Pharmacognostic Investigations and in Vitro Biological Profile of Illicium Angustisepalum

Pharmacognostic Investigations and in vitro Biological Profile of Illicium angustisepalum

KARINA MARIANNA SZYMULANSKA-RAMAMURTHY

Magister Farmacji, Jagiellonian University, Poland, 2009

DISSERTATION

Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacognosy in the Graduate College of the University of Illinois at Chicago, 2015

Chicago, Illinois

Doctoral Committee:

Dr. Chun-Tao Che, Chair/Advisor Dr. Birgit U. Jaki Dr. Hyun-Young Jeong, Department of Pharmacy Practice Dr. Jeremy J. Johnson Dr. Djaja D. Soejarto

DEDICATION

I dedicate this dissertation to my husband, Saikrishnan Ramamurthy, who has given me the motivation and strength to persevere beyond all the challenges that I have faced during the past five years. Without his presence, support and love, I could not have completed this dissertation. I love you forever.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Professor Chun-Tao Che, my advisor, for the opportunity to conduct my research work under his guidance, for providing essential environment to grow as a scientist and also for his generous support and inspiration throughout the past four years.

I extend my gratitude to the members of my dissertation committee:

Dr. Birgit U. Jaki for the thoughtful review of my work and for insightful comments that helped me to enrich my dissertation, and also for the encouragement and for the support throughout my tenure in the program.

Dr. Djaja D. Soejarto for providing me with opportunities to broaden my knowledge of medicinal plants and for teaching me about the plant taxonomy, and also for the insightful review of this work and for the encouragement while in the program.

Dr. Hyun-Young Jeong and Dr. Jeremy J. Johnson for the review of my dissertation and for their valuable comments and also for good advice.

Many thanks to Dr. Guido F. Pauli for providing me with the opportunity to participate in this program and to Dr. Jimmy Orjala for his guidance. And also, thanks to Dr. Brian T. Murphy and Dr. Birgit Dietz for their help and support.

Special thanks to Dr. Ming Zhao for training me in various research techniques and for his continuous support during my tenure as a graduate student.

III

ACKNOWLEDGEMENTS (continued)

I would also like to express my appreciation to the following people, without whose assistance my work would have not move forward:

Dr. Aleksjej Krunic and Dr. Benjamin Ramirez for providing me with the NMR training.

Dr. Jerry White, Caleb Nienow and Monika Lysakowska for providing mass spectra of the compounds isolated in the present study.

Dr. Arthur Anderson for help with GC-MS analysis used to establish the metabolite fingerprinting.

Wei-Lun Chen, Dr. Steven Swanson and Dr. Joanna Burdette for testing the compounds isolated in the present study in the cytotoxic assay.

Dr. Hyunwoo Lee, Joy Barranis and Dr. Tamiko Oguri for testing the compounds isolated in the present study in the antimicrobial assay.

Dr. Bernard Santarsiero for collecting x-ray diffraction crystallographic data.

Dr. Shi Lei and Dr. Paul Ip for testing compounds and fractions in the neural cell protection assay and in the promotion of neural growth assay.

Also thanks to all past and present members of Professor’s Che laboratory for their help.

Many thanks go to the faculty, staff and graduate students of the Department of Medicinal

Chemistry and Pharmacognosy for assistance and conversations.

ACKNOWLEDGEMENTS (continued)

I am very grateful to the Department of Medicinal Chemistry and Pharmacognosy for the teaching assistantships provided during my tenure as a graduate student.

I thank my friends, Dr. Edyta Grzelak, Rasika Phansalkar, Dr. Yu Zhang, Joy Barranis, Wei-

Lun Chen, Yang Liu, Sunaina Premkumar, Prabha Venkat, Meera Ranganathan, Dr. Sai Hari

Gandham, Vaishnav Vijayakumar, Naren Babu, Prashanthi Gandham and Amruth Rao for their friendship and support.

Last, and the most important, I thank my parents, Jozefa and Michal, who instilled in me the perseverance and passion for science and to my brothers, Michal, Daniel and Wojciech, who continuously supported me in my efforts despite the geographical distance between us. This work would not be possible without their love and caring support. I also thank my uncle,

Janusz Borek, who inspired me to pursue my career goals. I express my gratitude to my parents-in-law, Uma Maheswari and Ramamurthy, sisters in law, Uthara Prashant,

Magdalena Szymulanska, and Meng-Yi Wu for their encouragement and love.

- KSR

TABLE OF CONTENTS

CHAPTER 1 - LITERATURE INFORMATION ...... 1 INTRODUCTION ...... 1 LITERATURE REVIEW ON THE GENUS ILLICIUM ...... 4 Taxonomy of the genus Illicium ...... 4 Ethnomedical and other uses of species of Illicium ...... 7 Brief history of scientific investigations on the genus Illicium ...... 9 Biological activities of Illicium plants ...... 12 1.2.4.1 In vitro biological activities of species of Illicium ...... 12 1.2.4.1.1 Acetylcholinesterase inhibiting activity ...... 12 1.2.4.1.2 α-Glucosidase inhibiting activity ...... 16 1.2.4.1.3 Anti-inflammatory activity ...... 17 1.2.4.1.4 Antimicrobial activity ...... 20 1.2.4.1.5 Antioxidant activity ...... 21 1.2.4.1.6 Antiviral activity ...... 24 1.2.4.1.7 Chemopreventive activity...... 25 1.2.4.1.8 Cytotoxic activity ...... 26 1.2.4.1.9 Estrogenic activity ...... 29 1.2.4.1.10 Lipase inhibition activity ...... 30 1.2.4.1.11 Neural cell protection activity ...... 30 1.2.4.1.12 Promotion of neural growth activity ...... 31 1.2.4.1.13 Promotion of ChAT activity ...... 33 1.2.4.2 In vivo biological activities of Illicium plants ...... 34 1.2.4.2.1 Anti-depressant ...... 34 1.2.4.2.2 Anti-inflammatory ...... 34 1.2.4.2.3 Chemopreventive ...... 35 1.2.4.2.4 Fumigant ...... 36 Morphological description of I. angustisepalum ...... 36 Chemical constituents of I. angustisepalum ...... 37 OBJECTIVES OF THE STUDY ...... 40 CHAPTER 2 - METABOLITE FINGERPRINTING OF I. ANGUSTISEPALUM ...... 42 RATIONALE ...... 42

TABLE OF CONTENTS (continued)

EXPERIMENTAL PROCEDURES ...... 43 Plant material collection ...... 43 Plant material extraction ...... 44 2.2.3 GC-MS analysis, data processing and compound identification ...... 44 RESULTS ...... 46 Identification of volatile secondary metabolites ...... 46 Metabolite fingerprint of I. angustisepalum ...... 51 Comparative profiles between I. angustisepalum, I. lanceolatum and I. verum ...... 52 DISCUSSION ...... 54 CONCLUSIONS ...... 56 CHAPTER 3 - PHYTOCHEMICAL STUDY OF I. ANGUSTISEPALUM ...... 57 RATIONALE ...... 57 GENERAL EXPERIMENTAL PROCEDURES ...... 57 PLANT MATERIAL ...... 58 EXTRACTION OF THE PLANT MATERIAL ...... 58 FRACTIONATION OF THE ETHANOL EXTRACT ...... 58 ISOLATION AND STRUCTURE ELUCIDATION ...... 59 Thymol (1) ...... 68 (-)-T-Muurolol (2) ...... 68 2-Hydroxy-2-methyl-6-methyleneoct-7-en-3-yl benzoate (3) ...... 70 Angustanoic acid E (4)...... 74 Majusanic acid C (5) ...... 76 2, 6-Dimethoxychavicol (6) ...... 78 Angustanoic acid F (7) ...... 79 Angustanoic acid G (8) ...... 80 6β-Hydroxy-4-stigmasten-3-one (9) ...... 81 (2R, 3R)-3,5,7,3’,4’-Pentahydroxyflavonone (10) ...... 83 Angustisepalin (11) ...... 86 Clovane-2,9-diol (12) ...... 89 Angustanol (13) ...... 91 Majucin (14) ...... 92

TABLE OF CONTENTS (continued)

2-Hydroxy-7-methyl-hexan-1,5-olide (15) ...... 93 Majusanic acid B (16) ...... 95 DISCUSSION ...... 96 CONCLUSIONS ...... 99 CHAPTER 4 - BIOLOGICAL EVALUATIONS ...... 101 4.1 RATIONALE ...... 101 4.2 CYTOTOXICITY ASSAY ...... 101 4.2.1 Cell cultures ...... 102 4.2.2 Experimental procedures ...... 102 4.2.3 Results and discussion ...... 103 4.2.4 Conclusions ...... 106 4.3 ANTIMICROBIAL ASSAY ...... 106 4.3.1 Cell cultures ...... 106 4.3.2 Experimental procedures ...... 106 4.3.3 Results and discussion ...... 107 4.3.4 Conclusion ...... 110 4.4 NEURAL CELL PROTECTION ASSAY ...... 110 4.4.1 Cell cultures ...... 111 4.4.2 Experimental procedures ...... 111 4.4.3 Results and discussion ...... 112 4.4.4 Conclusions ...... 115 4.5 ACETYLCHOLINESTERASE INHIBITION ASSAYS ...... 116 4.5.1 TLC-bioautographic assay ...... 116 4.5.2 Experimental procedures ...... 117 4.5.3 Results ...... 118 4.5.4 Discussion ...... 122 4.5.5 Conclusion ...... 123 4.5.6 Ellman’s assay ...... 124 4.5.7 Experimental procedures ...... 124 4.5.8 Results ...... 125 4.5.9 Discussion ...... 126 4.5.10 Conclusion ...... 127

TABLE OF CONTENTS (continued)

4.6 PROMOTION OF NEURAL GROWTH ASSAY ...... 127 4.6.1 Isolation and culture of neurons ...... 128 4.6.2 Experimental procedures ...... 128 4.6.3 Results and discussion ...... 129 4.6.4 Conclusion ...... 132 CHAPTER 5 - CONCLUSIONS ...... 134 5.1 GENERAL CONCLUSIONS ...... 134 5.2 PERSPECTIVE ...... 137 5.2.1 Metabolite fingerprinting of I. angustisepalum ...... 137 5.2.2 Chemical and biological characterization of isolated compounds ... 138 CITED LITERATURE...... 139 APPENDICES ...... 162 CURRICULUM VITA...... 175

LIST OF TABLES

TABLE 1-1: APPLICATIONS OF ILLICIUM IN FOLK MEDICINE ...... 8

TABLE 1-2: IN VITRO BIOACTIVITIES OF ILLICIUM PLANTS REPORTED IN THE LITERATURE ...... 13

TABLE 1-3: IN VIVO BIOACTIVITIES OF ILLICIUM PLANTS REPORTED IN THE LITERATURE ...... 35

TABLE 1-4: COMPOUNDS REPORTED FROM I. ANGUSTISEPALUM ...... 38

TABLE 2-1: PLANT MATERIALS USED IN THE EXPERIMENTS...... 43

TABLE 2-2: CONDITIONS USED IN THE GC-MS ANALYSIS ...... 46

TABLE 2-3: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. ANGUSTISEPALUM EXTRACT ...... 47

TABLE 2-4: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. LANCEOLATUM EXTRACT ...... 49

TABLE 2-5: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. VERUM EXTRACT ...... 50

TABLE 3-1: YIELDS OF SOLVENT PARTITION FRACTIONS OBTAINED FROM I. ANGUSTISEPALUM EXTRACT ...... 59

TABLE 3-2: COMBINED FRACTIONS FROM FLASH CHROMATOGRAPHY OF EA-1.. 60

TABLE 3-3: GC-MS DATA ANALYSIS OF THE VOLATILE FRACTION FROM I. ANGUSTISEPALUM ...... 62

TABLE 3-4: 1H AND 13C NMR SPECTROSCOPIC DATA OF 2-HYDROXY-2-METHYL-6- METHYLENEOCT-7-EN-3-YL BENZOATE ...... 73

TABLE 3-5: 1H AND 13C NMR SPECTROSCOPIC DATA OF (2R, 3R)-3,5,7,3’4’- PENTAHYDROXYFLAVONONE ...... 84

TABLE 3-6: 1H AND 13C NMR SPECTROSCOPIC DATA OF 2-HYDROXY-7-METHYL- HEXAN-1,5-OLIDE ...... 94

TABLE 4-1: CYTOTOXIC ACTIVITY OF SELECTED COMPOUNDS ISOLATED FROM I. ANGUSTISEPALUM ...... 104

TABLE 4-2: ANTIMICROBIAL ACTIVITY OF FRACTIONS RECEIVED FROM I. ANGUSTISEPALUM ...... 108

LIST OF TABLES (continued)

TABLE 4-3: ANTIMICROBIAL ACTIVITY OF SELECTED COMPOUNDS FROM I. ANGUSTISEPALUM ...... 109

TABLE 4-4: ANTIMICROBIAL ACTIVITY OF ANGUSTANOIC ACID E ...... 109

TABLE 4-5: AChE INHIBITION OF THE ACTIVE COMPOUNDS FROM I. ANGUSTISEPALUM ...... 126

LIST OF FIGURES

Figure 1-1: Synthesis of oseltamivir from shikimic acid ...... 9

Figure 1-2: Structure of trans-anethole ...... 9

Figure 1-3: New compounds reported from Illicium (1952 – 2014) ...... 11

Figure 1-4: New compounds reported from Illicium (1952 – 2014) ...... 11

Figure 1-5: Structures of anisatin, neoanisatin and pseudoanisatin ...... 12

Figure 1-6: Structures of acetylcholinesterase inhibitors from Illicium ...... 16

Figure 1-7: Structures of anti-inflammatory compounds from Illicium ...... 18

Figure 1-8: Structures of compounds with antimicrobial activity ...... 21

Figure 1-9: Structures of antioxidant compounds from Illicium plants ...... 24

Figure 1-10: Structures of antiviral compounds from Illicium plants ...... 25

Figure 1-11: Structures of chemopreventive compounds from Illicium plants ...... 26

Figure 1-12: Structures of cytotoxic compounds from Illicium plants ...... 28

Figure 1-13: Structures of Illicium compounds with estrogen-like activity ...... 29

Figure 1-14: Structures of neuroprotective compounds from Illicium plants ...... 30

Figure 1-15: Structures of neurotrophic compounds from Illicium ...... 33

Figure 1-16: Structures of ChAT promoting compounds from Illicium ...... 33

Figure 1-17: Flowering twig of I. angustisepalum ...... 37

Figure 1-18: Structures of compounds isolated from I. angustisepalum ...... 39

Figure 2-1: Schematic representation of the processes in metabolite analysis ...... 43

Figure 2-3: Comparison of GC-MS total ion chromatograms of analyzed extracts from genus Illicium ...... 53

Figure 2-4: Frequency distribution indicating number of shared and unique compounds ...... 54

Figure 3-1: Fractionation of the ethanol extract of I. angustisepalum twigs ...... 59

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LIST OF FIGURES (continued)

Figure 3-2: GC-MS total ion chromatogram of the volatile fraction from I. angustisepalum ...... 62

Figure 3-3: Chemical structures of compounds isolated from ethyl acetate fractions of I. angustisepalum ...... 67

Figure 3-4: Structure of myrcenediol ...... 73

Figure 3-5: Ortep view of 6β-hydroxy-4-stigmasten-3-one ...... 83

Figure 3-6: Comparison of circular dichroism spectra of (2R, 3R)-3,5,7,3’,4’- pentahydroxyflavanone ...... 86

Figure 4-1: The cytotoxic effect of fractions from I. angustisepalum on PC12 cells . 113

Figure 4-2: The protective effect of fractions from I. angustisepalum on H202 induced neurotoxicity in PC12 cells...... 113

Figure 4-3: The effects of compounds isolated from I. angustisepalum ...... 114

Figure 4-4: The protective effects of compounds isolated from I. angustisepalum.... 115

Figure 4-5: Reaction of enzyme AChE with 1-naphtyl acetate and formation of the purple dye in the TLC-bioautographic assay ...... 118

Figure 4-6: Determination of detection limits...... 119

Figure 4-7: Analysis of essential oil ...... 120

Figure 4-8: Identification of active subfractions...... 121

Figure 4-9: Detection of AChE inhibition with the Ellman’s reagent ...... 125

Figure 4-10: Neurite outgrowth effects of selected compounds from I.angustisepalum ...... 129

Figure 4-11: Clovanemagnolol with distinguished clovane-2,9-diol moiety ...... 132

LIST OF ABBREVIATIONS

[α]D Specific optical rotation

ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)

Ac Acetone extract

AChE Acetylcholinesterase

A.C. Sm Smith, Albert Charles

ACTI Acetylthiocholine

AMDIS Automated Mass Spectral Deconvolution and Identification System

AOP-6A Antioxidant protein

APG Angiosperm Phylogeny Group

Aq Aqueous extract

A549 Human adenocarcinoma lung cancer cell line

A2780 Human ovarian carcinoma cell line ax Axial

B16 Human melanoma cancer cell

BDNF Brain-derived neurotrophic factor

Bel7402/5-FU 5-fluorouracil-resistant hepatocellular carcinoma cell line

Benth. Ex Kurz Bentham, George ex Kurz, Wilhelm Sulpiz bFGF Basic fibroblast growth factor

B. N. Chang Chang, Ben Neng c Concentration

Calcd Calculated

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LIST OF ABBREVIATIONS (continued)

CCD Charge coupled device

ChAT Choline acetyltransferase

CHL Luminol enhanced chemiluminescence

C18 Octadecasilyl

C8 Octasilyl

CDCl3 Deuterated chloroform

Ch Chloroform

Di Dichloromethane extract

COSY Homonuclear correlation spectroscopy

CPE Cytopathogenic effect assay

CUPRAC cupric reducing antioxidant capacity

CVB-3 Coxsackie virus B3

C13 NMR Carbon-13 nuclear magnetic resonance

1D, 2D One-, Two-Dimension

δH Proton chemical shift

δC Carbon-13 chemical shift

Daoy Medulloblastoma cell line

Diels Diels, Friedrich Ludwig Emil

DMSO-d6 Deuterated dimethylsulfoxide

DMPD N,N-dimethyl-p-phenylendiamine assay

DNA Deoxyribonucleic acid

DPPH 2,2-diphenyl-1-picryl-hydrazyl-hydrate

DTNB 5,5-dithio-bis-(2-nitrobenzoic acid)

LIST OF ABBREVIATIONS (continued)

Dunn Dunn, Stephen Troyte

Ea Ethyl acetate extract

EBV-EA Epstein–Barr virus early antigen

Eo Essential oil

ESI Electrospray ionization

Et Ethanol extract

EtOH Ethanol

E18 Primary rat hippocampal cells eV Electron volt

FDA United States Food and Drug Administration

Fe2+/g Ferrous iron per gram

FRAP Ferric ion reducing antioxidant power

5-FU 5-Fluorouracil

GAE Gallic acid equivalence method

GC-MS Gas chromatography–mass spectrometry

G. Don. Don, George

GBM Glioblastoma cell line

Ha Hydroalcoholic extract

HaCaT Human keratinocyte cell line

HBV Hepatitis B virus

HCT-8 Human colorectal adenocarcinoma cell line

He Hexane

HeLa Cervical-epithelioid carcinoma cell line

LIST OF ABBREVIATIONS (continued)

Hep G 2.2.15 Liver carcinoma tumor cell line

Hep2 Liver carcinoma tumor cell line

HMBC Heteronuclear Multiple-Bond Correlation

H1 NMR Proton Nuclear Magnetic Resonance

HOB Primary human osteoblast cell line

Hook. f. Hooker, Joseph Dalton

Hook. f. et Thoms Hooker, Joseph Dalton and Thomson, Thomas

HPLC High performance liquid chromatography

HRESIMS High resolution mass spectrometry using electrospray ionization

HSQC Heteronuclear Single Quantum Correlation

Hz Hertz

H3N2 Influenza A virus subtype

IC50 Half of the maximal inhibitory concentration

ICAM-1 Intercellular adhesion molecule-1

IFN-γ Interferon gamma

IL Interleukin

IT-TOF Hybrid ion trap and time of flight mass spectrometer

IR Infrared spectroscopy

IUPAC The International Union of Pure and Applied Chemistry

KI Kovat’s retention index

L Liter

L. Linnaeus, Carl von

λ (nm) Wavelength in nanometers

LIST OF ABBREVIATIONS (continued)

LC Liquid column chromatography

LPO Lipid Peroxidation

LPS Lipopolysaccharide

(Luc)-HEK 293 Glucocorticoid Receptor Pathway GAL4 Reporter

Maxim. Maximowicz, Carl Johann (Ivanovič)

MC/9 Murine mast cell line

MCF-7 Human breast adenocarcinoma cell line

MCF-7/ADR ADR resistant breast cancer cells

MCI Ion exchange resins

MDA Malondialdehyde

MDC/CCL22 Macrophage-derived chemokine

Me Methanol extract

MeOH Methanol mg/kg Milligrams per kilogram

[M] + Molecular Ion

[M+H]+ Protonated Molecular Ion

[M-H]- Deprotonated Molecular Ion

MHz Megahertz

Mill. Miller, Philip

MIC Minimum inhibition concentration mL Milliliter mM Millimole

MPP+ 1-methyl-4-phenylpyridinium

LIST OF ABBREVIATIONS (continued)

MS Mass spectrometry

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide m/z Mass-to-charge ratio

NCI-H460 Human non-small cell lung carcinoma

NC/Nga Mouse model for atopic dermatitis

NH3 Ammonia nM Nanomolar

NMR Nuclear Magnetic Resonance

NIST National Institute of Standards and Technology

NO Nitric oxide

NOESY Nuclear Overhauser Effect Spectroscopy

NF-B Nuclear Factor kappa B

NSCLC Non-small cell lung cancer

Nutt. Nuttall, Thomas

OMe O-Methyl

PAF Platelet-activating factor

PC-3 Prostate cancer cell line

PC12 Pheochromocytoma cell line

PGE2 Prostaglandin type

PMACI Phorbol-12-myristate 13-acetate plus calcium ionophore A23187

PMN Human polymorphonuclear neutrophils

PFK Perflourokerosene

P10 Rat septal neurons

LIST OF ABBREVIATIONS (continued)

qC Quaternary carbon

Q. Lin Lin, Qi

RAW 264.7 Mouse leukemic monocyte macrophage cell line

Rf Retention factor

Rt Retention time

ROESY Rotating-frame Nuclear Overhauser Spectroscopy

SH-SY5Y Neuroblastoma cell line

SK-OV-3 Human ovarian cancer

SKMEL-2 Melanoma cells

SMMC-7721 Human hepatoma cells

SOSA Superoxide anion radical scavenging assay

TARC/CCL17 Thymus and activation regulated chemokine

TBA Thiobarbituric acid

TLC Thin Layer Chromatography

TNF-α Tumor Necrosis Factor-alpha

TOCSY Total Correlation Spectroscopy

TPA 12-O-tetradecanoylphorbol-13-acetate

TPC Total Phenolic Content assay

TRAP Total radical-trapping antioxidant parameter assay

Tutcher Tutcher, William James

UIC University of Illinois at Chicago

UFLC Ultra-fast liquid chromatography

UV Ultraviolet light

LIST OF ABBREVIATIONS (continued)

U/mL Units per milliliter

ν (cm-1) Infrared absorption frequency in reciprocal centimeters

WiDr Colon adenocarcinoma

μg/mL Micrograms per milliliter

μL Microliter

μM Micromole

μM/mL Micromoles per milliliter

SUMMARY

The genus Illicium L. (Schisandraceae) consists of ca. 40 species that form one of the earliest evolutionary branches of angiosperms. It is represented by evergreen trees and shrubs disjunctively distributed in North America, Mexico, Peru, the West Indies and eastern

Asia - with the highest concentration of species in Northern Myanmar and Southern China.

Commonly Illicium is used as a source for shikimic acid in the production of oseltamivir

(Tamiflu), there is a long history of traditional use of this genus as analgesic, antiemetic, antiseptic, antispasmodic, antirheumatic, anxiolytic, carminative, digestive, and sedative agents. Early phytochemical studies of the Illicium genus focused on the extraction of essential oils but recently a variety of secondary metabolites were reported, including the structurally unique and rarely occurring in nature seco-prezizaane sesquiterpenes, diterpenes, triterpenes, prenylated phenylpropanes, lignans, and neolignans. The previous phytochemical and pharmacological investigations of genus Illicium and Illicium angustisepalum are comprehensively reviewed in Chapter 1.

The metabolite fingerprinting and comparison are discussed in Chapter 2.

I. angustisepalum is a sparingly studied plant species, whose classification and identification as a member of the genus Illicium has been based on the morphological characteristics of its flowers, fruits, leaves, pollen and seeds. In order to substantiate the chemical characteristics of this plant species, the metabolite fingerprint by GC-MS was established and compared with those of better-known species, I. verum and I. lanceolatum. Existing variations of the volatiles within this taxon were identified.

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SUMMARY (continued)

In this study, the extract of I. angustisepalum was subjected to phytochemical and biological evaluations. As a result of the phytochemical work, a total of sixteen compounds were isolated and identified, including two novel structures, 2-hydroxy-2-methyl-6- methyleneoct-7-en-3-yl benzoate (3) and 2-hydroxy-7-methyl-hexan-1,5-olide (15). Thymol

(1), (-)-T-muurolol (2), majusanic acid C (5), 6β-hydroxy-4-stigmasten-3-one (9), (2R, 3R)-

3,5,7,3’,4’-pentahydroxyflavonone (10), clovane-2,9-diol (12), majucin (14) and majusanic acid B (16) are identified in I. angustisepalum for the first time. Compounds angustanoic acid

E (4), 2,6-dimethoxychavicol (6), angustanoic acid F (7), angustanoic acid G (8), angustisepalin (11) and angustanol (13) were reported from I. angustisepalum in previous reports. Isolation and structural elucidation of these compounds are described in Chapter 3.

