Keratinophilic Fungi Isolated from the Soil in Saharanpur up of India

International Journal of Advanced Science and Technology Vol. 29, No. 04, (2020), pp. 11061-11068

Keratinophilic Fungi Isolated From The Soil In Saharanpur U.P Of India And In Vitro Evaluation Of Antifungal Activity Of The Cinnamon, Peppermint And Jasmine Essential Oils Against These Fungi

Neeraj Choudhary, Richa Sharma and Krishna Kumar Department of Agricultural and Veterinary Science Jayoti Vidyapeeth Women’s University, Jaipur- 303007, Rajasthan, India

Abstract The present study was undertaken to assess the prevalence of dermatophytes and related keratinophilic fungi isolated and characterized from different soil samples of India and to evaluate the antifungal potential of selected herbal extracts against seven dermatophytes. Soil is rich in pathogenic and non pathogenic keratinophilic fungi including dermatophytes. Most of these fungal species have already been reported as dermatophytes causing infections of skin, hair, etc. Two keratinous fragments, human hairs and animals’ hairs were used for the growth of fungi by the hair-baiting technique. 8 fungal samples were collected and cultured on Sabouraud dextrose agar, containing chloramphenicol and incubated at 25 to 27°C. Isolates were identified on the basis of colony characterization and the morphology of the fungal strains. Eight different fungal species, namely, Trichophyton tonsurans, Trichophyton mentagrophytes and Trichophyton equinum were isolated. Out of them, T. mentagrophytes is most common T. tonsures and T. equinum and M.canis in this region. Evaluation of antifungal activity was carried out with different essential oil, namely, cinnamon oil peppermint oil and jasmine oil by the well diffusion method. In this assay, essential oil cinnamon peppermint oil and jasmine oil showed maximum antifungal activity. Therefore, these could be used as an alternative medicine against infections caused by dermatophytes.

Introduction Cinnamon Essential Oil

. Figure 1: Cinnamon oil (Cinnamomum Verum)

Classification of Cinnamon Kingdom Platae Clade Angiosperms Clade Magnoliids Order Lauraceae Genus Cinnamomum Species C-verum Binomial Name Cinnamomum Verum Cinnamon bark essential oil is safe for topical, aromatic, and internal use cinnamon bark essential oil is known for causing skin sensitivity, so this oil should always be diluted before using topically. Cinnamon kills viruses. Cinnamon essential oil is touted as a natural remedy for health concerns ranging from cough and colds to constipation. In addition, cinnamon essential oil is said to stimulate reduce stress, relieve pain, fight off infection, improve digestion and protect against insects. Cinnamon leaf oil can be used as an additive in soaps to seasonings. When used in aromatherapy- diffused, applied topically or added to your bath water it can have health promoting effects. A small human study suggests that merely smelling cinnamon can improve performance several types of tasks. Cinnamon is a good source of the powerful antioxidant manganese. Two teaspoons of cinnamon provides about half the RDA of manganese, a powerful antioxidant that is crucial for brain.

Figure 2: Peppermint oil (Mentha Piperita) Classification of Peppermint Oil Kingdom Plantae Clade Angiosperms Clade Eudicots Clade Asterids Order Lamiales Family Lamiaceae Binomical Name Mentha Piperita Peppermint (Mentha piperita Linn. (M. piperita) belongs to the family of Lamiaceae. Till date, all the 600 kinds of mints are raised from 25 well-defined species. The two most important are peppermint (M. piperita) and spearmint (Mentha spicata). Spearmint bears the strong aroma of sweet character with a sharp

