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Research Article
 

In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis



Avinash Marwal, Surendra Meena, Subhash Chandra and Anima Sharma
 
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ABSTRACT

Dermatophytes as the name suggest are the fungus that feed on skin. The chief source of their growth is keratin which is widely available in skin, nails and hairs. Here we have evaluated an In vitro study of antidermatophytic activity of Mint (Mentha piperita) against two dermatophytes i.e., Trichophyton rubrum and Microsporum canis. The data in the manuscript is very much helpful in curing dermatophytic infections as an application from Biotechnological point of view. Distribution of Trichophyton rubrum and Microsporum canis was found to be 19.23 and 32.69%, respectively. At variable temperature Trichophyton rubrum and Microsporum canis showed maximum growth at 37°C (0.23 and 0.19 g dry weight of mycelium, respectively). At variable pH Trichophyton rubrum showed maximum growth at pH 7.5 and 8.0 (0.32 g dry weight of mycelium) and Microsporum canis showed maximum growth at pH 7.5 (0.39 g dry weight of mycelium). Mentha piperita highest keratinase activity against Microsporum canis was found to be 2.99 unit mL-1 with 2.91 mg mL-1 extracellular release of protein and in Trichophyton rubrum it was found to be 2.99 unit mL-1 with 2.75 mg mL-1 extracellular release of protein.

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  How to cite this article:

Avinash Marwal, Surendra Meena, Subhash Chandra and Anima Sharma, 2012. In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis. Journal of Medical Sciences, 12: 182-187.

DOI: 10.3923/jms.2012.182.187

URL: https://scialert.net/abstract/?doi=jms.2012.182.187
 
Received: December 24, 2012; Accepted: February 13, 2013; Published: April 24, 2013



INTRODUCTION

Trichophyton, Microsporum and Epidermophyton are the three genera in which all the available dermatophytes fall (Bassiri-Jahromi, 2013). Cases of dermatophytosis have increased over the past few decades. These infections are often recalcitrant to therapy (Weitzman and Summerbell, 1995). Number of antifungal products is present in the markets to cure dermatophytosis (Al-Howiriny, 2002; Aderogba et al., 2006). But these drugs possess side effects on the proper healing of the skin from infection leading to skin wrinkling, pealing and distortion. Therefore there is a shift towards natural plant extracts for unfolding the folded medicinal importance of some of the medicinally important plants i.e., Mentha piperita (Mint) which have no side effects (Siramon et al., 2013).

Dermatophytic infections are very common in tropical and subtropical regions of the world which supports the favorable conditions where dermatophytes prevail on their chief source keratin which is present in nails, hairs and skin (Soares et al., 2012; Sharma et al., 2012). Keratinases are proteolytic enzymes in nature secreted by dermatophytes to feed on keratin which is the main constituent of structures that grow from the skin (Barchiesi et al., 2001).

The present retrospective work was aimed at isolation of dermatophytes using Baiting technique and its growth at different physiological conditions on Potato Dextrose Broth. Further we have evaluated an In vitro study of antidermatophytic activity of Mint (Mentha piperita) against two dermatophytes i.e., Trichophyton rubrum and Microsporum canis followed by screening of crude extract and essential oils of Mentha piperita against the fungus for determination of Minimum Inhibitory Concentration (MIC). The effect of plant extract on keratinase activity was also determined. The present data is very much helpful in curing dermatophytic infections as an application from Biotechnological point of view.

MATERIALS AND METHODS

Collection of soil samples: The present study was conducted for isolation of dermatophytic fungi from 52 different soil locations in Jaipur city of Rajasthan province (India). All the soil samples were collected in sealed polythene bags using sterile spatula at a depth of 5 cm below the ground surface. Soil was passed through a 2 mm sieve and stored at 10°C until isolation of dermatophyes.

Isolation and identification of keratinophilic fungi from soil: The experimental work was done by using the To Ka Va hair Baiting technique in which defatted human hairs were used as a growth medium for isolation of dermatophytes from the soil samples (Deshmukh, 2004). In this technique petri plates were sterilized and half filled with the soil samples. Short strand of sterilize defatted human hairs were spread over the surface of the soil. 10-12 mL of sterile water was added to the petri plates and incubated at room temperature (20-25°C). For identification of fungus species, internal and external morphological features were studied. Many different funguses was identified and isolated from the soil samples. Out of which Trichophyton rubrum and Microsporum canis were used for the present study.