Evaluation of the biological activities of the isolated compounds is described in

Chapter 4. The biological potential of the isolated compounds was assessed in a battery of in vitro assays for cytotoxic, antimicrobial, neuroprotective, acetylcholinesterase inhibiting and neurotrophic activities, and led to the determination of an extensive in vitro biological profile of I. angustisepalum. In the cytotoxic assay, none of the tested compounds showed cytotoxic activity against human melanoma cancer (MDA-MB-435), human breast cancer

(MDA-MB-231) and human ovarian cancer (OVCAR3) cells. In the anti-microbial assay, three abietane diterpenes, angustanoic acid E (4), and G (8) and majusanic acid C (5), displayed moderate antibacterial activities against clinical isolates of selected bacteria. Angustanoic acid E (4) was the most active among these compounds, with MIC’s of 6.25 µg/mL against

E. coli, B. anthracis sterne and B. cereus 14579, and 12.5 µg/mL against S. aureus USA

300, and S. aureus MSSA. For the comparison, a well-known antibiotic, chloramphenicol has

SUMMARY (continued)

a MIC of 1 and 8 µg/mL against E. coli BW25113 ∆TolC and S. aureus USA 300, respectively. Also, vancomycin has a MIC of less than 2 µg/mL against S. aureus MSSA 476, ofloxacin has MIC of 0.8 µg/mL against B. anthracis sterne and doxycycline has MIC of 0.37

µg/mL against B. cereus 14579. On the other hand, angustanoic acid G (8) and (-)-T- muurolol (2) displayed a good protection of primary hippocampal neurons from the MPP+ damage in the neural cell protection assay at 0.4 µg/mL and 10 µg/mL, respectively. In the

TLC bioautographic assay for anticholinesterase activity, six active compounds were identified, including thymol (1), 3,5-dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’- pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-hydroxy-7- methyl-hexan-1,5-olide (15). Thymol (1) and clovane-2,9-diol (12) displayed moderate activity with IC50 values of 13.47 and 11.02 µg/mL, respectively. In the promotion of neural growth assay, thymol (1), majucin (14) and majusanic acid C (5) caused an increase of neurite length greater than that of BDNF. It was also found that compound angustanol (13) was slightly less effective than BDNF. At the dose of 20 μg/mL compounds showed neurotrophic activity in the following order, thymol > majucin > majusanic acid C > BDNF > angustanol > BDNF > angustanol.

In conclusion, the phytochemical and biological investigation of I. angustisepalum led to new insights into this under-studied species. The first in vitro biological profile for this plant was established.

CHAPTER 1

LITERATURE INFORMATION

INTRODUCTION

The Latin names of all plant species, the names of all plant families and also the plant taxonomic hierarchy, included and discussed in the herein dissertation were validated by data retrieved from the Tropicos.org. (1)

The term “Pharmacognosy” derives from two Greek words, “pharmakon” (drug) and

“gnosis” (knowledge). It is disputable who first coined it; but based on the available literature it could be credited to one of the three researchers: Johann Adam Schmidt (Lehrbuch der

Materia Medica, 1811), Anotheus Seydler (Analecta pharmacognostica, 1815) or Ulisse

Aldrovandi (Antidotarii Bononiensis Epitome (1574). (2) (3) The latter author, Ulisse

Aldrovandi, is reckoned as the father of natural history studies and also founded the Botanic

Garden of Bologna, Italy, in 1568. (3) Historically, the field of pharmacognosy has specialized in the macro- and microauthentication and quality assessment of crude drugs that were primarily of plant origin. According to modern definition, “Pharmacognosy” is the field of science which explores naturally occurring substances for their medicinal, ecological, or other functional qualities. It embraces a wide range of disciplines such as organic, medicinal and analytical chemistry, biochemistry, biotechnology, ethnobotany, molecular biology, pharmacology, taxonomy and toxicology. Natural products include crude, semi-purified and purified substances from biological kingdoms, such as plants, animals, marine organisms, fungi, and bacteria.

The use of plants as traditional therapeutics dates far back in the human history.

Palaeoanthropological studies of the burial at the Shanidar cave, localized in the Zagros

Mountains area of Kurdistan, have identified the pollen grains of flowers at the excavation site. (4) These findings suggest that over 60,000 years ago, Neanderthal tribes had knowledge of the therapeutic potentials of the plants. (4) Several ancient civilizations show records of using plants in their traditional healing systems. Probably the oldest known medical text, found in Mesopotamia, dates back to ca. 2600 B.C. The text describes nearly 1000 medicinal plants and plant based substances, and among them are Glycyrrhiza glabra L.

(licorice) (Fabaceae) and Papaver somniferum L. (poppy latex) (Papaveraceae). (5) The

Egyptian medicine, which dates between 3300 B.C. and 252 B.C., has accumulated an extensive knowledge about medicinal plants. The Ebers Papyrus, describes 876 prescriptions made of more than 500 substances, including plants and plant derived substances such as Cassia alexandrina Mill. (senna leaf) (Fabaceae) and Ricinus communis

L. (castor-oil seed) (Euphorbiaceae). (6) Ayurveda, which dates to 2500 and 500 B.C., is the

Indian system of traditional medicine which describes ca. 1250 plants. The earliest mentions of Ayurveda were found in Rig Veda and Atharva Veda, but Charaka Samhita, dated to ca.

900 B.C., was the first treaty fully devoted to concepts and practice of Ayurveda. Some of

Ayurvedic medicines include Centella asiatica (L.) Urb. (asiaticosides) (Apiaceae), Curcuma longa L. (curcumin) (Zingiberaceae), Rauvolfia serpentina (L.) Benth. Ex Kurz (reserpine)

(Apocynaceae). The Chinese Materia Medica is famous for the extensive description of medicinal plants and their applications. The first medicinal book Wu Shi Er Bing Fang, was written in 1100 B.C., and was then followed by the monograph Shen Nong Ben Cao

Jing (Shen Nong Materia Medica), assembbled during the Eastern Han dynasty (25–220

A.D.) (7) and the first national pharmacopoeia, Xin Xiu Ben Cao (Tang Ben Cao) in 659 A.D.

(8) Chinese medicinal books describe numerous herbs, such as Coptis chinensis Franch.

(coptis root) (Ranunculaceae) to treat diarrheoea, Ephedra sinica Stapf. (ephedra herb)

(Ephedraceae) was used as an anti-asthmatic and Melia azedarach L. (chinaberry seed)

(Meliaceae) as an antihelmintic remedies. (8) The therapeutic use of plants was also prominent in Greek and Roman traditions as evidenced by works of Hippocrates, Dioscorides and Galen who described several species of plants and animals that became a source for traditional medicines.

According to the World Health Organization (WHO), more than 75% of the population still rely on plant-based traditional medicines in developing countries. (9) It has also been reported that plants historically used in the traditional healing systems have been the main sources of medicines for the early stage drug discovery. Due to a wide range of biological and pharmacological properties, plants continue to lead as a source of naturally derived drug candidates. Out of the 150 top prescription drugs in the United States market, 118 are either directly or indirectly derived from natural sources and 87 of them are related to plant origin.

(10) Top selling drugs that have been developed from natural products include vincristine from Catharanthus roseus (L.) G. Don. (Apocynaceae), morphine from Papaver somniferum, and Taxol from Taxus brevifolia Nutt. (Taxaceae), to name a few.

The genus Illicium L. belongs to the family Schisandraceae; it comprises of ca. 35 plant species which “form one of the earliest evolutionary branches of the angiosperms”. (11)

(12) (13) “This particular genus is represented by evergreen trees and shrubs disjunctively distributed in North America, Mexico, Peru, the West Indies and eastern Asia - with the highest concentration of species in Northern Myanmar and Southern China”. (11) (12) (13)

Illicium angustisepalum A. C. Sm. remains an under-studied member of the

Schisandraceae family. Until now, only two peer-reviewed articles regarding its chemistry have been published, and no biological activities of this plant were reported or recorded. (14)

(15)

LITERATURE REVIEW ON THE GENUS ILLICIUM

The following subsections review in detail the existing literature pertaining to members of Illicium. This includes the taxonomy and morphological descriptions of genus Illicium, ethnomedical uses, and in vitro and in vivo biological activities. The morphological characterization and chemical profile of the Illicium angustisepalum are discussed as well.

Taxonomy of the genus Illicium

The genus Illicium was created by Carl von Linnaeus in his Systema Naturae (1759) with the description of I. anisatum L. (16) (17) Subsequently, a number of new Illicium species has been introduced. A. C. Smith revised the classification of Illicium and recognized 42 species distributed across “Eastern part of the North America, Mexico, and the West Indies

(5 species) as weel as Eastern Asia (37 species)”. (12) (17) Historically, the systematic position of the genus Illicium received significant attention and has been widely debated. (18)

(19) Based on the early morphological data it was classified as a member of the

Magnoliaceae family (20) and then revised and moved to the new Illiciaceae family. (21)

Recent technological advances, especially application of molecular data analysis (DNA sequences), provided new informative sets of data. As a result, APG I (1998) proposed a new classification for the families of flowering plants. (22) APG II classification (2003) allows

Illiciaceae to be treated as a part of Schisandraceae or as a single family of Illiciaceae. (23)

According to the most recent APG III system (2009) Illicium cannot be treated as an independent plant family and must be included in the Schisandraceae. (11)

At present, the genus Illicium belongs to the Schisandraceae family in the major group of Angiosperms (flowering plants), and it is classified as follows: (24)

Class: Equisetopsida

Subclass: Magnoliidae

Superorder: Austrobaileyanae

Order: Austrobaileyales

The taxonomic description of Illicium is presented as follows: (12)

“Glabrous shrubs or small trees (young branchlets very rarely obscurely puberulent); bud-scales often conspicuous at apices of young branchlets, imbricate, soon caduceus; leaves essentially alternate but often clustered or pseudoverticillate at distal nodes of branchlets, exstipulate, petiolate, the petioles canaliculated, sometimes deeply, usually rugulose when dried, the blades chartaceous to coriaceous, pinnate-veined, decurrent on the petiole, entire; flowers solitary, sometimes appearing to arise in twos or threes, axillary or supra-axillary, sometimes appearing subterminal, often crowded among leaves toward apices of branchlets, rarely lateral on branchlets below leaves, very rarely arising from complex glomerules on trunk or large branches, pedicellate; pedicels terete, sometimes 1- or 2-bracteolate, subtended by few or numerous imbricate bract, these usually caducous; flower hermaphrodite, hypogynous, with free and usually numerous parts; torus convex to

short-conical, terminating in an inconspicuous extension, this oblong to conical, often minutely papillose, usually concealed by the carpel-bases; perianth-segments numerous (7-

33), usually several seriate, often grandular, the outermost ones often small, sometimes bracteole-like, the inner ones gradually larger, ligulate and membranaceous or carnose and ovate to suborbicular, the innermost ones often reduced in size, occasionally transitional toward stamens numerous (4-41 or rarely to 50), 1- to several-seriate, erect, composed of ligulate to subterate filaments and basifixed oblong 4-sporangiate anthers, the connective often carnose, sometimes grandular usually subequal to or sometimes slightly exceeding the thecae in length, the thecae introrse-lateral, protuberant or subimmersed, dehiscing by longitudinal clefts for their entire length, carpels usually 7-15 (rarely 5-21), free, in a single whorl, often closely appressed laterally, obliquely attached to the torus by the broad base and lower part ventral side, erect or subspreading, composed of a laterally flattened ovoid or ellipsoid ovary distally attenuate into a slender or stout acute style, the style conduplicate and stigmatic ventrally along all or most of its length, the ovary uniocular, with a single anatropous ovule borne ventrally near the base; fruit a follicetum composed of a single whorl of free spreading follicles, these oblong to ovoid, broad at base and often ventrally subauriculate, dehiscing ventrally, the dorsal follicle-walls often coriaceous, the lateral walls often thin, the ventral suture thickened, the style more or less persistent; seed with subbasal hilum, usually ellipsoid or obovoid and laterally flattened, rounded on the dorsal edge, subacuate on the ventral edge, rounded at apex, obliquely truncate at base, the testa usually stramineous or brownish, smooth, glossy, brittle, the endosperm copious, oily, the embryo minute, near the hilum.“

Ethnomedical and other uses of species of Illicium

While the most well-known application of Illicium is its use “as a source for shikimic acid in the production of oseltamivir” (Tamiflu; Figure 1-1), (13) there is a long history of traditional use of this genus as analgesic, antiemetic, antiseptic, antispasmodic, antirheumatic, anxiolytic, carminative, digestive, and sedative remedies (Table 1-1). (17)

Due to the geographical distribution of Illicium, most of the traditional uses were recorded in Asia. In the genus, I. verum is the most commonly used Illicium plant. In many

Asian regions, it is added to food as an aromatic spice. The fruits of I. verum have been applied to treat indigestion, colic in infants, skin infections and anxiety. I. verum is also a source of star anise oil, distilled from the plant’s fruits. Thanks to a high content of trans- anethole (85% to 90%; Figure 1-2), star anise oil has been applied in Japan as carminative, digestive and in China as antirheumatic remedy. (17) (25) (26) Other Illicium species have been mostly taken to relieve the pain associated with inflammation and rheumatism. The

Pharmacopoeia of the People’s Republic of China, Vol. 1 (Pharmacopoeia Commission of the Ministry of Public Health, 1995) describes the application of the “dried stem bark of I. difengpi (cortex illicii) for the treatment of pain, explaining that the crude drug dispels “wind”, removes “damp”, and regulates the flow of qi”. (17) (27)

TABLE 1-1: APPLICATIONS OF SPECIES OF ILLICIUM IN FOLK MEDICINE (39)

Application Plant part Illicium Region

Stem bark I. difengpi B. N. Chang China I. brevistylum A. C. Sm. I. dunnianum Tutcher I. henryi Diels Root bark I. jiadifengpi B. N. Chang China Stem bark I. majus Hook. f. et Thoms. I. micranthum Dunn Analgesic I. parvifolium subsp. oligandrum (Merr. & Chun) Q. Lin

I. pachyphyllum A. C. Sm. Fruits I. macranthum A. C. Sm. China Leaves I. simonsii Maxim. Fruits I. verum Hook. f. China I. macranthum Fruits I. majus China Leaves I. simonsii Fruits I. verum China Antiemetic Stem bark I. difengpi China I. brevistylum I. dunnianum I. henryi Root bark I. jiadifengpi Antirheumatic China Stem bark I. majus I. micranthum I. parvifolium subsp. oligandrum I. pachyphyllum Fruits I. verum Indonesia Fruits I. verum Malaysia Antiseptic Fruits I. verum Mexico Fruits I. verum United States Antispasmodic Fruits I. verum Mexico Fruits I. verum United States Anxiolytic Fruits I. verum India Carminative Fruits I. ternstroemioides A. C. Sm. China Fruits I. verum India Digestive Fruits I. verum Indonesia Fruits I. verum Malaysia Sedative Fruits I. verum Mexico Fruits I. verum United States

FIGURE 1-1: Synthesis of oseltamivir from shikimic acid (28) (29)

FIGURE 1-2: Structure of trans-anethole

Brief history of scientific investigations on the genus Illicium

Although Illicium has a long history of use in folk medicine, scientists started to explore its chemical profile only in the mid of the 20th century. The investigational history of Illicium can be divided into three periods: period I (1952-1971), period II (1988-2002) and period III

(2007-2014), as shown on Figure 1-3.

During the period I, when isolation and structure elucidation of natural products’ were limited by phledligng separatory, crystallographic and spectroscopic techniques, early attempts were made to explore the chemistry of Illicium. Structure elucidation was a tedious process mainly relying on information derived from 1H NMR and IR spectra. Anisatin, was the first compound that was reported from genus Illicium. Lane et al. isolated anisatin from

Illicium anisatum L., in 1952. (30) While, Lane and his colleauges purified and proposed the partial structure of anisatin, Yamada et al. reported the complete scafflod of the compound in 1965 (Figure 1-5a). (31) During the following years, Takada et al. isolated and proposed the structure of neoanisatin in 1966 (Figure 1-5b) and Okigawa et al. proposed the structure of pseudoanisatin in 1971 (Figure 1-5c). (32) (33)

The period II started in 1988 when Morimoto et al. and Yang et al. applied 13C NMR and 2D COSY techniques, for the first time, to elucidate the structures of Illicium compounds.

(34) (35) The Japanese researchers resolved structures of new compounds by comparison of the measured NMR data with the data of known compounds. This investigational period can also be characterized with expansion of Illicium species that were studied. Between 1988 and 2002, eight additional Illicium species were studied, including I. angustisepalum, I. difengpi, I. dunnianum, I. majus, I. merrillianum A.C.Sm. (36), I. micranthum subsp. tsangii

(37), I. tashiroi (38) and I. verum. Also a significant increase in number of secondary compounds isolated from Illicium was noted; a total of eighty five new compounds were discovered, from chemical classes such as diterpenes, lignans and neolignans, prenylated phenylpropanes, sesquiterpenes and triterpenes.

The period III, is defined as the time when the 2D NMR spectroscopic techniques

(13C–1H COSY, 1H–1H COSY, NOESY and ROSEY) were routinely used for elucidating structures of Illicium compounds. Between 2007-2014, additional seven species were studied, I. arborescens, I. burmanicum, I. jiadifengpi, I. henryi, I. lanceolatum, I. simonsii and

I. parvifolium subsp. oligandrum. Also, a total of ninety five compounds were discovered.

Most of the studies resulted in isolation and characterization of sesquiterpene compounds

(38% of all Illicium isolates), as shown in Figure 1-4.

The data presented in the Figures 1-3 and 1-4 resulted from the bibliographic investigation carried out by analyzing online scientific databases (Pubmed, SciFinder,

Scopus and Web of Science).

Monoterpenes Sesquiterpenes Diterpenes Triterpenes Prenylated phenylpropanes Lignans and neolignans Other

2 Illicium

915 1 5 1 7 19 12 9 112 6 1114 3 3 2 8 reported from 2 1 5

1 1 1 2 1 1 2 2 2 1 1 2 2 1 2 3 2 3 1 3 Number new of compounds

Time (year)

FIGURE 1-3: New compounds reported from Illicium (1952 – 2014)

9% 6% Sesquiterpenes

38% Diterpenes Triterpenes Prenylated phenylpropanes 30% Lignans and neolignans Other 16%

FIGURE 1-4: New compounds reported from Illicium (1952 – 2014)

a b c

FIGURE 1-5: Structures of anisatin (a), neoanisatin (b) and pseudoanisatin (c)

Biological activities of Illicium plants

Illicium plants have been used in traditional medicine of many Asian regions. A broad range of applications in folk medicine encouraged researchers to explore the pharmacological profile of Illicium plants. The following subsections review and summarize both in vitro and in vivo (Tables 1-2 and 1-3, respectively) biological activities found for Illicium species.

1.2.4.1 In vitro biological activities of species of Illicium

In vitro pharmacological studies indicated that extracts and chemical compounds isolated from Illicium have acetylcholinesterase inhibiting, α-glucosidase inhibiting, anti- inflammatory, antimicrobial, antioxidant, antiviral, chemopreventive, cytotoxic, estrogenic, lipase inhibiting, neuroprotective, neural cell growth promoting and choline acetyltransferase activity. A comprehensive in vitro pharmacological profile of Illicium is presented below (Table

1-2).

1.2.4.1.1 Acetylcholinesterase inhibiting activity

The acetylcholinesterase inhibiting activity of the ethanol extract from the aerial parts of I. simonsii was tested in vitro using Ellman’s assay. Isolated terpene-sesquineolignans,

clovanedunnianol (Figure 1-6a), p-menthadunnianol (Figure 1-6b), mixture of sesquineolignans simonsol E (Figure 1-6c) and simonsin A (Figure 1-6d), and phenylpropane isodunnianol (Figure 1-6e) moderately inhibited acetylcholinesterase with IC50 values of

4.58 µM, 6.55 µM, and 10.34 µM, 13.0 µM respectively. Tacrine served as a positive control in both studies with IC50 value of 0.33 µM. (39) (40)

In another study, the standardized fruit extract of I. verum was tested in vitro using

TLC-bioautography and Ellman’s assay. In the TLC-bioautography study the extract showed weak acetylcholinesterase (AChE) inhibiting activity at 10 mg/mL, and the IC50 was 58.67

μg/mL. (41)

TABLE 1-2: IN VITRO BIOACTIVITIES OF ILLICIUM PLANTS REPORTED IN THE LITERATURE

Activity Species Test/Cell line/Organism Main constituents Sample Ref. Sesquineolignans,

I. simonsii Ellman’s assay terpene- Et (42) AChE inhibition sesquineolignans I. simonsii Ellman’s assay Phenylpropanes Et (40) TLC-bioautography I. verum 70% Et (43) Ellman’s assay α-glucosidase I. griffithii α-glucosidase inhibition Ea (44) inhibition I. verum α-glucosidase inhibition Me (45) Anti-inflammatory I. anisatum MTT/RAW 264.7 Eo (46) I. lanceolatum MTT/RAW 264.7 Phenylpropanes Et (47) Sesquiterpene Luciferase Assay/(Luc)- I. burmanicum lactones, 80% Me (48) HEK 293 sesquiterpenes I. floridanum CHL/PMNs Lignans Di (49) I. parvifolium Inhibition of β- Phenolic subsp. glucuronidase 95% Et (50) diglycosides oligandrum release/PMNs (51) I. verum MTT/HaCaT Monoterpenes 70% Et

Inhibition of β- I. dunnianum glucuronidase Sesquiterpenes 95% Et (52) release/PMNs

TABLE 1-2: IN VITRO BIOACTIVITIES OF ILLICIUM PLANTS REPORTED IN THE LITERATURE

Activity Species Test/Cell line/Organism Main constituents Sample Ref.

I. verum MTT/HaCaT Monoterpenes 70% Et (53) I. verum MTT/MC/9 70% Et (54) Inhibition of TNF-α and I. difengpi Triterpenes 80% Et (55) NF-B/RAW264 I. parvifolium Inhibition of β- Phenylpropanes, subsp. glucuronidase Et (56) sesquiterpenes oligandrum release/PMNs B. subtilis, E. coli, P. aeruginosa, P. vulgaris, S. aureus, Antimicrobial I. griffithii Ea (57) S. paratyphi B, V. parahaemolyticus, Y. enterocolitica, X. oryzae B. subtilis, E. coli, I. griffithii K. pneumoniae, He (57) P. vulgaris, S. aureus S. aureus, B. subtilis, I. griffithii K. pneumoniae, Me (57) X. oryzae A. viscosus, S. mutans, I. simonsii Phenylpropanes 95% Et (58) S. sanguis, A. naeslundii DPPH, CUPRAC, reducing Antioxidant I. griffithii power, LPO, OH- Ea (44) scavenging, DMPD, FRAP I. verum DPPH, ABTS Eo (59) Flavonols, I. verum DPPH, reducing power (60) anthocyanins I. henryi LPO in Fe2+-Cys system Phenylpropanes 95% Et (61) I. dunnianum LPO in Fe2+-Cys system Phenolic glycosides 95% Et (62) I. dunnianum LPO in Fe2+-Cys system Neolignans, lignans 95% Et (63) I. verum DPPH, TPC Me (45) I. difengpi LPO in Fe2+-Cys system Neolignans Me (64) LPO, reducing power, I. verum 50% Et (65) SOSA, TPC I. anisatum DPPH Eo (66) I. parvifolium Neolignan subsp. LPO in Fe2+-Cys system 95% Et (67) glycosides oligandrum LPO in linoleic acid and Phenolics, 80% Et I. verum β-carotene systems, (68) flavonoids Eo DPPH LPO in peroxide, TBA, p-anisidine, total carbonyl, I. verum Monoterpenes Eo (69) linoleic acid systems, reducing power, DPPH

TABLE 1-2: IN VITRO BIOACTIVITIES OF ILLICIUM PLANTS REPORTED IN THE LITERATURE

Activity Species Test/Cell line/Organism Main constituents Sample Ref. (70) I. verum LPO in peroxide system Ea

Sesquiterpene Antiviral I. henryi Anti-HBV 95% Et (71) lactones I. jiadifengpi CPE/Vero cells/CVB-3 Diterpenes 95% Et (72) I. majus CPE/Vero cells/CVB-3 Diterpenes 95% Et (73) I. parvifolium subsp. CVB-3, H3N2 Spirooliganones Ch (74) oligandrum Sesquiterpenes, I. jiadifengpi CPE/Vero cells/CVB-3 sesquiterpene 95% Et (75) glycosides I. jiadifengpi CPE/ Vero cells/CVB-3 Diterpenes 95% Et (76) I. henryi Anti-HBV/Hep G 2.2.15 Sesquiterpenes 95% Et (77) I. anisatum Test for anti-tumor Phenylpropanoids, Chemopreventive I. arborescens (78) promoters/ EBV-EA phytoquinoids I. tashiroi Ea Cytotoxic I. griffithii MTT/A549 He (44) Me MTT/NCI-H460, SMMC- I. simonsii Sesqui-neolignans 95% Et (79) 7721, MCF-7, BGC-823 MTT/ NCI-H460, SMMC- I. simonsii Sesquiterpenes 95% Et (80) 7721 MTT/MCF-7/ADR, Phenylpropanoids, I. simonsii 95% Et (81) Bel7402/5-FU phenol, in-dole I. parvifolium MTT /HCT-8, BGC-823, subsp. Phenylpropanes 95% Et (82) A549, A2780 oligandrum MTT /Hep-2, Daoy, MCF- I. arborescens Phenylpropanes Di/Ea (83) 7, WiDr MTT /HeLa, WiDr, Daoy, I. arborescens Sesquiterpenes Me (84) Hep2 Estrogenic I. arborescens MTT/HOB Phenylpropanes Ea (85) Lipase inhibition I. anisatum Pancreatic lipase inhibition Et (86) Neural cell MTT/Cortical neurons from I. simonsii Phenylpropanes Me (87) protection fetuses of E18 rats I. parvifolium Sesquiterpenes, Attenuation of damage subsp. erythro form of 95% Et (88) induced by H2O2/SH-SY5Y oligandrum anethole glycol Promotion of Germacrane I. lanceolatum SH-SY5Y Me (13) neuronal growth sesquiterpenes Primary cultured rat I. jiadifengpi Sesquiterpenes Me (89) cortical neurons Primary cultured rat I. anisatum Phenylpropanes Me (90) cortical neurons

TABLE 1-2: IN VITRO BIOACTIVITIES OF ILLICIUM PLANTS REPORTED IN THE LITERATURE

Activity Species Test/Cell line/Organism Main constituents Sample Ref. Primary cultured rat Sesquiterpenes I. jiadifengpi Me (91) cortical neurons Primary cultured rat I. anisatum Phenylpropanes Me (92) cortical neurons Primary cultured rat I. simonsii Phenylpropanes Me (93) cortical neurons Primary cultured rat I. jiadifengpi Sesquiterpenes Me (94) cortical neurons Primary cultured rat I. merrillianum Sesquiterpenes Me (95) cortical neurons Primary cultured rat I. merrillianum Sesquiterpenes Me (96) cortical neurons Promotion of ChAT activity/P10 rat I. tashiroi Phenylpropanes Me (97) ChAT activity septal neurons ChAT activity/P10 rat I. tashiroi Phenylpropanes Me (98) septal neurons ChAT activity/P10 rat I. tashiroi Phenylpropanes Me (99) septal neurons Ea: ethyl acetate extract; Eo: essential oil; Et: ethanol extract; Ch: chloroform extract, Di: dichloromethane extract; He: hexane extract; Me: methanol extract.

a b c d e

FIGURE 1-6: Structures of acetylcholinesterase inhibitors from Illicium

1.2.4.1.2 α-Glucosidase inhibiting activity

In an in vitro insulin secretion studies, the ethyl acetate extracts of I. griffithii Hook. f.

