Antifungal screening of 61 folkloric medicinal plant extracts against dermatophytic fungi Trichophyton rubrum

Article history: Received on: 04/02/2015 Revised on: 22/02/2015 Accepted on: 18/03/2015 Available online: 27/05/2015 A series 305 extracts from 61 traditional medicinal plants (belonging to 33 different families) used in treating skin diseases in Hyderabad Karnataka region were subjected to antidermatophytic screening against Trichophyton rubrum (MTCC 1344). Pet ether, chloroform, ethyl acetate, methanol and aqueous extracts of each plant were tested for their antifungal activity using agar well diffusion method at sample concentration of 5 & 2.5 mg/ml. The results indicated that out of 61 plants, 18 exhibited very effective antidermatophytic activity in methanolic extracts, effective activity observed in 13 plants in different extracts, whereas 26 plants showed moderate activity, 04 plants showed weak activity. The minimum inhibitory concentrations of 18 very effective plants were determined. On the basis of the results obtained, the crude extracts of Allium sativam Linn., Annona reticulata L., Annona squamosa L., Argemone mexicana L., Butea monosperma, Ceasalpinia bonducella, Citrus medica L., Corchorus oleterius L., Emblica officinalis, Euphorbia tirucalli L., Ficus racemosa L., Gymnosporia montana, Lawsonia inermis L., Solanum nigrum L., Sterculia foetida L., Tribulus terrestris L., Vitex negundo L., and Zingiber officinale exhibited significant antidermatophytic activity (T. rubrum) and properties that support folkloric use in the treatment of skin diseases as broad-spectrum antimycotic agents. This probably explains the use of these plants by the indigenous people against dermatological infections.


INTRODUCTION
Medicinal plants represent a rich source of antimicrobial agents (Mahesh & Satish, 2008).Many of the plant materials used in traditional medicine are readily available in rural areas at relatively cheaper than modern medicine (Mann et al., 2008).Plants generally produce many secondary metabolites which constitute an important source of microbicides, pesticides and many pharmaceutical drugs.Plant products still remain the principal source of pharmaceutical agents used in traditional medicine (Ibrahim, 1997;Ogundipe et al., 1998).This is because of a broader degree of chemical diversity and novelty of molecules found in natural products than that from another source.Fungal diseases have historically been a difficult clinical entity to effectively deal with.The available drugs to treat fungal infections have been limited.Furthermore, in this armamentarium there are problems with side effects, the rapid development of resistance, drug interactions and fungistatic mechanism of action.Thus, there is a need for the development of more efficacious antidermatophytic agents.Plants are the best source for identification of leads for the development of novel drug compounds (Raginee Verma, Satsangi G, 2011).
The antifungal activity of Indian medicinal plants has not been extensively studied and was described in only a few reports (Sasidharan, 1997).The dermatophytes are a group of closely related fungi that have the capacity to invade keratinized tissue (skin, hair and nails) of humans and other animals to produce an infection, dermatophytosis, commonly referred to as ringworm.They are classified in three anamorphic genera, Epidermophyton, Microsporum and Trichophyton.
There were no previous reports using folklore medicinal plants against T. rubrum from this region.Therefore, in the present report the antibiograme against a universal dermatophytic fungi T. rubrum response reported.

Plant materials
Plant materials were collected from various localities of Hyderabad Karnataka region and Identified with the help of Gulbarga district flora (Seetharam et al., 2000) the voucher specimens deposited in herbarium centre, Department of Botany, Gulbarga University, Karnataka, India.The collected plant materials were initially rinsed with distilled water to remove soil and other contaminants and dried on paper towel in the laboratory at 37 ± 2ºC for a week.

Preparation of the plant extracts
The selected plant part materials after shade drying were ground in a grinding machine in the laboratory.25g of shade dried powder was weighed and extracted successively with petroleum ether, chloroform, ethyl acetate, methanol and aqueous in soxhlet extractor for 48h.The extracts were concentrated under reduced pressure and preserved in refrigerator in airtight bottles for further use.

