secondary metabolites isolated from perovskia atriplicifolia and their in vitro binding affinity for

78 Pages·2015·2.91 MB·English

by  Jason Ritchie

SECONDARY METABOLITES ISOLATED FROM PEROVSKIA ATRIPLICIFOLIA
AND THEIR IN VITRO BINDING AFFINITY FOR HUMAN OPIOID AND
CANNABINOID RECEPTORS

by
Ashli Fitzpatrick

A thesis submitted to the faculty of The University of Mississippi in partial fulfillment of
the requirements of the Sally McDonnell Barksdale Honors College

Oxford, MS
December 2014

Approved by

____________________________

 Advisor:
 Dr.
 Stephen
 Cutler

____________________________
Reader: Dr. Francisco Leon

____________________________

 Reader: Dr. Donna West-Strum
©2014
Ashli Fitzpatrick
ALL RIGHTS RESERVED
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ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Stephen Cutler and Dr. Francisco Leon for all of
their devoted time, encouragement, and guidance through this meticulous process. I
greatly appreciate Dr. Cutler for allowing me to work in his laboratory and giving me the
resources I needed to complete this thesis project. I would like to thank Dr. Leon for
guiding me through the experimental procedures involved and explaining all of the
scientific literature and procedures in great detail.

I would like to thank Dr. Debra Young and the Sally McDonnell Barksdale Honors
College for giving me the opportunity to complete such an interesting research project
and providing resources and encouragement during stressful times.

I would lastly like to thank my family and friends for their unyielding support. I could not
have completed this thesis without them.

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ABSTRACT
ASHLI FITZPATRICK: Secondary Metabolites Isolated from Perovskia atriplicifolia
and Their In Vitro Binding Affinity for Human Opioid and Cannabinoid Receptors

(Under direction of Dr. Stephen Cutler)

Plants and their natural products have been utilized for thousands of years in the
treatment of many human diseases. Because of their structural diversity, natural products
are ideal lead compounds for drug development. In this thesis, the natural products of the
plant Perovskia atriplicifolia were investigated, specifically their in vitro binding affinity
to receptors in opioid and cannabinoid classes. Extracts that demonstrated good selective
opioid and cannabinoid receptor binding were subjected to bioassay-guided fractionation.
Four active compounds were identified from the crude extract in Perovskia atriplicifolia:
1) 5-hydroxy-3’,4’,6,7-tetramethoxy flavone, 2) 5,7-dihydroxy-3’,4’,6,-trimethoxy
flavone, 3) 5-hydroxy-4’,6,7-trimethoxy flavone, and 4) 5,7-dihydroxy-4’,6,-dimethoxy
flavone. Each compound exhibited good selective δ-opioid receptor binding affinity and
was characterized as a flavanoid.

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TABLE OF CONTENTS

LIST OF TABLES AND FIGURES………………………………………………….....vi

LIST OF ABBREVIATIONS………………………………………………………….viii

BACKGROUND………………………………………………………………….............1

MATERIALS AND METHODS………………………………………………………..30

RESULTS………………………………………………………………………………..35

CONCLUSION…………………………………………………………………………..47

BIBLIOGRAPHY………………………………………………………………………..48

APPENDIX………………………………………………………………………………53

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LIST OF TABLES AND FIGURES

Figure 1 Chemical structure of paclitaxel 1……………………...………………3

Figure 2 Some important natural products……………………………………….4

Figure 3 Chemical structure of penicillin core…………………………………..5

Figure 4 Chemical structure of some terpenoids and sterols isolated from
Perovskia species……………………………………………………...10

