ANTIFUNGAL EFFECTS OF TAPINANTHUS GLOBIFERUS GROWING ON VITEXDONIANA AGAINST SOME FUNGAL ISOLATES

Objective: Fungal infections are the major cause of many skin diseases, especially in developing countries. Natural products of medicinal value represent a potential source of chemotherapeutic agents. Tapinanthus globiferus has been used extensively in ethnomedicine for the treatment hypertension, ulcer, cancer, diabetes and fungal infections without a scientific basis. This work was aimed at screening the phytochemical constituents and evaluating the antifungal properties of methanol leaf extract its ethyl acetate and n-butanol fractions of T. globiferus against some clinical fungal isolates including Candida albicans, Trychophyton mentagrophytes, Trychophyton rubrum and Aspergillus niger using agar well diffusion and broth micro-dilution techniques. Methods: Preliminary screening of phytochemical constituents of extract and fractions of T. globiferus indicated the presence of carbohydrates, alkaloids, glycosides, tannins, flavonoids, saponins, steroids and triterpenes. Results: The methanol extract and its fractions demonstrated significant (P<0.05) antifungal effect against all the test organisms with mean zone of inhibition ranging from 27.83±0.16-14.46±0.29mm which was higher compared to that of the standard drug, Fluconazole (26.1±0.44 –18.49±0.16 mm). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extract ranged between 6.25–25.0 mg/ml; ethyl acetate fraction had 3.13 – 25.0 mg/ml while n-butanol fraction had the least MIC ranging from 0.39-12.5 mg/ml against the test organisms. Conclusion: Study concluded that T. globiferus have good antifungal activity validating the ethnomedicinal claim for the use of the plant in the treating fungal diseases.


INTRODUCTION
Fungal infection and their complications continue to affect many nations and have claimed the lives of many people especially in Africa. The commonly implicated fungal pathogens includes Aspergillus, Candida Cryptococcus, Pneumocystis spp. Statistics showed that the incident of fungal infection is becoming outrageous each year and claimed the lives of about 1.4 million people worldwide 1 . In 2001, the mortality rate of about 20,000 in sub Saharan African was due to skin diseases 2 . Thus, skin diseases manifest due to lack of supply of potable water, malnutrition and poor environmental sanitation, these factors contribute to the burden of mycotic disease in Africa 3,4,5 and Nigeria inclusive. Medicinal plants have been a rich source of secondary metabolites that are widely used for their therapeutic application; this has been attributed to their affordability, accessibility and lesser side effects 6,7 .
About 80% of the world population still relies on plantbased traditional medicines for some aspect of their primary health care. Most often, the search for many potent drug candidates used in modern clinical practice has been achieved via research and development on medicinal plants with therapeutic applications 8 . Tapinanthus globiferus (Loranthaceae) is a semi or hemi-parasitic that grows mostly on the branches of different trees including Citrus, Acacia, Aloe, Vitellaria paradoxa, Kola, Terminalia and Combretum, as host trees 9,10 . It is widely distributed throughout the tropical and subtropical regions of Western and Eastern African. The plant is used in ethnomedicine to treat itching 8 , tumor 7 , ulcers, hypertension, epilepsy, diabetes, promoting relaxation of muscles before delivery and weakness of vision 11 and it is also used to remove placenta after parturition 12

Preparation of plant material
The powdered leaf of T. globiferus (2.0 kg) was exhaustively extracted with 3 L of 90 % methanol for 6 days. The content was filtered using filter paper and the solvent was removed using vacuum rotary evaporator at 40℃ to afford crude methanol leaf extract (140 g). Some part of the extract (120 g) was partitioned using different solvents into n-butanol, ethyl acetate, chloroform and n-hexane fractions.

Preliminary Phytochemical Screening
The preliminary screening of phytochemical was performed on the methanol leaf extract T. globifeus and its ethyl acetate and n-butanol fractions in accordance with the procedures 17,18 to identify the presence of some secondary metabolites.

Antifungal studies Test organisms
Four clinical fungal isolates obtained from the Clinical Microbiology Department of Usmanu Danfodiyo University Teaching Hospital, Sokoto, includes

Candida albicans, Aspergillus niger, Trychophyton rubrum and Trychophyton mentagrophyte Preparation of test organisms
Test organisms were sub-cultured and grown on 10 ml SDA slants, it was eventually stored in the refrigerator at 2-8℃.