ISSN: 2005-4238 IJAST Copyright ⓒ 2020 SERSC 11062 International Journal of Advanced Science and Technology Vol. 29, No. 04, (2020), pp. 11061-11068 menthol undertone. Its oil constituents contain carvacrol, menthol, carvone, methyl acetate, limonene and menthone. The pharmacological action is due to menthol, a primary constituent of peppermint oil. At least 44% free menthol is present in peppermint oil. Components are sensitive to climate, latitude and maturity of the plant. Inhalation and application of menthol on skin causes a skin reaction. It is used in many liniments dosage form to relieve pain spasms and arthritic problems. Workers studies the peppermint oil for documented antispasmodic properties of oil make it a better choice during pain associated with the menstrual cycle and are also used in the treatment of irritable bowel syndrome. When superficially applied around the head and temple, it has very good action on headache. Further, itching due to various reasons like herpes blisters, ringworm infestation, scabies, poison oak, and ivy can also be relieved. It is observed that it can relieve many bacterial, fungal, and viral infections when inhaled or applied in the form of a vapor balm. Sinus and lung congestion are also known to be cleared from this oil. Much have been said and discussed about the menthe oil by many researchers for its various activities but its use in aroma therapy needs more efforts.

Figure 3: Jasmine oil (Jasminum Officinale) Classification of Jasmine Oil Kingdom Plantae Clade Angiosperms Clade Eudicots Clade Asterids Jasminum Order Lamiales Family Oleaceae Genus Jasminum Binomical Name Jasmine oil is excellent in treating dry dehydrated skin.. Jasmine oil is an essential oil derived from the white flowers of the common jasmine plant, also known as jasminum officnale. Jasmine oil besides acting as an excellent agent in fighting infections can also be used for its revitalizing effects on people. However, since it does have certain non sensitizing effects, it is not always pleasant to use on cracked or open wounds on the skin, as it can cause an allergic reaction, or irritation. Its aroma can prove effective in the treatment of insomnia and depression. It is a known as a popular aromatic herb jasmine essential oil has a sweet, exotic and richly floral smell. The more common species – jasminum grandiflorum is also used for essential oil extraction, but our 25%

ISSN: 2005-4238 IJAST Copyright ⓒ 2020 SERSC 11063 International Journal of Advanced Science and Technology Vol. 29, No. 04, (2020), pp. 11061-11068 blend is made from jasminum officinale. Jasmine oil facilitates delivery in childbirth, it hastens the birth by strengthening the contractions and at the same time relieves pain. It is effective in post –natal depression and promotes the flow of breast milk. Jasmine oil tones dry, greasy, irritated and sensitive skin, increases elasticity and is often used to assist with stretch marks and to reduce scarring. Materials and Methods Selection of the Study Area The study was carried out at the Jayoti Vidyapeeth Womens University, Vedant Gyan Valley, Jharna, Jaipur, Rajasthan and it is 475.5 km away from Saharanpur city (U.P Collection of Different Plants for Extraction of Essential Oil The essential oil was purchased from Aadhunik Ayurveda Vitals Khasra no 79, Mohbaewala Industrial area Dehradun–Uttrakhand. Cinnamon bark oil, Jasmine flower oil and peppermint leave oil. Antimycotic Studies of Essential Oils against Selected Fungi by Disc Diffusion Method Oil was screened for their antifungal activity against fungus by disc diffusion method Rios et al., 1988. SDA medium f or disc diffusion test was prepared. After sterilization it was poured in to sterilized petriplates and allowed to solidify. Then one day old fresh culture of keratinophilic were be used for inoculums preparation. A suspension was just tubid (~0.5 McFarland standard) by visual inspection was prepared by suspending fungus in 0.9% NaCl solution and the homogeneous suspension was used for inoculation. Using a sterile cotton swab, fungal cultures were swabbed on the surface of sterile Sabourauds dextrose agar plates. Sterilized filter paper discs were soaked in neat, undiluted (100%, 75%, 50%, 25%) concentration of oils. Using an ethanol dipped and flamed forceps, oil saturated discs of 100%, 75%, 50%, 25% concentration were aseptically placed over Sabourauds dextrose agar plates seeded with the respective test microorganism. The antibiotic discs of Clotrimazole and Ketoconazole (10 mcg disc-1) were also aseptically placed over the seeded Sabourauds dextrose agar plates as a standard drug for comparison of antifungal activity of Cinnamon oil, peppermint oil and Jasmine oils. The plates were incubated at 28ºC for 24 hr. The diameter of the inhibition zones was measured in mm. Three replicates were kept in each case and average values were calculated. The activity of oils was measured by the following formula. Inhibition zoneof sample AI Activityindex = Inhibition zoneof standard

