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A trypsin inhibitor- SNTI with antidandruff activity from Sapindus trifoliatus

2011; World Wide Journals; Volume: 3; Issue: 3 Linguagem: Inglês

10.15373/2249555x/mar2013/2

2249-555X

K. Vijaya Rachel, Y. Vimala Y. Vimala, D. Apta Chaitanya,

Insect Pest Control Strategies

A trypsin inhibitor, designated Soap Nut Trypsin inhibitor (SNTI) with both antifungal and antibacterial activity exhibiting a molecular mass of 29kDa on SDS–polyacrylamide gel electrophoresis, was isolated from soap nut seeds (Sapindus trifoliatus L) by a combination of ammonium sulfate precipitation, ion exchange chromatography and gel permeation chromatography on Sephadex G-100. It exerted potent antifungal activity against dermatophytic fungi Trichophyton rubrum and Malassezia furfur which are implicated in causing dandruff. SNTI exhibited antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris and Escherichia coli. The extracts of the pericarp which contained secondary metabolites also exhibited antifungal activity to a lesser extent compared to SNTI. The results obtained herein indicate that SNTI possess exploitable potentials for therapeutic applications. Introduction Protease inhibitors play an important role in the protection of plant tissues from pest and pathogen attack by virtue of an anti nutritional interaction. Protease inhibitors with antifungal activity have captured the attention of a number of researchers on account of their tremendous potential in protecting crops from invading fungi and thus have important economic implications. Antifungal proteins may contribute to both defense against predators such as insects (Murdock LL et.al., 1990) as well as pathogens such as fungi (Wang SY et.al., 2005;2006). It is well identified that there is a spectacular diversity of antifungal proteins produced by plants and have been classified into different groups based on their structure and/or functions (Ng TB, 2004). Sometimes, a combination of antifungal proteins is found in a single species of bean. For instance, proteins and peptides such as lysozyme, nonspecific lipid transfer protein, and protease inhibitor were isolated from mung bean, and all have fungal inhibition activity (Wang SY et.al., 2005;2006;2004). HIV protease inhibitors and SARS coronavirus protease inhibitors are used to fight against HIV and SARS virus, respectively (Ng TB et.al., 1997). Plant protease inhibitors may be involved in the regulation of programmed cell death in plants. One of the common type of protease inhibitors is trypsin inhibitors which have been isolated from both animal and plant tissues. Kunitz-type trypsin inhibitors also inhibit chymotrypsin, α-amylase and human plasmin, and block the conversion of prothrombin to thrombin (Birk Y, 2003; Zhao M et.al., 1996). Dermatophytes, a group of about 40 related fungi belong to three genera: Microsporum, Trichophyton and Epidermophyton cause cutaneous mycoses infecting only the keratinized tissue like skin, hair and nails. Dermatophytoses are among the most prevalent infections in the world. Species of Malassezia and Trichophyton have been implicated as contributors to seborrheic dermatitis or dandruff. This condition is partially alleviated by ketoconazole treatment (A Sanfilippo and JC. English III, 2006). Soap nuts are being considered for commercial use in cosmetics and detergents, among many other products. Reports on the antimicrobial activity of SNTI a trypsin inhibitor are not yet available in the literature. Hence, it is worthwhile to investigate the effect of SNTI on selected bacterial strains and dermatophytic fungi causing dandruff. Materials and Methods The Sapindus trifoliatus trees bearing soap nuts were selected from Horticulture Research Station, Pandirimamidi, Rampachodavaram, AP (INDIA). Fruits were collected at ripened stage. The endosperm is used for the isolation and purification of SNTI. Various chemicals used in the present investigation were purchased from the following sources. Bovine pancreatic α-chymotrypsin (3x crystallized, type II), N-acetyl-L-tyrosine ethyl ester (ATEE), dimethyl sulfoxide, elastase, elastin congo red , pronase, papain, pepsin, thermolysin, α-amylase, acrylamide, bis-acrylamide , protein markers and sephadex G-100 were obtained from Sigma Chemical company, St. Louis, Missouri, USA. The bacterial strains Bacillus subtilis (NCIM 2063), Staphylococcus aureus (NCIM 3021), Escherichia coli (NCIM 2066), Klebsiella pneumonia (NCIM 2957), Proteus vulgaris (NCIM 2027) were from NCIM Pune and fungal species Malassezia fur fur (MTCC 1374) was procured from MTCC, Chandigarh. Trichophyton rubrum was collected from Andhra Medical College, Visakhapatnam. Sample preparation The outer hard seed coat was removed and 25 g of kernel is homogenized with 200 ml of 50 mM phosphate buffer, pH 7.6 and then made up to 250 ml with the same buffer. The clear supernatant obtained after centrifugation at 2500 rpm was treated with 50% ice cold acetone and again centrifuged at 2500 rpm for 15 minutes at 4oC. The precipitate dissolved in 250 ml of 50 mM phosphate buffer, pH 7.6 was used for further investigations. Purification of SNTI Purification was carried out according to the procedure adopted by (Sai Annapurna et.al., 1991). All operations were carried out at 4oC unless otherwise stated. SDS-PAGE Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to the method of (Laemmli UK and Favre M, 1973). Preparation of pericarp extract for Antimicrobial activity The pericarp from soap nut was removed and air dried and then ground to powder using an electric mill. The powdered material (10g) was subjected to soxhlet extraction for 18h using water, methanol, ethanol, chloroform and hexane separately. The extracts were concentrated and further used for anti microbial activities.