Abstract
Fennel (Foeniculum Vulgare), is a flowering medicinal plant that belongs to the family Umbelliferae (Apiaceae). It is native to southern Europe and Mediterranean region with long history of use by humans as a spice, medicine, and fresh vegetable. Despite its popularity for its medicinal value, there are limited scientific studies about the structure and functions of proteins isolated from the seeds and their pharmacological activities. Yet majority of data on literature is based on the crude extracts and/or essential oils. Thus, this project explores the complete structure of Non-Specific Lipid transfer protein (NsLTP) isolated from fennel seeds along with their cytotoxic activities. The protein was extracted using Tris/HCl pH 8 buffer and purified by the combination gel filtration and reverse phase HPLC. The purity was confirmed by SDS-PAGE gel electrophoresis and intact mass analysis by MALDI-TOF mass spectrometry. The purified NsLTP-protein was modified by 4-vinyl pyridine followed by digestion with trypsin. The tryptic digest was then separated by reversed phase HPLC. The primary structure of NsLTP was established by combining N-terminal amino acid sequence of intact chain and tryptic peptides by Edman degradation in automated protein sequencing system. The data suggest that Fennel NsLTP is a monomeric protein of 10kDa based on SDS-PAGE electrophoresis. Multiple sequence alignment performed using Clustal Omega depicted a sequence similarity with Daucus carota and Actinidia chinensis. The MTT assay result revealed that Fennel NsLTP led to dose dependent cytotoxic effect in MCF-7 cells with an IC50 value of 98.1µg/ml. Besides, mRNA expression of the genes Bax, EGFR, VEGF, Caspase-3, Survivin, MMP, and Bcl-2 were modulated as assessed by real time PCR.