Investigating an Alternative Treatment to Cystic Fibrosis: Testing the Effect of Panax ginseng C.A. Meyer extracts on Pseudomonas aeruginosa

The predominant pathogen found in the lungs of cystic fibrosis (CF) patients is Pseudomonas aeruginosa. The success of the infection is partially due to virulence factor production, which is regulated by quorum sensing (QS) signaling. Currently, antibiotics are used to treat the infection, but resistant forms of P. aeruginosa have evolved, necessitating alternative treatments. Previous animal studies showed that treatment with extracts from the Chinese herb Panax ginseng C.A. Meyer reduced bacterial load resulting in a favorable immune response. It is hypothesized that ginsenosides, the major bioactive compounds in ginseng, is responsible for this effect. This study explores the role of ginseng extracts in attenuating P. aeruginosa virulence. A sequential extraction was performed using hexane, methylene chloride, methanol, and water. High performance liquid chromatography (HPLC) analysis showed the methanol and water ginseng extracts contained the known ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1• All extracts were tested on biomonitor strains of Agrobacterium tumefaciens,Chromobacterium violaceum, and P. aeruginosa. Antibacterial and anti-QS activity were assessed using a disc diffusion assay. This was then followed by thin layer chromatography (TLC) bioautographic assay to further separate active compounds. The hexane and dichloromethane extracts, that lacked ginsenosides, displayed antibacterial activity against C. violaceum, whereas methanol and water extracts had anti-QS activity. The results of the bioassay with the pure ginsenoside standards showed that they lack antibacterial or anti-QS activity. Our results indicate that there are bioactive compounds, other than ginsenosides, that are the cause of antibacterial effects and anti-QS in the ginseng extracts.

Antibacterial Activity of Extracellular Products from Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes that can be found in freshwater and marine environments as well as in soil. These organisms produce a variety of different biologically active compounds exhibiting anti-bacterial, anti-fungal and anti-cancer properties among others. In this study, cyanobacterial isolates were screened for their ability to produce extracellular antibacterial products. Cyanobacteria were isolated from fresh water and soil samples collected in the Pembroke Pines, FL area. Twenty- seven strains of cyanobacteria were isolated belonging to the following genera: Limnothrix, Nostoc, Fischerella, Anabaena, Pseudoanabaena, Lyngbya, Leptolyngbya, Tychonema, and Calothrix. Individual strains were grown in liquid culture in laboratory conditions. Following 14-day cultivation, the culture liquid was filtered and tested for activity against the following bacteria: Escherichia coli, Bacillus megatarium, Staphylococcus aureus, and Micrococcus luteus. Among all genera of cyanobacterial strains tested, Fischerella exhibited the greatest inhibitory activity. An attempt was made to isolate the active compound from the culture liquid of the active strains. Lipophilic extracts from culture liquid were obtained from three selected Fischerella strains. The extracts proved to have varying levels of activity against the tested bacteria. Inhibitory activity from all three Fischerella strains was detected against B. megatarium and M luteus. The only strain that was active against S. aureus was Fischerella sp. 114-12 while none of the extracts showed activity against E. coli. This kind of screening has potential pharmaceutical and agricultural benefits, including possible discovery of novel antibiotics.

Synthesis of a PbTx-2 photoaffinity and fluorescent probe and an alternative synthetic route to photoaffinity probes

A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.