Results and Discussion In the present study, the antifungal activity of peppermint oil, jasmine oil and cinnamon essential oils against dermatophytes were evaluated. The selected test fungi were T. mentagrophytes T. tonsurans, T. equinum and M. canis. In the current study, T. mentagrophytes T. tonsurans, T. equinum, and M. canis was found predominant dermatophytes isolated from soils. Disc diffusion method was employed for the screening of essential oils. The diameter of Inhibition at different concentrations 100%, 75%, 50% and 25% of peppermint oil, cinnamon oil and jasmine oil as shown in table no 4.20, 4.21 and 4.22 against selected dermatophytes the present results suggest that peppermint oil, cinnamon oil and jasmine oil exhibits strong antifungal activity. This is in agreement with the findings of Valero and Frances, 2006. Many Curcuma species are traditionally used for their medicinal properties. Antifungal, antibacterial and anti-inflammatory activity has been reported for species such as Curcuma longa, Curcuma zedoaria, Curcuma aromatic and Curcuma amada Apisariyakul et al, 1995. In the present study, essential oil of jasmine, cinnamon and peppermint exhibited the strong antimycotic activity

ISSN: 2005-4238 IJAST Copyright ⓒ 2020 SERSC 11064 International Journal of Advanced Science and Technology Vol. 29, No. 04, (2020), pp. 11061-11068 against. T. tonsurans and T. equinum. In screening of peppermint oil and jasmine oil the diameter of inhibition zone by disc diffusion method was found to be against T. tonsurans and T. equinum at 100% concentration of pure oil. Our work is in agreement with the observations of Wuthi-udomlert et al., 2000. Several antimycotic drugs are available in the market, but due to various side-effects, long duration of treatment and high cost of the drugs, treatments have not been successful in some cases. The traditional use of plants as medicines provide the basis for indicating which essential oils may be useful for specific medical conditions. It is important to investigate scientifically those plants which have been used in traditional medicines as potential sources of novel antimicrobial compounds. Who reported the antifungal activity of turmeric oil against 29 clinical strains of dermatophytes and in screening of turmeric oil, diameter of inhibition zone was found to vary from 26.1 mm to 46 mm against 29 clinical strains of dermatophytes. There are numerous scientific studies which are proves the inhibitory effect of the essential oils against different fungi Duarte et al, 2000 and Falahati et al, 2005. It is important to analyze that the plants which have been used in the medicines as a potential source of normal antimicrobial compounds Mitscher et al, 1987. The present work coincides with the work of Sharmopa et al 2011 who also reported the additive and inhibitory effect of Mentha piperita and jasminum essential oils against skin infections. The use of essential oils in treatment and prevention from infection has been in demand in the field of research from the past Sherry et al, 2001 reported that of these oils can be used for the development of the natural antifungal agents against T. mentagrophytes, T. tonsurans, T. equinum and M. canis to prevent dermatophytic infections. Antibiotics known to be effective against each of the test micro-organisms in their established doses were used as reference for comparison with the antidermatophytes activity of the test samples. These were ketoconazole and clotrimazole was used as a standard for T. mentagrophytes, T. tonsurans, T.equinum and M. canis table no1, 2, 3, 4 and 5. In the present work T. mentagrophytes, T tonsurans, T. equinum and M. canis was found main etiological agent of dermatophytes disease hence the antimycotic studies of essentinal oils also was carred out on about isolated fungi. The treatment of fungal disease is limited and part of the reason is due to the limited spectrum of the currently antifungal drugs and the expensive treatment particularly due to the need of prolonged therapy. Pawar et al., 2006 who also reported drugs and alternative therapies are necessary including natural products. Because of high volatility and lipophilicity of the essential oils they are readily attached to penetrate in to the cell membrane to exert their biological effect. Ali et al 2010 also reported conducted in Saudi Arabia was designed to investigate the antifungal activity of plants from the Jeddah region (Aindica,Z.spina-christi, and O.europaea), P. dactylifera seed neem oil. The tested M. canis and T.mentgrophyta were compared to ketoconazole. Lesher 1996 reported that the ketoconazole is antifungal drug with wide spectrum of antifungal activity effective against several of these superficial infections, such as candidiasis, pitiriasis versicolor, tinea curis and dermatophytosis. Shi et al 2006 who also reported antifungal mechanism is inhibits the synthesis of ergosterol in fungal and cholesterol in mammalian cells. The zone of inhibition of test organism growth around each well can be measured in order to enchange the detection limit the inoculated plates can be kept at a lower temperature for several hours before incubation. In this way compound diffusion can be over microbial growth and better Inhibition diameter is expected. The results showed the efficacy of peppermint oil, cinnamon oil and jasmine oil on the animals skin infection caused by T. tonsurans and T. mentagrophytes. Present study, peppermint oil exhibited the strong antimycotic activity against T. mentagrophytes, T. tonsurans, T. equinum and M. canis.

