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ORIGINAL ARTICLE
Year : 2003  |  Volume : 21  |  Issue : 2  |  Page : 98-101
 

Effect of azadirachta indica (neem) on the growth pattern of dermatophytes


Department of Microbiology, Rajah Muthiah Medical College, Annamalai University, Annamalainagar - 608 002, Tamil Nadu, India

Correspondence Address:
Department of Microbiology, Rajah Muthiah Medical College, Annamalai University, Annamalainagar - 608 002, Tamil Nadu, India

 ~ Abstract 

PURPOSE: To determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) for the extracts of the leaves and seeds of the plant Azadirachta indica against various dermatophytes. METHODS: Clinical isolates of dermatophytes(Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum nanum) were treated with extracts of leaves and seeds of the plant Azadirachta indica (neem) for antifungal activity by in vitro tube dilution technique. RESULTS: The MIC of neem seed extracts was 31g/mL for all the dermatophytes tested. The neem seed extract at 15g/mL concentration (below MIC) was observed to be sufficient for distorting the growth pattern of the organisms tested. CONCLUSIONS: The changes in growth curve of the treated dermatophytes were found to be statistically significant with reference to the untreated fungi.

How to cite this article:
Natarajan V, Venugopal P V, Menon T. Effect of azadirachta indica (neem) on the growth pattern of dermatophytes. Indian J Med Microbiol 2003;21:98-101


How to cite this URL:
Natarajan V, Venugopal P V, Menon T. Effect of azadirachta indica (neem) on the growth pattern of dermatophytes. Indian J Med Microbiol [serial online] 2003 [cited 2019 Aug 20];21:98-101. Available from: http://www.ijmm.org/text.asp?2003/21/2/98/7983


Dermatophytes are the major cause of superficial mycoses of man and remain a public health problem especially in tropical countries such as India.[1] The humid weather, over population and poor hygienic conditions are conducive to the growth of dermatophytes. Even though it responds to treatment with conventional antifungals, the disease has a tendency to recur at the same or at different sites.
Herbal medicines have been known to man for centuries. Therapeutic efficacy of many indigenous plants for several disorders has been described by practitioners of traditional medicine. Azadirachta indica (Neem) is a tree which has been used for a long time in agriculture and medicine. Azadirachta indica is an indigenous plant widely distributed in India. The medicinal properties of the plant Azadirchata indica were studied by several workers. The antipyretic effect,[2],[3] antimalarial effect,[4],[5] antitumour effect,[6] antiulcer effect,[7] antidiabetic effect,[8] antifertility effect,[9] effect on the central nervous system,[10],[11] and cadiovascular effect[12] were some of the studies of the earlier workers. Antimicrobial properties of Azadirchata indica were studied by several authors. Rao et al[13] reported the antimicrobial activity of the seed oil against a variety of pathogens. The antifungal effects were reported of gedunin against polyporous wood rot,[14] of a leaf extract against Alternaria alternata[15] and of a mixture of sulphorous compounds from the steam distillate of fresh matured leaves against Trichophyton mentagrophytes in a concentration of 125g/ml.[16] We planned the present study to find out the antidermatophyte activities of 'neem' leaves and seeds and their effect on the growth pattern on dermatophytes.

