.5 McFarland standard. Similarly inoculum was prepared for filamentous fungi (3-7 d old slant at 37 °C on potato dextrose agar). Byswabbing the pure colony (mixture of conidia and hyphal fragments) was suspended in 3-7 ml of sterile saline. The mixture was vortexed and heavy particles were allowed to settle. The homogenous suspension was adjusted to 0.5 McFarland standard.
Inoculation of drug containing tubes
The semisolid agar tubes containing known concentrations of test oils as well as oil-free controls, prepared in duplicate, were inoculated with one loopful (Himedia Flexiloop 4) of 0.5 McFarland adjusted culture by inserting the loop deep within the semisolid agar. The tubes were incubated at 37 °C for 72 h for dermatophytes. A loopful of the inoculum suspension was streaked onto Sabouraud dextrose agar to check for purity and viability.
End point determination
Endpoint determination was done according to the NCCLS/CLSI guidelines, M27-A and M38-A. Growth was compared to that of oil-free control and scored by visual inspection as follows: +4: growth same as control; +3: slight decrease in growth; +2: significant reduction in growth reduction 80% in yeast and 50% in filamentous);+1 slight growth or few visible hyphal fragments; 0: no growth.
MFC was recorded through slightly modification of SAAS method. After 72 hour observation of drug containing tubes for MIC, these tubes were further restored as same temperatureconditions for 3-4 more days. Observation were recorded for MFC.
Each parameter was tested in triplicate. Conventional statistical methods were used to calculated means and standard deviations. Statistical analysis (T-test) was applied to the data to determine differences (p<0.05).
T. vulgarisan important spice is widely used in various ailments. Lots of work has been done on the chemical composition of Thyme oil[20,22,29-30]. Thymol is major constituents of oil ranging from 42.6-57.8% according to collection time and cultivation place. Other components are α-cymene,carvacrol, α-thujene, α-pinene, β myrcene, trans-ocimene,γ terpinene, limonene.
Preliminary screening of theT. vulgaris essential oilfor antidermatophytic activity was carried outagainst six selected dermatophyticfungiusingsemi solid agar antifungal susceptibility method.Data incorporated in table 1 show MIC and MFC of Thyme essential oil.
Thyme oil exhibited excellent antifungal potential against all selected fungi. Microsproum gypseum was found most susceptible fungus (0.020±0.000μl/ml)followed by T.mentagrophytes(0.05±0.000 μl/ml),T. interdigitale(0.05±0.000 μl/ml) and T.soudanense (0.053±0.003μl/ml).M.fulvum(0.1±0.033 μl/ml) was less susceptible fungus as compared to other test fungi. MFC was same to MICin case ofM.gypseum(0.020±0.000 μl/ml)and T.mentagrophytes (0.05±0.003 μl/ml).MFC againstM. fulvum was found to be 0.2±0.000 μl/ml.
Table 1: Antifungal activity of Thymus vulgaris essential oil against dermatophytic fungi
|Trichophyton mentagrophytes MTCC 7687||0.05±0.000||0.05±0.003|
|Microsporum fulvum MTCC2837||0.10±0.033||0.2±0.000|
|Trichophyton rubrum MTCC 296||0.026±0.002||0.05±0.003|
|Microsporum gypseum MTCC 4524||0.020±0.000||0.020±0.00|
MIC=minimum inhibitory concentration,MFC=minimum fungicidal concentration, Statistical analysiswas carried out through T-test(p<0.05).
T. vulgaris L. (Lamiaceae) is a well-known medicinal shrub that contains important phenolic compounds such as thymol and carvacrol and it has been used in many pharmaceutical applications. Data incorporated in present studies revealed strong antidermatophyticactivity ofT. vulgaris essential oilagainst all selectedfilamentous fungi namely T. mentagrophytes, T. rubrum, T. interdigitale, T. soudanense, M. fulvum andM. gypseum. MIC was ranged between 0.020±0.000 to 0.1±0.033μl/ml. These promising antifungal activity against all selected pathogenic fungi may be due to presence of high concentration of phenolic compounds. Similar results were also contributed by other researchers.Shigeharu Inouye et al. studied the vapour activity of 72 essential oil on T. mentagrophytes. Oils containing phenol as the major constituentshowed the most prominent vapor activity, with an MFD of 1.56 μg/ml air. They suggestedthat essential oils such thyme thymol, wild thyme exhibited potent vapor activity againstT. mentagrophytes may be useful as room disinfectants when patients with tinea pedis may drop the living pathogens on the floor.