Microbial culture and growth conditions
Trichophyton rubrum (MTCC 1344) was used as test microorganism.Culture of T. rubrum grown on Sabouraud dextrose broth (HiMedia) at 28 •C for 48 h and it was maintained on agar slants at 4 • C.

Inoculum preparation
Stock inoculums of T. rubrum was prepared from 10-day cultures in PDA at 28 °C to induce sporulation.Fungal colonies were covered with 5 ml of sterile saline solution (NaCl 0.85 % w/v), the surface gently scraped with a sterile loop and the resultant mixture of fungal units was then transferred to a sterile tube.
The turbidity of the final inoculum was standardized according to a McFarland scale 0.5 tube and adjusted to a fungal population of 106 colony former units (CFU).The confirmation of inoculum quantification was done by plating 0.01 ml of inoculum suspension in Sabouraud dextrose agar (SDA).The dishes were incubated at 28 °C and examined daily for the presence of fungal colonies which were counted as soon as growth became visible (Santos et al., 2006;Hadacek, Greger, 2000).

Agar-well diffusion method
The assay was conducted by agar well diffusion method.About 15 to 20 ml of potato dextrose agar medium was poured in the sterilized petridishes and allowed to solidify.Fungal lawn was prepared using 5 days old culture strains.The fungal strains were suspended in a saline solution (0.85% NaCl) and adjusted to a turbidity of 0.5 Mac Farland standards (108 CFU/ml). 1 ml of fungal strain was spread over the medium using a sterilized glass spreader.Using flamed sterile borer, wells of 4 mm diameter were punctured in the culture medium and required concentrations of serially diluted extract (2.5, 5mg/ml) was added to the 20μl to each wells.
The plates thus prepared were left for diffusion of extracts into media for one hour in the refrigerator and then incubated at 30 o C.After incubation for 48h, the plates were observed for zone of inhibition.Diameter zone of inhibition was measured and expressed in millimeters.Dimethyl formamide (DMF) was used as a negative control.The experiments were conducted in triplicates (Magaldi, 2004)./

Minimum Inhibitory Concentration
One ml of sterile liquid Sabouraud medium was added to 08 sterile capped tubes, 1 ml of each solvent extracts suspension was added to tube 1.The contents were mixed and 1 ml was transferred to tube 2.
This serial dilution was repeated through to tube six and 1 ml was discarded from tube 6. Fifty μl of inoculum was added to tubes 1-8 and the contents were mixed.Medium control (no inoculum and no drug) and inoculum control (no drug) tubes were prepared.The final concentrations of each plant, solvent extract ranged from 05 mg/ml to 0.15 mg/ml.
The tubes were incubated at 30 o C for 96 h.The fungal growth in each tube was evaluated visually depending upon the turbidity in the tubes.MIC was defined as the drug concentration at which the turbidity of the medium was the same as the medium control.(NCCLS-1997)

Preliminary screening tests for secondary metabolites:
Preliminary tests, for the detection of secondary metabolites, were carried out for 305 extracts of 61 plants by adopting standard methods (Harborne, 1998).

Statistical Analysis
All the experiments were conducted in triplicate unless stated otherwise and statistical analysis of the data was performed by analysis of variance (ANOVA), using STATISTICA 5.5 (Stat Soft Inc, Tulsa, Oklahoma, USA) software.A probability value of difference p ~ 0.05 was considered to denote a statistically significant all data were presented as mean values ± standard deviation (SD).

