Sudan P, Goswami M, Singh J. Exploration of Antifungal Potential of Azadirachta Indica against Microsporum Gypseum. Biomed Pharmacol J 2020;13(2).

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Manuscript received on :06-02-2020
Manuscript accepted on :09-04-2020
Published online on: 09-05-2020

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Puneet Sudan^{1, 2*}, Manish Goswami¹ and Jitender Singh³

¹UIPS-Chandigarh University Gharuan-Mohali, Punjab, India

²Chandigarh College of Pharmacy-Landran Mohali, Punjab, India

³School of Pharmacy-Career point University-Hamirpur, Himachal Pradesh, India

Corresponding Author E-mail: cgc.ccp.ps@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/1960

Abstract

Throughout ancient times, human civilizations have greatly exploited plants which have directly or indirectly served the humanity for diagnosis and treatment of various ailments. Herbal drugs have the provision to endanger multifarious range of phytochemical and biochemical compounds which can be acclimated for the performance of diverse biological functions. Many of these phytoconstituents and herbal products have salutary effects on long-term health when consumed by the human and can be efficaciously used to treat various human ailments. The present investigation was conducted to evaluate the antifungal potential of various extracts of dried powder of leaves of Azadirachta indica by means of paper disc diffusion method, with petroleum ether, ethyl acetate, ethanol, and aqueous solvents in 25 μml, 50 μml and 100 μml concentrations against Microsporum gypseum. Clotrimazole was used as a standard. The findings from the research revealed that Azadirachta indica is a potent antifungal agent against Microsporum gypseum. The ethanol extract of fenugreek using 100 μml concentrations depicted the highest zone of inhibition of 20.520+ 0.84 mm and 47.720% of mycelial inhibition against a tested pathogen. While drug extracts in other solvents also revealed reasonable to least antifungal potential. This finding tells us that Azadirachta indica extracts tested proved to be a potent antifungal agent against Microsporum gypseum. It was found that ethanol extract of Azadirachta indica is best effective against tested strain. This exploration of Azadirachta indica extracts has confirmed its importance, particularly in the area of influence on dermatophytic fungal strain.

Keywords

Antifungal, Azadirachta Indica, Microsporum Gypseum

Introduction

The previous investigations about Azadirachta indica have been not only restricted to its known antifeedant activity on insects, but also as potent antifungal drug [1]. Fungi are a varied collection of saprophytic and parasitic eukaryotic microorganisms. Dermatophytoses is a skin disorder or skin infection which is caused by the fungi to the human skin. Superficial mycosis includes mild to chronic infections of scalp, skin, nail, and hair. Cutaneous infective agents like Trichophyton rubrum, Microsporum gypseum, and Epidermophyton floccosum are vital reasons responsible for dermatophytoses. Dampness, unhygienic conditions and sweat are major causative factors for the emergence of dermatophytic infection. The resistance and side effects of the synthetic drugs are the major concern and motivated many researchers for developing newer and also safer drugs of herbal origin [2, 3]. Roughly 25 % of the world population is infected from fungal infections of nails and skin, with a major prevalence in hot and humid areas [4]. There is progressive interest on the isolation of phytoconstituents with antifungal potential, from which terpenoids have revealed to produce either fungi static or fungicidal power on several harmful fungi.

Azadirachta indica was used as an air purifier and an ornamental tree in last century in arid zones of Africa and can be presently seen in many tropical Asian countries around the globe [5]. Azadirachta indica can be grown or cultivated in soil even with poor nutrition and also have capacity to survive in extreme temperatures of severe hot and even deep frost [6]. Azadirachta indica elaborates an immense array of phytoconstituents which are not only biologically active but are also chemically diverse and structurally variable with approximately 150 compounds isolated from different parts of the tree [7]. The leaves of Azadirachta indica constitutes 10-25% crude fibre, 45-50 % carbohydrates, 15-20 % crude protein, 2.5-7.0 % fat, 7.7-8.5% ash, 0.8-2.4% calcium, 0.13-0.24 % phosphorus [8] with amino acids  [9, 10]. Different varieties of sugars, carbohydrates, and polysaccharides have also been isolated from the bark and gum of Azadirachta indica [11, 12]. Carotenoids were also reported from a two-dimensional TLC profile of leaves extracts of neem [13]. Neem has multifarious pharmacological action on the human skin and soft tissue. It is clinically approved in many skin infections without any side effects.