Effect of variation of pH and temperature: For the study of growth and sporulation of dermatophytic fungi at different pH (Bhadauria and Sharma, 2001), temperature (Baxter and Illston, 1980) conditions and maintenance of fungal cultures Sabourauds Dextrose Agar/Broth Medium and Potato Dextrose Broth Medium was used with shaking for 15 days on rotation speed of 30 revolutions per minute. On sixteenth day of inoculation the mycelium was harvested for determining the growth and sporulation.

Inhibitory effect of crude extracts of plants on dermatophytes: Fresh leaves were collected and dried in shade. The dried leaves were ground to powder and suspended into petroleum ether and kept in refrigerator overnight for removing all the fatty substances. After overnight incubation, the supernatant was discarded and the residue was dried at room temperature. The residue was further divided into four parts and each part was suspended in methanol, ethanol, ethyl acetate and diethyl ether respectively in sterile 250 mL conical flasks and kept at 4°C overnight (Biswas et al., 2012). Each 100 g of powdered leaf material were soaked in 250 mL of methanol, ethanol, ethyl acetate and diethyl ether. After overnight incubation, the supernatant was filtered through whatman No.1 filter paper and the filtrate was dried to evaporate the organic solvent at room temperature. The sedimented extract was weighed and dissolved in 5% Dimethyl sulfoxide (DMSO). Filter diffusion method was used for determining the antidermatophyte effect of crude extracts of plants (Gould, 1952).

Determination of minimum inhibitory concentration (MIC): The essential oils of Mentha piperita were screened against these fungus species to measure their Minimum Inhibitory Concentration (MIC). MIC was determined by incorporating various concentrations of plant extracts in SD broth. Twenty microliter of standard fungal inoculum was added to each tube and incubated at room temperature for 21 days. Suitable controls were also included. SD broth with 20 μL of inoculum served as positive control. SD broth alone served as negative control (Nikkon et al., 2003). The tubes in duplicate for each agent were incubated at room temperature for 21 days. The MIC was regarded as the lowest concentration of the extract that did not show any viable growth after 21 days of incubation (compared with control) (Natarajan et al., 2003).

Keratinase activity: The effect of plant extracts on keratinase activity was determined by culturing fungal samples on Basal/Minimal Salt Medium (BSM/MSM) supplemented with human hairs and their total protein content, specific activity and percent inhibition of enzyme activity was measured (Fernandez-Torres et al., 2006).

RESULTS

Screening of 52 soil samples from different habitats of the Jaipur city, such as SMS Hospital, Gardens, Farmhouse, Colleges, Roadsides, Hostels, Malls, Airport, Colonies, Fort, Palaces etc. was carried out for the presence of dermatophytes. Many different funguses was identified and isolated from the soil samples. Out of which Trichophyton rubrum and Microsporum canis were used for the present study. They were identified on the bases of shape arrangement of spores and other structures through Scotch Tape Mount staining technique. Distribution of Trichophyton rubrum was found in 10 Petri plates (19.23%) and Microsporum canis was found in 17 petri plates (32.69%) out of 52 soil samples (Table 1). The color, texture, pigmentation on reverse surface of the colony and other characteristics were also recorded for fungal identification.

Environmental factors play an important role in the growth and sporulation of Keratinophilic fungi. Effect of variable temperature and pH on selected dermatophytic fungi was analyzed from weight of mycelium and spore count. These fungi were then subjected to growth at different physiological conditions such as temperature (5, 25, 30, 37, 45, 50 and 55°C) and pH (5, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5) on Potato Dextrose Broth (PDB). The growth index was then measured.

At variable temperature (Table 2) T. rubrum and M. canis showed maximum growth at 37°C (0.23 and 0.19 g dry weight of mycelium, respectively). At variable pH (Table 3) T. rubrum showed maximum growth at pH 7.5 and 8.0 (0.32 g dry weight of mycelium) and M. canis showed maximum growth at pH 7.5 (0.39 g dry weight of mycelium). The in-vitro antifungal activity of organic solvent extracts of leaves of Mentha piperita i.e., methanol, ethanol, ethyl acetate and diethyl ether (Table 4) was investigated against two selected dermatophytes i.e., Trichophyton rubrum and Microsporum canis.

The ethanolic extract of leaves of Mentha piperita exhibited the strongest activity against all the tested dermatophytes. For Trichophyton rubrum it showed inhibition zone of 24 mm and activity index of 0.75. Microsporum canis exhibited inhibition zone of 13 mm and activity index of 0.52 against ethanolic extract. El-Ghorab et al. (2003) demonstrated that the ethanolic leaf extract of Mentha piperita is known to have a very low toxicity.

