Evaluation of anti-dermatophytic activity of medicinal plants derived products

INTRODUCTION

Dermatophytosis is most common superficial skin infection in India. Dermatophytes are fungi that invade within keratinized tissues; skin, hair and nails and causes infection. On the basis of their genera, dermatophytes can be classified into three groups: Trichophyton, Epidermophyton and Microsporum. Dermatophyte can be classified on the basis of their natural habitat like anthrophilic which spread from one person to others, geophilic associated with soil which transfer to human from soil and zoophilic which infect animals and easily get transfer from animals to human beings. Clinically dermatophyte infections have been classified into Tinea capitis, Tinea faciei, Tinea barbae, Tinea corporis, Tinea mannum, Tinea cruris, Tinea pedis, and Tinea unguium. Other clinical variants are Tinea imbricata, Tinea pseudoimbricata, and Majocchi granuloma.¹

In last 10 years the prevalence of dermatophyte has increased. Hot and humid weather, working conditions of people and other predisposing factor play important role to increase the infection but development of resistant strains to a range of antifungal drugs is also an important factor to increase the prevalence of infection.²

From last many decades many antifungal drugs were used to treat the dermatophytosis like griseofulvin used from last six decades and imidazole and ketoconazole were used from last four decades. Many other antifungal drugs like terbinafine, fluconazloe, itraconazole etc. are also popular in clinical practice.³

These antifungal agents are effective for treatment of dermatophytosis but also have many side effects for the patients. These drugs need longer periods of treatment for cure and with time some dermatophyte species acquire resistance from these drugs, therefore there is need to search new alternatives which have anti-dermatophyte activity and not harmful for human health and are also cost effective. Different traditional medicinal plants are used from centuries in many parts of the world for the treatment of dermatophytes.⁴

Many studies reported that some medicinal plants are very useful to treat various skin diseases including dermatophyte infections. As these medicinal plants are natural so have low cost, high availability, fewer side effects and valuable resources.

Development of antifungal compounds with diverse chemical structures and novel mechanisms of action is necessary because there has been an alarming increase in the incidence of new and re-emerging infectious diseases as well as resistance to currently used drugs. The investigations on new antifungal substances should be continued and all possible strategies and techniques need to be explored further.

The study aims at finding the anti-dermatophyte activity of medicinal plants like Melaleuca alternifolia, Zingiber officinale, Allium sativum, Azadirachta indica, Citrus limonum, Curcuma longa and Cocos nucifera.

METHODS

The present research was conducted in the centre for Interdisciplinary biomedical research, Adesh university, Bathinda in the period of June 2017-June 2019. The sample was collected from the out-patient department of dermatology, Adesh hospital, Bathinda. Total 320 patients were included in this study in which 272 patients were having skin fungal infection and 48 patients were having nail fungal infection. After taking an informed consent/assent from all the participants and recording their socio-demographic information a detailed history was taken including age, sex, occupation, any allergy, duration of infection and other demographic profile. Patient unwilling to give consent were excluded from the study. The specimens were taken from the edge of the infected area and collected in a sufficient amount in 2 ml Eppendorf by using blunt edge of a sterile surgical blade. The collected specimens were labelled with name, gender, age and OPD number

Extraction of medicinal plant- In present study different medicinal plant extract was evaluated through agar well method. Medicinal plants Melaleuca alternifolia (tea-tree oil), Azadirachta indica (neem oil), Curcuma longa (turmeric), Allium sativum (garlic extract), Zingiber officinale (ginger extract), Cocos nucifera (coconut oil) and Citrus limonum (lemon oil) were used.

Fresh rhizomes of turmeric, ginger and garlic were purchased from local market. All the papery skin from rhizomes was removed and washed properly to remove all the soil. The fresh rhizomes were then peeled and sliced. The aqueous extract of turmeric, garlic and ginger was prepared freshly.