Sokovićet al. studied the chemical composition of T. vulgaris essential oil and their antifungal activity against 17 micromycetal food poisoning, plant, animal and human pathogens. MIC and MLCwere tested againstfivedermatophytic fungi namely Microsporum canis, Epidermophyton floccosum, Trichophyton rubrum, T. mentagrophytes and T. tonsurans. The essential oil of T. vulgaris showed very strong antifungal activity at low concentrations, 0.05-1.0 μl/ml. Pina-Vaz et al. screened Thymus vulgaris, T. zygis subspecies zygis and T. mastichina subspecies mastichina essential oils and their major components (carvacrol, thymol, p-cymene and 1,8-cineole) against different strain and species of Candida. The essential oils of T. vulgaris and T. zygis showed similar antifungalactivity, which was greater than T. mastichina. MIC and MLC values were similar for all thecompounds tested.
During present investigation, M. gypseum was found to be most susceptible fungus, where MICand MFC were found similar. MIC of T.vulgaris against T.rubrum was also 0.026±0.002 μl/ml but MFC was slightly increased (0.05±0.003 μl/ml). MFC of essential oil was slightly increased as compared to MIC against all filamentous fungi. MFC were ranged from 0.020±0.00μl/ml to 0.2±0.000μl/ml.Adams et al. described that the high antifungal activity ofThyme essential oil are due to presence ofthe high percentage of phenol components. It believed that phenol components may interfere with cell wall enzymes like chitin synthase/chitinase as well as with the α-and β-glucanases of the fungus. Consequently, the high content of phenol components may account forthe high antifungal activity of oils . Rota et al. explained thatpresence ofphenolic compounds thymol and carvacrolare responsible factors of excellent antifungal activities of T. vulgaris oil.The antifungal activity of the oil is mostly incorporatedwith theinteraction of phenolic compounds, thymol, carvacrol, and p-cymene with cytoplasmic membraneergostrol[22,32]. It is believed that the hydroxyl group on these compounds interact on the cytoplasmic membrane andchanges its permeability by affecting lipid ordering and stability of its bilayer. Therefore proton passive flux across the membrane increased,whichdisruptedthecytoplasmic membrane and leading toleakage of cellular contents[35-38].Antifungal activity of the essential oil of T. vulgarisL. and Thymol on experimentally induced dermatomycoses was carried outby Sokovi et al. and found excellent results.
This study is a preliminary evaluation of antimicrobial activity of theThymus plant essential oil.
Present study concluded that the essential oil of T. vulgarisishighly active as fungitoxicants and could safely be used as natural preservatives to replace synthetic allopathicpreparationsin the prevention and cure ofsuperficial skin infection.The plant presented broad spectra of activity, may help to discover new antibiotics that could serve as selective agents for the maintenance of animal or human health and provide biochemical tools for the study of infectious diseases. This versatile medicinal plant is the unique source of varioustypes of chemical compounds, which are responsible for the various activities of the plant. Hencethe extensive investigation is needed to exploit their therapeutic utility to combat diseases.
Authors are thankful to University Grant Commission, New Delhi, India for providing financial assistance during research work (Award letter no 15-34/12(SA-II)dated 23.1.2012). The authors are also thankful to Head of the Botany Department, University of Rajasthan, Jaipur for providing laboratory facilities.
Neetu Jain researched and wrote the article while Meenakshi Sharma provided guidance, critical review, and revision. All authors read and approved the final version of present manuscript.
No conflict of interest
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How to cite this article
Neetu Jain, Meenakshi Sharma. Screening of Thymus vulgaris essential oil against fungi causing dermatophytosis in human beings.Int J Pharm Pharm Sci 2017;9(10):236-239.