Figure 5 Photo of Perovskia atriplicifolia……………………………………....11

Figure 6 Major monoterpenes identified in P. atriplicifolia…………………....12

Figure 7 Perovstatone A isolated from P. atriplicifolia…………………...……12

Figure 8 Selective opioid receptor agonists and antagonists……………………16

Figure 9 Opioid receptor mechanism…………………………………………...18

Figure 10 Major sites of opioid receptors and endogenous opioid production…..20

Figure 11 Chemical structure of Δ9-tetrahydrocannabinol (THC)……………....23

Figure 12 Cannabinoid receptor agonists and antagonists……………………….23

Figure 13 Location of cannabinoid receptors in the human brain………………..25

Figure 14 Structure of cannabinoid receptors, CB and CB …………………….26
1 2

Figure 15 Initial steps and activity for P. atriplicifolia………………………......36

Figure 16 Fractionation of P. atriplicifolia………………………………………38

Figure 17 TLC plate for fraction 4………………………………………………39

Figure 18 Fractionation for fraction 5 of P. atriplicifolia………………………..41

Figure 19 1H NMR spectrum of 5-hydroxy-3’,4’,6,7-tetramethoxy flavone…….42

Figure 20 13C NMR spectrum of 5-hydroxy-3’,4’,6,7-tetramethoxy flavone…...43

Figure 21 5-hydroxy-3’,4’,6,7-tetramethoxy flavone, compound A…………….44

Figure 22 5,7-dihydroxy-3’,4’,6,-trimethoxy flavone, compound B………….…44
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Figure 23 1H NMR spectrum of 5-hydroxy-4’,6,7-trimethoxy flavone…………45

Figure 24 5-hydroxy-4’,6,7-trimethoxy flavone, compound C………………….45

Figure 25 5,7-dihydroxy-3’,4’,6,-trimethoxy flavone, compound D…………....46

Table 1 Selective opioid receptor ligands……………………………………...17

Table 2 % of displacement of radioligands for subfractions obtained for fraction
4 of P. atriplicifolia……………………………………………………39

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LIST OF ABBREVIATIONS

ADH Antidiuretic Hormone

BCE Before Christian Era

CNS Central Nervous System

COSY Correlation Spectroscopy

DEPT Distortionless Enhancement by Polarization Transfer

DMEM Dublecco’s Modified Eagle Medium

DNA Deoxyribonucleic acid

DOR Delta opioid receptors

EtOAc Ethyl Acetate

FDA Food and Drug Administration

GPCRs G-protein Coupled Receptors

HMBC Heteronuclear Multiple Bond Coherence

HMQC Heteronuclear Multiple Quantum Coherence

HMRS High-resolution mass spectra

IASP International Association for the Study of Pain

KOR Kappa opioid receptor

MeOH Methanol

MOR Mu opioid receptor

NCE New Chemical Entity

NCI National Cancer Institute

NIH National Institute of Health

NMR Nuclear Magnetic Resonance

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NOESY Nuclear Overhauser Effect Spectroscopy

NOR Nociceptin opioid receptor

OGFr Opioid growth factor receptor

THC Δ9-tetrahydrocannabinol

TLC Thin-layer Chromatography

TMS Trimethylsilane

TOSCY Total Correlated Spectroscopy

VLC Vacuum Liquid Chromatography

WHO World Health Organization

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Background

I. Natural Products as a Resource for Drug Discovery

Natural products, also known as secondary metabolites, refer to chemical
compounds or substances produced by living organisms such as plants, animals, or
microbes (Clement, 2005). Primary metabolites, which include glucose, DNA, and amino
acids, are chemically identical in nearly all organisms and are required for normal
development and growth of the organisms (Clement, 2005). Unlike primary metabolites,
secondary metabolites are not directly required by an organism, are typically unique to a
specific species or subset of a species, and can offer novelty in structure or function
(Amsler et al., 2011). Secondary metabolites are synthesized primarily from the
compounds acetate, mevalonate, shikimate, and various amino acids (Gao, 2010).
Because these compounds are also used in the synthesis of essential primary metabolites,
it is theorized that the excess compounds were utilized to create non-essential natural
products (Gao, 2010). Natural products are created in nature and have naturally evolved
with their producers, resulting in biological activity that is increasingly useful and
effective (Gao, 2010). Natural product structures are extremely diverse and have high
binding affinities to their specific receptor type (Gao, 2010). Because of their structural
diversity and receptor binding activity, natural products are ideal bioactive lead
compounds for drug development (Kingston, 2012).
Plants and their natural products have formed the foundation of complex traditional
medicine systems and have been a major source of curative products for thousands of
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