Allium sativum aqueous extract was prepared from garlic cloves. All the papery skin was removed and it was washed in sterilized MilliQ water. Then cloves were grinded into paste in sterile ceramic mortar pestle followed by the addition of 10 ml MilliQ water. The mixture was filtered and final concentration was stored in sterile container and explored for the sensitivity test.

Zingiber officinale extracts were also prepared from ginger. The rhizomes were washed with sterilized MilliQ water and peeled properly, and then crushed into mortar pestle to make paste. The paste was dissolved in 10 ml MilliQ water and then filtered. Thus, the aqueous extracts obtained were explored against the dermatophytes.

Curcuma longa extracts were prepared from the turmeric rhizomes. They were washed, peeled and crushed into mortar pestle to make paste. The paste was dissolved in 10 ml MilliQ water and then filtered. Thus, the aqueous extracts obtained were explored against the dermatophytes.

Every time fresh prepared garlic, turmeric and ginger extract was used for antifungal susceptibility test

Melaleuca alternifolia (tea-tree oil) 10 ml was used from ‘The body shop’. It is an essential oil that comes from steaming the leaves of the Australian tea tree.

Cocos nucifera (coconut oil), bio-organic extra virgin Coconut oil was used.

Citrus limonum (lemon oil), PRZ essential lemon oil was used.

Azadirachta indica (neem oil) 30 ml was used from ‘juicy chemistry’. It is a vegetable oil pressed from the fruits and seeds of the neem.

Procedure of antifungal susceptibility test: The isolates from Sabouraud dextrose agar (SDA) plates were sub cultured in 100 ml Sabouraud dextrose broth to enhance sporulation for 5 days at room temperature. After fungal growth, the suspensions were mixed on spinner and then allowed to sediment for 30 minutes. With a pipette 100 µl clear culture suspensions were taken from the side of the tube and were streaked evenly over the surface of Petri dishes containing Sabouraud dextrose agar. Lids were left open for 2-3 minutes in a laminar air flow to allow the excess moisture absorbed into the agar before antifungal mixture was applied. After that wells of 5 mm in diameter, 4 mm deep and 2 cm apart were punched in with a sterile cork-borer. Then 100 µl of medicinal plants extracts were loaded into each well in sterilize conditions and plates were kept at 4°C for 30 min for diffusion of supernatant into agar. The plates were incubated at 25°C for 5-10 days. The zone of inhibition around the wells were measured in mm and noted.

The statistical analysis was performed using the statistical package for social sciences (SPSS) software version 20.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were presented as measures of central tendency such as mean and standard deviation and categorical variables were presented as absolute numbers and percentage. Observations were analyzed using the Chi square test, Z test and proportion test, wherever applicable. For all statistical tests, a p<0.05 was considered significant.

RESULTS

In present study total 320 cases of dermatophytes were reported in which Trichophyton mentagrophytes was the most common isolated fungi followed by Trichophyton rubrum, Epidermophyton floccosum, Trichophyton tonsurans, Microsporum gypsesum, Trichophyton verrucosum and Microsporum canis.

The anti-dermatophyte activity was carried out on these etilogical agents. Medicinal plant such as Melaleuca alternifolia, Zingiber officinale, Allium sativum, Azadirachta indica, Citrus limonum, Curcuma longa and Cocos nucifera were investigated in this study as anti-dermatophyte. All dermatophyte species were found sensitive for Melaleuca alternifolia, Zingiber officinale and Allium sativum. These medicinal plants showed very good results as antifungal against dermatophytes while Azadirachta indica and Citrus limonum were moderate sensitive and Curcuma longa and Cocos nucifera did not show any zone of inhibition around the well (Table 1).