RESULTS AND DISCUSSION
The plant extracts and their level of activity against Trichophyton rubrum was listed in Table 1.A series of 305 extracts from 61 ethno medicinal plants (parts were shown in figure.1) Belonging to 33 different families were used in treating Aloe vera, Milletia pinnata (petroleum ether).Whereas the weak activity observed in 04 plants, i.e., Lantana camara, Mentha viridis, Tridax procumbens and Zizyphus jujuba.There was no inhibition recorded from the negative control (DMSO), while the standard drug, Ketoconazole significantly inhibited (28.66±1.15 to 12. 33±1.52 mm) the growth of the test dermatophyte.
The maximum activity of very effective and effective plants extracts shown in figure 5 & 6 respectively.The minimum inhibitory concentrations of very effective 18 plants were determined, among the 18 plants extracts 04 i.e., Allium cepa Linn., Euphorbia tirucalli L., Lawsonia inermis, Tribulus terrestris L., were showed highest MIC at 0.31 mg/ml conc.(Figure 4).
In the present report the effective activity observed in 18 plants of methanolic extract concentrations of between 05 and 2.5 mg/ml, this result is in line with the work of Shinkafi and Manga, (2011), who reported that the aqueous and organic leaf extracts of Mitracarpus scaber and Pergularia tomentosa exhibited significant anti-fungal activities against T. mentagrophytes, T. rubrum and M. gypseum at extracts concentrations of between 80 and 160mg/ml.The activities of the methanol extract were higher, though not significant (P>0.05) when compared with the aqueous extract.The reason for this slight difference may be attributed to the solubility level of the phytoconstituents in the ext-racting solvents.It means that the methanol dissolved more of more of the active ingredients than aqueous solvent.
This reason is supported by Cowan (1999), who reported that organic solvent were better extraction solvent over water.The response of dermatophyte to treatment with various plant extracts varied from solvent extract to solvent extract; nevertheless it was shown to be dose dependent as greater .inhibition of growth was observed as the concentrations of the extracts increased.This is supported by the work of (Bharti and Vidyasagar, 2012;Shivakumar and Vidyasagar, 2014), where they revealed that, ethyl acetate extract showed antimycotic activity against T. rubrum and T. mentagrophytes.
Chloroform and methanol extracts showed activity at concentration of 50mg/ml where as ethyl acetate showed activity at high concentration of 150mg/ml.The aqueous extracts of Allium sativum and Ocimum sanctum at 10% conc.were more pronounced antifungal properties against the dermatophytic fungus Microsporum gypseum.(N.C.Sowjanya and C.Manohara Chary, 2012).
The MIC of 18 effective plants of methanolic extracts showed different values was recorded (Figure.4).The similar to that from the work of Ali-Shtayeh and Abu-Ghdeib (1999), who reported that aqueous extracts of 22 plants recorded wide variations in their MIC values against Microsporum canis, Trichophyton mentagrophytes and Trichophyton violaceum.This could be attributed to the variations in the phytochemical properties of the plants and differences among the fungal species.
In the present report the weak activity was observed in 04 plants, i.e., Lantana camara, Mentha viridis, Tridax procumbens and Zizyphus jujuba.Whereas in previous report Lantana camara was recorded as a most active in the 61plants series against pathogenic bacteria (V.Prashanth Kumar, Neelam S. 2006).This could probably suggest that certain phytochemicals exhibit their antifungal action only with other phytoconstituents in antibacterial.According to the previous similar report among the 21 plant methanolic extracts showed effective activity against two bacteria, but 20 plants were shown activity against two fungi (R. S. Taylor et al., 1995).
The methanolic and ethyalacetate solvent extracts were very effective and effective respectively in the present study.The similar results reported by Mehmood Z et al., (1999) ethanol extracts showed an inhibitory effect against the three Trichophyton spp.Preliminary phytochemical investigation revealed the presence of saponins, glycosides, tannins, alkaloids and flavonoids, as indicated in Table 2.
The crude successive extracts of 61 traditional medicinal plants were qualitatively screened for the occurrence of various secondary metabolites such as phenols (Lead acetate test), flavonoids (NaOH test), tannins (Ferric chloride test), alkaloids (Dragendroff's test), Saponins Foam test), glycosides (Keller-Killiani test).The reactions with these reagents have shown the presence of metabolites and record in the Table -4.
The present study suggests that the effective extracts of these plants is a potential source of natural antifungal agents.After this screening experiment, further work should be performed to describe the antifungal activities in more detail as well as their activity in-vivo.In addition, phytochemical studies will be necessary to isolate the active constituents and evaluate the antidermatophytic activities against a wide range of fungi population.

Table 2 :
Preliminary phytochemical screening for the detection of secondary metabolites in 18 effective folkoric plant extracts.