In King George Medical College, Lucknow-India, Azadirachta indica lotion was formulated from extract of dried leaves and clinical trial was conducted, results were promising and beneficial in the treatment of ring worm and scabies. Extracts of Neem oil and some pure isolates (Nimbidin, Nimbidiol, and Nimbin) can also inhibit fungal growth on animals [14, 15, 16] and humans [17]. Leaf extracts were shown good improvement within 15-20 days in chronic cases [18]. The present investigation aimed to compare the antifungal activity of  various extracts of dried leaves of Azadirachta indica by means of paper disc diffusion method, with petroleum ether, ethyl acetate, ethanol, and aqueous solvents in 25 μml, 50 μml and 100 μml concentrations against Microsporum gypseum. 

Materials and Methods

Preparation of Plant Extract

Azadirachta indica leaves were collected from local areas of Chandigarh and Mohali. Leaves were dried in shade and kept safely in dry area. The dried Azadirachta indica leaves were grounded to form a powder with the help of a mechanical grinder. Herbarium sheet is submitted with the Pharmacognosy department Chandigarh college of Pharmacy- Landran Mohali with voucher no. CCP/AI/070. Extracts were prepared by extracting Azadirachta indica leaves powder successively with petroleum ether, ethyl acetate, ethanol, and aqueous solvents and tested against Microsporum gypseum. The prepared extract was weighed and stored in airtight sample bottles. The filtered extracts were tested against dermatophytes at three different concentrations viz. 25 μml, 50 μml and 100 μml.

Procedure and Procurement of Strain 

The antifungal potential of an extract of dried Azadirachta indica leaves were evaluated by the Paper disc diffusion method. The test organisms used were the dermatophyte strains of Microsporum gypseum, which was procured from IMTECH, Chandigarh. MTCC No. was 2829. Sabouraud Dextrose agar was used as a culture media according to the manufacturer’s direction. The dermatophyte cultures were aseptically inoculated on Sabouraud agar plate and subjected to incubation at 28^oC for approximately 3 days.

Phytochemical-Screening

Test for alkaloids

The ethanolic extract of leaves of Azadirachta indica was evaporated to dryness and the residue was subjected to indirect heat on a boiling water-bath with 2% HCL. After cooling, the mixture was filtered and few drops of Meyer’s reagent were added. Turbidity or yellow precipitation confirms the presence of alkaloids.

Test for glycosides

2-5 ml of extract of leaves of Azadirachta indica, 3 ml of glacial acetic acid and 1 drop of 5% ferric chloride were taken in a test tube. 0.5 ml of concentrated H₂SO₄ was added towards the walls of the test tube. Formation of blue colour in the acetic acid layer indicates the presence of cardiac glycosides.

Test for flavonoids

4 mg of extract of leaves of Azadirachta indica was treated with 1.5 ml of 50% methanol solution. The solution was warmed and metal magnesium was added. To this solution, 5-6 drops of concentrated hydrochloric acid was integrated and orange or red colour was observed for presence of flavonoids.

Test for reducing sugars

To 0.5 ml of extract solution, 1 ml of di hydrogen monoxide and 5-8 drops of Fehling’s solution was integrated at boiling and observed for brick red precipitate.

Test for saponins

5 ml of extract was shaken vigorously with 5 ml of distilled water in a test tube and heated. The formation of stable foam was accepted as an indication of the presence of saponins.

