Research Article BIO-CONTROL OF CLINICAL FUNGAL ISOLATES ASSOCIATED WITH FUNGAL KERATITIS USING MEDICINAL PLANT EXTRACT SARIKA GUPTA 2 , KULSHRESHTH KIRAN 3 AND APARNA DATTA 1 1 Dr. B.Lal Institute of Biotechnology, Jaipur, 2 Department of Biotechnology, Dr. B.Lal Institute of Biotechnology, Jaipur, 3 Rani Durgawati Vishwavidyalaya, Jabalpur, M.P. Email: blalbiotech@gmail.com Received: 07 Jun 2012, Revised and Accepted: 13 July 2012 ABSTRACT Most common causes of corneal ulcer is mycotic infestation, it damage cornea and iris. On the culture incidence of Penicillium sp. is high followed by Fusarium sp., Microsporium sp., Aspergillus sp. Plants are reservoir of biological active compounds to combat various pathogens, aqueous and methanolic extracts of four plants Thuja occidentalis, Catharanthus roseus, Withania sominifera, Lawsonia inermis were tested for their antifungal activity against fungal clinical isolates from fungal keratitis as Fusarium sp., Microsporium sp., Aspergillus sp. and Penicillium sp. Maximum inhibition was recorded by methanolic extracts in comparison to aqueous extracts. Methanolic extract of Thuja occidentalis (10% v/v) were found to be significantly inhibiting all clinical isolates as Fusarium sp. (89.05%), Microsporium sp. (72.22%), Aspergillus sp. (90.59%) and Penicillium sp. (90.0%) whereas, 10% methanolic extract of Lawsonia inermis showed mycelial inhibition of Microsporium sp. (81.33%) and Fusarium sp. (93.08%). Percent inhibition by Withania sominifera extract significantly reduces Fusarium sp (85.45%) and Microsporium sp. (89.6%) by10% Catharanthus roseus extract. It was recorded that Aspergillus flavus was only inhibited by methanolic extracts of Thuja occidentalis. Aqueous extracts could not significantly control the incidence of the test fungi but only 10% aqueous extract of Withania sominifera control Fusarium sp. (83.69%). Keywords: Bio-control, Clinical fungal isolates, Fungal keratitis, Medicinal plant INTRODUCTION Fungal keratitis was first described by Leber in 1879. Fungal keratitis is a suppurative, ulcerative, and sight-threatening infection of the cornea that sometimes leads to loss of the eye. It represents one of the major causes of infectious keratitis in tropical areas of the world. Corneal ulcer is an open sore on the cornea, the thin clear structure overlying the iris, which is the colored part of eye. Damage may occur as a result of injury, a foreign body in the eye, or excessive or inappropriate wearing of contact lenses. The infections that can result in ulceration of the cornea may be caused by viruses, bacteria, fungi or acanthamoeba. Fungal keratitis is caused mainly due to infection by Candida sp., Fusarium sp., Aspergillus sp., Penicillium sp., Cephalosporium sp. and mycosis fungoides species 5 . Most of the chemical formulations used against fungal keratitis contain azole, trienes and flucocytosine. Medicinal plants synthesize a vast array of secondary metabolites that are important for human life 7 . For medicinal purposes, antimicrobial activity of substances derived from plant extracts have been recognized for many years. The present study aims at evaluating the antifungal activity of plant extract (aqueous and methanolic) of medicinal plant against the clinically isolated fungi from fungal keratitis. MATERIALS & METHODS Test Plant Four wild medicinal plants extracts of Catharanthus roseus, Thuja occidentalis, Withania sominifera and Lawsonia inermis were used. Fresh leaves were collected washed thoroughly 2-3 times with running tap water and once with sterile distilled water, air dried at room temperature on a sterile blotter and used for preparation of extracts 9 . Solvent extraction The dried powdered leaves were subjected to two types of extraction (methanolic and aqueous). Plant extract were prepared by 15 grams fine powder of leaves was filled in the thimble and extracted successively with water and methanol for 48 hours at 55 ⁰ C (methanolic) and 85oC (aqueous). All the solvent extracts were concentrated using rotary flash evaporator under reduced pressure. The extracts were preserved in airtight brown bottle until further use 10,6. A series of concentration of plants extract were prepared as 2.5%, 5%, 7.5%, 10% were taken to find the significant control of the clinically isolated fungi by the food poisoning method12,13. Test fungi Fungal cultures were collected from Dr. B. Lal Clinical Laboratory, Jaipur. Samples were collected from mycotic ulcer and all the samples were subjected to culture on Sabouraud Dextrose Agar medium (SDA). Four species of clinically isolated fungi from fungal keratitis as Fusarium sp., Microsporium sp., Aspergillus sp. and Penicillium sp. were used as test fungi for antifungal activity assay. Antifungal activity assay by poisoned food technique Solvent extract One gram of each of the solvent extract was dissolved in 10ml of respective solvents, which served as the mother solvent extracts. Sabouraud Dextrose Agar medium (SDA) with different concentration of each of the solvent extracts viz., 2.5%, 5.0%, 7.5% and 10% were prepared. SDA medium amended with the same concentrations of these respective solvents served as control. Five mm mycelial discs from the margins of seven day old cultures of test fungi were placed in the center of SDA medium. The plates were incubated at 25±1 o C for seven days and ten replicates were maintained for each treatment. The percent inhibition of mycelial growth was determined 12,13 . The data were subjected to statistical analysis by ANOVA and Tukey’s HSD. RESULTS The clinical samples (50) suspected for mycotic keratitis were collected from Dr. B. Lal clinical Laboratory. The relative percent occurrence (RPO) of Fusarium sp. (22), is high followed by Aspergillus sp. (12), Microsporium sp. (4), Trichophyton sp. (4), Penicillium sp. (2) (Fig. 1). Antifungal activity assay by poisoned food technique Among the four different plant extract (Aqueous and methanolic) against clinically isolated fungi from the mycotic keratitis maximum inhibition was recorded by methanolic extracts in comparison to aqueous extracts. Maximum inhibition of the test fungi was recorded at 10% concentration (v/v) out of 2.5%, 5.0%, Percent inhibition = Mycellial growth (C) – Mycellial growth (T) X 100 Mycellial growth (C)