Table 1: Antifungal activity of Cinnamon oil against selected isolated fungal Concentration IZ of AI AI S. No Oil Test strain

of oil sample ketoconazole Clotrimazole

100% 59mm 5.36 2.36 75% 50mm 4.54 2

1. T. 50% 30mm 2.72 1.2

25% 20mm 1.81 0.8 mentagrophytes

100% 60mm 2.4 3

2. 75% 46mm 1.84 2.3

50% 31mm 1.24 1.55 T. T. tonsurans 25% 19mm 0.76 0.95

100% 58mm 2.23 5.27

3. 75% 45mm 1.73 4.09 Cinnamomum Verum oil

T. T. equinum 50% 31mm 1.19 2.81 25% 19mm 0.73 1.72

100% 51mm 2.55 4.25 4. 75% 40mm 2 3.33

M. M. canis 50% 30mm 1.5 2.5 25% 11mm 0.55 0.91 Here, IZ= Inhibition zone (in mm) including the diameter of disc (6mm), AI (Activity index) Table 2: Antifungal activity of peppermint oil against selected isolated fungal S. Concentration ZO of AI AI Oil Test strain

No of oil sample ketoconazole Clotrimazole

100% 60mm 5.45 2.4 75% 50mm 4.54 2

1. T. 50% 29mm 2.63 1.16

25% 12mm 1.09 0.48 mentagrophytes

100% 67mm 2.68 3.35 2. 75% 40mm 1.6 2

50% 30mm 1.2 1.5 T. T. tonsurans

Mentha piperita Oil 25% 15mm 0.6 0.75

100% 50mm 1.92 4.54 3. 75% 39mm 1.5 3.54 T. T. equinum 50% 29mm 1.11 2.63

25% 15mm 0.57 1.36

100% 59mm 2.59 4.91 4. 75% 40mm 2 3.33

M. M. canis 50% 30mm 1.5 2.5 25% 11mm 0.55 0.92 Here, IZ= Inhibition zone (in mm) including the diameter of disc (6mm), AI (Activity index) Table 3: Antifungal activity of Jasmine oil against selected isolated fungal Concentration IZ of AI AI S. No Oil Test strain