 ~ Materials and methods Top

Plant extract preparation
The plant materials used in this study were collected from Annamalainagar, Tamilnadu. It was identified and authenticated by the Department of Botany, Annamalai University. Fresh leaves and ripened fruits were collected and dried in shade. The dried leaves were ground to powder and suspended in 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 in to three parts and each part was suspended in ethanol, ethyl acetate and hexane respectively in sterile 250 mL conical flasks and kept at 4oC overnight. Each 100 gms of powdered leaf material were soaked in 250 mL of ethanol, ethyl acetate and Hexane.
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).
In the case of seed, the seed coat was removed and 1 gm of seed was ground in 100 mL of 5% DMSO aseptically so as to achieve 1000 g/mL concentration and the filtrate was used.
Fungal Inoculum preparation
Twelve strains of Trichophyton rubrum which were isolated from clinical cases of tinea cruris, tinea corporis and tinea unguium, 10 strains of T.mentagrophytes which were isolated from tinea pedis, tinea corporis and tinea capitis and one strain of Microsporum nanum isolated from tinea corporis were tested.
These organisms were grown on Sabouraud dextrose (SD) agar. The 21 day old culture was scraped with a sterile scalpel and macerated in 10mL sterile distilled water. The suspension was adjusted spectrophotometrically to an absorbance of 0.600 at 450nm. Each tube was inoculated with 20 L of fungal suspension.
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)
MIC and MFC were determined according to the method described earlier.[17] MIC was determined by incorporating various concentrations of extracts (1000 g/mL to 31 g/ml) in SD broth. 20 l 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 20l of inoculum served as positive control. SD broth alone served as negative control. 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).
The MFC was determined using the method of Rotimi et al.[18] The tubes which showed no visible growth after 21 days incubation were subcultured on extract free SDA plates and incubated at room temperature for 21 days. The MFC was regarded as the lowest concentration of the extract that prevented the growth of any fungal colony on the solid medium.
Growth Pattern
The growth pattern of the organisms (T. mentagrophytes, T. rubrum, M. nanum) at dilutions of 15g/mL (below MIC) of 'neem' seed extract in SD agar were compared with the control. One millilitre of the standard suspension of the organism was inoculated on test and control plates. Both the plates were incubated at room temperature and observed for growth on every third day upto 30 days. Three sets of replicate test was done. A growth curve was plotted using time interval on X axis and extent of growth on (cm) Y axis. The results of the test were compared with that of the control. The statistical significance of the growth pattern of the test with reference to control was done by using two-way analysis of variance.

 ~ Results Top

The ethanol extracts of 'neem' leaves showed MIC and MFC at 250 g/mL concentration for all the strains of T. rubrum and M. nanum tested. MIC and MFC recorded for stains of T. mentagrophytes was 125 g/mL. The ethyl acetate extract of 'neem' leaf showed MIC and MFC at 125 g/mL for all the stains of T. rubrum, T. mentagrophytes and 250 g/mL for M. nanum. Hexane extracts of 'neem' leaf showed MIC and MFC at 500 g/mL for all the strains of T. rubrum, T. mentagrophytes and M. nanum. The 'neem' seed extract showed MIC and MFC at 31 g/mL for all the dermatophytes tested [Table].
The growth pattern of T. rubrum treated with 15 g/mL (below MIC) of 'neem' seed extract is shown in [Figure - 1]. The log phase was regular and continuous till the 18th day with 0.5cm growth in diameter on every third day in the control.
The maximum growth in diameter-recorded on 18th day was 3.5 cms. After the 18th day the growth was observed to become stationary. In contrast, distorted growth pattern was recorded for T. rubrum in the test plate. The log phase continued till the 12th day with a growth of 1cm diameter on 12th day after which the growth became stationary. The growth patterns of T. mentagrophytes and M. nanum on 'neem' seed extract and control plates are shown in [Figure - 2] and 3 respectively.
Statistical analysis showed that the growth pattern changes in the test were significantly different from the growth pattern in untreated culture.

 ~ Discussion Top

The MIC and MFC of the 'neem' seed extract were similar, which shows that MIC is sufficient for measuring fungicidal activity. 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.[19],[20] The present study showed that 'neem' seed extract has high antidermatophytic properties. 'Neem' seed extract at a concentration of 15g/mL (below MIC) was observed to distort the growth pattern of the organisms tested. This finding supports the use of 'neem' oil in the treatment of various skin infections by alternative systems of medicine. 

