Identification of Dermatophytes by DNA Markers and Their Antimycotic Studies Using Essential Oils

A medicinal plant is used by people for medicinal purposes and to build or maintain health. Traditions of healthcare must always been features of human societies and from the available evidences it is found that plants are playing a lead role in the therapy (Srivastava, 2005). Herbal medicine even today plays an important role in rural areas and various locally produce drugs are still being used as household remedies for various diseases especially in these areas for different ailments (Qureshi & Ghufran, 2005).

The investigators of Central Institute of Medicinal and Aromatic plants (CIMAP) Lucknow have screened about 3051 plants for their biological activity (Dhawan et al. , 1980). Only 10 plants exhibited activity against pathogen of superficial mycosis. Antimicrobial properties of essential oils obtained from aerial parts and seeds of aromatic plants such as oregano, thyme, lavender, rosemary, cinnamon, clove, basil, ginger, coriander, cilantro, eucalyptus, funnel and dill are well documented (Elgayyar et al. , 2001, Delaquis et al. , 2002, Kwon et al. , 2002 ; Jirovetz et al. , 2003, Lo Cantore et al. , 2004 and Soylu et al. , 2006). Examination of indigenous local herbs and plant materials have also been reported from around the world e. g. India (Ahmad & Beg, 2001), Argentina (Penna et al. , 2001) and Finland (Rauhaef al. , 2000).

Extract of Rhuburb was reported to be effective against Trichophyton, Microsporum and Epidermophyton (Itsuo, 1985). McCutchen et al. , (1994) screened more than one hundred methanolic plants extracts for antifungal activity and 30 extracts showed activity against 4 or more of the fungi assayed. Essential oils and extracts from aromatic plants have long been used for a wide variety of medicinal and domestic purposes (Brown, 1995). Basically they are mixture of 30 to 40 components of varying concentrations. Out of these only two to three compound are in major concentration and rest of all are in trace amount only. For example, linalool is the major component in Coriandrum sativum, menthol and menthone are the major component in Mentha piperita, carvacrol and thymol are the major component in Origanum compactum etc. Generally these major components of essential oil determine biological properties of that oil Pichersky et al. , 2006 and Ipek et al. , 2005. But in some studies (Santana Rios et al. , 2001 and Hoet et al. , 2006) it was revealed that biological activity of essential oil is not due to a particular major component but it was due to synergism of both major and minor component and even there are some cases where the minor component play important role then major component in the given activity (Franzios et al. , 1997).

So it is more important to study the whole composition of essential oil instead of one or two major component. Thakur et al. , (1982) have also reported the higher incidence of ringworm infection in goats during the months of November to March. Several investigators have contributed much on antimycotic activity of medicinal plants, yet the work is very fragmentary and meagre (Pattnaik et al. , 1996; Rai and Acharya, 2000). The exceptional enzymatic capabilities of aquatic fungi have led to their broad use in biotechnological applications (Adrio & Demain, 2003), including the degradation and detoxification of many organic and inorganic water pollutants. For example, studies on the aquatic hyphomycete Heliscus lugdunesis suggest a role of aquatic fungi in then environmental fate of aquatic pollutants (Augustin et al. , 2006). Itisha Singh, 2004 survey 60 samples of various types of soil from Himachal Pradesh and isolated 257 KF isolates, most from forest soil (86) followed by garden soil (44) and playground soil (39). This study attributed high dominance of Keratinophilic Fungi in such cold climate due to addition of keratin material from birds and animals.

Chrysosporium indicum is a most predominant of all fungal species. Jain and Sharma, 2012 did a wide survey for Keratinophilic fungi presence in and near University of Rajasthan, Jaipur and collected 67 samples from site like library, ICICI bank, Canteen road side area, PG study centre, hostels, garden etc. which yielded 192 isolates belonging to 21 species of Keratinophilic fungi and dermatophytes. Additionally they also concluded that Keratinophilic fungi presence dominates from natural to slightly alkaline pH. Kumawat et al. , 2013 surveyed keratin degrading fungal flora in industrial area of Jaipur and isolated 12 fungal strain from 58 soil samples in which Trichophyton mentagrophytes is most predominant (12. 5%), followed by Microsporum canis (10%). But the interesting part of study is the absence of Microsporum canis from samples which are collected from poultry farm soil. Deshmukh and Verekam, 2014 showed 25 samples from and different sites of Sanjay Gandhi National Park and isolated 73 isolates, Chrysosporium indicum (16. 0%) being the most predominant followed by Trichophyton mentagrophytes (12. 0%). These fungal identification is done both morphologically as well through ITS sequencing of ribosomal DNA. Mahariya and Sharma, 2017 performed an interesting study about fungal succession on kerationous nail and hair best of human origin and concluded that initial phase of fungal growth is highly dominated by non-keratinous fungi like Fusarium but as we progress from 75 days to 90 days of fungal growth, nearly all non-keratinophilc fungus eliminates and only 4 Keratinophilic Fungi C. tropicum, Trichophyton tonsurans, Trichophyton soudanses and Chrysosporium indicum remain persistent.

