Evaluation of Pacific Evaluation of Pacific Northwest Native Plants For Northwest Native Plants For

the Presence of ACE the Presence of ACE InhibitorsInhibitors

Andrea M GarciaAndrea M Garcia

Working with Dr. Joe KarchesyWorking with Dr. Joe Karchesy

What is ACEWhat is ACE

ACE, Angiotensin ACE, Angiotensin Converting EnzymeConverting Enzyme

An exopeptidase An exopeptidase

that catalyses the that catalyses the conversion of conversion of Angiotensin I to Angiotensin I to Angiotensin II, in Angiotensin II, in the Renin- the Renin- Angiotensin Angiotensin pathwaypathway

Renin- Angiotensin pathwayRenin- Angiotensin pathwayhttp://www.hypertensiononline.orghttp://www.hypertensiononline.org

What is the Renin- Angiotensin What is the Renin- Angiotensin pathwaypathway

AngiotensinogenAngiotensinogen

ReninRenin

Angiotensin IAngiotensin I

ACEACE

Angiotensin IIAngiotensin II

HypertensionHypertension

High blood pressure is High blood pressure is considered to be a silent killer.considered to be a silent killer.

Nearly one in three U.S. adults have Nearly one in three U.S. adults have high blood pressure.high blood pressure.

Uncontrolled high blood pressure can Uncontrolled high blood pressure can lead to stroke, heart attack, heart lead to stroke, heart attack, heart failure or kidney failure. failure or kidney failure.

Once high blood pressure develops, it Once high blood pressure develops, it usually lasts a lifetime and it can be usually lasts a lifetime and it can be prevented and controlled by taking prevented and controlled by taking action.action.

Preventive measuresPreventive measures

Synthetic drugsSynthetic drugs Exercise and Exercise and

good eating good eating habits.habits.

Use of natural Use of natural products, teas or products, teas or supplements.supplements.

Plant products Plant products that exhibit ACE that exhibit ACE inhibitioninhibition..

hawthornberrieshawthornberries

What are ACE inhibitors?What are ACE inhibitors?

A group of A group of pharmaceuticals or pharmaceuticals or compounds that compounds that slow or inhibit the slow or inhibit the activity of ACE.activity of ACE.

Based on research Based on research from the venom of a from the venom of a Brazilian pit viper Brazilian pit viper that led to the that led to the development of the development of the first ACE inhibitor first ACE inhibitor drug to treat drug to treat hypertension.hypertension.

Ace inhibitors in plantsAce inhibitors in plants

There are various secondary metabolites There are various secondary metabolites with inhibitory activity towards ACE. with inhibitory activity towards ACE.

Found in flavonoids, proanthocyanidins Found in flavonoids, proanthocyanidins and small peptides.and small peptides.

There are well known plant extracts that There are well known plant extracts that have been used by indigenous peoples.have been used by indigenous peoples.

Reason for investigating ACE Reason for investigating ACE inhibition in plantsinhibition in plants

Herb drug interactions.Herb drug interactions. Low cost compared to synthetic drugs.Low cost compared to synthetic drugs. They have a wide range of therapeutic They have a wide range of therapeutic

use and are suitable for chronic use and are suitable for chronic treatmentstreatments

Find better and more selective Find better and more selective compounds. compounds.

Possibility of indicating new molecular Possibility of indicating new molecular structure with ACE activity.structure with ACE activity.

List of plants under study that we List of plants under study that we think may contain ACE inhibitionthink may contain ACE inhibition

Black CohoshBlack Cohosh Cimicifuga elataCimicifuga elata

BaneberryBaneberry Actea RubraActea Rubra

Prince PinePrince Pine PipsisewaPipsisewa

Ocean SprayOcean Spray Holodiscis discolorHolodiscis discolor

Red AlderRed Alder Alnus RubraAlnus Rubra

Devil’s ClubDevil’s Club Oplopanax Oplopanax HorridumHorridum

SoapberrySoapberry Shepherdia Shepherdia canadeniscanadenis

BuckbrushBuckbrush Amelanchier Amelanchier AlnifoliaAlnifolia

In Vitro Screening of In Vitro Screening of InhibitorsInhibitors

Collect specimens of plants to be Collect specimens of plants to be studied. studied.

Isolate compounds.Isolate compounds. React plant ACE inhibitors with rabbit React plant ACE inhibitors with rabbit

lung enzyme assay.lung enzyme assay. Test for ACE activity, using the HPLC Test for ACE activity, using the HPLC

method (Hansen et al. 1995).method (Hansen et al. 1995). Further testing for the plants that Further testing for the plants that

exhibit ACE compounds will be done exhibit ACE compounds will be done for us through the college of for us through the college of pharmacypharmacy..

ReactionReaction

Use a Titer plateUse a Titer plate– Rabbit lung ACE Rabbit lung ACE

enzymeenzyme– Plant solutionPlant solution– Dansyltriglycine Dansyltriglycine

substratesubstrate IncubateIncubate Stop the reaction Stop the reaction

and analyze with and analyze with HPLC.HPLC.

ACE – Catalyzed Cleavage of ACE – Catalyzed Cleavage of Dansylytriglycine.Dansylytriglycine.

Dansyltriglycine Dansyltriglycine (dansyl-Gly-Gly-Gly), (dansyl-Gly-Gly-Gly), UV labeled tripeptide.UV labeled tripeptide.

Presence of ACE Presence of ACE

Dansylglycine (dansyl-Dansylglycine (dansyl-Gly)Gly)

Diglycine (Gly-Gly)Diglycine (Gly-Gly)

From Elbl and Wagner, 1991From Elbl and Wagner, 1991

High Performance Liquid High Performance Liquid ChromatographyChromatography

Dansyl-L-glutamine: Dansyl-L-glutamine: the internal standardthe internal standard

Dansylglycine, product Dansylglycine, product of enzyme reaction of enzyme reaction

Dansyltriglycine, Dansyltriglycine, substrate of enzyme substrate of enzyme reaction.reaction.

Ratio of the two is the Ratio of the two is the measurement of measurement of inhibition.inhibition.

From Hansen etal, 1995From Hansen etal, 1995

StatusStatus

Analysis currently underway for ACE Analysis currently underway for ACE inhibitioninhibition

Promising compounds will be Promising compounds will be forwarded to Pharmacy for further forwarded to Pharmacy for further characterization.characterization.

AcknowledgementsAcknowledgements Jaworski Scholarship, HHMI, Garden Jaworski Scholarship, HHMI, Garden

Club of AmericaClub of America

Joe Karchesy, Kevin Ahern, Debbie Joe Karchesy, Kevin Ahern, Debbie MaynardMaynard