Antifungal Activity

In this research, Paper disc diffusion method was employed, and some amount of Sabouraud Dextrose agar was dispersed in Petri dishes, which were allowed to solidify. A micropipette was employed to introduce 0.1 ml. Spores on agar medium and was spread with the help of glass rod spreader under aseptic conditions. Sterilized discs (5 mm, Whatman No. 1 filter paper) were prepared by soaking in different concentrations of the extract 25 μml, 50 μml and 100 μml for approximately 5- 6 hour. After this duration, discs were removed and then allowed to dry. To evaluate the antifungal potential of dried leaf extracts of neem, various discs impregnated with different concentrations of the dried neem leaves extracts were positioned on the fungal spore or mycelium with the help of sterilized forceps. The Petri dishes incubated at 28 ºC for 72 hours. The antifungal potential was determined by measuring the zone of inhibition (ZOI) around the discs and percentage inhibition after the period of incubation [19].

Data Analysis

Data from antifungal screening was analyzed with the help of simple statistics from Microsoft Excel and recorded in appropriate tables as a mean standard deviation of the mean.

Results and Discussions

Antifungal potential of extracts of leaves of Azadirachta indica against the tested fungal strain Microsporum gypseum can be seen in Table 1. The Pet ether extract of Azadirachta indica showed 7.69 mm ZOI at 25 μml concentration. 50 μml concentrations were moderately effective with 12.21 mm zone of inhibition. At 100 μml, the zone of inhibition was observed to be as 14.51 mm. The ethyl acetate extract showed a 9.89 mm inhibition zone at 25 μml concentration. 50 μml concentrations were effective with 15.13 mm inhibition zone. 18.36 mm inhibition zone was observed at 100 μml. The ethanol extract showed a 13.21 mm inhibition zone at 25 μml concentration. 50 μml concentrations were moderately effective with 17.10 mm inhibition zone. At 100 μml, the inhibition zone was observed to be as 20.52 mm. While it’s aqueous extract showed a 4.56 mm inhibition zone at 25 μml concentration. 50 μml concentrations were effective with a 10.56 mm inhibition zone. 12.10 mm inhibition zone was observed at 100 μml concentration. The antifungal potential was determined by comparing the activity of extracts with the Clotrimazole, in which the zone of inhibition was 43mm. Percentage inhibition was also calculated, which was 47.72 % with 100 μml ethanol extract depicted in Table 2. The results of this study showed that of all the extracts screened, Azadirachta indica ethanol extract had higher inhibitory activity against the test organism in comparison to the standard drug. This could be as a result of better extraction with ethanol solvent. Other solvents also showed promising effects against the tested pathogen. As per results from previous studies, it was reported that topical application of a lotion prepared from 70% alcoholic extract of neem leaves was found to be effectual in chronic skin infections like ringworm infection, scabies and also eczema [18]. Antidermatophytic potential of neem leaf extract has also been reported against different species of dermatophytes including Trichophyton rubrum, Microsporum gypseum and Epidermophyton floccosum [20]. Different extracts of neem leaf are effective against variety of fungal strains, like Trichophyton rubrum, Microsporum gypseum, and Epidermophyton floccosum [17]. Aqueous extract of leaves of Azadirachta indica showed minimal percentage of inhibition against Candida albicans and Microsporum gypseum [21].On other hand, as per results of my research the ethanol extract is showing a high zone of inhibition. Standardization is a significant consideration to make sure of the relevance between the phytoconstituents and its multifarious pharmacological activities. The phytochemical tests performed, and results were recorded in Table 3.

Table 1: Mean Zone of Inhibition in different solvents (mm) of the Azadirachta indica

Table 2: Percentage inhibition (%) of various extracts of the Azadirachta indica

Table 3: Phytochemical components of different extracts (+ low concentration, ++ moderate concentration, +++ high concentration)

Conclusion

The present research confirmed the antifungal potential of crude extracts of Azadirachta indica leaves against Microsporum gypseum. The extent of antifungal potential varied depending on the polarity of the solvent utilized in the extraction process. From the study, it can be depicted that ethanol extract of neem leaves are promising in comparisons to other solvents used. Furthermore, quantitative phytochemical analysis can be conducted in the future to isolate and identify the phytoconstituents liable for the antifungal potential.

Acknowledgments

The author(s) received no specific funding for this work.

Conflict of Interests

There is no conflict of interest.