 ~ References Top

1.Venugopal PV, Venugopal TV. Antimycotic susceptibility testing of dermatophytes. Indian J Med Microbiol 1993;11(2):151-154.  Back to cited text no. 1    
2.Okpanyi SN, Ezeukwk GC. Anti-inflammatory and antipyretic activities of Azadirachta indica. Planta Medica 1981;41:34-49.  Back to cited text no. 2    
3.Khattak SG, Gilani SN, Ikram M. Antipyretic studies on some indigenous Pakistani Medicinal Plants. J Ethno Pharmacol 1985;14:45-51.  Back to cited text no. 3  [PUBMED]  
4.Tella A. The effects of Azadirachta indica in acute Plasmodium berghei malaria. Nigerian Medical Journal 1977;7:258-263.  Back to cited text no. 4    
5.Rochankij S, Thebraranonth.Y, Yenjal C, Yuthavong. Nimbolide a constituent of Azadirachta indica inhibits Plasmodium falciparum in culture. South East Asian J Trop Medi Public Health 1985;16:66-72.  Back to cited text no. 5    
6.Fujiwara T, Takeka TO, Gihara Y, Shimizu M, Nomura T, Tomuta Y. Studies on the structure of Polysaccharides from the bark of Medica Azardichta. Chemical Pharmaceu Bull 1982;30:4025-4030.  Back to cited text no. 6    
7.Pillai NR, Santhakumari G. Toxicity studies on nimbidin, a potential antiulcer drug. 1984 b;50:146-148.  Back to cited text no. 7    
8.Shukla R, Singh S, Bhandari CR. Preliminary clinical trial on antidiabetic actions of Azadirachta indica. Medicine and Surgery 1973;13:11-12.  Back to cited text no. 8    
9.Sinha KC, Riar SS, Tiwary RS, Dhawan AK, Bhadhan J, Thomas P, Kain AK, Jain RK. Neem oil as a vaginal contraceptive. Indian J Med Research 1984;79:131-136.  Back to cited text no. 9    
10.Pillai NR, Shanthakumari G. Effect of nimbidin as acute and chronic gastro duodenal ulcer models in experimental animals. Planta Medica 1984;50:143-146.  Back to cited text no. 10    
11.Singh PP, Junnarkar AY, Reddi GS, Singh KV. Azadirachta indica neuro-psychopharmacological and antimicrobial studies. Fitoterapia 1987;58:233-238.  Back to cited text no. 11    
12.Thompson EB, Anderson CC. Cardiovascular effects of Azadirachta indica extract. J Pharmaceu Sci 1978;67:1476-1478.  Back to cited text no. 12    
13.Rao DVK, Singh K, Chopra P, Chabra PC, Ramanujalu G. In vitro antibactericidal activity of neem oil. Indian J Med Research 1986;84:314-316.  Back to cited text no. 13    
14.Sundarasivarao B, Nazma, Madhusudhanarao J. Antifungal activity of gedunin. Curr Sci 1977;46:714-716.  Back to cited text no. 14    
15.Bhomick BN, Chooudhary BK. Antifungal activity of leaf extracts of medicine plants on Alternaria alternata. Indian Botanical Reporter 1982;1:164-165.  Back to cited text no. 15    
16.Pant N, Garg HS, Madhusudhanan KP, Bhakuni DS. Sulpharous compounds from Azadirachta indica leaves. Fitoterapia 1986;57:302-304 .  Back to cited text no. 16    
17.Irobi ON, Daramola SO. Antifungal activities of crude extracts of Mitra carpus villosus (Rubiaceae). J Ethno Pharmacol 1993;40:137-140.  Back to cited text no. 17    
18.Rotimi VO, Lanhon BE, Bartlet JS, Mosadomi HA. Activities of Nigerian chewing sticks extracts against Bacteriodes gingivalis and Bacteriodes melaninogenicus. Antimicrob agents chemother 1988;32:598-600.   Back to cited text no. 18    
19.Dixit SN, Srivastava HS, Tripathi RD. Lawsone, the antifungal antibiotic from the leaves of Lawsonia inermis and some aspects of its mode of action. Indian phytopathol 1980;31:131-133.   Back to cited text no. 19    
20.Natarajan MR, Lalithakumar D. Leaf extracts of Lawsonia inermis as antifungal agent. Curr Sci 1987; 56:1021-1022.  Back to cited text no. 20    
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2004 - Indian Journal of Medical Microbiology
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