One might conclude that the Mentha piperita would probably produce less side effects toxicity compared with conventional chemotherapeutic agent. Moreover essential oils of Mentha piperita were also screened against these fungus species to measure their minimum inhibitory concentration (Table 5). The minimum inhibitory concentration of essential oils of Mentha piperita against Trichophyton rubrum is 70% and against Microsporum canis is 90%. This suggested that at this value the growth of fungus was totally retarded.

In vitro degradation of keratin (human hair) was found to be associated with the release of extracellular keratinase into culture medium by the fungal species. Both the fungal strains (Trichophyton rubrum and Microsporum canis) were able to produce keratinase at variable conditions but only in specific environmental conditions. Data obtained were analyzed and represented and percentage activity was determined.

It was found that Trichophyton rubrum and Microsporum canis grow best at temperature 37°C and pH 7.5 conditions. At this temperature and pH highest keratinase activity against Microsporum canis was found to be 2.99 unit mL-1 with 2.91 mg mL-1 extracellular release of protein (Table 6) and in Trichophyton rubrum it was found to be 2.99 unit mL-1 with 2.75 mg mL-1 extracellular release of protein (Table 7).

DISCUSSION

Here, we have checked the growth pattern of two dermatophytes i.e., Trichophyton rubrum and Microsporum canis at variable temperature and pH, showing their excellent growth at pH 7.5 and 37°C (Table 2, 3). This holds the same favorable condition of human skin where the dermatopytic infections are common. A number of naturally derived therapeutic agents are in currently use against dermatophytes and one of them is of Mentha piperita. Traditionally Mentha piperita has been extensively used for the treatment of stomach ache, common cold, aromatherapy, antioxidants, etc (Zheng and Wang, 2001).

Table 1: Abundance and external morphology of the mixed dermatophytic fungi from different soil samples of Jaipur
Image for - In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis
-: No growth, +: Poor growth, ++: Fair growth, +++: Good growth, ++++: Excellent growth

Table 2: Growth of Trichophyton rubrum and Microsporum canis at different temperature range
Image for - In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis
+: Poor growth, ++: Fair growth, +++: Good growth, ++++: Excellent growth

Table 3: Growth of Trichophyton rubrum and Microsporum canis at different pH range
Image for - In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis
+: Poor growth, ++: Fair growth, +++: Good growth, ++++: Excellent growth

Table 4: Screening of different organic solvents extracts of Mentha piperita against Trichophyton rubrum and Microsporum canis
Image for - In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis
Activity Index (AI) = Inhibition zone of sample/ Inhibition zone of standard, Inhibition zone of standard Ketoconazole against Trichophyton rubrum = 32 mm, Inhibition zone of standard Ketoconazole against Microsporum canis = 25 mm

Table 5: Minimum inhibitory concentration of essential oils of Mentha piperita against Trichophyton rubrum and Microsporum canis
Image for - In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis
-: no growth of fungus

Table 6: Effect of different concentration of leaf extract of Mentha piperita on Keratinase activity of Microsporum canis
Image for - In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis
Specific Activity (enzyme unit/mg protein) = Keratinase Activity (unit mL-1)/Protein (mg mL-1), Inhibition of Enzyme Activity (%) = 100/Control concentration X concentration of enzyme activity-100

Table 7: Effect of different concentration of leaf extract of Mentha piperita on Keratinase activity of Trichophyton rubrum
Image for - In vitro Study of Antidermatophytic Activity of Mint (Mentha Piperita) Against Trichophyton rubrum and Microsporum canis

The crude extracts and the essential oils of Mentha piperita had shown strong antifungal activity against the two isolated fungus from Jaipur city, their details are cited in the Table 4 and 5. The growth of Trichophyton rubrum was totally retarded at 70% concentration of plant extract, whereas in the case of Microsporum canis 90% concentration was found effective. The mint extracts has antifungal activity and suggested its therapeutic use in curing dermatophytosis.

CONCLUSION

The Mentha piperita extracts were found to be effective against Trichophyton rubrum and Microsporum canis. The results of the analysis of the essential oils of Mint were qualitative and quantitative. This study shows potential uses of extracts for antidermatophytic application. Use of plant extracts in the treatment regimen of various diseases are gaining importance as antimicrobial, antibacterial, antiviral and antifungal activities of many plants are reported. Antimicrobial properties of plant extracts are now recognized by several workers. Dose response studies are not needed because there are no side effects of natural products. Natural antifungals play an important role by restoring the barrier function of skin and allowing the skin to naturally replace itself. The present data is very much helpful in curing dermatophytic infections as an application from Biotechnological point of view, thus, this In vitro testing can help to determine the activities of new drugs and to find the right therapy.

