Ethnobotany: Chemistry and Medicinal Properties of Plants (Lecture 3) Paclitaxel Pacific Yew (Taxus brevifolia) Richard Proenneke did exactly what Chris McCandless did--- but lived in the wilderness for 30 years filming and serving as a naturalist Left Twin Lakes, Alaska in 1999 at age 82 and died at 86 in 2004 Terpenes Class organic chemicals that plants produce and are thought to lend protection towards plants. Composed of C5H8 or multiples of that i.e. 2 isoprene units or 3 isoprene units Used as building blocks by plants to make steroids which are used in cell walls Often the terpenes will have a strong odor, aromatic odor Essential oils: hydrophobic compounds with an aroma. They are the more volatile compounds found in plants and made up of terpenes Terpenes Isoprene Isoprenyl pyrophosphate Dimethylallyl pyrophosphate Synthesize thousands of different molecules from these, ranging from steroids to carotenes, serve as a building block for plants to synthesize molecules they require. Referred to as terpenes if only have carbon & hydrogen If add a functional group i.e. alcohol group, then referred to as a terpenoid Terpenes Hemiterpenes: only one terpene unit is used and the only compound in this class is isoprene itself. Molecular formula of C5H10 Example of a hemiterpenoid: prenol Still has five carbons but # hydrogens will change Monoterpenes Monoterpenes: use two terpene units and will have 10 carbons Example of a monoterpene: Pinene Example of a monoterpenoid: Camphor Triterpenes Triterpenes composed of six isoprene units, so will have 30 carbons. Use triterpines to make steroids Squalene is converted into the steroid lanosterol which is converted by plants into other steroids such as the brassinolides and ecdysteroids Selected Eighteen Plants Used for Medicinal Purposes by American Indians Genus & Species Common Name Traditional Medicinal Use (example) Ageratina altissima Snake root Stomachache (root) Grindelia squarrosa Gum weed Sores (boiled flowers & leaves) Amorpha canescens Lead plant Stomachache (leaves for tea) Monarda fistulosa Horse mint / Bee balm Fever or colds (tea) Mahonia repens Oregon grape iIfections, liver, digestive (root) Dalea purpurea Purple clover Heart trouble Arctostaphylos uva-ursi Bearberry Urinary problems (tea) Artemisia ludoviciana White Sage Dermitalogical aid Pulsatilla patens Pasque flower Speed childbirth (tea) Heuchera richardsonii Alum root Diarrhea (tea) Ipomoea leptophylla Bush morning glory Nervousness (root smoked) Liatris punctata Dotted gay feather iIprove appetite (root) Ratibida columnifera Prairie coneflower Stomach & headaches (tea) Sphaeralcea coccinea Scarlet globe mallow External sores Yucca glauca Yucca Stomachache (roots) Cynoglossum officinale Hounds tongue / Begger’s lice Burns, wounds, rashes (salve) Chorispora tenella Blue (purple) mustard None known--Trial test plant Artemesia frigida Woman’s Sage Headaches—aroma therapy Plant Extraction Process: Sequential Gradient Extraction Procedure Antimicrobial Testing Methods Tube Turbidity Assay 96-Well Microtiter Plate Tube Turbidity Assay is visual, easy to run but consumes large quantities of extracts/compounds Microtiter Plate Assay: minature version of Tube Turbidity Assay, 200 uliters vs 4-5 ml 620 nm light is used and not transmitted through solution when the microbes have grown and compound is inactive. Soln. clear when active. White Sage Structure ● Mass Spectroscopy: ● Calculated Molecular Mass: 154.1356 ● Molecular Formula: C10H18O ● Similar to endo-borneol ● Infrared Spectroscopy: Endo-borneol ● No alcohol present ● Bridged or bicyclic compound ● Ether ● NMR (1H and 13C) Spectroscopy: ● Aliphatic hydrocarbon ● Ether adjacent carbon with one proton (hydrogen) ● All three methyl groups adjacent to tertiary carbons --Phytochemistry 69(2008) 1732-1738 --Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 9 (2): 136 –142. White Sage Bicyclic Ether Species ~I90 S. aureus (BH Strain) 73 mM S. aureus (ATC 29213-weak