& Thomson (100) and I. verum fruits were studied. Both extracts showed a moderate α- glucosidase inhibiting activity with IC50 of 810.32 µg/mL for I. verum, for both extracts.

Acarbose was used as a positive control with the IC50 value of 31.00 µg/mL. (44) (101)

1.2.4.1.3 Anti-inflammatory activity

The use of Illicium plants as anti-inflammatory agents is well documented. The essential oil from I. anisatum was found to be an effective inhibitor of LPS-induced NO and

PGE2 production in RAW 264.7 cells. (102) Bio-assay guided fractionation of the ethanol extract from aerial parts of I. lanceolatum lead to isolation of phenylpropanes, 5,5′-diallyl-

2,2′,3′-trimethoxydiphenyl ether (Figure 1-7a), 4′,5-diallyl-2-hydroxy-3-methoxybiphenyl ether

(Figure 1-7b) and difengpin (Figure 1-7c). Compounds inhibited LPS-induced NO production in a dose-dependent manner with IC50 values of 27.58, 26.59 and 34.35 μg/mL, respectively.

Aminoguanidine served as a positive control with IC50 values of 15.94 μg/mL. (47)

In another study, the anti-inflammatory activity of compounds isolated from the 80% methanol extract from stem bark of I. burmanicum was evaluated by measuring the enzymatic activity of luciferase in NF-κB reporters in a (Luc)-HEK 293 cell line treated with lipopolysaccharide. Sesquiterpene lactones burmanicumolide B (Figure 1-7d), burmanicumolide C (Figure 1-7e), burmanicolide D (Figure 1-7f), and sesquiterpene 4β, 10β- dihydroxyaromadendrane (Figure 1-7g) displayed moderate inhibitory activity with an IC50 value of 8.49, 9.80, 10.7, and 4.33 μM, respectively, vs. 6.20 μM for parthenolide. (48)

a b c d

e f g h

i j k

m, R= (24 R)-OH n, R= (24S)-OH

l m, n

o p q r

FIGURE 1-7: Structures of anti-inflammatory compounds from Illicium

Tetrahydrofuran type lignan, di-O-methyltetrahydrofuroguaiacin B (Figure 1-7h), isolated from the dichloromethane fruits and leaves extract of I. floridanum J. Ellis had influence on luminol enhanced chemiluminescence induced by different stimuli in human polymorphonuclear neutrophils. Lignan strongly inhibited chemiluminescence induced by N- formyl-methionyl-leucyl-phenylalanine at concentrations below 1 μM. Quercetin was used as a positive control showed the IC50 of 0.5 μM. (103)

Phenolic glycoside, illoliganoside D (Figure 1-7i), isolated from the 95% ethanol root extract from I. parvifolium subsp. oligandrum Merr. & Chun (104) moderately inhibited the release of the enzyme β-glucuronidase from the polymorphonuclear leukocytes (PMN) triggered with the platelet activating factor (PAF). The inhibitory ratio of this compound was

25.7% at a concentration of 10 μM, respectively, vs. ginkgolide B inhibitory ratio of 81.5% at

10 μM. (105)

Investigations of I. verum extracts indicated a significant anti-inflammatory activity and therapeutic potential. In one study, 70% ethanol extract from fruits of I. verum significantly inhibited IFN-γ-induced mRNA and protein expression of ICAM-1. In addition, both extract and its constituents, p-anisaldehyde (Figure 1-7j) and trans-anethole (Figure 1-2), effectively suppressed IFN-γ-induced adherence of Jurkat T cells to HaCaT cells and ICAM-1 expression on the cell surface. (51) In another study, 70% ethanol extract from fruits of

I. verum caused “inhibition of the expression of TNF-α/IFN-γ-induced mRNA and protein expression of thymus and activation-regulated chemokine (TARC/CCL17), macrophage- derived chemokine (MDC/CCL22), interleukin (IL)-6, and IL-1β. (106) Furthermore, extract contained 2.14% trans-anethole which showed significant anti-inflammatory activities. (107)

It was also found that the I. verum extract inhibits histamine release from PMACI-stimulated

MC/9 cells at concentrations of 50-200 μg/mL and suppresses production of inflammatory cytokines (IL-4, Il-6 and TNF-α). (108)

Monocyclofarnesane sesquiterpene, 8′-oxo-6-hydroxy-dihydrophaseic acid (Figure 1-

7k) and sesquiterpene tashironin (Figure 1-7l) isolated from the root extract of I. dunnianum, were found to be potent inhibitors of the β-glucuronidase release from rat polymorphonuclear

leukocytes (PMN) triggered by platelet-activating factor, with IC50 of 2.10 and 1.93 μM, respectively. Ginkgolide B was used as a positive control with the IC50 of 2.92 μM. (109)

3,4;9,10-Seco-cycloartane type triterpenoid stereoisomerides, Illiciumolide A (Figure

1-7m) and Illiciumolide B (Figure 1-7n), and (all-E)-2,6,10,15,19,23-hexamethyl-

2,6,10,14,18,22-tetracosahexaene (Figure 1-7o) isolated from I. difengpi showed significant inhibition of TNF-α production in the inhibition of tumor necrosis factor-alpha (TNF-α) assay.

The compounds reduced the concentration of TNF-α in the RAW 264.7 cells by 90%, 85%, and 91%, respectively. Tripterygium tablets (TRT) and total glucosides of paenia (TGP) used as positive controls inhibited LPS-induced RAW 264.7 cells by 59% and 49%, respectively.

Compounds also inhibited the release of the nuclear factor-B (NF-B) in RAW264. 7 cells induced by LPS at 10 and 20, 20 and 90 μg/mL, respectively. (55)

Phenylpropanes Illioliganones B (Figure 1-7p) and C (Figure 1-7q) and sesquiterpene lactone neomajucin (Figure 1-7r) isolated from stem bark of I. parvifolium subsp. oligandrum inhibited platelet-activating factor (PAF) induced release of the β-glucuronidase release in rat polymorphonuclear leukocytes (PMNs). While the inhibitory ratio of ginkgolide B, used in the study as a positive control, was 80.5% at 10 µM, the inhibitory ratios of tested compounds were 33.8, 20.1 and 22.4%. (110)

1.2.4.1.4 Antimicrobial activity

Several studies have been conducted to evaluate the antibacterial effects of Illicium plants. So far, I. griffithii and I. simonsii have been studied. I. griffithii extracts were found to be active against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, P. vulgaris, Staphylococcus aureus, Salmonella paratyphi B, Vibrio

parahaemolyticus, Yersinia enterocolitica, and Xanthomonas oryzae. (111) Phenylpropanes, simonin A (Figure 1-8a), dunnianol (Figure 1-8b), macranthol (Figure 1-8c), isodunnianol

(Figure 1-6e) and manolol (Figure 1-8d) isolated from I. simonsii were active against oral bacteria Actinomyces viscosus, Streptococcus mutans, Streptococcus sanguis and

Actinomyces naeslundii with MIC values between 1.95 to 31.25 µg/mL. Triclosan was used as a positive control with MIC values between 3.9 to 7.8 µg/mL. (112)

a b c d

FIGURE 1-8: Structures of compounds with antimicrobial activity

1.2.4.1.5 Antioxidant activity

The review of the literature demonstrated that the genus Illicium is a good source of natural antioxidants. Anti-oxidant activity was found in I. anisatum, I. difengpi, I. dunnianum,

I. griffithii, I. henryi, I. parvifolium subsp. oligandrum and I. verum.

The ethyl acetate extract from I. griffithii was found to be a potent free radicals scavenger at the dose of 1,000 μg/ml in DPPH (91.12 %) and in CUPRAC (2.384). The extract also showed potent activities in the three assays including reducing power (0.847), lipid peroxidation (55.52 %), hydroxyl (75.83) and also DMPD (76.12 %). I. griffithii extract also showed maximum activity at th dose of 300 μg/ml in the total antioxidant activity (0.290

GAE mg/g) and FRAP (2.150 mM Fe2+/g) assays. (44)

The essential oil obtained from fruits of I. anisatum exhibited moderate DPPH scavenging activity with IC50, 3.83 mg/mL. (102) Essential oil from the fruits of I. verum showed potent antioxidant activity in DPPH and ABTS assays. (59) In another study I. verum

−6 extract had good antioxidant activity with IC50 2.89 x 10 mg/ml which is 100-fold more efficient than antioxidant activity of quercetin. (60) It was also reported that I. verum extract showed DPPH free radical scavenging activity of 87.22% at a concentration of 1 mg/ml and inhibition of FeCl3 induced LPO at IC50 175 μg/mL. (45) I. verum extracts showed potent antioxidant activity in three assays including linoleic acid peroxidation, β-carotene-linoleate and 1, 1-diphenyl-2-picryl hydrazyl (DPPH). In the linoleic acid peroxidation assay, the petroleum ether, ethanol/water and water extracts showed inhibition at 53.0%, 40.6%, 76.3% and 56.7%, respectively. On the other hand, in the β-carotene-linoleate assay, the ethanol/water extract exerted the highest antioxidant activity, followed by water and the petroleum ether extracts. In the, DPPH assay ethanol/water was more active then the petroleum ether extract, at 1 mg/ml. (68)

Phenylpropane, illihenryipyranol A (Figure 1-9a) isolated from roots of I. henryi exhibited strong antioxidant activity with IC50 value of 2.97 μM. Vitamin E was used as a positive control with IC50 of 51.69 μM. (113)

Phenolic glycosides, dunnianosides A–E (Figure 1-9b-9f), G (Figure 1-9g) and 6′-O- vanilloyltachioside (Figure 1-9h), isolated from root of I. dunnianum, exhibited potent antioxidant activities against Fe2+-cystine-induced rat liver microsomal lipid peroxidation, with

IC50 values ranging from 3.8 to 23.0 μM. Vitamin E was used as a positive control with IC50 of 23.40 μM. (114)

Neolignans (–)-dehydrodiconiferyl alcohol (Figure 1-9i) and simulanol (Figure 1-9j) and lignans (–)-matairesinol (9k), aviculin (9l), and (–)-secoisolariciresinol-O-a-L- rhamnopyranoside isolated from the roots of I. dunnianum, exhibited potent antioxidant

2+ activities against Fe -cysteine-induced rat liver microsomal lipid peroxidation, with IC50 values ranging from 4.2 to 38.4 µM. Vitamin E was used as a positive control with IC50 of

23.40 μM. (115)

Neolignan (7R, 8S)-4,7, 9-trihydroxy-3,5,3′,5′-tetramethoxy-8-O-4′-neolignan-8′-ene

(Figure 1-9m) isolated from I. difengpi displayed antioxidant activity with an IC50 value of

42.3 μM. (64)

(7R,8S)-9-O-shikimoyl-4-O-β-D-glucopyranosyldihydrodehydrodiconiferyl alcohol

(Figure 9n) and also (7S,8R)-1-[4-O-(β-D-glucopyranosyl)-3-methoxyphenyl]-2-[4-(3- hydroxypropyl)-2,6-methoxyphenoxy]-1,3-propanediol (Figure 1-9o) isolated from I. parvifolium subsp. oligandrum , were found to have a moderate antioxidant properties with the inhibitory rates of MDA of 13.30 % and 9.30 % at 1.0 × 10-5 M. The inhibition rate for vitamin E was 21.90%. (67)

a b, R1= OCH3 R 2= OCH3 e, R1= H R 2= H c, R1= OCH3 R2= H h, R1= OCH3 R2= H d, R1= H R2= H

i f, R1= H R2= H R3=OCH3 j g, R1= OCH3 R2= OCH3 R3=OH

k l m

n o

FIGURE 1- 9: Structures of antioxidant compounds from Illicium plants

1.2.4.1.6 Antiviral activity

The antiviral activity was reported in I. jiadifengpi, I. henryi, I. majus and I. parvifolium subsp. oligandrum . Tashironin (Figure 1-7m), isolated from I. henryi, inhibited HBV surface antigen (HBsAg) secretion at IC50 of 0.48 mM and also HBV antigen (HBeAg) secretion at

IC50 of 0.15 mM. Lamivudine was used as a positive control with IC50 of 16.0 mM (HBsAg) and 20.0 mM (HBeAg). (116)

Spirooliganones A (Figure 1-10a) and B (Figure 1-10b), isolated from I. parvifolium subsp. oligandrum , were found to exhibit potent activities against Coxsackie virus B3 with

IC50 of 11.11 and 3.70 μM, respectively. Spirooliganone A was also active against influenza

A (H3N3) with an IC50 value of 5.05 μM. Ribavirin and oseltamivir were used as a positive controls with IC50 of 2120.4 μM and 4.39 μM, respectively. (74)

Abietane diterpenes, isolated from I. jiadifengpi, exhibited reasonable activity against

CVB3, with IC50 values of 7.0-22.2 µM/mL vs. ribavirin 1.25 µM/mL. (72) Similarly, abietane diterpenes isolated from I. majus also displayed antiviral activity against the Coxsackie B3 virus, with IC50 values of 3.3−51.7 μM/mL vs. ribavirin 0.9 µM/mL. (73) Abietane diterpenes from I. jiadifengpi were active against Coxsackie B2, B3, B4 and B6 viruses in African green monkey kidney cells (Vero cells) using a cytopathogenic effect (CPE) assay most potent with

IC50 values all lower than 10 μM/ml vs. ribavirin 1.10-2.21 μM/ml. (117)

a b

FIGURE 1-10: Structures of antiviral compounds from Illicium plants

1.2.4.1.7 Chemopreventive activity

Phenylpropanoids isolated from I. anisatum, I. arborescens and I. tashiroi were found to be valuable as potential cancer chemopreventive agents. Noteworthy, the IC50 values of these compounds were lower when compared to a vitamin A precursor, β-carotene (IC50 400

M ratio/TPA), commonly used as a reference in the cancer prevention studies. Interestingly,

phenylpropanoids with the prenyl group such as 4-allyl-2-methoxy-6-(3methyl-2- butenyl)phenol (Figure 1-11a) and 4-allyl-2,6-dimethoxy-3-(3-methyl-2-butenyl) phenol

(Figure 1-11b) were the most potent anti-tumor promoters (IC50 224 and 217 M ratio/TPA, respectively). (118)

a, R1= OCH3 R2= OH R3= prenyl R4=H b, R1= OCH3 R2= OH R3= OCH3 R4= prenyl

FIGURE 1-11: Structures of chemopreventive compounds from Illicium plants

1.2.4.1.8 Cytotoxic activity

Extracts and compounds from I. arborescens, I. griffithii, I. simonsii, I. parvifolium subsp. oligandrum were found to have good anticancer activities.

I. griffithii fruit extracts had good cytotoxic activity against A549 human adenocarcinoma lung cancer cell line. The hexane extract showed 56.7% activity at the dose of 500 µg/mL with IC50 value of 400 µg/mL. Ethyl acetate showed 78.7% activity at the dose of 500 µg/mL with IC50 value of 300 µg/mL. Methanol had 68.9% activity at 500 µg/mL with

IC50 value of 400 µg/mL. (44)

Sesqui-neolignans, simonol A (Figure 1-12a) and simonol B (Figure 1-12b), isolated from I. simonsii fruit extract showed strong activities comparable to 5-Fluorouracil.

Compounds showed activity against NCI-H460 cell line with IC50 values 17.69, 33.23 μM,

SMMC-7721 cell line with IC50 values 16.74, 25.16 μM, MCF-7 cell line with IC50 values 46.15,

62.46 μM, BGC-823 cell line with IC50 values 36.25, 47.08 μM. 5-FU activities were 11.04,

14.51, 12.38 and 15.43 μM. (79)

Seco-prezizaane type sesquiterpenes anisatin (Figure 1-5a) and (1S)-minwanenone

(Figure 1-12c), from fruits of I. simonsii were found to have potent cytotoxic activities against

NCI-H460 and SMMC-7721, with IC50 values of 16.77, 21.84 μM and 12.06, 24.66 μM. 5-FU was used as a positive control with 11.04 and 14.51 μM. (119)

In another study compounds isolated from fruit extract of I. simonsii were evaluated for their ability to sensitize tumor multidrug resistant cell lines, MCF-7/ADR and Bel7402/5-

FU, to anti-neoplastic agents. Phenylpropanoids (Figures 1-12d and 1-12e), a simple permethoxylated phenol (Figure 1-12f), and one in-dole (Figure 1-12g) exhibited the most potent reversal potential. Verapamil used as a positive control showed weak reversal potential.

(120)

a b c d

e f g h

i j k

FIGURE 1-12: Structures of cytotoxic compounds from Illicium plants

Phenylpropanes from roots of I. parvifolium subsp. oligandrum (2S, 4S)-illicinone D

(Figure 1-12h) exhibited potent cytotoxic activity against HCT-8, BGC-823, A549, and A2780, with IC50 values of 0.30–2.57 μM. 4R-illicinone C (Figure 1-12i) showed moderate selective cytotoxicity against sensitive A2780 cells with IC50 value of 1.38 μM vs. adriamicin 0.32 μM.

(121)

Illicaborin B (Figure 1-12j), from fruit extract of I. arborescens, exhibited a moderate cytotoxic activity against panel of cancer cell lines, Hep-2 (human laryngeal carcinoma),

Daoy (human medulloblastoma), MCF-7 (human breast adenocarcinoma), and WiDr (human colon adenocarcinoma) tumor cell lines with IC50 values of 10.32 μg/mL, 14.52 μg/mL, 16.82

μg/mL, 17.16 μg/mL. (122)

Investigation of aerial parts of I. arborescens lead to isolation of minwanensin-type sesquiterpene, 14-O-benzoylminwanensin (Figure 1-12k) and anisatin-type sesquiterpene,

(3b)-3-(acetyloxy)-14-O-benzoyl-10-deoxyfloridanolide (Figure 1-12l). Compounds showed weak cytotoxicity against HeLa, WiDr, Daoy and Hep2 tumor cell lines with IC50 values for

14-O-benzoylminwanensin as 9.0, 7.1, 11.2 and 10.9 mg/mL respectively and for (3b)-3-

(acetyloxy)-14-O-benzoyl-10-deoxyfloridanolide as 5.1, 6.3, 10.9, 6.24 mg/mL respectively.

(123)

1.2.4.1.9 Estrogenic activity

Phenylpropanes, illicarborene A (13a), illioliganfunone D (13b), 1-allyl-3, 5-dimethoxy-

4-(3-methylbut-2-enyloxy) benzene (13c), (−)-illicinone A (13d) and (−)-illicinone B (13e) isolated from fruits of I. arborescens showed estrogen-like activity. Compounds increased the proliferative activity in primary cell culture of osteoblast cells over 130% at 1000 nM.

Illicarborene A, (−)-illicinone A and (−)-illicinone B evaluated in MTT assay increased osteoblasts proliferation by 146% at 50 nM, 153% at 10 nM and 164% at 5 nM, respectively.

Positive control was 17β-estradiol 140.6% at 1.0 nM. (85)

a b c

d e

FIGURE 1-13: Structures of Illicium compounds with estrogen-like activity

1.2.4.1.10 Lipase inhibition activity

Pancreatic lipase inhibition was determined in 50% ethanol extract of stem from I. anisatum with IC50 of 21.9 µg/mL. Orlistat was used as a positive control with IC50 of 0.75

µg/mL. (86)

1.2.4.1.11 Neural cell protection activity

Macranthol (Figure 1-8c) isolated from fruit extract of I. simonsii was found to have neuroprotective activity at 5-10 µM in rat cortical neurons. Basic fibroblast growth factor

(bFGF) at 10ng/mL was used as a positive control. (124)

Anislactone B (Figure 1-14a) and the erythro form of anethole glycol (14b), isolated from I. parvifolium subsp. oligandrum, attenuated the damage induced by H2O2 in SH-SY5Y cells. Compounds were effective at 1 µM and at 10 µM which produced increase of cell survival of 12.55% and 7.29%, respectively. The positive control α-tocopherol (vitamin E) caused 7.16% increase in cell viability at 10 µM. (125)

a b

FIGURE 1-14: Structures of neuroprotective compounds from Illicium plants

1.2.4.1.12 Promotion of neural growth activity

Promotion of neural growth activity has been widely described in Illicium plants.

Activity was found in sesquiterpenes and phenylpropanes in several Illicium species such as

I. lanceolatum, I. jiadifengpi, I. anisatum, I. simonsii and I. merrillianum.

I. lanceolatum was recently found to have neurotrophic potential. (1S,5R,7R)-1,5-

Dihydroxygermacra-4(15),10(14),11(12)-triene (at 62.5 µM; Figure 1-15a) and (1R,5R,7R)-

1,5-dihydroxygermacra-4(15),10(14),11(12)-triene (at 15.6 µM; Figure 1-15b) induced proliferation of the neuroblastoma (SH-SY5Y) cell line by 36.2% and 45.8%, respectively, indicating potential neurotrophic activity. (13)

Seco-prezizaane-type sesquiterpene, isolated from I. jiadifengpi, (2R)-hydroxy- norneomajucin (Figure 1-15c) exhibited a significant neurotrophic activity. This compound promoted outgrowth of the primary cultured rat cortical neurons at concentrations between

1 to 10 μM/L. Basic fibroblast growth factor (bFGF) at 10 ng/mL was used as a positive control. (126)

Phenylpropane, 4-allyl-2-methoxy-6-(2-methylbut-3-en-2-yl)phenol (Figure 1-15d), isolated from I. anisatum moderately promoted outgrowth of the primary cultured rat cortical neurons at 10 µM. Basic fibroblast growth factor (bFGF) at 10 ng/mL was used as a positive control. (90)

In another study two seco-prezizaane-type sesquiterpenoids, jiadifenolide (Figure 1-

15e), jiadifenoxolane A (Figure 1-15f), isolated from I. jiadifengpi strongly promoted the outgrowth of primary cultured rat cortical neurons at 0.01 to 10 μM. bFGF at 10 ng/mL was used as a positive control. (127) Illicinin A (Figure 1-15g) and 4-allyl-2,6-dimethoxy-3-(3-

methylbut-2-enyl)phenol (Figure 1-15h), from I. anisatum, promoted the outgrowth of primary cultured rat cortical neurons at 0.1 to 10 μM. bFGF at 40 ng/mL was used as a positive control

(128)

Sesqui-neolignan, isodunnianol (Figure 1-6e) from I. simonsii also promoted the growth of the primary rat cortical neurons activity at 0.1 to 10 μM. bFGF at 10 ng/mL was used as a positive control. (129)

Jiadifenin (Figure 1-15i) and (2S)-hydroxy-3,4-dehydroneomajucin (1-15j), from I. jiadifengpi, significantly promoted the neurite outgrowth in primary cultures of fetal rat cortical neurons at 0.1 to 10 μM. bFGF at 10 ng/mL was used as a positive control. (94)

11-O-Debenzoyltashironin (Figure 1-15k), isolated from I. merrillianum, showed neurotrophic activity in the primary culture of rat derived cortical neurons at 0.1−10 μM. (130)

Merrilactone A (1-15l), also discovered from I. merrillianum, exhibited potent neurotrophic activity, by promoting the outgrowth of the primary cultured rat cortical neurons at 10 μM/L to 0.1 μM/L. (96)

a b c d

e f g h

i j k l

FIGURE 1-15: Structures of neurotrophic compounds from Illicium

1.2.4.1.13 Promotion of ChAT activity

Promotion of the choline acetyltransferase (ChAT) activity was displayed only in one

Illicium species, namely I. tashiroi. A number of phenylpropanoids such as tricycloillicinone

(Figure 1-16a), Bicycloillicinone asarone acetal (Figure 1-16b) and 2(R)-12-chloro-2,3- dihydroillicinone E (Figure 1-16c) increased activity of the enzyme choline acetyltransferase in the culture of P10 rat septal neurons by 143% at 30 µM for tricycloillicinone and 228% at

30 µM for 2(R)-12-chloro-2,3-dihydroillicinone E. (131)

a b c

FIGURE 1-16: Structures of ChAT promoting compounds from Illicium

1.2.4.2 In vivo biological activities of Illicium plants

A comprehensive in vivo pharmacological profile of Illicium is discussed in the sections below Table 1-3 presents the in vivo bioactivities of Illicium plants reported in the literature

1.2.4.2.1 Anti-depressant

An anti-depressant effect was described in I. dunnianum. In brief, after treatment with macranthol (10, 20 and 40 mg/kg) mice were subjected to three assays including the forced swimming test, tail suspension test and chronic unpredictable mild stress. (132) In the forced swimming and in the tail suspension tests, macranthol decreased the immobility time. This triphenyl lignan also reversed the reduction of sucrose preference in the chronic unpredictable mild stress assay. Based on these findings it was concluded that macranthol exerted an antidepressant-like effects. In addition, authors suggested that the antidepressant-like effcts might be mediated by neuroendocrine and serotonergic systems.