Coriander Coriandrum sativum belongs to family Umbelliferacea/Apiaceae and cultivated throughout the world with the help of seeds (Mhemdi et al. , 2011). When we talk about its overall production then India is the biggest producer, consumer and exporter of Coriander, with an annual production of 3 lakh tonne annually. The main component of both seeds and leaves of this plant are the essential oil and fatty acids (Coskuner and Karababa, 2007). Essential oil content in this plant varies from 0. 03% to 2. 6%, which is quiet good ration when we compare it with other plants of similar nature (Nadeem et al. , 2013). Knio et al. , 2007 used Coriandrum sativum essential oil against Ochlerotatus caspius (Mosquito, Larvae) with a concentration gradient of LC 50 15-158 ppm and find out some good results. From above example it is clearly evident that C. sativum oil also shows cidal activity in addition to antimicrobial activity. Three species of-Juniperus communis, J. oxycedrus and J. turbinate showed excellent results against most of the keratinophilic fungi like T. rubrum, T. mentagrophytes, M. canis, M. gypseum and E. flocossum.

These results were showed by both berry (at a concentration of 0. 08-10 ul/ml) and leaf (at a concentration of 0. 32-20 ul/ml (Cavaleiro et al. , 2006). Similarly Pyun and Shin (2006) used three species of Allium against Trichophyton at a concentration of 64 ug/ml and find excellent results. Oils extracted from fifteen medicinal plants were screened by Bansod and Rai (2008) for their activity against A. fumigatus and A. niger by disc diffusion method. Minimum inhibitory concentrations (MICs) of oils (%v/v) against A. fumigatus and A. niger done by agar dilution method while minimum cidal concentration (MCCs) data (%v/v) obtained by the broth micro dilution method. The results showed that the maximum antimycotic activity was demonstrated by oils of Cymbopogon martinii, Eucalyptyus globulus and Cinnamomum zeylenicum as compared to control, followed by Cymbopogon citratus which showed activity similar to control (miconazole nitrate).

The oils of Mentha spicata, Azadiracta indica, Eugenia caryophyllata, Withania somnifera and Zingiber offinale exhibited moderate activity. The oils of Cuminum cyminum, Allium sativum, Ocimum sanctum, Trachyspermum copticum, Foenicumlum vulgare and Elettaria cardamomum demonstrated comparatively low activity against A. fumigatus and A. niger as compared to control. Mixed oils showed maximum activity as compared to standard. These results support the plant oils can be used to cure mycotic infections and plant oils may have role as pharmaceutical and preservatives. The Nigella sativa essential oil was effective against Collectotrichum capsici and Sclerotinia trifoliorum while seeds are ineffective against A. fumigatus and A. niger. Nazmi-islam et al. , (1989) reported the antifungal activities of the oil of N. sativa seeds against twenty fungi (including pathogenic and industrial strains). All the oils were found to have significant activities against the fungi, but the volatile oil showed stronger and wider range of antifungal activities. MIC values of the Coriandrum sativum volatile oil against two pathogenic fungi viz. A. fumigatus and A. niger is maximum and minimum value recorded against A. flavus. Higher and aromatic plants have traditionally been used in folk medicine as well as to extend the shelf life of foods, showing inhibition against bacteria, fungi and yeasts (Hulin et al. , 1998). Most of their properties are due to essential oils produced by their secondary metabolism (Adam et al. , 1998).

Plant-derived essential oils have long been used as flavouring agents in food and beverages and, due to the presence of antimicrobial compounds; they have potential as natural agents for food preservation (Helander et al. , 1998). Essential oils are complex volatile compounds produced in different plant parts, which are known to have good antimicrobial activity (Kim et al. , 2001 and Singh, 2003). Goubran and Holmes, 1993, examined the keratinolytic properties of Coriandrum sativum for various functions in plants including conferring pest and disease resistance. The complexity in essential oils is due to terpene hydrocarbons as well as their oxygenated derivatives, such as alcohols, aldehydes, ketones, acids and esters (Wijesekara et al. , 1997).

The essential oils contents in different species is influenced by their genetic material and as well as environment (Charles and Simon, 1990) and finally, by crop and post-crop processing (Paakkonen et al. , 1990). Essential oils from higher plants are variously used in the preparation of cosmetics, perfumes, antiseptics and in food as a flavouring agent. These being natural products their demand are increasing day by day. Various essential oils are used as drugs, such as skin stimulants, antichloristic agents, sedatives, expectorants, stomachers, carminatives, diuretics, antiseptics and disinfectants besides their common uses.

These volatile oils are also used in the pharmacy to mask the disagreeable taste of certain medicines (Claus et al. , 1970; Steinegger and Hansel, 1972). Essential oils of aromatic and medicinal plants present a great potential of application as antimicrobial agents and their use as remedies has been recognised for long time (Kim et al. , 1995). Ibrahim et al. , 2015 screened 26 plants derived commercial essential oil against four dermatophytic and its related keratinophilic fungi and found that essential oil of Prunusarmeniaca, Prunusdulcis var. amara, Oleaeuro pea and Mentha piperita were the most potent of all. Additionally studied the comparative account of commercial essential oil with that of pure extracted oil and found that both have similar efficacy.