Structural characterization and in vitro lipid binding studies of non-specific lipid transfer protein 1 (nsLTP1) from fennel (Foeniculum vulgare) seeds.

Non-specific lipid transfer proteins (nsLTPs) are cationic proteins involved in intracellular lipid shuttling in growth and reproduction, as well as in defense against pathogenic microbes. Even though the primary and spatial structures of some nsLTPs from different plants indicate their similar features, they exhibit distinct lipid-binding specificities signifying their various biological roles that dictate further structural study. The present study determined the complete amino acid sequence, in silico 3D structure modeling, and the antiproliferative activity of nsLTP1 from fennel (Foeniculum vulgare) seeds. Fennel is a member of the family Umbelliferae (Apiaceae) native to southern Europe and the Mediterranean region. It is used as a spice medicine and fresh vegetable. Fennel nsLTP1 was purified using the combination of gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). Its homogeneity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The purified nsLTP1 was treated with 4-vinyl pyridine, and the modified protein was then digested with trypsin. The complete amino acid sequence of nsLTP1 established by intact protein sequence up to 28 residues, overlapping tryptic peptides, and cyanogen bromide (CNBr) peptides. Hence, it is confirmed that fennel nsLTP1 is a 9433 Da single polypeptide chain consisting of 91 amino acids with eight conserved cysteines. Moreover, the 3D structure is predicted to have four α-helices interlinked by three loops and a long C-terminal tail. The lipid-binding property of fennel nsLTP1 is examined in vitro using fluorescent 2-p-toluidinonaphthalene-6-sulfonate (TNS) and validated using a molecular docking study with AutoDock Vina. Both of the binding studies confirmed the order of binding efficiency among the four studied fatty acids linoleic acid > linolenic acid > Stearic acid > Palmitic acid. A preliminary screening of fennel nsLTP1 suppressed the growth of MCF-7 human breast cancer cells in a dose-dependent manner with an IC50 value of 6.98 µM after 48 h treatment.

Structural elucidation of some antimicrobial constituents from the leaf latex of Aloe trigonantha L.C. Leach.

BACKGROUND: The incidents of drug resistant microorganisms and the need of treatments for newly emerging pathogens are of great concern to the global community. Our ability to treat infectious diseases is dependent on the development of new pharmaceuticals, and one potential source being medicinal plants with traditional claims. The leaves of Aloe trigonantha L.C. Leach, an endemic Ethiopian plant, are locally used for the treatment of infectious and inflammatory diseases. This study explores the potential of the latex of this plant and compounds isolated thereof for their in vitro antibacterial and antifungal properties. METHODS: Analytical RP-HPLC and silica gel preparative TLC were used for identification and isolation of active constituents, respectively. Characterization of the compounds was based on UV, IR, HR-ESIMS, (1)H and (13)C NMR, and 2D-NMR spectral assignments. Antimicrobial activity studies were carried out against 21 pathogenic bacterial and 4 fungal strains using the disk diffusion method. Minimum inhibitory concentrations (MICs) were determined by the broth dilution method. RESULTS: A C-glycosylated chromone identified as aloesin, and three C-glycosylated anthrones characterized as 8-O-methy-7-hydroxyaloin A/B, aloin A/B and aloin-6'-O-acetate A/B were isolated. The latex and isolated compounds exhibited in vitro antibacterial activity against the tested pathogens. In some cases the activity of the isolated compounds (MIC = 10 µg/mL) was comparable with that of the standard drug ciprofloxacin, particularly against some of the Gram-negative bacterial strains tested. However, their activity towards the fungal pathogens tested was relatively weaker showing maximum activity against Candida albicans with MIC value of 400 µg/mL. CONCLUSION: The present findings can be used for further research aimed at the development of new antibacterial agents, and may also justify the ethnomedicinal claim of the plant for the treatment of infectious diseases.