(132)

1.2.4.2.2 Anti-inflammatory

The ethanol extract of the leaves and stems of I. lanceolatum was tested for the ability to reduce dimethyl benzene-induced edema in the mouse ear. The ethanol extract significantly suppressed dimethyl benzene-induced edema in mice at an oral dose of 800 mg/kg administration and caused 44.44% edema inhibition. (47)

The topical application of the 70% ethanol extract derived from I. verum caused supression of the atopic dermatitis in NC/Nga mice. The histopathological evaluation

revealed that the epidermis was thinner and that dermis was infiltrated by inflammatory cells.

(133)

1.2.4.2.3 Chemopreventive

I. verum extract was found to have a chemopreventive effect in N-nitrosodiethylamine initiated and phenobarbital promoted hepato-carcinogenesis. Star anise extract reduced the tumor burden, lowered oxidative stress and increased the level of phase II enzymes, which may all contribute to its anti-carcinogenic potential. (134)

TABLE 1-3: IN VIVO BIOACTIVITIES OF ILLICIUM PLANTS REPORTED IN THE LITERATURE

Activity Species Main constituents Sample Ref.

Anti-depressant I. dunnianum Macranthol 95% Et (135) Anti-inflammatory I. lanceolatum Et (47) I. verum Anethole Eo (133) I. verum 70% Et (108) Chemopreventive I. verum 50% Et (136) Fumigant I. verum (137) trans-ρ-Mentha-18-dien-2-ol,d-limonene, I. pachyphyllum Eo (138) caryophyllene oxide I. verum Eo (139) α-Terpineol, carvone, d-limonene, trans-carveol, I. simonsii Eo (140) trans-pinocarveol I. difengpi Safrole, linalool Eo (141) β-Caryophyllene, ∆-cadinene, methyl eugenol, I. simonsii Eo (142) β-elemene, α-amorphene I. verum Eo (143) I. verum trans-Anethole Eo (144) I. verum Me (145) I. verum Eo (146) I. verum Me (147) I. verum trans-Anethole Me (148) Eo: essential oil; Et: ethanol extract; Me: methanol extract.

1.2.4.2.4 Fumigant

The fumigant activity is very well discussed in the literature. Most of the studies report fumigant activity of essential oils, extracts and their monoterpene components. A number of Illicium species was evaluated for their fumigant activity such as I. difengpi, I. simonsii, I. pachyphyllum and I. verum.

Morphological description of I. angustisepalum

The morphological description of I. angustisepalum is as follows (149):

“Trees to 11 m tall. Perules oblong to ovate, to 7 mm. Leaves in clusters of 3-6 at distal nodes; petiole 1-2.5 cm; leaf blade oblong-elliptic to elliptic, (5.5-) 7-12(-20) × 2-4(-7.5) cm, thinly leathery to leathery, midvein adaxially slightly prominent, secondary veins 6-9 on each side of midvein, abaxially prominent, and adaxially prominent or slightly impressed, base attenuate or decurrent along petiole, apex subacute to acuminate. Flowers axillary or subterminal. Flower peduncle 0.5-2 cm. Tepals (17-) 22-33, white or pale yellowish, elliptic

(outer) to elliptic-oblong (largest), 14-16 × 3-3.5 mm (largest), thinly papery to submembranous. Stamens 22-25, 2.5-3.2 mm; filaments 1.2-2 mm; connectives truncate to slightly cuspidate; anthers 1.1-1.6 mm; pollen grains tricolpate. Carpels 11-16, 3.5-4 mm; ovary 1.3-1.7 mm; style 1.5-3 mm. Fruit peduncle 1.2-3 cm. Fruit with 11-16 follicles; follicles

1-2.2 × 0.5-1.5 × 0.3-0.8 cm. Seeds 5-8.5 × 4-6 × 2-4 mm. Fl. Feb-Apr, fr. Sep-Oct.” An image of the flowering twigs of I. angustisepalum is shown in Figure 1-17.

FIGURE 1-17: I. angustisepalum, the whole plant, at the collection site in Hong Kong

Chemical constituents of I. angustisepalum

I. angustisepalum is a sparingly studied plant species. Up to date, only nineteen compounds have been reported from this member of Illicium. Most of the reported compounds are abietane diterpenes although a rare prezizaane sesquiterpene have also been found (Table 1-4 and Figure 1-18).

TABLE 1-4: COMPOUNDS REPORTED FROM I. ANGUSTISEPALUM

Compound Type Ref.

Angustanal (a) Abietane diterpene (15) Angustanoic acid A (b) Abietane diterpene (15) Angustanoic acid B (c) Abietane diterpene (15) Angustanoic acid C (d) Abietane diterpene (15) Angustanoic acid D (e) Abietane diterpene (15) Angustanoic acid E (f) Abietane diterpene (15) Angustanoic acid F (g) Abietane diterpene (15) Angustanoic acid G (h) Abietane diterpene (15) Angustanoic acid H (i) Abietane diterpene (15) Angustanoic acid I (j) Abietane diterpene (15) Angustanol (k) Abietane diterpene (15) Angustisepalin (l) Prezizaane sesquiterpene (14) Caryophyllene oxide (m) Sesquiterpene (15) Methoxy eugenol (n) Phenylpropene (15) Geraniol Benzoyl Ester (o) Monoterpene (15) 4-epi-Palustric acid-9r,13r-endoperoxide (p) Abietane diterpene (15) Epi-Palustric acid (q) Abietane diterpene (15) 4-epi-Sandaracopimaric acid (r) Abietane diterpene (15) β-Sitosterol (s) Sterol (15)

a b c d

e f g h

g i j k

l m n o

p q r

FIGURE 1-18: Structures of compounds isolated from I. angustisepalum

OBJECTIVES OF THE STUDY

The three major objectives of the present research work are outlined below.

Aim 1. To create a metabolite fingerprint of Illicium angustisepalum and identify variations of the volatiles within genus Illicium

I. angustisepalum is a sparingly studied plant species, whose classification and identification as a member of the genus Illicium has been based on the morphological characteristics of its flowers, fruits, leaves, pollen and seeds. (150) In order to substantiate the chemical characteristics of this plant species, it is desirable to establish the metabolite fingerprint by GC-MS and to compare this metabolite profile with those of better-known species, I. verum and I. lanceolatum and also to identify any existing variations of the volatiles within this taxon.

Aim 2. To isolate and elucidate the structures of secondary metabolites from I. angustisepalum

In order to obtain an insight of the chemical composition of the plant species and to search for novel and/or biologically active metabolites, the twig of I. angustisepalum will be explored. The structures of isolated secondary metabolites will be elucidated using a combination of spectroscopic techniques, such as 1D and 2D NMR and HRESIMS, and also by comparisons with the published data.

Aim 3. To evaluate the biological activities of the fractions and secondary metabolites from I. angustisepalum

Despite many biological studies of Illicium, I. angustisepalum has not been subjected to any evaluations, yet. Present study determined the in vitro biological profile of

I. angustisepalum. The isolated compounds will be tested in a battery of in vitro bioassay

systems for cytotoxic, antimicrobial, neuroprotective, acetylcholinesterase inhibiting and neurotrophic activities. Bioassays included the use of human melanoma cancer (MDA-MB-

435), human breast cancer (MDA-MB-231) and human ovarian cancer (OVCAR3) cells, clinical isolates of bacterial strains Acinetobacter calcoaceticus, Bacillus anthracis sterne,

Bacillus cereus 14579, Enterococus faecalis V583, E. coli MG1655, E. coli BW25113 ∆TolC,

Streptococcus aureus USA 300, S. aureus MSSA 476, pheochromocytoma (PC12) and primary cortical rat neurons.

CHAPTER 2

METABOLITE FINGERPRINTING OF I. ANGUSTISEPALU

RATIONALE

Metabolomics can be defined as the identification of all metabolites present in one system. At present it is not entirely possible as none of the currently available analytical techniques is selective and sensitive enough to completely identify of all metabolites present in a given sample. (151)

Depending on the problem addressed, researchers use specific metabolomic approaches or their combinations such as: metabolite fingerprinting and metabolite profiling.

While metabolite fingerprinting applies analytic technologies to discover some major differences between samples, metabolite profiling identifies from hundreds to thousands of metabolites and demands an efficient pipeline for extraction, separation and data analysis to robustly and quantitatively measure these metabolites in the complex mixture of chemicals

(‘matrix’) found in cellular extracts. (151)

Metabolite fingerprinting is a rapid and effective tool applied in authentication and quality control of plant material as well as in chemotaxonomic discrimination between plant species. Figure 2-1 outlines the general approach to metabolic analysis.

As plant volatiles are key targets in chemotaxonomy studies, the plant volatile secondary metabolite profiles for I. angustisepalum, I. lanceolatum and I. verum were created and compared.

Plant tissue

Extraction

Data aquisition

Analysis

FIGURE 2-1: Schematic representation of the processes in metabolite analysis

EXPERIMENTAL PROCEDURES

The following subsections present the experimental procedures which led to creation of metabolite fingerprints of I. angustisepalum and the two better studied Illicium species such as I. lanceolatum and I. verum. A detail comparison of all three species has also been discussed.

Plant material collection

Fresh twigs of Illicium species were collected on March 20th 2011, from the Hong Kong region (Table 2-1). Twigs were dried and then milled into a fine powder. A total of 100 g of fresh twigs from each of species were cut into small pieces, dried and milled into fine powder.

Samples were stored at -20°C.

TABLE 2-1: PLANT MATERIALS USED IN THE EXPERIMENTS

Illicium Origin Code

I. angustisepalum Lantau Peak, Hong Kong IAT I. lanceolatum Lantau Peak, Hong Kong ILT I. verum Lantau Peak, Hong Kong IVT

Plant material extraction

Plant material samples were percolated individually with 90% (v/v) ethanol at room temperature, filtered and then dried in vacuo. A total of about 20g of each of the extracts was received and used in GC-MS analysis.

2.2.3 GC-MS analysis, data processing and compound identification

The metabolite fingerprints of I. angustisepalum, I. lanceolatum and I. verum were generated by direct analysis of the respective twig extract solutions using gas chromatography coupled with mass spectrometry (GC-MS) which is a rapid and reproducible technique suitable for the identification of volatile secondary metabolites in complex matrices such as the plant’s extract. (152) Experiments were carried as previously described in the literature. (153) JEOL GCMate II (JEOL USA, Peabody MA) gas chromatograph/ mass spectrometer (GC-MS) was used in these experiments. The gas chromatograph was the

Agilent 6890Plus (Wilmington DE) supplied with a G1513A auto-injector and 100 vials sample tray connected to the G1512A controller. The GC column was a fused silica capillary column coated with a nonpolar 5% phenyl 95% dimethylpolysiloxane phase (Agilent HP-5ms

Ultra Inert), 30 meters long, 0.25 mm internal diameter and 0.25 um film thickness. The carrier gas was Helium (99.9995% Research Grade) run through a STG triple filter (Restek

Corp.) at a constant flow rate of 1.2 mL/min. The injector was held at 250°C and was fitted with an Agilent 4 mm ID single taper split liner containing deactivated glass wool. 1ul of solution was injected at a split ratio of 1:1. The initial oven temperature was 40°C held for 2 min, raised to 280°C at a rate of 3°C per min, then held for 0 min. Total run time was 82 min.

The mass spectrometer was a benchtop magnetic sector operating at a nominal resolving power of 500 using an accelerating voltage of 2500 volts. The spectrometer was operated in

45 full scan EI mode (+eV) with the filament operating at 70eV scanning from m/z 10 to m/z 850 using a linear magnet scan. The scan speed was 0.2 sec per scan. The solvent delay was

4.0 min. Data analysis was carried out with the TSS Pro software (Shrader Analytical &

Consulting Laboratories, Inc., Detroit MI) provided with the spectrometer. Total ion current

(TIC) chromatographic peaks were used for quantitation. Mass calibration was performed using perflourokerosene (PFK). GC-MS conditions applied in the analysis are summarized in Table 2-2.

The Automated Mass Spectral Deconvolution and Identification System (AMDIS) was applied for peak identification and deconvolution of the chromatogram. AMDIS method retrieved mass spectra and related information (relative abundance, retention time, integrated signal and base peak) for each peak from raw GC-MS data.

According to IUPAC Kovat’s retention index (KI) comes from the interpolation that relates the adjusted retention time (or volume) of the component detected in the sample to the adjusted retention times (or volumes) of the two reference compounds eluted before and after the component detected in the sample. (154) KIs of each of the peaks were calculated using the retention time (Rt) values of alkane standards C7–C25 and the Retention Index

Calculator. (155) Identification of volatile compounds was achieved by matching of the calculated KIs with the KIs and mass spectra of compounds retrieved from the NIST library in the MS Search Program and AMDIS. The match required a spectral fit score of at least

70.0 and KI within five units of the reported values. The KI used to identify compounds is based on the non-polar capillary column (DB-5) similar to the HP-5 MS column used for GC-

MS analysis in the present study.

TABLE 2-2: CONDITIONS USED IN THE GC-MS ANALYSIS

Acceleration voltage 2500 HP-5 MS (30 m x 0.25 mm non-polar capillary column, 0.25 µm film Column thickness) Carrier gas Helium (1.2 mL/min) Instrument JEOL GCMate II (JEOL USA, Peabody MA) Ion source temperature 230 °C Ionization energy Electron impact, 70 eV Mass resolution 500 Oven temperature program 40 °C initial held for 1.45 min, then increased at a rate of 3 °C/min to 280 °C Run time 82 min Sample injection volume 1 µL Split injection 1:1 Scan range 10-850 amu

RESULTS

The following subsections discuss the identification of volatile secondary metabolites in the twig extracts of the three analyzed Illicium species. The metabolite fingerprints for

I. angustisepalum, I. lanceolatum and I. verum are presented and compared.

Identification of volatile secondary metabolites

Confirmation of the identity of organic compounds typically demands implementation of at least two analytical techniques. (155) In studies involving plant volatiles analysis, matching of mass spectral data with retention indices is convenient as both can be obtained during a single run. In addition, KIs are highly reproducible and allow for identification of stereoisomers that are produced often.

The qualitative analysis of GC-MS data of I. angustisepalum led to identification of 44 organic volatile compounds which all are reported from I. angustisepalum for the first time.

Most of the detected compounds are chemically classified as terpenoids, including 18 monoterpenes and 21 sesquiterpenes. In addition to that, the analyzed I. angustisepalum compounds had similar mass spectra but distinctly separate in the chromatographic domain.

(156) The volatile secondary metabolites were identified by comparing mass spectra and the

KIs of unknowns with data stored in publicly available databases such as NIST database in the MS Search Program and AMDIS. The Rt, KI of unknowns and the values of nearest KIs are listed for each of the analyzed Illicium species in Tables 2-3, 2-4 and 2-5.

The analysis of GC-MS data of I. lanceolatum led to identification of 56 distinct components. Similar to I. angustisepalum, majority of the identified compounds are terpenoids, including 19 monoterpenes and 27 sesquiterpenes. In addition, the I. lanceolatum sample also contained 4 phenylpropanes, an organic aldehyde, 2 organic alcohols, an organic long chain aldehyde and an organic long chain ester.

TABLE 2-3: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. ANGUSTISEPALUM EXTRACT

Rt (min) of KI of Nearest KI from NIST compound with KI nearest Ref. unknown unknown NIST to unknown 6.219 831 835 isovaleric acid (157) 6.727 847 853 (E)-2-hexenal (158) 7.459 867 868 n-hexanol (157) 9.590 920 926 tricyclene (159) 10.117 933 932 α-thujene (160) 12.135 978 978 sabinene (161) 13.378 1001 1001 n-octanal (157) 13.520 1005 1004 a-phellandrene (160) 14.086 1019 1019 α-terpinene (161) 14.478 1028 1027 o-cymene (161) 14.653 1032 1031 limonene (157) 16.096 1062 1062 y-terpinene (161) 17.857 1096 1096 trans-sabinene hydrate (159) 18.097 1100 1099 linalool (158)

TABLE 2-3: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. ANGUSTISEPALUM EXTRACT

Rt (min) of KI of Nearest KI from NIST compound with KI nearest Ref. unknown unknown NIST to unknown 20.013 1144 1144 camphor (159) 21.659 1178 1178 terpene-4-ol (157) 22.283 1190 1190 a-terpineol (158) 23.643 1220 1219 trans-carveol (161) 24.061 1230 1230 cis-carveol (159) 26.625 1285 1286 borneol acetate (161) 30.556 1376 1376 a-copaene (159) 30.857 1383 1383 (E)-β-damascenone (157) 32.376 1419 1419 β-caryophyllene (162) 34.056 1460 1459 (E)-β-farnesene (160) 34.744 1477 1476 g-muurolene (163) 35.703 1499 1499 a-muurolene (160) 36.236 1513 1513 g-cadinene (159) 36.601 1523 1523 d-cadinene (159) 37.355 1543 1543 a-calacorene (164) 38.683 1577 1576 spathulenol (159) 38.886 1582 1582 caryophyllene oxide (158) 39.761 1604 1607 b-oplopenone (163) 40.618 1628 1628 1-epi-cubenol (163) 40.886 1636 1633 y-eudesmol (161) 40.913 1636 1639 hinesol (163) 41.135 1643 1642 cubenol (163) 41.292 1647 1646 a-muurolol (159) 41.602 1655 1653 a-cadinol (159) 41.705 1658 1658 1-epi-a-eudesmol (163) 42.195 1671 1668 bulnesol (163) 42.329 1675 1675 methyl-epi-jasmonate (163) 44.571 1737 1735 oplopanone (163) 45.815 1773 1761 benzyl-benzoate (157) 49.056 1869 1867 flourensiadiol (163)

KI: Retention index, NIST: National Institute of Standards and Technology Virtual Library, Rt: Retention time.

TABLE 2-4: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. LANCEOLATUM EXTRACT

Rt (min) of KI of Nearest KI from NIST compound with KI nearest Ref. unknown unknown NIST to unknown 6.82 850 853 (E)-2-hexenal (158) 7.35 864 861 (Z)-3-hexenol (162) 7.46 867 868 n-hexanol (157) 9.58 919 926 tricyclene (159) 10.38 939 939 α-pinene (157) 13.56 1006 1004 a-phellandrene (160) 14.43 1026 1025 p-cymene (163) 14.45 1027 1027 o-cymene (161) 14.75 1034 1033 1,8-cineole (163) 16.99 1080 1083 artemisia alcohol (163) 17.35 1087 1087 p-mentha-2,4(8)-diene (163) 18.10 1101 1099 linalool (158) 19.80 1139 1139 trans-pinocarveol (163) 20.01 1144 1144 camphor (159) 21.66 1178 1178 terpene-4-ol (157) 21.97 1184 1184 p-cymen-8-ol (157) 22.30 1190 1190 a-terpineol (158) 23.60 1219 1219 trans-carveol (161) 24.01 1229 1229 nerol (157) 24.07 1230 1230 cis-carveol (159) 24.74 1245 1245 ethyl-ester of benzene acetic acid (157) 26.12 1275 1274 p-menth-1-en-7-al (161) 26.73 1288 1287 p-cymen-7-ol (164) 28.98 1340 1339 d-elemene (158) 29.46 1351 1352 a-cubebene (163) 29.79 1359 1357 eugenol (157) 30.56 1376 1376 a-copaene (159) 31.85 1405 1405 β-isocomene (161) 32.36 1418 1419 β-caryophyllene (162) 34.74 1477 1476 g-muurolene (163) 35.70 1499 1499 a-muurolene (160) 35.89 1504 1506 d-selinene (163) 36.24 1513 1513 g-cadinene (159) 36.62 1524 1523 d-cadinene (159) 37.36 1543 1543 a-calacorene (164) 37.72 1553 1552 elemicin (158) 38.02 1560 1557 germacrene B (163) 38.14 1563 1564 β-calacorene (164) 38.32 1568 1565 (E)-nerolidol (161) 38.69 1577 1577 himachalene epoxide (161) 38.88 1582 1578 spathulenol (161) 38.89 1582 1582 caryophyllene oxide (158) 39.15 1589 1590 viridflorol (163) 39.65 1601 1607 b-oplopenone (163) 39.91 1608 1608 humulene epoxide II (164) 40.03 1612 1612 tetradecanal (157) 41.11 1642 1642 cubenol (163) 41.30 1647 1646 a-muurolol (159) 41.56 1654 1653 a-cadinol (159)

TABLE 2-4: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. LANCEOLATUM EXTRACT

Rt (min) of KI of Nearest KI from NIST compound with KI nearest Ref. unknown unknown NIST to unknown 41.71 1658 1658 1-epi-a-eudesmol (163) 42.66 1684 1684 epi-a-bisabolol (161) 42.89 1690 1686 8-cedren-13-ol (163) 44.56 1737 1735 oplopanone (163) 45.04 1751 1749 xanthorrizol (163) 45.82 1773 1761 benzyl-benzoate (157) 47.22 1813 1827 isopropyl tetradecanoate (157)

KI: Retention index, NIST: National Institute of Standards and Technology Virtual Library, Rt: Retention time.

The analysis of the GC-MS data of I. verum revealed the presence of 31 volatile compounds. Most of identified components are classified as terpenes, including 11 monoterpenes and 16 sesquiterpenes. In addition, I. verum sample contained a phenylpropane, 2 organic aldehydes and an organic alcohol.

TABLE 2-5: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. VERUM EXTRACT

Rt (min) of KI of Nearest KI from NIST compound with KI nearest Ref. unknown unknown NIST to unknown 6.94 853 853 (E)-2-hexenal (158) 7.46 867 868 n-hexanol (157) 13.30 1000 1001 n-octanal (157) 14.44 1027 1025 p-cymene (163) 19.98 1143 1143 cis-pinene hydrate (158) 22.48 1194 1194 cis-piperitol (159) 22.76 1199 1196 methylchavicol (158) 24.08 1230 1230 cis-carveol (159) 25.17 1255 1254 piperitone (158) 26.18 1276 1277 trans-carvone oxide (161) 26.45 1282 1282 a-terpinen-7-al (161) 26.63 1286 1286 borneol acetate (161) 29.09 1343 1339 d-elemene (158) 29.96 1363 1365 neryl acetate (163) 30.49 1375 1373 a-ylangene (163) 30.91 1384 1384 (E)-β-damascenone (164) 31.50 1397 1396 Z-jasmone (164) 32.63 1425 1420 β-caroyophyllene (161) 32.91 1432 1430 β-copaene (161) 33.40 1444 1440 a-guaiene (158) 33.87 1456 1455 a-humulene (164) 35.63 1497 1495 a-zingiberene (160) 36.10 1510 1509 β-bisabolene (160)

TABLE 2-5: VOLATILE SECONDARY METABOLITES IDENTIFIED IN THE I. VERUM EXTRACT

Rt (min) of KI of Nearest KI from NIST compound with KI nearest Ref. unknown unknown NIST to unknown 38.32 1568 1565 (E)-nerolidol (161) 38.47 1572 1574 prenopsan-8-ol (161) 41.10 1642 1642 cubenol (163) 41.31 1647 1646 a-muurolol (159) 41.58 1655 1653 a-cadinol (159) 42.28 1674 1674 cadalene (164) 42.52 1680 1682 a-bisabolol (163) 49.06 1869 1867 flourensadiol (163)

KI: Retention index, NIST: National Institute of Standards and Technology Virtual Library, Rt: Retention time.