Preparation of reference antifungal agent
About 50 mg of fluconazole powder was dissolved in 10 ml dimethyl sulfoxide to prepare a stock concentration of 5 mg/ml, from which 0.05 mg/ml (50 μg/ml) working concentration was also prepared.

Preparation of plant extract/fractions
A 100 mg/ml Stock concentration was prepared when 0.5g of methanol extract and its fractions (ethyl acetate and n-butanol) was dissolved in 5 ml of 10% DMSO and eventually two-fold serial dilution was carried out to obtain three more concentrations of 50, 25 and 12.5 mg/ml.

Preparation of culture media
The sabouraud dextrose agar (SDA) and broth as growth media were weighed and prepared with distilled water according to the manufacturer's specifications. SDA was gently heated to aid its dissolution, it was transferred into an already sterilized Petri dishes, it was allowed to cool and solidify. These were kept aseptically until ready for use.

Determination of the antifungal activity of T. globiferus Standardization and Culturing of the fungal isolates
A suspension of solid culture of Candida albican (18 h) in Sabo broth was prepared. The standardization was performed according to method by Clinical Laboratory Standard Institute guidelines 19 by inoculating in normal saline and adjusting its turbidity to match that of 0.5 McFarland standard which is equal to 1.0×10 6 CFU/ml. Aspergillus niger and Trichophyton spp were subcultured from (6 days old) SDA slant, the suspension was adjusted to 1.0x10 6 CFU/ml at 530nm of a spectrophotometer.

Antifungal screening of T. globiferus
The antifungal activity of the plant ant its ethyl acetate and n-butanol fractions were carried out according to the method 20 . Sabouraud dextrose agar (SDA) as the media for organism growth was prepared according to instructions by the Manufacturer and was autoclaved at 121 0 C for 15 min, the media was transferred into sterile dishes and allowed to cool and solidify. A cork borer of 8 mm in diameter was used to punch wells on the plates. 0.1ml of the inoculum was seeded on the media and cotton swab was used to spray the inoculum on the surface of the media. About 200 μl of the graded concentration of extract and its fractions was transferred into each well of the micro plate. 0.05 mg/ml Fluconazole which served as positive control, 10% DMSO was also used as negative control, plate was incubated at 27 0 C for 48-72 h, zone of inhibition was measured using transparent ruler. Each experiment was performed in triplicates.

Determination of minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) was determined using a 96 wells micro plate as previously described 21  each micro well of the micro plate. Extract of 100μl and its fractions was transferred into well-1 making up 200μl total volume. Mixture (extract/fractions) and media of 100μl was taken from well-1 to well-2 and serially diluted (2-fold) up to well-10 where 100 μl finally discarded from the last well, well 11 (extract blank) served as negative control and well-12 (media and inoculum) which served as a positive control. About 100 μl of the fungal inoculum approximately (10 6 CFU/ml -1 ) was transferred into each well except for well-11 of the microplate. The microplate was covered with aluminum foil and allowed to stand for 30 min before incubating at 27°C for 72h. The experiment was performed in triplicate. The MIC of the extract/fraction is the lowest concentration that caused growth inhibition of more than 90% after 48 h of incubation 22 .

Determination of Minimum fungicidal concentration (MFC)
Twenty (20μl) of each well which exhibited no visible or apparent growth after MIC determination was subcultured onto the solid media (SDA) and was incubated at 27°C for 48 h. The lowest concentration of the extract and fractions that does not yield any fungal growth on the solid medium used was taken as the MFC.  Values are mean inhibition zone (mm) ± S.E of three replicates; Key; M.E=methanol extract, EAF=ethyl acetate fraction, BTF=n-butanol fraction

Statistical Analysis
The results obtained were expressed as mean±standard error of mean and it was analyzed for significant using analysis of variance (ANOVA); values were considered significant at P<0.05.   The fungicidal effect of extract may be as a result of the inhibition of protein synthesis or nucleic acids metabolism of the organisms 33 . Fungicidal effect of the plant extract could also be as a result of the damage it caused to the cell membrane of the organism 34 .

CONCLUSION
The use of Tapinanthus globiferus as antifungal agent is promising as the methanol leaf extract and its fractions showed excellent antifungal activity against some selected fungal species with n-butanol fraction being the most active. This study indicated that T. globiferus has demonstrated good antifungal activity validating the ethno medicinal claim for the use of the plant in the treatment of fungal infections.