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

Isoprenyl pyrophosphate

Dimethylallyl pyrophosphate

Isoprene

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:

Example of a monoterpenoid: Camphor

Pinene

Triterpenes

Triterpenes composed of six isoprene units, so will have 30 carbons.Use triterpines to make steroids

Squalene is converted into the steroid lanosterolwhich is converted by plants into other steroidssuch as the brassinolides and ecdysteroids

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

Selected Eighteen Plants Used for Medicinal Purposes by American Indians

Plant Extraction Process: Sequential Gradient Extraction Procedure

Tube Turbidity Assay 96-Well Microtiter Plate

Antimicrobial Testing Methods

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:● 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

Endo-borneol

--Phytochemistry 69(2008) 1732-1738--Boletín Latinoamericano y del Caribe de Plantas

Medicinales y Aromáticas 9 (2): 136 –142.

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

White Sage Bicyclic Ether

Ampicillin

2.3 x 10 -8 M on S. aureus

Bee Balm (Monarda fistulosa)

S. aureus strain Resistant To:

ATCC 29213 weak β-lactamase

ATCC 43300 resistant to β-lactam

ATCC BAA-977 Erythromycin

(Screening Rate: crude extract `1mg / mL)

Thymol

S. aureus strain Results

ATCC 29213 Inactive @ 1 mM

ATCC 43300 Inactive @ 1mM

Thymol Testing on S. aureus

Bee Balm HPLC

8.1

8.1

HPLC – C18 Column(Gradient)-MeOH:H2O 70:30—100% MeOHover 10 min and 100% MeOH 5 min-1mL per min

Thymol

Bee Balm Extract following FlashChromatography (SiO2/ CH2Cl2: EtOAc)

Farmatsevtichnii Zhurnal (Kiev, Ukraine) (2010), (5), 89-93—Thymol 43% essential oilInt. J. Essential Oil Therapeutics (2008) 139-144—Trichophyton activity thymolFEMS 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

Plasmodium cultures suspended in CM and A+

blood

Cultures checked daily for parasitemia by means of Giemsa

stain

Immature trophozoite(ring stage)

TrophozoiteSchizont

Cultures are centrifuged, old media sucked off and then “re-fed”, gassed

and placed in incubation

Cultures showing high parasitemiaand synchronization (pref. ring stage)

Checked in 24-48 hours depending on growth

In vitro compound testing

MIC assay

LDH assay

SYBR Green

Culturing

• Nutrient base (RPMI 1640)

• Human serum (A+)

• Whole human blood (A+)

• Gas mixture (5% O2, 5% CO2, and 90% N2)• Incubation (37° C)

Complete medium (CM)

Malaria: Culturing Method

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 • Large dynamic range

•Exact binding mechanism unknown• Intercalculation vs external?• When bound to DNA fluorescent output is greater by many orders of magnitude

Blue light Green light

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

(Fringed sage)

(Silver sagebrush)

(Common Wormwoood)

O

O

OH3C

O

H H

O

CH3H

HPLC Analysis of Extracts

HPLC Chromatograph of A. Frigida CH2Cl2 Extract(C18-MeOH:H2O 70-100% 10min /10 min@ 100%)

Retention Time on HPLCSimilar to that for Artemisinin

Artemisinin