β-lactamase) 73 mM S. aureus (ATC 43300 resistant to β-lactam) 73 mM S. Aureus ( BAA-976 weak macrolide efflux) 73 mM 2.3 x 10 -8 M on S. aureus Ampicillin Bee Balm (Monarda fistulosa) (Screening Rate: crude extract `1mg / mL) S. aureus strain Resistant To: ATCC 29213 weak β-lactamase ATCC 43300 resistant to β-lactam ATCC BAA-977 Erythromycin Thymol Testing on S. aureus S. aureus strain Results Thymol ATCC 29213 Inactive @ 1 mM ATCC 43300 Inactive @ 1mM Bee Balm HPLC 8.1 Thymol Bee Balm Extract following Flash Chromatography (SiO2/ CH2Cl2: EtOAc) 8.1 HPLC – C18 Column(Gradient) -MeOH:H2O 70:30—100% MeOH over 10 min and 100% MeOH 5 min -1mL per min Farmatsevtichnii Zhurnal (Kiev, Ukraine) (2010), (5), 89-93—Thymol 43% essential oil Int. J. Essential Oil Therapeutics (2008) 139-144—Trichophyton activity thymol FEMS Microbiology Letters (2004) 191-195—S.aureus 43300 Malaria • With an estimated 250-500 million cases annually, malaria continues to remain a global health concern • 2.5 million deaths per year, most those deaths occurring in African children below the age of five •Transmitted by female Anopheles mosquito • Species of protozoan parasites in the genus Plasmodium) • United by an apicoplast • More than 100 species that infect a wide range of organisms Malaria: Culturing Method Plasmodium cultures Cultures are centrifuged, old media Checked in 24-48 hours depending suspended in CM and A+ sucked off and then “re-fed”, gassed on growth blood and placed in incubation Culturing • Nutrient base (RPMI 1640) • Human serum (A+) Complete medium (CM) • Whole human blood (A+) • Gas mixture (5% O2, 5% CO2, and 90% N2) • Incubation (37° C) Cultures checked daily for parasitemia by means of Giemsa Cultures showing high parasitemia In vitro compound testing stain and synchronization (pref. ring stage) MIC assay LDH assay Schizont Trophozoite Immature trophozoite (ring stage) SYBR Green Malaria: SYBR Green I (SGI) • SGI is a sensitive nucleic acid stain that is used for detection of double- stranded DNA (dsDNA): Excite @ 497 nm and emission @ 520 nm • High affinity for dsDNA • Low toxicity Green light • Large dynamic range Blue light •Exact binding mechanism unknown • Intercalculation vs external? • When bound to DNA fluorescent output is greater by many orders of magnitude Plants, Culture and Medicine: Plants and Extraction • Four species of sage from the genus Artemisia (Asteraceae family) were selected for extraction and testing: Spectroscopy and Structure Determination NMR (Nuclear Magnetic Resonance): 1) No radionuclide involved, no radioisotope that will emit high energy particles is used 2) Used to determine the skeleton or “super structure” of a molecule. It will tell you how the atoms are connected together. 3) NMR led to the development of MRI (magnetic resonance imaging) Spectroscopy and Structure Determination Infrared Spectroscopy (IR): 1) Yields information on functional groups present in molecules 2) Functional groups are atoms or collection of atoms responsible for interacting with amino acids on an enzyme or receptor. Carbon is the backbone or skeleton of the structure that holds the functional groups in place. Spectroscopy and Structure Determination Mass spectrometry (MS): 1) Yields information on the molecular weight of a structure 2) From the molecular weight the molecular formula can be generated 3) Identifies no. carbons, hydrogens, oxygens, nitrogen 4) CSI (Crime Scene Investigation): often used to determine if a drug or toxin was found in the blood of a victim. Also used by the “Doping” organizations to ensure athletes are not using steroids, growth hormones etc……. (Floyd Landis 2006 Tour de France winner) SYBR Green I (SGI) Assay of Artemisia Extracts (Common Wormwoood) (Fringed sage) (Silver sagebrush) HPLC Analysis of Extracts Artemisinin CH3 H O O H3C O H H O O Retention Time on HPLC Similar to that for Artemisinin HPLC Chromatograph of A. Frigida CH2Cl2 Extract (C18-MeOH:H2O 70-100% 10min /10 min@ 100%).