DISCUSSION

In spite of the development of science and technology, it has failed to develop a novel and efficient antifungal drug and there is need of more efficient antifungal drugs which can cure the specific fungi without any side effects on human beings. The duration of treatment depends upon the site, type of infection and symptom. The dosage depends on the clinical site and severity of infection and more important efficacy of the drugs.⁵

Sagar and Vidyasagar, also reported the use of suitable medicinal plants for dermatophyte infections in their study.⁶ The improved and better responses of these home remedies, minimum side effects, and cost effectiveness make these therapies favourable choice to combat fungal infection including dermatophytes.

In present study the medicinal plants were used as antifungal substance were Melaleuca alternifolia, Zingiber officinale, Allium sativum, Azadirachta indica, Citrus limonum, Curcuma longa and Cocos nucifera against different dermatophyte species like Trichophyton mentgophytes, Trichophyton rubrum, Epidermophton floccosum, Trichophyton tonsurans, Mycosporum gypsum, Trichophyton verricosum and Mycosporum.

Sharma and Malik, reported that Zingiber officinale, Citrus limonum and Curcuma longa essentials oils alone and in combination showed good antifungal activity against Trichophyton mentgophytes, Trichophyton rubrum, Mycosporum gypsum and Mycosporum canis.⁷ They used oils of these medicinal plants while in current study their extract were used as antifungal agents and zone of inhibition was also higher in this study.

Bokhari, reported that methanol extract and ethyl extract of lemon grass (Cymbopogon citrates DC.), lantana (Lantana camara L.), nerium (Nerium oleander L) basil (Ocimum basilicum L.) and olive leaves (Olea europaea L.) showed highest activities against Trichophyton rubrum followed by Microsporum canis, Micosporum gypseum, and Trichophyton mentagrophytes.⁸

Dilig et al, conducted a study of antifungal activity with twenty different medicinal plants and result showed that garlic juice and methanol extract of calamansi were inhibitory to Microsporum canis and Trichophyton rubrum.⁹

Adiguzel et al, reported that the methanol extract essential oil of Satureja hortensis plant has a strong antimicrobial activity against eight bacteria and three fungi.¹⁰

According to Abinu et al, scientists are more interested now days to use more plants extract for antimicrobial activities and identifying the compounds responsible for the antimicrobial properties.¹¹

Shaikh et al reported that Allium sativum, Zingiber officinalis, Glycyrrhiza glabra, Curcuma longa and Azadirachta indica have potent antifungal properties.¹²

Shahitha et al, reported that the medicinal plant Lawsoniainermis (henna) show good results against dermatophyte. It showed maximum zone of inhibition against Mycosporum canis (41 mm), Trichophyton equinum (30 mm), Trichophyton rubrum (27 mm) and Mycosporum gypsum (24 mm).¹³

Imo and Za’aku, reported in their review article that Garlic (Allium sativum) and Ginger (Zingiber officinale) has many medicinal properties and antifungal activity is one of them.¹⁴ Similarly current study reported that garlic and ginger show good antifungal activity as compare to other medicinal plants against seven different dermatophyte species.

CONCLUSION

The antimicrobial properties of medicinal plant have been observed and documented all over the world. In present work the medicinal plants Melaleuca alternifolia, Zingiber officinale, Allium sativum, Azadirachta indica has shown good antifungal activity against different dermatophyte species. This study showed that the plant extract and oils from plants possesses active ingredients that could be employed in their purified form to cure dermatophytes. Natural plant-derived fungicides may be a source of new alternative active compounds, in particular with antifungal activity. These preliminary results, obtained from in vitro experiments, may be supplemented by other more comprehensive studies in vivo, both in controlled greenhouse conditions and in open field to practically evaluate the use of these extract in the frame of an integrated pest management system. Subsequently, this can help researchers to look for new bio stimulatory agents.

ACKNOWLEDGEMENTS

We are extremely grateful to Dr. Ramgopal Saini, Head of Department of Centre for Interdisciplinary Biomedical research for his patience and motivation. He played a pivotal role in this work with his unerring accuracy and ability to solve the problems promptly.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the institutional ethics committee

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