of oil sample ketoconazole Clotrimazole

100% 59mm 5.36 2.36 75% 46mm 4.18 1.84

1. T. 50% 36mm 3.27 1.44

25% 17mm 1.54 0.68 mentagrophytes

100% 60mm 2.4 3 2. 75% 49mm 1.96 2.45

50% 35mm 1.4 1.75

T. T. tonsurans

25% 16mm 0.64 0.8

100% 61mm 2.34 5.54 Jasminum Oil 3. 75% 40mm 1.53 3.63

T. T. equinum 50% 30mm 1.15 2.72 25% 13mm 0.5 1.18

100% 58mm 2.9 4.83 4. 75% 40mm 2 3.33

M. M. canis 50% 30mm 1.5 2.5 25% 15mm 0.75 1.25 Here, IZ= Inhibition zone (in mm) including the diameter of disc (6mm), AI (Activity index) Table 4: Antifungal activity of Ketoconazole drug against selected isolated fungal S. No drug Test strain IZ of sample 1. Ketoconazole T. mentagrophytes 11mm 2. Ketoconazole T. tonsurans 25mm 3. Ketoconazole T. equinum 26mm 4. Ketoconazole M. canis 20mm Here, IZ= Inhibition zone (in mm) including the diameter of disc (6mm), AI (Activity index)

Table 5: Antifungal activity of Clotrimazole drug against selected isolated fungal S. No Drug Test strain IZO of sample 1. Clotrimazole T. mentagrophytes 25mm 2. Clotrimazole T. tonsurans 20mm 3. Clotrimazole T. equinum 11mm 4. Clotrimazole M. canis 12mm Here, IZ= Inhibition zone (in mm) including the diameter of disc (6mm), AI (Activity index) Conclusion It was concluded that volatile vapours of essential oils possessed fungicidal activity at high dose level, preventing to resume growth after removal of essential oils. Therefore antifungal activity of volatile vapours of essential oils could be widely applicable in the variety of fungal infections, treatment and prevention. The positive results obtained in vitro suggest the use of these essential oils as a means of struggle against wood decay and consequently as a method of preservation of the wood. References 1. Apisariyakul, A. Vanittanakom, N. and D. Buddhasukh (1995). Antifungal activity of turmeric oil from Curcuma longa (Zingiberaceae). Journal of Ethnopharmacology. 49: 163–169. 2. Sierra, P. Guillot, J. Jacob, H. Bussiéras, S. and R. Chermette (2000). Fungal flora on cutaneous and mucosal surfaces of cats infected with feline immunodeficiency virus or feline leukemia virus. Am J Vet Res. 61:158–61. 3. Marcella, R. and R. Mercantini (1986). Keratinophilic fungi isolated from soils of the Abruzzo National Park Italy.Mycopathologia.94:97-107. 4. DeBoer, D. J. and K.A. Moriello (2006). Cutaneous fungal infections. In Greene CE (ed): Infectious diseases of the dog and cat. Elsevier Saunders, St Louis, Missouri. 555-569. 5. Fillipello, M.V. (2000). Keratinophilic fungi: Their role in nature and degradation of keratinic substrates. In: Biology of dermatophytes and other keratinophilic fungi. Kushawaha RKS, Guarri J, (eds), Spain, Revista Iberoamericana de Micologia. 7: 77-85. 6. Valero, M. and E. Francés (1995). Synergistic Bactericidal Effect of Carvacrol, Cinnamaldehyde or Thymol and Refrigeration to Inhibit Bacillus cereus in Carrot Broth.Food Microbiology,.23: 68-73. 7. Jain, S. K. and Agrawal, S.C. (2002). Fungistatic activity of some perfumes against otomycotic pathogens. Mycoses, 45: 88. 8. Alizadeh, A., Zamani, E., Sharaifi, R., Javan-Nikkah, M. and Nazari, S. (2010). Antifungal activity of some essential oils against toxigenic Aspergillus species. Commununications in Agricultural and Applied Biological Sciences, 75(4): 761-7. Annussek, G. (2001). Sesame oil. In: Gale encyclopedia of 9. Pawar, V.C. and Thaker, V.S. (2006). In vitro efficacy of 75 essential oils against A. niger. Mycoses, 49: 316-323. Pinto, E., Pina-Vaz, C. and Salgueirro, L. (2006). Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and Dermatophyte species. Journal of Medical Microbiology, 55: 1367-1373.