Funding Source

There is no funding source.

References

1. Govindachari TR, Suresh G, Gopalakrishnan G, Banumathy B, Masilamani S, Identification of Antifungal Compounds from the Seed Oil of Azadirachta indica. Phytoparasitica. 26(2):109-116 (1998).
2. Pawar PL, Nabar BM, Effect of Plant Extracts Formulated in Different Ointment Bases on MDR Strains. Indian J Pharm Sci. 72(3): 397-401(2010).
3. Olila D, Olwa-Odyek, Opuda-Asibo J, Antibacterial and antifungal activities of extracts of Zanthoxylum chalybeum and Warburgia ugandensis, Ugandan medicinal plants. Health Sci 1(2):66-72 (2001).
4. Nagabhushan, Raveesha KA, Shrisha DL. Antidermatophytic Activity of Eclipta prostrata against Human Infective Trichophyton and Microsporum spp. Int J Chem Anal Sci 4(2):136-138 (2013).
5. Lewis WH, Elvin-Lewis MPF. Neem Cultivation in Haiti. Bot. 7:69-70 (1983).
6. Radwanski S. Neem tree. 1. Commercial potential, characteristics and distribution. World crops Livest. 29:62-63, 65-66 (1977).
7. Subapriya R, Nagini S. Medicinal properties of neem leaves: a review. Med. Chem. Anticancer Agents 5(2): 149-160 (2005).
8. Keher, Nagi SS. Neem leaves as a feed for livestock. sci. (Bangalore), 18:325 (1949).
9. Dakshinamurthi K. The aminoacids in the leaf of Azadirachta indica (Melia). Sci. (Bangalore) 23: 125-126 (1954).
10. Mitra CR, Misra PS. Aminoacids of processed seed meal proteins. Agric. Food Chem 15: 697-700 (1967).
11. Naik BR, Paatabiraman TN. Studies on plant gums. Isolation and characterization of a high molecular weight glycoprotein from neem (Azadirachta indica) gum. Indian J. Biochem. Biophys 18: 202-205 (1981).
12. Fujiwara T, Takeda T, Ogihara Y, Shimizu T, Tomita Y. Studies on the structure of polysaccharides from the bark of Melia azadirachta. Pharm. Bull. (Tokyo), 30: 4025-4030 (1982).
13. Tirimanna ASL. Surveying the chemical constituents of the neem leaf by two-dimensional thin layer chromatography. IN proceedings of the 2nd international neem conference, Rauischholzhausen, West Germany 25, 67-74 (1984).
14. Murthy SP, Sirsi M. Pharmacological studies on Melia azadirachta Indian J. Physiol. Pharmacol 2: 456-46 (1958).
15. David SN. The antifungal activity of neem oil and its constituents. Mediscope 8: 322-325 (1965).
16. Thind TS, Dahiya MS. Inhibitory effects of essential oils of four medicinal plants against keratiniohilic fungi. Pharm 20: 147-148 (1977)
17. Khan M, Wassilew SW. The effect of raw material from the neem tree, neem oil and neem extracts on fungi pathogenic to humans. In Proceedings of the 3rd International Neem Conference, Nairobi, Kenya 10: 645-650 (1987).
18. Singh N, Misra N, Singh SP, Kohli RP. Melia azadirachta in some common skin disorders, a clinical evaluation. Antiseptic 76: 677-679 (1979).
19. Rawal, P., Adhikari, R. S. Evaluation of the antifungal activity of Zingiber officinale against Fusarium oxysporum f. Sp. lycopersici. Advances in Applied Science Research 7(2): 5-9 (2016).
20. Natarajan V, Venugopal PV, Menon T. Effect of Azadirachta indica (neem) on the growth pattern of dermatophytes. Ind J of Med Microbio 21 (2): 98-101 (2003).
21. Mahmoud DA, Hassanein NM, Youssef KA, AbouZeid MA. Antifungal activity of different Neem leaf extracts and the Nimonol against some important human pathogens. Brazilian Journal of Microbiology 42: 1007-1016 (2011).