IN-VITRO STUDY OF ANTIFUNGAL ACTIVITY OF VARIOUS ...

International Journal of Pharma and Bio Sciences RESEARCH ARTICLE

MICROBIOLOGY

IN-VITRO STUDY OF ANTIFUNGAL ACTIVITY OF VARIOUS COMMERCIALLY AVAILABLE ITRA (VOLATILE PLANT OIL) AGAINST THE KERATINOPHILIC FUNGI ISOLATED FROM SOIL PRABAL SINGH1, RUCHI BUNDIWALE2 AND LK DWIVEDI1* 1

Institute of Biomedical Sciences, Bundelkhand University, Jhansi-284128, UP, India 2 Department of Microbiology, College of Life Sciences Cancer Hospital and Research Institute Gwalior-474009, M.P., India

LK DWIVEDI Institute of Biomedical Sciences, Bundelkhand University, Jhansi-284128, UP, India

*Corresponding author

ABSTRACT Keratophilic fungi, a type of dermatophytes cause infection to hair, glabrous skin and nails of human beings and animals. Soil is well known to be supporting the transient existence of them. The volatile plant oils have been of concern recently to develop a new antifungal agent. The present study attempts to evaluate four different commercially available ItraBella (Lonicera x bella zabel), Kewda (Pandanus odoratissimus), Rajnigandha (Polianthes tuberosa) and Mogra (Jasminum sambac) for their antifungal activity against the Aspergillus flavus, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton verrucosum, Epidermatophyton floccosum and Microsporum nanum isolated from soil. A few among the selected Itra were observed and were found to be showing better antifungal activity than existing antifungal drugs like Terbinafine, Itraconazole and Fluconazole (taken as control). The maximum effect was shown by Bella against T.tosurans with inhibitory zone of 47mm. The paper concludes that the Itra can be used as antifungal agent against prevalent keratinophilic fungi.

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KEY WORDS Keratinophillic fungi, Lonicera x bella zabel (Bella), Pandanus odoratissimus (Kewda), Polianthes tuberosa (Rajnigandha) and Jasminum sambac (Mogra), Terbinafine, Itraconazole and Fluconazole

INTRODUCTION Keratinophilic fungi which have the ability to degrade the keratinous substrates have got attention in recent days. Several new species with pathogenic ability are emerging rapidly. Interestingly, the latest reports of teleomorphic development of these fungi on soil hair in almost all habitat of world are an additional outbreak. Approximately 270 keratinophic fungal floras are reported in India too 1 whose effect on human beings is a matter of concern. The public places like railway station, bus station, hospital, temple and even houses also which generally remain crowded, mostly get dust on the floor. The dust becomes seriously contaminated from various sources like shoes, barefoot, domestic animals and indoor air floras which subsequently affect humans and 2 cause dermatophytosis etc . The dermatophytosis is caused by dermatophytes which are responsible for most superficial fungal infections 3 and estimated to be the cause of acquiring a dermatophyte infection between 10-20% 4. Itra is the volatile oil obtained from plant parts (flowers, buds, leaves, twigs, bark, herbs, wood, fruits and root etc). it is used for purest non-alcoholic natural fragrances in many parts of the world. The word Itra is derived from Arabic word 'Itr' meaning a perfume and is generally used to signify a concentrate essence of fragrant material. These are very pleasant, stirring and easy to use as they combine the qualities of the whole plant in their process of creation. Since, due to increasing changes in environmental and biological constituents, the azoles and other antifungal drugs often fail to

stop the infections. Hence, there has been a need to search for an alternative antifungal agent which can work effectively. Supportively, few studies have reported the plant oil as an antifungal agent. Therefore, in the present investigation, efforts are made to evaluate the antifungal activity of four different widely preferred Itra- Lonicera x bella zabel (Bella), Pandanus odoratissimus (Kewda), Polianthes tuberosa (Rajnigandha) and Jasminum sambac (Mogra) against the keratinophillic fungi isolated from soil.

METHODS The soil samples were collected from different locations around the temples using UV sterilized brush, gloves and polythene and then inoculated with human & horse hair and human nail for two weeks at 25ºC to observe the fungal growth. The observed fungus on hairs and nails was scratched with needle and taken on streptomycin added (added to inhibit bacterial growth) Sabaraud’s Dextrose Agar (SDA) plates and incubated at 25ºC for 24 hours for colony development. The grown fungal strains observed after incubation were identified as Trichophyton verrucosum, T.mentagrophytes, T.tonsurans, Aspergillus flavus, A. niger, E.floccosum, M.nanum. The strains were identified by the morphological study of culture and microscopic analysis of developed colonies. The age of culture, growth rate, topography (flat or heaped), texture (yeast like powdery, granular, velvety, cottony etc.) and surface pigmentation of fungal growth were considered for culture study. Microscopically, the cellular morphology


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(unicellular/hyphal: septate/aseptate, branching or non -branching) General morphology (True mycelium or pseudo mycelium) and spore morphology (smooth/rough, shape attachment, conidiophores/ sporangiophore /arthospore /chlamydospore) were studied to identify the type of fungus grown on SDA plate. Now the antifungal activity of Itra was tested against isolated fungi using agar well diffusion method. In which 20 ml of Mueller–Hinton Agar (MHA) containing respective test fungal strain was prepared on sterilized petridish followed by punching of 4 wells of 7 mm each on every plate. 40 µl of every selected Itra (Bella, Kewda, Rajnigandha and Mogra) was then poured into each well and incubated at 28±2ºC for 48 hrs. A set of control containing Terbinafine, Itraconazole and Fluconazole, the known antifungal drugs was run in the similar fashion against each test organism. The diameter of zone of inhibition of Itra and control drugs were measured three times each with antibiotic zone measuring scale (Hi Media). The zone of inhibition ≥10mm diameter was considered as positive result.