ACKNOWLEDGMENT

The authors would like to acknowledge a vote of thanks to Department of Science and Technology, Rajasthan (India) for their financial support.

REFERENCES

1:  Al-Howiriny, T.A., 2002. Chemical composition and antimicrobial activity of essential oil of Salvia verbenaca. Biotechnology, 1: 45-48.
CrossRef  |  Direct Link  |  

2:  Aderogba, M.A., E.K. Okoh, I.N. Okeke, A.O. Olajide and A.O. Ogundaini, 2006. Antimicrobial and anti-inflammatory effects of Piliostigma reticulatum leaf extract. Int. J. Pharmacol., 2: 70-74.
CrossRef  |  Direct Link  |  

3:  Barchiesi, F., D. Arzeni, V. Camiletti, O. Simonetti, A. Cellini, A.M. Offidani and G. Scalise, 2001. In vitro activity of posaconazole against clinical isolates of dermatophytes. J. Clin. Microbiol., 39: 4208-4209.
CrossRef  |  Direct Link  |  

4:  Bassiri-Jahromi, S., 2013. Epidemiological trends in zoophilic and geophilic fungi in Iran. Clin. Exp. Dermatol., 38: 13-19.
CrossRef  |  PubMed  |  Direct Link  |  

5:  Baxter, M. and G.M. Illston, 1980. Temperature relationships of fungi isolated at low temperatures from soil and other substrates. Mycopathologia, 72: 21-25.
CrossRef  |  Direct Link  |  

6:  Bhadauria, S. and M. Sharma, 2001. Soil borne keratinophilic fungi in relation to habitat pH. J. Environ. Pollut., 8: 245-248.

7:  Biswas, S.M., N. Chakraborty, P. Chakraborty and S. Sarkar, 2012. Antioxidant and antimicrobial activities of hot pungent chabbarin are responsible for the medicinal properties of Piper chaba, Hunter. Res. J. Med. Plant, 6: 574-586.
CrossRef  |  Direct Link  |  

8:  Deshmukh, S.K., 2004. Isolation of dermatophytes and other keratinophilic fungi from the vicinity of salt pan soils of Mumbai, India. Mycopathologia, 157: 265-267.
CrossRef  |  

9:  El-Ghorab, A.H., K.F. El-Massry, F. Marx and H.M. Fadel, 2003. Antioxidant activity of Egyptian Eucalyptus camaldulensis var. brevirostrisleaf extracts. Food/Nahrung, 47: 41-45.
CrossRef  |  Direct Link  |  

10:  Fernandez-Torres, B., A. Carrillo-Munoz, I. Inza and J. Guarro, 2006. Effect of culture medium on the disk diffusion method for determining antifungal susceptibilities of dermatophytes. Antimicrob. Agents Chemother., 50: 2222-2224.
CrossRef  |  Direct Link  |  

11:  Gould, J.C., 1952. The determination of bacterial sensitivity of antibiotics. Edinburgh Med. J., 59: 178-199.
PubMed  |  

12:  Natarajan, V., P.V. Venugopal and T. Menon, 2003. Effect of Azadirachta indica (neem) on the growth pattern of dermatophytes. Indian J. Med. Microbiol., 21: 98-101.
PubMed  |  Direct Link  |  

13:  Nikkon, F., Z.A. Saud, M.H. Rahman and M.E. Haque, 2003. In vitro antimicrobial activity of the compound isolated from chloroform extract of Moringa oleifera Lam. Pak. J. Biol. Sci., 6: 1888-1890.
CrossRef  |  Direct Link  |  

14:  Sharma, A., S. Chandra and M. Sharma, 2012. Difference in keratinase activity of dermatophytes at different environmental conditions is an attribute of adaptation to parasitism. Mycoses, 55: 410-415.
CrossRef  |  Direct Link  |  

15:  Siramon, P., Y. Ohtani and H. Ichiura, 2013. Chemical composition and antifungal property of Eucalyptus camaldulensis leaf oils from Thailand. Rec. Nat. Prod., 7: 49-53.

16:  Soares, B.V., S.M. Morais, R.O. dos Santos Fontenelle, V.A. Queiroz and N.S. Vila-Nova et al., 2012. Antifungal activity, toxicity and chemical composition of the essential oil of Coriandrum sativum L. fruits. Molecules, 17: 8439-8448.
CrossRef  |  Direct Link  |  

17:  Weitzman, I. and R.C. Summerbell, 1995. The dermatophytes. Clin. Microbiol. Rev., 8: 240-259.
PubMed  |  

18:  Zheng, W. and S.Y. Wang, 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem., 49: 5165-5170.
CrossRef  |  PubMed  |  

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