Metabolite fingerprint of I. angustisepalum

The qualitative analysis of GC-MS data of I. angustisepalum led to the identification of 44 organic volatile compounds which all are reported from I. angustisepalum for the first time. These identified compounds can be classified into two major chemical groups, monoterpenes (18 compounds) and sesquiterpenes (21 compounds), as shown on Figure

2-2. In addition, the I. angustisepalum sample contained isovaleric acid (simple fatty acid),

(E)-2-hexenal and n-octanal (organic aldehydes), n-hexanol (organic alcohol) and benzyl- benzoate (phenylpropanoid).

FIGURE 2-2: Total ion chromatogram of I. angustisepalum

Comparative profiles between I. angustisepalum, I. lanceolatum and I. verum

Plants species used in the present study belong to a single genus, Illicium, and were collected on the same day in the Hong Kong region. GC-MS analysis requires volatile and thermally stable analytes such as short chain alcohols and mono- and sesquiterpenes which dominated in the tested extracts. Careful investigation of total ion chromatograms of analyzed extracts distinguished the two major regions (as shown on Figure 2-3). Compounds eluting in the first region (Rt of 7.4-30.8 min for IAT; Rt of 9.5-26.7 min for ILT; Rt of 14.4-31.5 min for

IVT) were found to be monoterpenes and compounds eluting in the second region (Rt of 32.7-

49.0 min for IAT; Rt of 28.9-44.5 min for ILT; Rt of 32.6-49.0 min for IVT) were classified as sesquiterpenes. A total of 131 volatile secondary metabolites were identified from the analyzed samples by GC-MS. It was also found that there were only 7 compounds shared by all analyzed extracts; they are, namely, (E)-2-hexenal, a-cadinol, a-muurolol, cis-carveol, cubenol, n-hexanol and β-caryophyllene. There were also 20 compounds shared by extracts of I. angustisepalum and I. lanceolatum, including 1-epi-a-eudesmol, a-calacorene,

53 a-copaene, a-muurolene, a-phellandrene, a-terpineol, benzyl-benzoate, b-oplopenone, camphor, caryophyllene oxide, d-cadinene, g-cadinene, g-muurolene, linalool, o-cymene, oplopanone, spathulenol, terpene-4-ol, trans-carveoland tricyclene. Extracts of

I. angustisepalum and I. verum had 4 compounds in common, including (E)-β-damascenone, borneol acetate, flourensiadiol and n-octanal. Extracts of I. lanceolatum and I. verum shared

3 compounds, (E)-nerolidol, d-elemene and p-cymene. The frequency distribution indicating number of shared and unique compounds is depicted on Figure 2-4

FIGURE 2-3: Comparison of GC-MS total ion chromatograms of analyzed extracts from three species of Illicium collected in Hong Kong

FIGURE 2-4: Frequency distribution indicating number of shared and unique compounds

DISCUSSION

Illicium species are a rich source of secondary metabolites. There are at least 183 various compounds reported from Illicium so far (Figure 1-3). According to the literature information, previous reports on the intraspecific variations of secondary metabolites in

Illicium have detailed differences within individual species, between species from a given population and also amid populations from geographically remote regions. There are two reports that support incorporation of genus Illicium into Schisandraceae family. Brown et al. described three cycloartanes, schizandronic acid, schizandrolic acid and magniferolic acid, and ring-A cleaved cycloartane, 3,4-seco-(24Z)-cycloart-4(28),24-diene-3,26-dioic acid 3- methyl ester, in the extract I. dunnnianum. (165) In another study by Sy and Brown, they found nigranoic acid and the 3,26-dimethyl ester of nigranoic acid in the I. verum extract.

(166) Both these latter compounds are also known from Schisandra nigra Maxim (167). The common possession of the cycloartanes and seco-cycloartanes in I. dunnianum and I. verum and the Schisandraceae family (in the genera Schisandra Michx. and Kadsura Juss.) supports that the extremely rare 3,4-seco-cycloartane class of triterpenes can be regarded as chemotaxonomic markers.

In this study the chemotaxonomic discrimination between I. angustisepalum,

I. lanceolatum and I. verum is based on the comparison of the volatile secondary metabolites fingerprints. As the GC-MS is a fast and reliable method applied in previous GC-MS analyzes of Illicium, it was selcted for this study. Noteworthy, most if not all former GC-MS analyzes of

Illicium, focused on the fruits of two species, namely I. anisatum and I. verum. This significant amount of attention was dedicated to their fruits as this particular part of the plant is associated with numerous cases of accidental poisoning. (168) (169) (170) (171) In brief, the fruits of I. verum, which are known for their flavor and digestive properties often used in the folk medicine, are difficult to visually distinguish from the fruits of the toxic species,

I. anisatum. For this reason many scientist attempted to find a reliable method that would differentiate between fruits of these two species. (172) (173) (174) (175)

As most of the previous GC-MS research of Illicium plants gave a little or none attention to twigs, this particular study has undertaken a careful analysis of this plant part. In addition, current study presents first GC-MS based fingerprint for I. angustisepalum and comparison with toxic I. lanceolatum and edible I. verum species. The following are the key findings of the study: (1) a total of 131 volatile secondary metabolites are identified, (2) two major regions with mono- and sesquiterpenes can be distinguished in fingerprints of all three species (3), seven compounds, namely, (E)-2-hexenal, a-cadinol, a-muurolol, cis-carveol,

56 cubenol, n-hexanol and β-caryophyllene, are present in metabolite fingerprints of all species,

(4) metabolite fingerprint of I. angustisepalum is more similar to I. lanceolatum than I. verum.

The above observations are of importance as they help to understand the general and specific metabolite patterns between the three analyzed Illicium species.

CONCLUSIONS

The present study describes the variation of the volatile secondary metabolites between I. angustisepalum, I. lanceolatum and I. verum using metabolite fingerprinting. The volatile compounds shared by the analyzed Illicium species, (E)-2-hexenal (organic aldehyde), a-cadinol (sesquiterpene), a-muurolol (sesquiterpene), cis-carveol

(monoterpene), cubenol (sesquiterpene), n-hexanol (organic alcohol) and β-caryophyllene

(sesquiterpene), can be considered as chemotaxonomic markers of Illicium. Terpenes, including mono- and sesquiterpenes may have the potential to produce new insights into the historical debate concerning systematic position of genus Illicium and its incorporation into

Schisandraceae family.

CHAPTER 3

PHYTOCHEMICAL STUDY OF I. ANGUSTISEPALUM

RATIONALE

Plants are complex biological systems capable of producing a wide range of chemical compounds called primary and secondary metabolites. Primary metabolites, such as carbohydrates, lipids, proteins, and nucleic acids, support essential functions of the plant such as growth and development. Primary metabolites are present in all plants. Secondary metabolites can be classified into a number of chemical classes based on their unique chemical scaffolds. Secondary metabolites have specialized function and most commonly they serve as chemical defense system. Distribution and the role of secondary metabolites vary across plant kingdom. Secondary metabolites can serve as potential drug leads to combat various ailments. Therefore it is important to continue exploration of the plant kingdom as an approach to search for new drugs.

GENERAL EXPERIMENTAL PROCEDURES

All solvents used were analytical or HPLC grade. TLC: Merck aluminium backed sheets coated with 60F254 silica gel or 60F254 RP-silica gel; visualization by using an UV lamp

(ʎmax 254 nm), and spraying with Komarowsky reagent (a mixture of 2% 4- hydroxybenzaldehyde MeOH soln. and 5% H2SO4/EtOH soln. 10 : 1 (v/v)), followed by heating. Open column chromatography (CC): silica gel (SiO2), MCI gel CPH20P (Supelco,

Sigma-Aldrich, United States) or Sephadex LH-20 (GE Healthcare Bio-Sciences AB,

Sweden). For HPLC purification, a C18 semi-prep. HPLC column (Phenomenex C18 column,

250x10 mm, 5 mm) and a Shimadzu UFLC system were used; the UV detection wavelength

and flow rate were set at 254 nm and 4 mL/min, respectively. Optical rotations: at Na D line;

Perkin-Elmer 241 digital polarimeter using quartz cell with a path length of 100 mm at r.t.; concentrations (c) in g/100 mL. NMR Spectra: Bruker DPX-400 spectrometer; chemical shifts

1 (d) in ppm using residual solvent as the internal standard (DMSO-d6: 2.50 ppm for H- and

13 1 13 39.51 ppm for C-NMR; CDCl3: 7.24 ppm for H- and 77.23 ppm for C-NMR; methanol-d4:

3.31, 4.78 ppm for 1H- and 49.2 ppm for 13C-NMR); coupling constants (J) in Hz. HRESI-MS:

Shimadzu LCMS-IT-TOF mass spectrometer. X-Ray diffraction experiments were carried out on a Bruker Kappa APEXII DUO diffractometer with a CCD area detector using CuKa X-ray source.

PLANT MATERIAL

Twigs of I. angustisepalum were collected in spring 2011 from Hong Kong, and authenticated by Ms. Yu-Ying Zong. The specimen voucher of I. angustisepalum was deposited at the School of Chinese Medicine, Chinese University of Hong Kong. A total of

1 kg of fresh twigs were cut into small pieces, dried and then milled into fine powder.

EXTRACTION OF THE PLANT MATERIAL

The pulverized plant material was exhaustively extracted by percolation with 90% (v/v) ethanol at room temperature and then dried in vacuo. A total of 231.71g of extract was obtained.

FRACTIONATION OF THE ETHANOL EXTRACT

The concentrated ethanol extract was suspended in 2 L of distilled water and partitioned against petroleum ether (4 x 2 L). The petroleum ether layers were pulled together and dried in vacuo. The remaining, de-fatted, water layer was subsequently partitioned

against ethyl acetate (4 x 2L) and then butanol (4 x 2L), and then similar layers were combined and concentrated, affording ethyl acetate and butanol fractions. Fractionation was summarized in Figure 3-1 and the yields are given in Table 3-1.

FIGURE 3-1: Fractionation of the ethanol extract of I. angustisepalum twigs

TABLE 3-1: YIELDS OF SOLVENT PARTITION FRACTIONS OBTAINED FROM I. ANGUSTISEPALUM EXTRACT

Fraction Weight (g)

Pe (Petroleum ether) 41.00

Ea (Ethyl acetate) 49.85

Bu (Butanol) 63.36

Wa (Water) 77.50

ISOLATION AND STRUCTURE ELUCIDATION

The ethyl acetate (Ea) fraction was subjected to further separation. The Ea fraction was initially fractionated using flash chromatography column (Ea-1). In brief, the fraction was reconstituted in methanol, filtered and mixed with silica gel (100 g). The mixture was dried under reduced pressure and then applied under the vacuum conditions inside a

chromatographic column with tightly packed layer of silica gel (100 g). Elution was initiated with 100% petroleum ether, and progressed via step gradient to reach 100% ethyl acetate.

A total of 47 fractions were collected. All fractions were monitored on TLC plates. Fractions with similar TLC profiles were combined to afford sixteen combined subfractions (Table 3-2).

TABLE 3-2: COMBINED FRACTIONS FROM THE FLASH CHROMATOGRAPHY OF EA-1

Fraction Mobile phase (%) Weight (g) Isolate Weight (mg) 0.1865 1 Pe 0.0706 2 – 5 Pe 0.6681 6 Pe:Ea (9:1) 1.9917 Majusanic acid C 11.4 7 – 8 Pe:Ea (9:1) Angustanoic acid E 9.2

(-)-T-Muurolol 6.8

2-Hydroxy-2-methyl-6- 2.3 methyleneoct-7-en-3-yl benzoate

9 – 10 Pe:Ea (9:1) 0.4225 2,6-dimethoxychavicol 30.6 0.5484 11 Pe:Ea (8:2) 1.3378 12 Pe:Ea (8:2) 1.7770 Angustanoic acid F 10.2 13 – 14 Pe:Ea (8:2) Angustanoic acid G 27.5

6β-hydroxy-4-stigmasten-3-one 2.7

1.3070 15 – 17 Pe:Ea (8:2) 1.9137 18 – 19 Pe:Ea (7:3) 1.9320 20 – 21 Pe:Ea (7:3) 2.3046 (2R, 3R)-3,5,7,3’4’-Pentahydroxy 57.4 22 Pe:Ea (7:3) flavononol Clovane-2,9-diol 58.1

Angustisepalin 30.2

TABLE 3-2: COMBINED FRACTIONS FROM THE FLASH CHROMATOGRAPHY OF EA-1

Fraction Mobile phase (%) Weight (g) Isolate Weight (mg) Angustanol 24.0

Majucin 9.8 2-Hydroxy-7-methyl-hexan-1,5- 23 – 27 Pe:Ea (6:4) 9.4439 1.7 olide Majusanic acid B 8.0 10.4481 28 – 32 Pe:Ea (5:5) 10.3055 33 – 39 Pe:Ea (4:6) 38.8035 40 – 47 Ea

The ethyl acetate fraction was subjected to mix drying under reduced pressure conditions. During this process a volatile fraction was obtained as a byproduct. The TLC analysis of the volatile fraction indicated presence of at least three major spots with Rf values of 0.4, 0.9 and 0.92 after developing the plate in petroleum ether–ethyl acetate (6:4 v/v). By means of preparative chromatography, GC-MS analysis and TLC co-chromatography with the standard, the spot at the 0.92 was isolated (1 mg) and identified as thymol (1). The remaining spots were not isolated, their identification is based on the comparison of GC-MS analysis of the volatile fraction with the compounds from on-line database NIST (Figure 3-2 and Table 3-3).

FIGURE 3-2: GC-MS total ion chromatogram of the volatile fraction from I. angustisepalum

TABLE 3-3: GC-MS DATA ANALYSIS OF THE VOLATILE FRACTION FROM I. ANGUSTISEPALUM

Compound Rt (min) CAS#

2-Pentanone, 4-hydroxy-4-methyl- 5.45 123-42-2

1-Octen-3-ol 7.82 3391-86-4

1,6-Octadien-3-ol, 3,7-dimethyl-, acetate 9.82 115-95-7

Bicyclo[3.1.0]hexan-2-ol, 2-methyl-5-(1-methylethyl)-, (1à,2à,5à)- 11.1 17699-16-0

2,5-Cyclohexadiene-1,4-dione, 2-methyl-5-(1-methylethyl)- 12.15 490-91-5

Thymol 12.67 89-83-8

Phenol, 2-methyl-5-(1-methylethyl)- 12.81 499-75-2

2,5-Cyclohexadiene-1,4-dione, 2-methyl-5-(1-methylethyl)- 16.06 1076-55-7

1,3-Benzodioxol-5-ol,6-(7-methoxy-3,4(2H)-dihydro-1-benzopyran-3-yl)- 17.73 490-91-5

(1-Benzo[1,3]dioxol-5-yl-1H-tetrazol-5-ylsulfanyl)-acetic acid hydrazide 18.64

Rt: Retention time, CAS#: Unique identifier assigned by Chemical Abstracts Service (CAS)

Subfraction 7-8 from Ea -1 was reconstituted in 1 mL of ethyl acetate, mixed with silica gel (5 g) and dried under reduced pressure. The dry mixture was subjected to chromatographic separation over silica gel (50 g). A total of 58 fractions were obtained.

Fractions showing similar TLC patterns were pooled together to give 21 combined fractions.

TLC of combined fraction 6-8 showed a major purple spot at Rf 0.6 [petroleum ether-acetone

(8:2)] which was isolated in two steps. First, fraction 6-8 (2 g) was separated on silica gel (70 g) into 279 fractions. Similar fractions were pooled together into 45 combined fractions.

Combined fraction 60-117 (46.7 mg) was further purified on MCI resin (20 g) to afford (-)-T- muurolol (2). TLC profile of combined fraction 13-18, revealed presence of two spots, a pink spot at Rf 0.58, and a blue spot at Rf 0.6 [petroleum ether-acetone (8:2)]. Fraction 13-18 was separated on MCI resin (20 g) into 14 fractions. Similar fractions were combined into 7 subfractions (1-8, 9, 10, 11/12, 13, 14, 15). Subfraction 10 yielded 2-Hydroxy-2-methyl-6- methyleneoct-7-en-3-yl benzoate (3) and fraction 14 gave angustanoic acid E (4). Combined fraction 41-44 was further separated on MCI resin (20 g) into 20 subfractions. Subfraction 18 gave majusanic acid C (5). Further purification was required by HPLC, using methanol- water/0.1% formic acid at different ratios to elute desired compound (2; 6.8 mg; Rt 13.2 min in 80% methanol-water), (3; 2.3 mg; Rt 10.6 min in 75% methanol-water), (4; 9.2 mg; Rt 10.3 min in 80% methanol-water) and (5; 11.4 mg; Rt 6.6 min in 80% methanol-water).

Subfraction 9-10 from Ea-1 was chromatographed under vacuum conditions over a flash column with silica gel (20 g). Elution started with 100% petroleum ether and continued through increasing step gradient to reach 100% ethyl acetate. A total of 22 fractions were collected. Each fraction was monitored on TLC plates. Similar fractions were pooled to obtain

5 combined fractions (1-2, 3, 4-6, 7-17 and 18-22). Combined fraction 7-17 was further

separated over MCI resin (20 g) and lead to isolation of 2,6-dimethoxychavicol (6). Final purification was performed by HPLC, using methanol-water (6; 30.6 mg; Rt 22.5 min in 90% methanol-water).

Subfraction 13-14 from Ea-1 was reconstituted in 1 mL of methanol and separated on a chromatographic column with MCI resin (60 g). Elution started with 36% aqueous methanol and continued to reach 100% methanol. A total of 72 fractions were collected. Similar fractions were combined to afford 31 combined fractions. Subfraction 32, after standing overnight at room temperature in methanol, produced crystalline particles. The precipitate was purified by means of recrystallization to afford 10.2 mg of angustanoic acid F (7).

Similarly, combined fractions 54-56 and 57-58, after standing overnight at room temperature in the methanol, gave pale crystals. The precipitate was collected by filtration and repeatedly crystallized from hot methanol to yield 27.5mg of angustanoic acid G (8). TLC analysis of the combined subfraction 66-68 displayed a yellow spot at Rf 0.2 [methanol-water (90:10)].

Subfraction 66-68 (59.7 mg) was separated on MCI (20 g) into 8 subfractions, which were pooled together into 3 combined fractions (1-4, 5-7, 8). Subfractions 5-7 and 8 gave pale crystals after standing overnight at room temperature. The precipitate was purified by means of recrystallization to afford 2.7 mg of 6β-hydroxy-4-stigmasten-3-one (9).

Subfraction 22 from Ea-1 was redissolved in methanol, mixed with silica gel (5 g) and dried under reduced pressure. The mixture was applied under vacuum conditions on a chromatographic column with tightly packed layer of silica gel (50 g). The column was eluted by step gradient starting at 100% petroleum ether and continuing up to 100% ethyl acetate.

A total of 19 fractions were collected and monitored by TLC. Similar fractions were pooled together resulting in 6 combined fractions (1-2, 3-4, 5-7, 6-11, 12-14, 15-18). Fraction 6-11

was further separated using MCI resin (20 g). Elution started with 30% aqueous methanol and continued through increasing gradient to 100% methanol. A total of 46 fractions were collected. Combination of similar fractions resulted in 9 combined fractions (1-4, 5-18, 19-22,

23-24, 25-27, 28-29, 30-31, 32-35, and 36-46). TLC of fraction 5-18 displayed an orange spot at Rf 0.3 [methanol-water (30:70)] which was purified on Sephadex LH-20 to yield (2R, 3R)-

3,5,7,3’4’-pentahydroxyflavonone (10). Analysis of TLC profile of fraction 32-35 indicated the presence of three major spots, a brown spot at Rf 0.6 and two almost overlapping purple spots at Rf 0.45 and 0.40 [methanol-water (60:40)]. The two purple spots were isolated by chromatographic separation of fraction 32-35 on MCI resin. Separation produced 24 fractions that were later combined into 6 combined fractions (1, 2, 3, 4-6, 7-13 and 14-17). Fraction 4-

6 yielded angustisepalin (11) and fraction 14/17 yielded clovane-2,9-diol (12). The brown spot was obtained by chromatographic analysis of fraction 2, which was separated into 21 fractions. Similar fractions were pooled together yielding 3 combined fractions (1-9, 10-12 and 14-21). Fraction 14-21 gave angustanol (13). Further purification was required for compounds 10-13 by HPLC, using AcCN-water/0.1% formic acid in water at different ratios to elute the compounds (10; 57.4 mg; Rt 7.7 min in 25%AcCN-0.1% formic acid in water),

(11; 30.2 mg; Rt 23.0 min in 90%AcCN- water/0.1% formic acid in water), (12; 58.1 mg; Rt

29.7min in 90%AcCN- water/0.1% formic acid in water), (13; 24.0 mg; Rt 23.2min in

90%water/0.1% formic acid).

Subfraction 23-27 from Ea-1 was redissolved in 5 mL of methanol, mixed with silica gel (10 g) and dried under reduced pressure. The mixture was applied under vacuum conditions on chromatographic column containing tightly packed silica gel (200 g). Elution started at 100% petroleum ether, continuing via increasing step gradient up to 100% ethyl

acetate. A total of 29 fractions were obtained. Fraction 22, after standing overnight at room temperature in methanol, produced crystalline particles. The precipitate was purified by means of recrystallization to afford 9.8mg of majucin (14). TLC profile of fractions 11 to 20 was similar and they were combined. Further analysis of the TLC of combined fraction 11-20 indicated the presence of a major purple spot at Rf 0.5 [methanol-water (50:50)] which was isolated in two steps. Fraction 11-20 was first separated on C18 resin starting at 5%, continuing through increasing concentration of methanol up to 100%. A total of 80 fractions were collected. Similar fractions were pooled to obtain 21 combined fractions. Combined fraction 1-4 (197 mg) was further separated on chromatographic column packed with silica gel (10 g). A total of 17 fractions were collected. Similar fractions were combined into 4 combined fractions. Fraction 2-4 afforded 2-Hydroxy-7-methyl-hexan-1,5-olide (15). Further purification was required for compound 15 by HPLC, using methanol-water at different ratios to elute the desired compound (15; 1.7 mg; Rt 7.71 min in 70% water/0.1% formic acid).

Fractions 59-64 were also combined as their TLC profiles were similar. The composition of fraction 59-64 was simple thus it was subjected to separation of semi-preparative HPLC which resulted in isolation of majusanic acid B (16) eluted at Rt 8.45 minutes (70% MeOH- water/0.1% formic acid).

The chemical structures of all compounds isolated from I. angustisepalum are presented in Figure 3-3.

(1) (2) (3) (6) (9)

(10) (11) (12) (14) (15)

Majusanic acid B (16) R1=COOH, R2=CH2OH, R3=OH Majusanic acid C (5) R1=COOH, R2=CH3, R3=OCH3 Angustanoic acid E (4) R1=COOH, R2=R3=CH2 Angustanoic acid F (7) R1=COOH, R2=CH3, R3=OH

FIGURE 3-3: Chemical structures of compounds isolated from ethyl acetate fractions of I. angustisepalum

Thymol (1)

+ White, crystal; GC-MS 70eV, m/z 150.0000 ([M] , calcd for C10H14O, 150.1045). Thymol (1) is a monoterpene alcohol, reported from I. angustisepalum for the first time. The identity of thymol was confirmed by means of TLC co-chromatography with a reference standard purchased from Sigma-Aldrich and also by analysis of GC-MS data. In the TLC analysis, the Rf of thymol and the reference standard were both 0.9 after elution with petroleum ether–ethyl acetate (6:4 v/v). The GC chromatogram showed that the retention times of thymol and the reference standard were 12.67 and 12.66 minutes, respectively.

Further analysis of GC-MS fragmentation data revealed the presence of ion [M]+ at m/z

150.0000 in both compounds.

(-)-T-Muurolol (2)

2 HMBC correlations for 2

25 o Brown, oil; [α]D -70.0 (c 0.1, DCM); IR (film) vmax 3345, 2957, 2932, 2907, 2870,

-1 1 1713, 1668, 1454, 1369, 1019, 952 cm ; H NMR (CDCl3, 400 MHz, multiplicities of some signals were not clear due to poor peak shape) δH 5.63 (1H, d, J = 5.6 Hz, H-5), 2.41 (1H, m,

H-6), 2.03 (1H, m, H-11), 1.82 (2H, m, H-3), 1.59 (3H, s, H-15), 1.50-1.45 (1H, m, H-8b),

1.50-1.40 (2H, m, H-2), 1.45-1.35 (1H, m, H-9b), 1.50-1.40 (1H, m, H-1), 1.30-1.20 (1H, m,

H-7), 1.30-1.25 (1H, m, H-9a), 1.30-1.20 (1H, m, H-8a), 0.99 (3H, s, H-14), 0.86 (3H, d, J =

13 7.0 Hz, H-12), 0.85 (3H, d, J = 7.0 Hz, H-13); C NMR (CDCl3, 100 MHz) δC 133.5 (C, C-4),

124.8 (CH, C-5), 72.4 (C, C-10), 46.1 (CH, C-1), 43.9 (CH, C-7), 34.6 (CH2, C-9), 34.5 (CH,

C-6), 31.2 (CH2, C-3), 29.2 (CH3, C-14), 26.6 (CH, C-11), 23.6 (CH3, C-15), 21.6 (CH3, C-

12), 20.9 (CH2, C-2), 19.3 (CH2, C-8), 15.3 (CH3, C-13); (+)-HRESIMS m/z 205.1948 ([M-

+ H2O+H] , calcd for C15H25, 205.1951).