RESULTS The diameter of zone of inhibition formed by Bella, Rajnigandha, Kewda and Mogra against T.tonsurans were observed 47mm, 34mm, 17 mm and 30mm respectively (Fig 2, Table-1) while it was 30mm, 22mm,15mm and 17mm against the M.nanum (Fig 3, Table-1). The activity was observed quite low against the Aspergillus flavus with 17mm, 15mm, 11mm and 13mm inhibitory zone shown by Bella, Rajnigandha, Kewda and Mogra respectively (Fig 4, Table-1). While it was intermediate against the Epidermatophyton floccosum with 27mm, 20mm, 21mm and 15mm of zone inhibition (Fig 5, Table-1). Thus, the maximum antifungal effect was shown by Bella with 47mm inhibitory zone against the T.tosurans, (Fig 2) and the minimum was 11mm shown by Kewda against the Aspergillus flavus (Fig 4). Significantly, the antifungal activity shown by few Itra was comparatively better than control (antifungal drugs, Terbinafine, Itraconazole and Fluconazole) (Table-1).

Table 1 Comparative data of antifungal activity shown by controlled and selected Itra against different fungal isolates. Zone of inhibition (in mm) Control

Test organisms Aspergillus flavus Microsporum nanum Epidermatophyton floccosum Tricophyton tonsurans

Treated

Terbinafine

Itraconazole

Fluconazole

Pandanus odoratissimus (Kewda)

Jasminum sambac (Mogra)

Polianthes tuberosa (Rajnigandha)

Lonicera x bella zabel (Bella)

11

14

28

11

13

15

17

45

-

28

15

17

22

30

19

14

-

15

21

20

27

13

20

-

17

30

34

47


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Z o n e o f in hib itio n(m m )

Antifungal activity of itra against different fungi 50 40

A.flavus

30 20

M.nanum E.floccosum

10

T.tonsurans

0

ac us bel o sa lla za tuber inum samb attsim a x be anus odor polianthus r m e s c ja e Lon pand Itra

Figure - 1 The antifungal activity of Itra against different fungi isolated from soil.

Figure – 2 The antifungal activity shown by (A) Bella, (B) Mogra, (C) Kewda and (D) Rajnigandha in comparison to (T) Terbinafine (the antifungal drug taken as control) against the Trichophyton tonsurans

A

B

C

D

Figure – 3 The antifungal activity shown by (A) Bella, (B) Mogra, (C) Kewda and (D) Rajnigandha in comparison to (F) Fluconazole (the antifungal drug taken as control) against the Microsporum nanum This article can be downloaded from www.ijpbs.net

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A

C

B

D

Figure – 4 The antifungal activity shown by (A) Bella, (B) Mogra, (C) Kewda and (D) Rajnigandha in comparison to (T) Terbinafine (the antifungal drug taken as control) against the Aspergillus flavus

Figure – 5 The antifungal activity shown by (A) Bella, (B) Mogra, (C) Kewda and (D) Rajnigandha in comparison to (I) Itraconazole (the antifungal drug taken as control) against the Epidermatophyton floccosum

DISCUSSION Keratinophilic fungus, being natural decomposer of keratinized residues 5 is one of the ecologically important microorganisms. Similar to dermatophytes they cause skin and hair infections in human 6 and animals 7. Due to lack of efficacy, side effects and resistance of the existing antifungal drugs 8 new antifungal agents are being explored by scientists. In continuation of that, the Itra (plant based perfumes) especially Bella (Lonicera x bella zabel) tested in our study have proved to be a good antifungal agent against the keratinophilic fungi which may be due to phenols, aliphatic acid, aldehydes, terpenes and alcohols 9 found

as active components in plant based perfumes. Moreover, they have also been shown to possess antibacterial 10, 11, 12 antiviral 10, insecticidal 13 and antioxidant properties 14. The significant difference in the tolerance of tested fungi towards selected volatile plant compounds was due to the differences in their metabolic capabilities. Their inhibitory effects were interesting in terms of drop in concentration and mold growth which could be used instead of synthetic products.

CONCLUSIONS Though, the results of other similar studies may fluctuate due to composition of extracted


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smell. Moreover, the selected plants’ extracts may work as an excellent agent for development of remedies against many fungal diseases including mycosis and dermatophysis.

oil of selected plants, which most of the time depends on the geographical conditions, variety & age of plant, method of extraction etc, the volatile plant oils, currently used as perfumes may be used to get rid of keratinophilic fungal infection along with body

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