ISOLATION, CHARACTERIZATION AND CYTOTOXIC ACTIVITY OF NON- SPECIFIC LIPID TRANSFER PROTEIN (nsLTP) FROM FENNEL (FOENICULUM VULGARE Mill.) SEEDS

Cancer is one of the prevalent diseases comprising over 100 types of malignancy that is known to be the major burden of chronic disease in the world. Due to its multifactorial etiology, it's difficult to understand the mechanism of the disease, making it difficult to cure. Despite all the efforts, it continues to be one of the greatest health problems owing to toxicity, off-targets, drug resistance, and limited bioavailability. Hence, it is not erroneous to rely on medicinal plants to get new drugs, with higher potency and specificity, as they are factories of a series of bioactive compounds. Among the recent natural products displaying anticancer activity are proteins isolated from plants. Hence, this project aims at exploring the cytotoxic activity and primary structure of nsLTP isolated from fennel (Foeniculum vulgare). Fennel is a medicinal plant of Umbelliferae (Apiaceae) family, one of the therapeutic plants proved to add a remarkable contribution in the research and development of new drugs. While most of the authentic studies associated with this plant focus on activities of essential oils or organic crude extracts, the structure-function relationship of biologically active proteins and peptides isolated from fennel seeds, remained unexplored. The protein was extracted using Tris/HCl pH 8 buffer and purified by the combination gel filtration and reverse phase HPLC. Purity was confirmed by SDS-PAGE 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 RP-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.