(-)-T-Muurolol (muurol-4-en-10β-ol, 2) (176) was obtained as a brown oil. The

+ HRESIMS showed a quasi-molecular ion at 205.1948 m/z [M-H2O+H] (calcd for C15H25,

205.1951), suggesting a molecular formula C15H26O with three indices of hydrogen deficiency when 13C NMR spectroscopic data were taken into consideration. The IR broad stretch at

3345 cm-1 indicated the presence of a hydroxyl group. The 1H NMR spectrum displayed one olefinic proton at δH 5.63 (d, J = 5.6 Hz, H-5) and four methyl groups at δH 1.59 (s, H-15), δH

13 0.99 (s, H-14), δH 0.86 (d, J = 7.0 Hz, H-12) and δH 0.85 (d, J = 7.0 Hz, H-13). The C NMR spectrum exhibited 15 carbon signals corresponding to a four methyls, four methylene, five methine (one olefinic and four aliphatic), an oxygenated tertiary carbon and one quaternary carbon. The presence of one double bond accounted for one degree of unsaturation, the remaining two were therefore deduced from bicyclic structure in the molecule. All of the proton signals were assignable to their attached carbons through an HSQC experiment. In

1 1 the H- H COSY data, signal at δH 1.50-1.40 (m, H-1) displayed correlations with signals at

δH 1.50-1.40 (m, H-2) and δH 2.41 (m, H-6) and also proton signal at δH 1.30-1.20 (m, H-7) showed correlations with δH 2.41 (m, H-6), δH 1.30-1.20 (m, H-8a), δH 1.50-1.45 (m, H-8b)

and δH 2.03 (m, H-11). In addition, correlations between H-2 (δH 1.50-1.40)/H-3 (δH 1.82), H-

5 (δH 5.63)/H-6 (δH 2.41),H-8 (δH 1.50-1.45, δH 1.30-1.20)/H-9 (δH 1.45-1.35, δH 1.30-1.25),

H-11 (δH 2.03)/H-12 (δH 0.86), and H-11 (δH 2.03)/H-13 (δH 0.85) were also observed allowing the assignment of connectivities of these fragments. Interpretation of the HMBC data then led to the proposed structure of 2. The connection between the A- and B-rings was confirmed by HMBC correlations observed for H-1 and C-2, C-6 and C-10. The hydroxyl group was assigned to the oxygenated tertiary carbon C-10 (δC 72.4), based on its HMBC correlations with H-1 and H-9. The isopropyl group was established by correlations between C-11 (δC

26.6), C-12 (δC 21.6), C-13 (δC 15.3) and methine proton at H-7 (δH 1.30-1.20). All available evidence led to the determination of the structure 2 as shown. The relative configuration of 2 was then assigned on the basis of NOESY analysis, in which the following key correlations were observed: H-1 (δH 1.50-1.40) with H-6 (δH 2.41), H-6 (δH 2.41) and H-11 (δH 2.03), and between H-7 (δH 1.30-1.20) and H-14 (δH 0.99). Consequently, compound 2 was elucidated to be: muurol-4-en-10β-ol [(-)-T-Muurolol]. The experimental data and elucidation of 2 were in agreement with the literature. (176) (177) (178) T-Muurolol, belongs to the muurolane type sesquiterpene class. Present study reports T-Muurolol for the first time from

I. angustisepalum.

2-Hydroxy-2-methyl-6-methyleneoct-7-en-3-yl benzoate (3)

3 HMBC correlations for 3

25 o White, amorphous; [α]D +1.4 (c 0.07, MeOH); IR (film) vmax 3429, 2971, 2936, 2366,

1 13 2342, 1713, 1275, 1119, 716; H (methanol-d4, 400 MHz) and C NMR (methanol-d4, 100

+ MHz) data, see Table 3-4; (+)-HRESIMS m/z 275.1569 ([M+H] , calcd for C17H23O3,

275.1647).

2-Hydroxy-2-methyl-6-methyleneoct-7-en-3-yl benzoate (3) was obtained as a white amorphous powder. The HRESIMS of 3 displayed a quasi-molecular ion [M+H]+ at m/z

275.1569 (calcd for C17H23O3, 275.1647), corresponding to the molecular formula of

13 C17H22O3 with 7 indices of hydrogen deficiency when C NMR data were taken into account.

The IR broad stretch at 3429 cm-1 suggested the presence of the hydroxyl moiety and the broad stretch at 1713 cm-1 indicated presence of a benzoyl carbonyl moiety. The 1H NMR spectrum displayed olefinic protons at δH 6.34 (dd, J = 11.0, 10.8, H-7), 5.16 (d, J = 17.7, H-

8a), 5.02 (br s, H-8b), 5.0, (br s, H-9) and two methyl groups at δH 1.22 (s, H-1/10) (Table

13). The 13C NMR and DEPT135 spectra of 3 (Table 3-4) exhibited 17 carbons signals corresponding to two methyl, four methylenes (including two olefinic and two aliphatic), seven methines (including five aromatic, one olefinic, one aliphatic), an oxygenated tertiary carbon, carbonyl carbon and two quaternary carbons (Table 3-4). The presence of two double bonds accounted for two degrees of unsaturation, the remaining five were then deducted from an aromatic ring structure in the molecule. Inspection of the HSQC spectrum allowed for assigning of proton signals with carbons attached to them (Table 3-4). In the 1H-1H COSY spectrum, signal at δH 7.61 (t, J = 7.3 Hz, H-5’) displayed correlations with the overlapped signal at δH 7.49 (H-4’/6’), and then δH 7.49 (H-4’/6’) exhibited correlation with overlapped signal at δH 8.07 (dd, J = 1.3 Hz H-3’/7’) suggesting presence of an aromatic system in the molecule. The correlations of δH 2.23, 1.87 (m, H-4) with signals at δH 5.09 (dd, J = 2.3, 2.2

Hz, H-3), δH 2.02 (m, H-5a), δH 1.28 (d, J = 3.2 Hz, H-5b) and also cross peaks between δH

6.34 (dd, J = 11.0, 10.8 Hz, H-7) and δH 5.16, (d, J = 17.7 Hz, H-8a) and also δH 5.02 (5.02, br s, H-8b) were observed, allowing the assignment of connectivities of these fragments.

Subsequently, interpretation of the HMBC data led to the proposed structure 3. It is noteworthy that both methine proton at δH 5.09 (H-3) and aromatic protons at δH 8.07 (H-

3’/7’) displayed long-range correlations with carbonyl carbon at C-1’ (δC 168.0). This particular correlation was helpful in establishing of an ester bond between aromatic ring and the monoterpene part of the molecule. The hydroxyl group was assigned to the oxygenated tertiary carbon C-2 (δC 72.9) based on its HMBC correlations with H-1, H-3 and H-10. The position of the two terminal double bonds was assigned based on the following HMBC evidence: long-range correlations between δH 5.16 (d, J = 17.7Hz, 8a) and δH 5.02 (br s, H-

8b) with C-6 (δC 147.2) and C-7 (dd, J = 11.0, 10.8 Hz) and also cross peaks between δH 5.0

(br s, H-9) with C-5 (δC 29.6), C6 (δC 147.2) and C-7 (δC 139.7). Comparison of the 1D NMR data with literature data confirmed close similarity of the monoterpene part of the molecule to myrcenediol (Figure 3-4). (179) (180) Assembly of all fragments led to the determination of the planar structure of 3 as 2-hydroxy-2-methyl-6-methyleneoct-7-en-3-yl benzoate, therefore compound 3 is formally an ester of benzoic acid and myrcenediol. The evaluation

25 o of the optical rotaton of 3 configuration of 3 (α]D +1.4 ; c 0.07, MeOH) indicated that this particular compound exisits in a racemic mixture of the two enantiomers, R and S. To the best of our knowledge, compound 3 is a new structure isolated from natural source.

FIGURE 3-4: Structure of Myrcenediol

TABLE 3-4: 1H AND 13C NMR SPECTROSCOPIC DATA OF 2-HYDROXY-2-METHYL- 6-METHYLENEOCT-7-EN-3-YL BENZOATE (Δ IN PPM, J IN HZ)A

b Position δC, type δH, mult. (J in Hz) HMBC (H  C)

10, 3, 2 1 25.5, CH3 1.22, s

2 72.9, C

3 81.3, CH 5.09, dd (J = 2.3, 2.2) 1’, 4

4a, 2.23, m (overlap) 6, 5 4 29.3, CH2 4b, 1.87, m (overlap) 5, 3

5a, 2.02, m (overlap) 9,7, 4 5 29.6, CH2 5b, 1.28, d (J = 3.2) 9, 7, 4

6 147.2, qC

7 139.7, CH 6.34, dd (J = 11.0, 10.8) 9, 6, 5

8a, 5.16, d (J = 17.7) 6, 7 8 113.7, CH2 8b, 5.02, br s (overlap) 6, 7 4, 5, 6, 7 9 116.7, CH2 5.0, br s (overlap) 3, 2, 1 10 26.4, CH3 1.22, s

1’ 168.0, C

2’ 131.6, qC

3’ 130.6, CH 8.07, dd (J = 1.3) 1’, 6’, 5’, 4’, 2’

TABLE 3-4: 1H AND 13C NMR SPECTROSCOPIC DATA OF 2-HYDROXY-2-METHYL- 6-METHYLENEOCT-7-EN-3-YL BENZOATE (Δ IN PPM, J IN HZ)A

b Position δC, type δH, mult. (J in Hz) HMBC (H  C)

4’ 129.6, CH 7.49, t (J = 7.5) 7’, 6’, 4’, 3’

5’ 134.3, CH 7.61, t (J = 7.3) 7’, 6’, 4’, 3’

6’ 129.6, CH 7.49, t (J = 7.5) 7’, 6’, 4’, 3’

7’ 130.6, CH 8.07, dd (J = 1.3) 1’, 6’, 5’, 4’, 2’ a 1 13 Data measured in methanol-d4; 400 MHz for H NMR and 100 MHz for C NMR; J: Coupling constant. b Data recorded at 400 MHz.

Angustanoic acid E (4)

4 HMBC correlations for 4

25 o White, amorphous; [α]D +22.1 (c 0.19, MeOH); IR (film) vmax 2956, 2931, 2850,

-1 1 1695, 1437, 1231, 108, 950, 888 cm ; H NMR (methanol-d4, 400 MHz) δH 7.22 (2H, d, J =

3.0 Hz, H-11/12), 7.11 (1H, s, H-14), 5.31 (1H, s, H-16b), 4.99 (1H, t, J = 1.4 Hz, H-16a), 2.89

(1H, m, H-7b), 2.79 (1H, td, J = 6.0, 6.0, 6.0 Hz, H-7a), 2.31 (1H, m, H-1b), 2.24 (1H, m, H-

3b), 2.23 (1H, m, H-6b), 2.19 (1H, m, H-6a), 2.09 (3H, s, H-17),1.62 (1H, m, H-2b), 1.58 (1H, m, H-2a), 1.55 (1H, dd, J = 1.6, 1.6 Hz, H-5), 1.36 (1H, m, H-1a), 1.14 (3H, s, H-20), 1.11

13 (1H, m, H-3a), 1.30 (3H, s, H-18); C NMR (methanol-d4, 100 MHz) δC 181.5, (C, C-19),

148.7 (C, C-9), 144.6 (C, C-15), 139.4 (C, C-13), 136.1 (C, C-8), 126.9 (CH, C-14); 126.4

(CH, C-11), 124.0 (CH, C-12), 111.6 (CH2, C-16), 54.2 (CH, C-5), 44.9 (C, C-4), 40.7 (CH2,

C-1), 39.6 (C, C-10), 38.8 (CH2, C-3), 33.2 (CH2, C-7), 29.2 (CH3, C-18), 23.6 (CH3, C-20),

+ 22.3 (CH2, C-6), 22.0 (CH3, C-17), 21.1 (CH2, C-2); (+)-HRESIMS m/z 299.2004 ([M+H] , calcd for C20H27O2, 299.2006).

Angustanoic acid E (4) was obtained as a white amorphous powder. The HRESIMS

+ of 4 displayed the quasi-molecular ion at m/z 299.2006 [M+H] (calcd for C20H27O2,

299.2006), corresponding to the molecular formula of C20H26O2 with eight degrees of unsaturation. Detailed examination of the 13C NMR and DEPT135 spectra of 4 exhibited 20 carbons signals corresponding to three methyls, six methylenes (including one olefinic and five aliphatic), four methines (including three aromatic and one aliphatic), a carbonyl carbon and six quaternary carbons. Further inspection of the 13C NMR spectra demonstrated presence of carboxylic acid group at δC 181.5 (C-19) and an aromatic system (C-13, δC 139.4;

C-9, δC 148.7; C-8, δC 136.1; C-14, δC 126.9; C-11, δC 126.4; C-12, δC 124.0). All of the proton signals were assigned to their attached carbons through an HSQC experiment. Then the substitution pattern for the aromatic ring C in 4 was recognized from the inspection of its 1H

NMR spectrum (C-12, δH 7.22, d, J = 3.0 Hz; C-11, δH 7.22, d, J = 3.00 Hz; C-14, δH 7.11, s).

1 1 Based on the H- H COSY data, three spin systems were established at H-1 (δH 2.31,

1.36)/H-2 (δH 1.62, 1.58)/H-3 (δH 2.24, 1.11), H-5 (δH 1.55)/H-6 (δH 2.23, 2.19)/H-7 (δH 2.89,

2.79) and H-11/12 (δH 7.22). Interpretation of the HMBC data then led to the proposed structure of 4. The terminal double bond at C-15 (C-15, δC 144.6 and C-16, δC 111.6) in angustanoic acid E (4)(15) is a characteristic moiety that distinguished compound 4 from the remaining abietanes isolated in this study. The information derived from NOESY experiment, allowed for the determination of the relative configuration at C-4, C-5 and C-10. The following key NOESY correlations were observed: H-20 (δH 1.14) with H-1b (δH 2.31), H-20 (δH 1.14) with H-6a (δH 2.19) and H-20 (δH 1.14) with H-11 (δH 7.22), H-20 (δH 1.14) with H-2a (δH 1.58)

and between H-18 (δH 1.30) and H-3b (δH 2.24), H-18 (δH 1.30) with H-5 (δH 1.55) and H-18

(δH 1.30) with H-6b (δH 2.23). This analysis indicated that the methyl group at C-4 was on the

R-face of the molecule, that is, equatorial (C-18); and that the carboxylic acid was therefore axial (C-19). Interestingly, in over 20 abietanes with a carboxylic acid moiety at the C-4 position are known as natural products, only a few contain an axial carboxylic acid. (181)

(182) Based on the available experimental data compound 4 was elucidated to be angustanoic acid E. Experimental data and elucidation were in agreement with the literature.

(15) Angustanoic acid E (4) was first isolated by Sy and Brown from I. angustisepalum. (15)

At that time it was the first phytochemical investigation of this plant species and it resulted in the isolation of fourteen abietane type diterpenes.

In the present study six similar compounds were found. Angustanoic acids E (4), F

(7), G (8), angustanol (12) and majusanic acids B (15) and C (5) were identified to be diterpene abietane derivatives. All of them contained the abietane skeleton with an aromatized ring C and a carboxyl group attached to C-4. The NMR spectroscopic assignments for the remaining aromatic abietanes compounds where established in the same manner.

Majusanic acid C (5)

5 HMBC correlations for 5

25 o White, amorphous; [α]D +88.0 (c 0.1, MeOH); IR vmax 2964, 2931, 1692, 1471, 1256,

-1 1 1171, 1147, 1071, 755 cm ; H NMR (CDCl3, 400 MHz) δH 7.21 (1H, d, J = 8.0 Hz, H-12),

7.14 (1H, d, J = 8.0 Hz, H-11), 7.02 (1H, s, H-14), 3.06 (3H, s, OCH3-15), 2.91 (1H, m, H-

7b), 2.82 (1H, m, H-7a), 2.26 (1H, m, H-6b), 2.25 (1H, m, H-3b), 2.05 (1H, m, H-6a), 2.03

(1H, m, H-2b), 1.60 (1H, m, H-2a), 1.58 (1H, br dd, H-5), 1.51 (6H, s, H-16/17), 1.40 (1H, m,

13 H-1a), 1.35 (3H, s, H-18), 1.13 (3H, s, H-20), 1.09 (1H, m, H-3a); C NMR (CDCl3, 100 MHz)

δC 183.8 (C, C-19), 146.6 (C, C-13), 142.7 (C, C-9), 135.0 (C, C-8), 126.4 (CH, C-14), 125.5

(CH, C-11), 123.6 (CH, C-12), 76.7 (C, C-15), 52.9 (CH, C-5), 50.7 (OCH3, C-15), 44.0 (C,

C-4), 39.5 (CH2, C-1), 38.5 (C, C-10), 37.7 (CH2, C-3), 32.4 (CH2, C-7), 28.9 (CH3, C-18),

28.0 (CH3, C-17), 28.0 (CH3, C-16), 23.3 (CH3, C-20), 21.2 (CH2, C-6), 20.0 (CH2, C-2); (+)-

+ HRESIMS m/z 330.2195 ([M] ; calcd for C21 H30 O3, 330.2189).

Majusanic acid B (5), was obtained as a white amorphous powder. Based on the

+ 13 HRESIMS data (m/z 330.4689 [M] ; calcd for C21 H30 O3, 330.2189) and C NMR data it has a molecular formula C21H30O3 (seven degrees of unsaturation). Its 1D NMR data are similar to those of angustanoic acid E (4), with major differences observed at C-15 and C-16.

Namely, in angustanoic acid E (4) carbons at C-15 (δC 144.6; C) and C-16 (δC 111.6; δH 5.31, s, H-16b; δH 4.99, t, J = 1.4 Hz, H-16a) correspond to a double bond and in majusanic acid

B (5) carbons at C-15 (δC 76.7; C-OCH3) and C-16 (δC 28.0; δH 1.51, s, H-16) correspond to tertiary oxygenated carbon with attached methoxyl group and methyl group, respectively.

The remaining parts of structure of 5 and also the relative configuration are same as in compound 4. Majusanic acid B was first isolated from I. majus. (183) Experimental data for majusanic acid B were in agreement with those reported in the literature. (15)

2, 6-Dimethoxychavicol (6)

6 HMBC correlations for 6

25 o Brown, oil; [α]D -0.3 (c 0.69, MeOH); IR (film) vmax 3443, 2937, 1613, 1515, 1459,

-1 1 1428, 1328, 1239, 1213, 1119 cm ; H NMR (methanol-d4, 400 MHz) δH 6.40 (1H, s, H-2’),

5.96 (2H, m, H-3/5), 5.4 (1H, br d, H-3a’), 5.10 (1H, br s, H-3b’), 3.80 (6H, m, OCH3-2/3), 3.30

13 (2H, s, H-1’); C NMR (methanol-d4, 100 MHz) δC 149.1 (C, C-2/6), 139.2 (CH, C-3/5), 134.9

(C, C-4), 132.9 (C, C-1), 115.5 (CH2, C-3’), 106.6 (CH, C-2’), 56.6 (OCH3, C-2/6), 41.1 (CH2,

+ C-1’); (+)-HRESIMS m/z 194.0943 ([M] , calcd for C11H14O3, 194.0937).

2,6-Dimethoxychavicol (6) was obtained as a yellowish oil. It has a molecular formula

+ of C11H14O3 as determined by HRESIMS data (m/z 194.0000 [M] ; calcd for C11H14O3,

194.0937) and 13C NMR spectroscopic data. The 1H, 13C and DEPT NMR spectra indicted the presence of one two methylenes (one olefinic and one aliphatic), three methines (two aromatic and one olefinic), three oxygenated tertiary carbons and one aromatic quaternary carbon. The IR broad stretch at 3443 cm-1 suggested the presence of the hydroxyl moiety.

The 1H NMR spectrum displayed an overlapped signal from two equivalent aromatic protons at δH 5.96 (m, H-3/5), two olefinic protons at δH 6.40 (s, H-2’), δH 5.4 (br d, H-3a’), δH 5.10 (br s, H-3b’) and one methylene group at δH 3.30 (s, H-1’). The presence of one double bond accounted for one degree of unsaturation, the remaining four were therefore deduced from an aromatic ring structure in the molecule. Inspection of the 13C NMR spectrum confirmed the presence of an aromatic system (C-2/6, δC 149.1; C-3/5, δC 139.2; C-4, δC 134.9; C-1, δC

132.9) and a double bond (C-3’, δC 115.5; C-2’, δC 106.6). Evaluation of the HSQC spectrum allowed for assigning of proton signals with carbons attached to them. Further analysis of the

NMR spectroscopic spectra suggested that presence of three oxygen bearing carbons including overlapped signal from the two equivalent methoxyl groups (C-2/6, δC 149.1, δH

1 1 3.30). In the H- H COSY spectrum signal from the proton at δH 3.30 (s, H-1’) displayed correlation with the olefinic proton at δH 6.40 (s, H-2’). The connection between the side chain and the aromatic ring was established based on the cross peaks between H-1’ and C-

3/5 (δC 139.2) and C-4 (δC 134.9). The hydroxyl group was established by assigning the oxygenated tertiary carbon at δC 132.9 to C-2 based on its HMBC correlations with H-3/5.

And the two equivalent methoxyl groups were established by assigning the remaining two equivalent oxygenated carbons at δC 149.1 to C-2/6. Subsequently, interpretation of the

HMBC data led to the proposed structure 6. All available evidence led to the determination of the structure 6 as shown. Experimental data were in agreement with the literature. (184)

Angustanoic acid F (7)

7 HMBC correlations for 7

25 o White, crystal; [α]D +88.0 (c 0.1, MeOH); IR (film) vmax 3400, 2962, 2930, 1706,

-1 1 1436, 1230, 1155, 1017, 951 cm ; H NMR (methanol-d4, 400 MHz) δH 7.22 (1H, br d, H-

11), 7.19 (1H, br dd, H-12), 7.13 (1H, s, H-14), 2.89 (1H, m, H-7b), 2.81 (1H, td, J = 6.0, 6.0,

6.0 Hz, H-7a), 2.31 (1H, m, H-1b), 2.24 (1H, m, H-3b), 2.22 (1H, m, H-6b), 2.08 (1H, m, H-

2b), 1.60 (1H, m, H-2a), 1.54 (1H, m, H-6a), 1.49 (6H, s, H-16/17), 1.35 (1H, m, H-1a), 1.30

13 (3H, s, H-18), 1.12 (3H, s, H-20), 1.11 (1H, d, J = 4.0 Hz, H-3a); C NMR (methanol-d4, 100

MHz) δC 181.5 (C, C-19), 147.5 (C, C-9), 147.4 (C, C-13), 135.9 (C, C-8), 126.2 (CH, C-14),

125.9 (CH, C-11), 123.2 (CH, C-12), 72.7 (C, C-15), 54.3 (CH, C-5), 44.8 (C, C-4), 40.8 (CH2,

C-1), 39.5 (C, C-10), 38.8 (CH2, C-3), 33.3 (CH2, C-7), 31.8 (CH3, C-17), 31.8 (CH3, C-16),

29.9 (CH3, C-18), 23.7 (CH3, C-20), 22.3 (CH2, C-6), 21.1 (CH2, C-2); (-)-HRESIMS m/z

- 315.1954 ([M-H] , calcd for C20H27O3, 315.1966).

Angustanoic acid F (7) was obtained in the form of crystalline needles. Compound 7 has the molecular formula of C20H28O3 with seven degrees of unsaturation, which was established based on the HRESIMS quasi-molecular ion [M-H]- at m/z 315.1954 (calcd for

C20H27O3, 315.1966). Based the experimental and literature data, it was identified as a diterpene with aromatized abietane skeleton and a carboxyl group attached to C-4. 1D NMR spectroscopic data are similar to previously discussed compounds 4 and 5, although the hydroxyl group at C-15 distinguishes this particular compound from them. Noteworthy, the terminal double bond at C-15 in angustanoic acid E was replaced by a C-15 hydroxyl group

(C-15, δc 72.7) in angustanoic acid F. Compound 7 was first isolated from I. angustisepalum.

(15) Experimental data reported here are in agreement with the data from the literature. (15)

Angustanoic acid G (8)

8 HMBC correlations for 8

25 o White, crystal; [α]D +135.3 (c 0.17, MeOH); IR (film) vmax 2932, 1706, 1677, 1271,

-1 1 1017, 951 cm ; H NMR (methanol-d4, 400 MHz) δH 7.67 (1H, s, H-14), 7.72 (1H, dd, J =

8.0Hz, 1.9, H-12), 7.42 (1H, d, J = 8.4 Hz, H-11), 2.98 (1H, m H-7b), 2.85 (1H, td, J = 6.0 Hz,

6.0, 6.0, H-7a), 2.56 (3H,s, H-16), 2.35 (1H, m, H-1a), 2.25 (1H, m, H-3b), 2.25 (1H, m, H-

6a), 2.08 (1H, m, H-2b), 2.08 (1H, m, H-6b), 1.63 (1H, m, H-2a), 1.57 (1H, dd, J = 1.6 Hz,

1.6, H-5), 1.38 (1H, td, 4.00, 4.0, 4.0 Hz, H-1b), 1.31 (3H, s, H-18), 1.17 (3H, s, H-20), 1.12

13 (1H, td, 4.00, 4.0, 4.0 Hz, H-3a); C NMR (methanol-d4, 100 MHz) δC 200.6 (C, C-15), 181.2

(C, C-19), 155.4 (C, C-9), 137.1 (C, C-8), 135.5 (C, C-13), 130.5 (CH, C-14), 127.1 (CH, C-

11), 126.8 (CH, C-12), 53.7 (CH, C-5), 44.8 (C, C-4), 40.3 (C, C-10), 40.3 (CH2, C-1), 38.6

(CH2, C-3), 33.0 (CH2, C-7), 29.1 (CH3, C-18), 26.6 (CH3, C-17), 23.4 (CH3, C-20), 22.1 (CH2,

+ C-6), 21.0 (CH2, C-2); (+)-HRESIMS m/z 301.1786 ([M+H] , calcd for C19H25O3, 301.1798).

Angustanoic acid G (8) was obtained in the form of crystalline needles. Angustanoic acid G has the molecular formula of C19H24O3 (eight degrees of unsaturation), which was

+ established by HRESIMS quasi-molecular ion [M+H] at m/z 301.1786 (calcd for C19H25O3,

301.1798). Angustanoic acid G (8) is a norabietane, which may be derived by oxidative cleavage of the terminal double bond found in angustanoic acid E (4). Compound 8 was first isolated from I. angustisepalum. (15) Experimental data and the elucidation for 8 were in agreement with the literature. (15)

6β-Hydroxy-4-stigmasten-3-one (9)

9 HMBC correlations for 9

25 o White, crystal; [α]D +1.42 (c 0.07, MeOH); IR (film) vmax 3400, 2918, 2849, 2359,

-1 1 1341 vmax cm ; H NMR (methanol-d4, 400 MHz, multiplicities of some signals were not clear

due to overlapping peaks) δH 5.8 (1H, s, H-4), 4.28 (1H, br t, H-6), 4.58 (1H, td, J = 4.9 Hz,

4.9, 4.9, H-1a), 2.33 (1H, br dt, H-1b), 0.72 (3H, s, H-18), 0.82-1.12 (m, H-21, 26, 27, 29),

13 1.18 (3H, s, H-19); C NMR (methanol-d4, 100 MHz) δC 203.1 (C, C-3), 171.9 (C, C-5),

126.6 (CH, C-4), 73.7 (CH, C-6), 57.4 (CH, C-14), 57.2 (CH, C-17), 55.1 (CH, C-9), 47.2 (CH,

C-24), 43.6 (C, C-13), 41.0 (CH2, C-12), 39.7 (CH2, C-11), 39.3 (C, C-10), 38.3 (CH2, C-7),

37.4 (CH, C-20), 35.0 (CH2, C-1/2), 31.0 (CH, C-25), 30.3 (CH, C-8), 29.3 (CH2, C-22), 27.1

(CH2, C-23), 25.2 (CH2, C-15), 24.1 (CH2, C-16), 22.0 (CH2, C-28), 20.2 (CH3, C-26), 19.6

(CH3, C-27), 19.3 (CH3, C-21), 19.2 (CH3, C-19), 12.4 (CH3, C-18), 12.3 (CH3, C-29); (+)-

+ HRESIMS m/z 429.3586 ([M+H] , calcd for C29H49O2, 429.3727).

6β-Hydroxy-4-stigmasten-3-one (9) was obtained in the form of crystalline needles.

The mass spectral data of 9 gave a molecular formula of C29H48O, which was supported by the 13C NMR spectral data. The 1D NMR spectra (1H and 13C) of 9 indicated the presence of six methyls, ten methylenes, nine methines (one olefinic and eight aliphatic), one carbonyl, as well as three quaternary carbons. Analysis of the DEPT spectrum with the aid of 2D NMR determined the planar structure of 9, with the carbonyl group at δC 203.1 (C-3) and the methine carbon at δC 73.7 (C-6). The double bond was located between carbon C-4 (δC

126.6) and C-5 (δC 171.9) as determined by the presence of the HMBC correlations of the proton at 5.8 (s, H-4) with carbons at C-3, C-6 and C-10. The B-ring was established based on the HMBC correlations between the methyl group at δH 1.18 (H-19) with carbons at C-5

(δC 171.9), C-9 (δC 55.1) and C-10 (δC 39.3). The C-ring was determined based on the long range correlations between δH 0.72 (H-18) and C-12 (δC 41.0), C-13 (δC 43.6) and C-14 (δC

57.4).The side chain was assigned based on the long-range correlations between methyl groups at δH 0.82-1.12 (m, H-21) and carbons C-20 (δC 37.4), C-22 (δC 29.3) and between

δH 0.82-1.12 (m, H-29) and carbons C-24 (δC 47.2) and C-28 (δC 22.0). Thus, the planar structure of 9 was assigned as shown. Finally, the structure of 9 including the relative configuration was confirmed by single-crystal X-ray diffraction (Figure 3-5). The physical and spectral data are consistent to those in the reported literature. (185)

FIGURE 3-5: ORTEP view of 6β-Hydroxy-4-stigmasten-3-one (9). The C-atom labeling shown here is different from the IUPAC labeling for sterols

(2R, 3R)-3,5,7,3’,4’-Pentahydroxyflavonone (10)

10 HMBC correlations for 10

25 o Yellow, amorphous; [α]D +18.0 (c 0.5, acetone); IR (film) vmax 3344, 1638, 1469,

-1 1 13 1283, 1162, 1086 cm ; H (DMSO-d6, methanol-d4, 400 MHz) and C (DMSO-d6, methanol-

- d4, 100 MHz) NMR data, see Table 3-5; (-)-HRESIMS m/z 303.0502 ([M-H] , calcd for

C15H11O7, 303.0510).

TABLE 3-5: 1H AND 13C NMR SPECTROSCOPIC DATA OF (2R, 3R)-3,5,7,3’4’- PENTAHYDROXYFLAVONONE (Δ IN PPM, J IN HZ)

Literature data (500 MHz) Experimental data (400 MHz for 1H NMR and 100 MHz for 13C NMR) (186)

a b a b b b Position δC, type δC, type δH, mult. (J in Hz) δH, mult. (J in Hz) δC, type δH, mult. (J in Hz)

2 83.0., CH 85.0, CH 4.97, d, J = 11.1 4.86, d, J = 11.6 85.1, CH 4.90, d, J = 11.4

3 71.6, CH 73.5, CH 4.48, dd, J = 11.1, 5.8 4.45, d, J = 11.6 73.7, CH 4.49, d, J = 11.4

4 197.8, C 198.2, C 198.4, C

5 163.3, C 165.1, C 165.3, C

6 96.0, CH 97.2, CH 5.90, d, J = 2.0 5.87, d, J = 1.9 97.3, CH 5.91, d, J = 2.4

7 166.8, C 168.5, C 168.8, C

8 95.0, CH 96.2, CH 5.85, d, J = 1.8 5.83, d, J = 1.9 96.3, CH 5.88, d, J = 2.4

9 162.6, C 164.4, qC 164.5, qC

10 100.5, C 101.7, qC 101.8, qC

1’ 128.0, C 129.8, qC 129.9, qC

116.1, 6.84, dd, J = 8.4, 2’ 115.3, CH 116.0, CH 6.87, br s 6.92, d, J = 1.4 CH 1.8

3’ 145.0, C 146.2, C 146.3, C

4’ 145.8, C 147.0, C 147.2, C

5’ 115.1, CH 115.8, CH 6.73, br s 6.84, d, J = 8.1 115.9, C 6.79, d, J=8.4

(overlapped H-5’ and 120.9, 6.95, d, J = 1.8 6’ 119.4, CH 120.8, CH 6.86, dd, J = 8.1, 1.6 H-6’) CH

3-OH 5.73, d, J = 6.1

5-OH 11.88, s 11.64 s a b Data measured in DMSO-d6, Data measured in methanol-d4, J: Coupling constant.

(2R, 3R)-3,5,7,3’4’-Pentahydroxyflavonone (10) was obtained as yellow amorphous

powder. The HRESIMS displayed quasi-molecular ion [M-H]- at m/z 303.0502,

corresponding to the molecular formula of C15H12O7 with 10 indices of hydrogen

deficiency after taking into account 13C NMR data. The IR broad stretch at 3344 cm-1 suggested the presence of the hydroxyl moiety and the stretch at 1638 cm-1 indicated presence of a carbonyl moiety in the molecule. The 1H NMR spectrum displayed presence of H-2 and H-3 protons of the dihydroflavonol at δH 4.86 (d, J = 11.6 Hz) and

δH 4.45, (d, J = 11.6 Hz). The 13C NMR spectrum exhibited 15 carbon signals corresponding to seven methines (five aromatic and two aliphatic), four oxygenated tertiary carbons, a carbonyl carbon, and three quaternary carbons. Inspection of the

HSQC spectrum allowed for assigning of proton signals with carbons attached to them.

In the 1H-1H COSY spectrum, signal at δH 6.84 (d, J = 8.1 Hz, C-5’) displayed correlation with δH 6.86 (dd, J = 8.1, 1.6 Hz) suggesting 1,3,4-trisubstitution pattern in the ring B.

Further analysis of the 1H-NMR spectra confirmed a 1,3,4-trisubstituted B ring with signals in the aromatic region at δH 6.92 (C-2’, d, J = 1.4 Hz), δH 6.86 (C-6’, dd, J = 8.1, 1.6 Hz) and δH 6.84, (C-5’, d, J = 8.1 Hz). The position of the carbonyl carbon at C-4 (δC 198.27) was established based on the displayed HMBC long-range correlations with H-2 and H-

3. The substitution pattern in the ring A was established based on the following evidence:

δH 5.87 (d, J = 1.9, H-6) and δH 5.83 (d, J = 1.9, H-8). Subsequently, interpretation of the

HMBC data led to the proposed planar structure of 10. The comparison of available NMR data with the literature confirmed the structure as taxifolin. (187) (187) The relative configuration of 10 was established based on the comparison of the optical rotation of 10 with the circular dichroism (CD) spectra of similar flavonols. The coupling constant between 2-H and 3-H (J = 11.1 Hz) indicated the presence of trans-conformation.

Analysis of the circular dichroism (CD) spectra of similar flavonones (Figure 3-6) in view

25 o of measured optical rotation [α]D +18.0 (c 0.5, acetone) resulted in the identification of

compound 10 to be (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavanone. The experimental data

and elucidation of 10 were in agreement with the literature. (187) (2R, 3R)-3,5,7,3’4’-

Pentahydroxyflavonone, belongs to the chemical class of flavonols. Present study reports

(2R, 3R)-3,5,7,3’4’-pentahydroxyflavonone for the first time from I. angustisepalum. Also,

10 is the first flavonoid compound reported from I. angustisepalum so far.

FIGURE 3-6: Comparison of circular dichroism spectra of (2R, 3R)-3,5,7,3’,4’- pentahydroxyflavanone (10) (187)

Angustisepalin (11)

11 Key HMBC correlations for 11

25 o White, amorphous; [α]D - 33.3 (c 0.06, CHCl3); IR (film) vmax 3480, 2955, 1785, 1754,

-1 1 1731, 1451, 1372, 1250, 1178, 1091, 711 cm ; H NMR (methanol-d4, 400 MHz) δH 7.99

(2H, d, J = 7.5 Hz, H-3'/7'), 7.67 (1H, t, J = 7.5 Hz, H-5'), 7.54 (2H, t, J = 7.5 Hz, H-4'/6'), 5.96

(1H, s, H-10), 4.66 (1H, t, J = 2.9 Hz, H-7), 4.36 (1H, d, J = 11.2 Hz, H-14a), 4.16 (1H, d, J =

11.2 Hz, H-14b), 3.66 (1H, br s, OH-6), 2.64 (1H, m, H-1), 2.62 (1H, dd, J = 15.2, 2.9 Hz, H-

8b), 2.30 (1H, br s, OH-4), 2.14 (1H, dd, J = 15.2, 2.9 Hz, H-8a), 2.05 (1H, m, H-2b), 2.03

(1H, m, H-3a), 1.87 (1H, m, H-3b), 1.37 (3H, s, H-13), 1.25 (1H, m, H-2a), 0.95 (3H, d, J =

13 7.5 Hz, H-15); C NMR (methanol-d4, 100 MHz) δC 177.9 (C, C-12), 170.1 (C, C-11), 165.9

(C, C-1’), 135.1 (CH, C-5’), 130.5 (CH, C-3’/7’), 130.4 (C, C-2’), 130.0 (CH, C-4’/6’), 84.1 (C,

C-4), 81.4 (CH, C-7), 79.6 (C, C-6), 73.0 (CH2, C-14), 71.3 (CH, C-10), 52.1 (C, C-9), 47.9

(C, C-5), 39.5 (CH, C-1), 31.8 (CH2, C-3), 30.6 (CH2, C-2), 27.4 (CH2, C-8), 21.17 (CH3, C-

+ 13), 13.8 (CH3, C-15); (+)-HRESIMS m/z 417.1532 ([M+H] , calcd for C22H25O8, 417.1544).

Angustisepalin (11) was obtained as white amorphous power. The analysis of

HRESIMS displayed quasi-molecular ion [M+H]+ at m/z 417.1532 corresponding to the molecular formula of C22H24O8 with 6 indices of hydrogen deficiency after taking into account

13C NMR spectroscopic data. The IR broad stretch at 3480 cm-1 suggested the presence of the hydroxyl moiety and the three stretches at 1785, 1754, 1731 cm-1 indicated presence of a carbonyl moieties in the molecule. The 13C NMR spectrum exhibited 22 carbon signals corresponding to two methyls, four methylenes, eight methines (five aromatic and three aliphatic), two oxygenated tertiary carbons, three carbonyl carbons, and three quaternary carbons. Further analysis of the 13C NMR spectrum of 11 showed two carbon signals of double intensity (C-3’/7’, δC 130.5; C-4’/6’, δC 130.09) consistent with the presence of a mono- substituted benzene group. Inspection of the HSQC spectrum allowed for assigning of proton signals with carbons attached to them. Based on the 1H-1H COSY experiment, three spin systems corresponding to H-1 (δH 2.64)/H-2 (δH 2.05, 1.25)/H-3 (δH 2.03, 1.87), H-7 (δH

4.66)/H-8 (δH 2.62, 2.14), H-3'/7' (δH 7.99)/H-5' (7.67)/H-4'/6' (7.54) were established,

allowing the assignment of connectivities of these fragments. Interpretation of the HMBC data then led to the proposed structure of 11. It is noteworthy that carbonyl carbon at C-1’ (δC

165.9) displayed long-range correlations with H-3'/H-7' (δH 7.5) and with the proton of an oxygen-bearing methine group (δH 5.96, H-10) in the sesquiterpene moiety of angustisepalin.

This particular correlation was helpful in establishing of an ester bond between aromatic ring and the sesquiterpene part of the molecule. The hydroxyl groups were established by assigning the oxygenated tertiary carbons at δC 84.1 and δC 79.6 to C-4 and C-6, respectively. The HMBC correlations between δC 84.1 and δH 2.30 (br s, OH-4) and also between δC 79.6 and δH 3.66 (br s, OH-6), supported the proposed assignments for hydroxyl groups. The γ-lactone moiety was established by assigning the quaternary carbon at δC 47.9 to C-5 based on its HMBC correlations with H-7 and H-14. Other carbons in this part of the molecule, including C-5 (δC 47.9), C-6 (δC 79.6) and C-14 (δC 73.0), showed long range correlations with methyl at δH 1.37 (s, H-13). The δ-lactone moiety was established based on the following evidence: HMBC correlations between oxygenated methine carbon at H-10 (δH

5.96) and the carbons C-8 (δC 27.4), C-9 (δC 52.1) and C-11 (δC 170.1). The key NOESY correlations were: H-3 with H-13, H-8b with H-10 and hydroxyl group at C-4, H-15 with H-10,

H-14a with H-13 and H-14b with H-2b. All available evidence led to determination of compound 11 as the benzoyl ester of a prezizaane sesquiterpene. Experimental data were in agreement with the literature. (14)

Clovane-2,9-diol (12)

12 HMBC correlations for 12

25 o White, amorphous; [α]D –5.0 (c 0.08, CHCl3); IR (film) vmax 3366, 2920, 2851, 1744,

-1 1 1718, 1463, 1367, 1245, 1073 cm ; H NMR (DMSO-d6, 400 MHz) δH 3.55 (1H, dd, J = 10.0,

5.0 Hz, H-2), 3.09 (1H, s, H-9), 1.79 (1H, m, H-10b), 1.64 (1H, m, H-11b), 1.52 (1H, m, H-

12b), 1.50 (1H, m, H-3b), 1.44 (1H, m, H-10a), 1.40 (1H, m, H-3a), 1.36 (1H, m, H-6a), 1.33

(1H, m, H-6b), 1.30 (1H, m, H-5), 1.25 (1H, m, H-7b), 1.05 (1H, m, H-7a), 0.96 (3H, s, H-14),

0.87 (1H, m, H-11a), 0.84 (3H, s, H-15), 0.79 (3H, s, H-13), 0.72 (1H, d, J = 12.0 Hz, H-12a);

13 C NMR (DMSO-d6, 100 MHz) δC 78.9 (CH, C-2), 73.0 (CH, C-9), 50.1 (CH, C-5), 47.3 (CH2,

C-3), 43.9 (C, C-1), 36.5 (C, C-4), 35.5 (CH2, C-12), 34.3 (C, C-8), 32.9 (CH2, C-7), 31.4

(CH3, C-14), 28.8 (CH3, C-15), 26.6 (CH2, C-11), 26.0 (CH2, C-10), 25.3 (CH3, C-13), 20.4

+ (CH2, C-6); (+)-HRESIMS m/z 238.1933 ([M] , calcd for C15H26O2, 238.1927).

Clovane-2,9-diol (12), was obtained as a white amorphous powder. The HRESIMS of

+ 12 demonstrated HRESIMS quasi-molecular ion [M] at m/z 238.3700 (calcd for C15H26O2,

238.1927), corresponding to a molecular formula C15H26O2 with three degrees of

-1 unsaturation. The IR broad stretch at 3366 cm indicated the presence of a hydroxyl group.

Investigation of the 13C/DEPT-135 NMR spectra indicated the presence of 15 carbon signals corresponding to a three methyls, six methylene, three methine and three quaternary carbon.

Careful analysis of their chemical shifts suggested the presence of two oxygenated methine

1 1 carbons at δC 78.9 (C-2) and δC 73.0 (C-9). Based on the information from H− H COSY,

three spin systems corresponding to H-2 (δH 3.55)/H-3 (δH 1.50, 1.40), H-5 (δH 1.30)/H-6 (δH

1.36, 1.33)/H-7 (δH 1.25, 1.05) and H-9 (δH 3.09)/H-10 (δH 1.79, 1.44)/H-11 (δH 1.64, 0.87) were established. All of the proton signals were assignable to their attached carbons through an HSQC experiment. The long range correlations helped to determine the structure of 12.

The cross peaks between δH 1.30 (H-5) and C-1 (δC 43.9) supported the connectivity between the A- and B-rings. The HMBC correlations between methyl proton at δH 0.84 (H-

15) and carbons at C-7 (δC 32.9), C-8 (δC 34.3), C-9 (δC 73.0) and C-12 (δC 35.5) further clarified the partial structures and established the methylene bridge between C-1 and C-8.

The HMBC correlations between the methyl proton signal at δH 0.79 (H-13) with C-3 (δC 47.3) and methyl proton signal at δH 0.96 (H-14) with C-4 (δC 36.5) and C-5 (δC 50.1) established the ring B. Confirmation of the partial structure’s resulted in construction of bicyclononane skeleton. Subsequently, 12 was elucidated to be: clovane-2,9-diol (12). The relative configuration of 12 was assigned based on the NOESY analysis, in which the following key correlations were observed: H-2 with H-3a and H-3b, H-6a with H-6b and H-7b, then H-9 with H-10a and H-10b, then H-11a with H-10a, H-10b, H-11b, and also between H-12a with

H-12b. Experimental data and elucidation were in agreement with the literature. (188) (189)

Clovane-2,9-diol (12) is a rare clovane-type sesquiterpene, isolated for the first time from

Baeckea frutescens L. (Myrtaceae). (190) (188) (191) Present study reports clovane-2,9-diol for the first time from I. angustisepalum.

Angustanol (13)

13 HMBC correlations for 13

25 o White, amorphous; [α]D + 9.0 (c 0.1, acetone); IR (film) vmax 3357, 2960, 2931, 2906,

-1 1 2871, 1697, 1455, 1375, 1139, 1041 cm ; H NMR (DMSO-d6, 400 MHz) δH 7.18 (1H, d, J

= 8.5 Hz, H-11), 7.13 (1H, d, J = 8.5 Hz, H-12), 7.07 (1H, s, H-14), 3.35 (1H, br s, H-19a),

2.80 (1H, dd, J = 4.7, 4.2 Hz, H-7b), 2.70 (1H, m, H-7a), 2.24 (1H, d, J = 12.5 Hz, H-1b), 2.11

(1H, m, H-6b), 2.10 (1H, m, H-3b), 2.06 (1H, m, H-6a), 1.93 (1H, td, J = 3.9, 4.5, 4.2 Hz, H-

2b), 1.50 (1H, m, H-2a), 1.43 (1H, d, J = 12.0 Hz, H-5), 1.33 (3H, s, H-18), 1.23 (1H, d, J =

5.0 Hz, H-1a), 1.19 (6H, s, H-16/17), 1.03 (3H, s, H-20), 0.98 (1H, dd, J = 3.7, 3.0 Hz, H-3a);

13 C NMR (DMSO-d6, 100 MHz) δC 145.8 (C, C-13), 144.0 (C, C-9), 139.9 (C, C-8), 125.7

(CH, C-14), 124.5 (CH, C-11), 123.0 (CH, C-12), 73.3 (C, C-15), 70.4 (CH2, C-19), 52.1 (CH,

C-5), 43.0 (C, C-4), 39.1 (CH2, C-1), 37.8 (C, C-10), 37.3 (CH2, C-3), 31.7 (CH2, C-7), 28.5

(CH3, C-16/17), 26.0 (CH3, C-18), 23.1 (CH3, C-20), 20.8 (CH2, C-6), 19.8 (CH2, C-2); (+)-

+ HRESIMS m/z 302.2244 ([M] , calcd for C20H30O2, 302.2240).

Angustanol (13) was isolated as white amorphous powder. Based the experimental and literature data it was identified as a diterpene with aromatized abietane skeleton and an alcohol group at C-19. Angustanol has the molecular formula C20H30O2 (six degrees of unsaturation), which was established by HRESIMS quasi-molecular ion [M]+ at m/z 302.1988

(calcd for C20H30O2, 302.2240). Formally, angustanol (13) is derivative of angustanoic acid F

(7) where carboxyl group at C-4 is replaced by CH2 -OH group. Angustanol (13) was first

isolated from I. angustisepalum. (15) Experimental data and elucidation were in agreement with the literature. (15)

Majucin (14)

14 Key HMBC correlations for 14

25 o White, crystal; [α]D -71.4 (c 0.22, MeOH); IR (film) vmax 3453, 2939, 2874, 1770,

-1 1 1731, 1510, 1453, 1372, 1211, 1123, 1008 cm ; H NMR (DMSO-d6, 400 MHz) δH 4.42 (1H, br t, H-7), 4.29 (1H, q, J = 5.0 Hz, H-3), 4.11 (1H, d, J = 5.5 Hz, H-10), 4.07 (1H, d, J = 4.0

Hz, H-14b), 3.91 (1H, d, J = 11.0 Hz, H-14a), 2.49 (1H, m, H-1), 2.36 (1H, br dd, H-8b), 1.91

(1H, dd, J = 3.3, 3.2 Hz, H-8a), 1.70 (1H, m, H-2b), 1.50 (1H, m, H-2a), 1.22 (3H, s, H-13),

13 0.86 (3H, J = d, 6.0 Hz, H-15); C NMR (DMSO-d6, 100 MHz) δC 176.8 (C, C-12), 173.7 (C,

C-11), 81.0 (C, C-4), 78.8 (CH, C-7), 78.4 (C, C-6), 71.4 (CH, C-3), 71.3 (CH2, C-14), 69.0

(CH, C-10), 50.2 (C, C-9), 46.0 (C, C-5), 41.3 (CH2, C-2), 36.6 (CH, C-1), 25.5 (CH2, C-8),

+ 19.6 (CH3, C-13), 13.7 (CH3, C-15); (+)-HRESIMS m/z 328.1158 ([M] , calcd for C15H20O8,

328.1153).

Majucin (14) was obtained in the form of crystalline needles. The HRESIMS molecular

+ quasi-molecular ion [M] at m/z 328.3175 (calcd for C15H20O8, 328.1153), corresponded to a molecular formula of C15H20O8 with six degrees of unsaturation. The IR spectrum exhibited absorptions at 1770 and 1731 cm1 characteristic for the γ-lactone and δ-lactone, respectively.

The 13C NMR spectrum exhibited 15 carbon signals corresponding to two methyls, three methylenes, four methines, two oxygenated tertiary carbons, two carbonyl carbons, and two

quaternary carbons. Inspection of the HSQC spectrum allowed for assigning of proton signals with carbons attached to them. The 1H-1H COSY experiment helped to establish two spin systems corresponding to H-1 (δH 2.49)/H-2 (δH 1.70, 1.50)/H-3/H-15 (δH 4.29) and H-7

(δH 4.42)/H-8 (δH 2.36, 1.91), which allowed the assignment of connectivities of these fragments. Interpretation of the HMBC data then led to the proposed structure of 14. The hydroxyl groups were established by assigning the carbons at δC 71.4, δC 81.0, δC 78.4 and

δC 69.0 to C-3, C-4, C-6 and C-10, respectively. The long range correlations observed between the methyl group at δH 1.22 (s, H-13) and carbons at C-5 (δC 46.0), C-6 (δC 78.4) and C-14 (71.3) helped to establish the γ-lactone ring. On the other hand, the correlations between methine at δH 4.42 (H-7, br t) and carbons C-11 (δC 173.7), C-9 (δC 50.2) and C-8

(δC 25.5) confirmed the presence of a δ-lactone ring in the molecule. Assembly of these fragments confirmed the structure of majucin (14). The key NOESY correlations include: H-

15 with hydroxyl at C-10, then hydroxyl at C-3 with hydroxyl at C-4, H-13 with hydroxyl at C-

6. Experimental data and elucidation were in agreement with the literature. (192) Comparison with literature also revealed close similarity to neomajucin which is identical to majucin except for the missing hydroxyl group at C-3. (192) Present study reports majucin (14) for the first time from I. angustisepalum.

2-Hydroxy-7-methyl-hexan-1,5-olide (15)

15 Key HMBC correlations for 15

25 Brown, oily; [α]D -2.2 (c 0.28, MeOH) ; IR (film) vmax 3366, 2970, 2933, 1779, 1705,

-1 1 13 1378, 1247, 1122 cm ; H (methanol-d4, 400 MHz) and C (methanol-d4, 100 MHz) NMR

+ data, see Table 3-6; (+)-HRESIMS m/z 145.0786 ([M+H] , calcd for C7H13O3, 145.0859).

2-Hydroxy-7-methyl-hexan-1,5-olide (15) was obtained as a brown oil. The HRESIMS

+ quasi-molecular ion [M+H] at m/z 145.4095 (calcd for C7H13O3, 145.0859) indicated a

13 molecular formula of C7H12O3 with two indices of hydrogen deficiency after C NMR spectroscopic data were taken into consideration.

TABLE 3-6: 1H AND 13C NMR SPECTROSCOPIC DATA OF 2-HYDROXY-7-METHYL- HEXAN-1,5-OLIDE (Δ IN PPM, J IN HZ)A

b Position δC, type δH, mult. (J in Hz) HMBC (H C)

1 174.0, C

2 68.5, C

4a, 2.53, d (J = 1.92) 3 44.4, CH2 2, 3, 5, 7 4b, 2.51, s

5a, 1.90, dt (J = 2.40, 2.30) 3, 4 4 43.8, CH2 5b 1.64, dd (J = 11.9, 11.7) 1, 4, 6

5 75.6, CH 4.77, m 2, 6

6 21.8, CH3 1.37, d (J = 6.43) 1, 2, 5

7 29.8, CH3 1.30, s 2, 3, 4 a 1 13 Data measured in methanol-d4; 400 MHz for H NMR and 100 MHz for C NMR; J: Coupling constant. b Data recorded at 600 MHz.

The IR absorption at 1779 cm1 indicated the presence of δ-lactone moiety.

Interpretation of the 13C NMR and DEPT135 spectra of 15 (Table 3-6) indicated the presence of seven carbons in the structure, including two CH3 groups (C-7, δC 29.8; C-6, δC 21.8), two

CH2 groups (C-3, δC 44.4; C-4, δC 43.8), CH (C-5, δC 75.6), a tertiary oxygenated carbon (C-

2, δC 68.5) and a carbonyl carbon (C-1, δC 174.0). The presence of carbonyl group accounted for one degree of unsaturation, the remaining one was deducted from the pyran ring structure in the molecule. Inspection of the HSQC spectrum allowed for linking of carbon signals with their respective protons (C-7, δH 1.30, s; C-6, δH 1.37, d, J = 6.43 Hz; C-3a, δH 2.53, d, J =

1.92 Hz; C-3b, δH 2.51, s; C-4a, δH 1.90, dt, J = 2.40, 2.30 Hz; C-4b, δH 1.64, dd, J = 11.9,

1 1 11.7 Hz; C-5, δH, 4.77, m) (Table 3-6). The H- H COSY spectrum showed correlations between H-3 and H-4 suggesting that the two methylene groups are directly connected. The

COSY experiment also exhibited correlations between the methine proton at C-5 (δH 4.77, m) and the methyl protons at C-6 (δH 1.37, d, J = 6.43), as well as between the methylene protons attached to C-4. Subsequently, interpretation of the HMBC data led to the proposed structure 15. It is noteworthy that quaternary carbon at δC 174.0 (C-1) displayed long-range correlations with the methine proton at δH 4.77 (m, C-5). This particular correlation was helpful in establishing of the closed ring structure of 15. All available evidence led to the determination of the structure of 15 as depicted. To the best of our knowledge, compound 15 is a new structure isolated from the natural source.

Majusanic acid B (16)

16 HMBC correlations for 16

25 o White, amorphous; [α]D +75.75 (c 0.8, MeOH); IR (film) vmax 3400, 2958, 2934,

-1 1 1701, 1046, 1025, 1000cm ; H NMR (DMSO-d6, 400 MHz) δH 7.17 (1H, d, J = 7.5 Hz, H-

11), 7.13 (1H, d, J = 8.6 Hz, H-12), 7.06 (1H, s, H-14), 3.34 (3H, br d, H-16), 2.79 (1H, m, H-

7b), 2.72 (1H, m, H-7a), 2.24 (1H, m, H-1b), 2.10 (1H, d, J = 10.0 Hz, H-3b), 2.08 (1H, m, H-

6a), 2.08 (1H, m, H-6b), 1.92 (1H, m, H-2b), 1.51 (1H, m, H-2a), 1.47 (1H, d, J = 12.0 Hz, H-

5), 1.33 (3H, s, H-18), 1.24 (1H, m, H-1a), 1.20 (3H, s, H-17), 1.03 (3H, s, H-20), 1.00 (1H,

13 m, H-3a); C NMR (DMSO-d6, 100 MHz) δC 178.5 (C, C-19), 145.6 (C, C-13), 144.0 (C, C-

9), 133.8 (C, C-8), 125.7 (CH, C-14), 124.5 (CH, C-11), 123.1 (CH, C-12), 73.3 (C, C-15),

70.4 (CH2, C-16), 52.0 (CH, C-5), 43.0 (C, C-4), 38.9 (CH2, C-1), 37.8 (C, C-10), 37.2 (CH2,

C-3), 31.7 (CH2, C-7), 28.3 (CH3, C-17), 26.0 (CH3, C-18), 23.0 (CH3, C-20), 20.8 (CH2, C-

+ 6), 19.7 (CH2, C-2); (+)-HRESIMS m/z 332.1988 ([M] , calcd for C20 H28 O4, 332.1982).

Majusanic acid B (16) was obtained as white amorphous powder. Majusanic acid B has the molecular formula C20 H28 O4 (seven degrees of unsaturation), which was established

+ by HRESIMS quasi-molecular ion [M] at m/z 332.4428 (calcd for C20 H28 O4, 332.1982). Its

1D NMR data are similar to those of angustanoic acid F (7), with major difference observed at C-16. Namely, one of the protons from the methyl group at δC 31.8 (δC 1.49, s, H-16) in 7 was replaced with a hydroxyl group in 16. The remaining parts of structure of 16 and also the relative configuration are same as in the rest of isolated abietanes. Majusanic acid B (16) was first isolated from I. majus. (183) Experimental data and elucidation were in agreement with the literature. (15)

DISCUSSION

Phytochemical analysis led to isolation of a total of 16 compounds. 2-Hydroxy-2- methyl-6-methyleneoct-7-en-3-yl benzoate (3) and 2-hydroxy-2-methyl-hexane 1,5-olide (15) are isolated as natural products for the first time. Compounds 1, 2, 5, 9, 10, 12, 14 and 16 are identified in I. angustisepalum for the first time. Compounds 4, 6, 7, 8, 11 and 13 were reported from I. angustisepalum previously.

Among the isolated compounds six major chemical classes can be distinguished including monoterpenes, sesquiterpenes, diterpenes, flavonoids and phenylpropanes. In addition to these derivatives of monoterpenes and pyran were also isolated.

Thymol (1) is an example of monoterpene that is a common component of the plant essential oils. It is not surprising to be found in the volatile fraction of Illicium species among which the most known source of essential oils is the fruit of I. verum. On the other hand, 2-

Hydroxy-2-methyl-6-methyleneoct-7-en-3-yl benzoate (3), is an ester derivative of a myrcenediol, (180) and it is reported in this study for the first time as a natural product.

Interestingly, myrcenediol was previously isolated from Bidens graveolens Mart.

(Asteraceae) (193) and from the flowers of Tanacetum annuum L. (Asteraceae). (194)

Aromatic abietane diterpenes continue to dominate the chemical profile of

I. angustisepalum. An array of abietanes reported from I. angustisepalum (15) was enriched in the present study. From fifteen isolated compounds, six are abietanes including angustanoic acid E (4), majusanic acid C (5), angustanoic acid F (7), angustanol (13) and majusanic acid B (16). Interestingly, these aromatic abietanes are structurally unique in that they all are oxygenated at axial C-19 methyl of the gem-dimethyl group rather than the equatorial C-18 position. This chemical class was identified in three Illicium species,

I. angustisepalum, I. jiadifengpi and I. majus and therefore can be interpreted as the chemical proof supporting the close taxonomic relationship between these species. In fact,

I. angustisepalum, I. jiadifengpi have been taxonomically arguable. (17) Specifically,

I. angustisepalum (195) and I. jiadifengpi (196) share same synonymic name Illicium jiadifengpi f. minwanense (B.N. Chang & S.D. Zhang) Q. Lin.

6β-hydroxy-4-stigmasten-3-one (9) is a sterol with an array of previously reported activities such as significant hypoglycemic (197), antiarrhythmic (198) and pronounced antituberculosis (199) activities. Present study reports 9 for the first time in I. angustisepalum.

The common flavonols widely occurring in the plant kingdom were also found in genus

Illicium. Literature indicates that kaempferol along with its glycosides and quercetin and its glycosides were isolated from the fruits of I. verum. (200) In this study (2R, 3R)-3,5,7,3’4’- pentahydroxyflavonone (10) was identified in the twig of I. angustisepalum.

Sesquiterpenes are very characteristic for Illicium plants. According to Figure 1-4, sesquiterpenes constitute 38% of all Illicium isolates. Most of them are produced by fruits but there are also reports of sesquiterpenes from other plant parts such as twigs or roots. (201)

Three types of sesquiterpenes including muurolane, clovane and seco-prezizaane type sesquiterpenes were also isolated from this plant. (-)-T-Muurolol (2) isolated from genus

Illicium and I. angustisepalum for the first time albeit it is also found in other plant species like for example Pinus sylvestris L. Surprisingly, this plant metabolite was also reported from marine bacterial species of Streptomyces sp. M491. (176) Clovane-2,9-diol (12) belongs to a rare clovane-type and as it is found in I. angustisepalum for the first time. Only few clovane derivatives have been found in genus Illicium. One example is a terpene-sesquineolignans,

clovanedunnianol (39) (Figure 1-6a), from I. simonsii. However clovanemagnolol, from

Magnolia obovata Thunb. (Magnoliaceae), is a combination of a sesquiterpene and neolignan, in which the sesquiterpene moiety is same as of clovane-2,9-diol (12). (202) (203)

One particular type of sesquiterpenes, seco-prezizaanes, are postulated by many authors as the chemotaxonomic markers of genus Illicium. Seco-prezizaane sesquiterpenes rarely occur in nature and can be divided into six subtypes: anisatin, cycloparvifloralone, majucin, minwanensin, pseudoanisatin and pseudomajucin. (17). From all prezizaanes reported so far, majority were isolated from Illicium plants (I. angustisepalum, I. anisatum, I. dunnianum,

I. majus, I. jiadifengpi, I. micranthum. (14) (204) (205) (52) (192) (206) (207) The existence of similar majucin-type seco-prezizaane sesquiterpenes indicate the close chemotaxonomic relationships among I. angustisepalum, I. anisatum, I. dunnianum, I. majus, I. jiadifengpi and

I. micranthum. In this study two seco-prezizaane type sesquiterpenes were isolated, majucin

(14) and angustisepalin (11). Angustisepalin is formally the 10-benzoyl ester of neomajucin.

Isolation of one additional seco-prezizaane sesquiterpene from this under-studied species, supports that seco-prezizaane sesquiterpenes could be considered as chemotaxonomic markers of Illicium.

CONCLUSIONS

100

Among the isolated compounds, a few major chemical classes can be distinguished which is a testament to the chemical diversity that is characteristic for Illicium plants. In addition, based on the observed chemical classes and patterns it can be concluded that aromatic abietanes oxygenated at axial C-19 methyl of the gem-dimethyl group and seco- prezizaane sesquiterpenes are chemical markers of Illicium. Therefore findings of the present study can be interpreted as a support to taxonomic studies.

CHAPTER 4

BIOLOGICAL EVALUATIONS

4.1 RATIONALE

Illicium plants have been used in th e folk medicine of many Asian regions. Illicium plants have been applied as analgesic, antiemetic, antiseptic, antispasmodic, antirheumatic, anxiolytic, carminative, digestive, and sedative (Table 1-1). (17)

Pharmacological studies show that extracts and chemical compounds isolated from

Illicium have acetylcholinesterase inhibiting, α-glucosidase inhibiting, anti-inflammatory, antimicrobial, antioxidant, antiviral, chemopreventive, cytotoxic, estrogenic, lipase inhibiting, neuroprotective, promoting neural cell growth and choline acetyltransferase activity. A comprehensive in vitro and in vivo pharmacological profiles of Illicium are presented in Tables

1-2 and 1-3, respectively.

Despite a number of pharmacological studies of species of Illicium, I. angustisepalum has not been subjected to biological evaluations yet. Present study successfully created the in vitro biological profile of I. angustisepalum.

4.2 CYTOTOXICITY ASSAY

According to the literature information, the extracts and compounds obtained from

Illicium plants such as I. arborescens, I. griffithii, I. simonsii and I. parvifolium subsp. oligandrum, possess cytotoxic activities (Table 1-2). The following subsections present the evaluation of the cytotoxicity of compounds 1-15 isolated from I. angustisepalum.

101

102

4.2.1 Cell cultures

In the cytotoxicity assay three tumor cell lines were used: human melanoma cancer

(MDA-MB-435), human breast cancer (MDA-MB-231) and human ovarian cancer

(OVCAR3).

4.2.2 Experimental procedures

The cytotoxicity assay was performed at the University of Illinois at Chicago (UIC),

Chicago, Illinois. As previously described in the literature, (208) the cells were purchased from the American Type Culture Collection (Manassas, VA) and then propagated at 37°C in

5% CO2 in RPMI 1640 medium, supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (100 µg/mL). Then the cells were harvested by trypsinization and then washed to get rid of the residual enzyme. A total of 5,000 cells were seeded into each well of a 96-well assay plate (Microtest 96®, Falcon) and then allowed for overnight incubation (37°C in 5% CO2). Compounds dissolved in DMSO were diluted and then added to the respective wells (at concentrations: 25 µg/mL, 5 µg/mL, 1 µg/mL, 0.2 µg/mL, 0.04

µg/mL; the total volume was: 100 µL; DMSO: 0.5%). The cells were incubated together with the test substance for 72 h at 37°C and tested for viability using the absorbance assay

(CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega Corp, Madison, WI).

IC50 values are given in µg/mL relative to the solvent (DMSO) control. Vinblastine, a drug applied in the cancer therapy was used as a positive control.

103

4.2.3 Results and discussion

The cytotoxicity of the isolates (1-15) has been evaluated against a small panel of cancer cell lines: MDA-MB-435, MDA-MB-231 and OVCAR3. The results summarized in

Table 4-1 indicate that compounds do not have any significant cytotoxic activity.

Based on the literature information, the extracts and compounds isolated from Illicium plants such as I. arborescens, I. griffithii, I. simonsii and I. parvifolium subsp. oligandrum, possess cytotoxic activities (Table 1-2). Evaluation of Illicaborin B (Figure 1-12j), from the fruit extract of I. arborescens, exhibited a moderate cytotoxic activity against four cancer cell lines: human laryngeal carcinoma (Hep-2), human medulloblastoma (Daoy), human breast adenocarcinoma (MCF-7), and human colon adenocarcinoma (WiDr). (122) On the other hand, investigation of minwanensin-type sesquiterpene, 14-O-benzoylminwanensin (Figure

1-12k) and anisatin-type sesquiterpene, (3b)-3-(acetyloxy)-14-O-benzoyl-10- deoxyfloridanolide (Figure 1-12l), from the aerial parts of I. arborescens, exhibited weak cytotoxic activity against cervical cancer (HeLa), human colon adenocarcinoma (WiDr), human medulloblastoma (Daoy) and human laryngeal carcinoma (Hep-2).(123) I. griffithii fruit extracts exhibted cytotoxic activity against human adenocarcinoma lung cancer (A549).

(44)

104

TABLE 4-1: CYTOTOXIC ACTIVITY OF SELECTED COMPOUNDS ISOLATED FROM

I. ANGUSTISEPALUM

IC50 (µg/mL)

Compound MDA-MB-435a MDA-MB-231b OVCAR3c

Thymol (1) > 25 > 25 > 25

(-)-T-Muurolol (2) > 25 > 25 > 25

2-Hydroxy-2-methyl-6-methyleneoct-7-en-3-yl > 25 > 25 > 25 benzoate (3)

Angustanoic acid E (4) > 25 > 25 > 25

Majusanic acid C (5) > 25 > 25 > 25

2,6-dimethoxyxhavicol (6) > 25 > 25 > 25

Angustanoic acid F (7) > 25 > 25 > 25

Angustanoic acid G (8) > 25 > 25 > 25

6β-hydroxy-4-stigmasten-3-one (9) > 25 > 25 > 25

(2R, 3R)-3,5,7,3’4’-Pentahydroxy flavonone (10) > 25 > 25 > 25

Angustisepalin (11) > 25 > 25 > 25

Clovane-2,9-diol (12) > 25 > 25 > 25

Angustanol (13) > 25 > 25 > 25

Majucin (14) > 25 > 25 > 25

2-Hydroxy-7-methyl-hexan-1,5-olide (15) > 25 > 25 > 25

Vinblastined 0.49 8.78 1.82 a Human melanoma cancer cells, b Human breast cancer cells, c Human ovarian cancer cells, d Positive control [IC50 (nM)]

Sesqui-neolignans, simonol A (Figure 1-12a) and simonol B (Figure 1-12b), furnished from I. simonsii, displayed moderate cytotoxic activity against human non-small cell lung carcinoma (NCI-H460), human hepatoma (SMMC-7721), breast cancer (MCF-7), and human gastric cancer (BGC-823). (79) Seco-prezizaane type sesquiterpenes, anisatin

(Figure 1-5a) and (1S)-minwanenone (Figure 1-12c), also isolated from I. simonsii showed

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cytotoxicity comparable to 5-FU, an anti-cancer drug used in skin cancer therapy, against human non-small cell lung carcinoma (NCI-H460) and human hepatoma (SMMC-7721).

(119) In another study, phenylpropanoids (Figures 1-12d and 1-12e), a simple permethoxylated phenol (Figure 1-12f), and one indole (Figure 1-12g) from the fruit extract of I. simonsii, exhibited potent ability to sensitize tumor multidrug resistant cell lines: MCF-

7/ADR and Bel7402/5-FU, to anti-neoplastic agents. (120) Also, phenylpropanes from the roots of I. parvifolium subsp. oligandrum demonstrated significant cytotoxic potential. And while (2S, 4S)-illicinone D (Figure 1-12h) exhibited significant cytotoxicity against human colorectal adenocarcinoma (HCT-8), human gastric cancer (BGC-823), human adenocarcinoma lung cancer (A549), and human ovarian carcinoma (A2780), the 4R- illicinone C (Figure 1-12i) showed moderate selective cytotoxicity against sensitive A2780.

(121)

According to the literature, (2R, 3R)-3,5,7,3’4’-pentahydroxyflavonone (10), was evaluated against four human tumor cell lines, human adenocarcinoma lung cancer (A549), human ovarian cancer (SK-OV-3), melanoma (SKMEL-2) and human colorectal adenocarcinoma (HCT-15) but showed no significant cytotoxicity, at concentrations below 30

μg/mL. (209) Flavononol was also evaluated for its in vitro growth inhibitory activity against human adenocarcinoma lung cancer (A549), non-small cell lung cancer (NSCLC), U373 glioblastoma (GBM) and prostate cancer (PC-3). (2R, 3R)-3,5,7,3’4’-Pentahydroxyflavonone

(10), was found to be selective inhibitor of the NSCLC cell line with an IC50 of 2.3 μM. (210)

Despite the previous studies showed cytotoxic potential of Illicium plants, in our hands, however, none of the isolates (1-15) displayed significant cytotoxic activity.

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4.2.4 Conclusions

Compounds isolated from I. angustisepalum were tested for cytotoxic activity against the MDA-MB-435, MDA-MB-231 and OVCAR3 cell lines. None of them showed significant cytotoxic activity.

4.3 ANTIMICROBIAL ASSAY

Illicium plants are known for their antibacterial properties. The fruits of I. verum have been applied in the folk medicine as antiseptic. In addition, extracts obtained from I. griffithii were previously reported as antibacterial against both Gram-positive and Gram-negative bacteria. These findings suggested antimicrobial potential of Illicium. The following subsections present the evaluation of antimicrobial activities of the selected compounds from

I. angustisepalum.

4.3.1 Cell cultures

In the antimicrobial assay seven bacterial strains were used: Acinetobacter calcoaceticus, Bacillus anthracis sterne, Bacillus cereus 14579, Enterococcus faecalis V583,

Escherichia coli MG1655, Escherichia coli BW25113 ∆TolC, Staphylococcus aureus USA

300 and Staphylococcus aureus MSSA 476.

4.3.2 Experimental procedures

The antibacterial assay was performed at the University of Illinois at Chicago (UIC),

Chicago, Illinois. According to the report prepared by researcher from UIC, the bacterial strains, A. calcoaceticus, B. anthracis sterne, B. cereus 14579, E. faecalis V583, E. coli

MG1655, E. coli BW25113 ∆TolC, S. aureus USA 300, S. aureus MSSA 476, were grown

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overnight (~16 hours) at 37°C. The next day, strains were diluted 100-fold and grown to OD

600 = 0.4-0.6. Then, the OD600 was adjusted to 0.001. Then, 100 µL of the culture was added to an entire row (12 wells) of a 96-well plate. In the 12th well, 200 µL was added instead of 100 µL. The compound was added to the 12th well to a final concentration of 200

µg/mL. This was serially-diluted 2-fold into the remaining 11 wells (but leaving the first well with no compound as the control). As a result only wells 2-12 contain the compound in different concentrations. This was then incubated overnight at 37° C. The next day, the MIC for each tested compound was determined.

4.3.3 Results and discussion

Genus Illicium is known for its antibacterial activity. Many in vitro reports showed that extracts and isolated compounds possess antibacterial activities. For example, I. griffithii extracts were found to be active against Gram-positive bacteria such as Bacillus subtilis,

S. aureus, Yersinia enterocolitica, and Gram-negative bacteria such as E. coli, Klebsiella pneumonia, P. aeruginosa, P. vulgaris, Salmonella paratyphi B, Vibrio parahaemolyticus,

Y. enterocolitica and Xanthomonas oryzae. (111) Also phenylpropanes, simonin A (Figure 1-

8a), dunnianol (Figure 1-8b), macranthol (Figure 1-8c), isodunnianol (Figure 1-6e) and manolol (Figure 1-8d) isolated from I. simonsii were active against oral bacteria Gram- positive Actinomyces viscosus, Streptococcus mutans, Streptococcus sanguis and

Actinomyces naeslundii with MIC values ranging from 1.95 to 31.25 µg/mL. (112)

Fractions and aromatic abietanes isolated from I. angustisepalum were tested against clinical isolates of antibiotic resistant strains such as Gram-positive, S. aureus USA 300, S. aureus MSSA 476, and Gram-negative, E. coli MG1655, E. coli BW25113 ∆TolC, bacteria.

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Angustanoic acid was also tested against additional Gram-positive strains including Bacillus anthracis sterne, Bacillus cereus 14579 and Enterococcus faecalis V583 and Gram-negative

Acinetobacter calcoaceticus.

Fractions obtained from I. angustisepalum did not show antibacterial activity against tested microbial strains (Table 4-2). Among all tested compounds, angustanoic acid E was the most active with MICs of 6.25, 12.5 and 12.5 µg/mL against E. coli BW25113 ∆TolC, S. aureus USA 300, S. aureus MSSA 476, respectively. Majusanic acid C and angustanoic acid

G had MIC of 25 µg/mL and angustanoic acid F had 50 µg/mL against strain E. coli BW25113

∆TolC (Table 4-3). For the comparison, a well-known antibiotic, chloramphenicol has a MIC of 1 and 8 µg/mL against E. coli BW25113 ∆TolC and S. aureus USA 300, respectively. (211)

(212) Also, vancomycin has a MIC of less than 2 µg/mL against S. aureus MSSA 476. (213)

TABLE 4-2: ANTIMICROBIAL ACTIVITY OF FRACTIONS RECEIVED FROM

I. ANGUSTISEPALUM

MIC (µg/mL)

Fraction BW25113 ∆ToICa MSSA476b

Pe 200 NA

Ea NA NA

Bu NA NA

Wa NA 200 a E. coli BW25113 ∆TolC, b S. aureus MSSA 476. NA: not active.

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TABLE 4-3: ANTIMICROBIAL ACTIVITY OF SELECTED COMPOUNDS FROM I. ANGUSTISEPALUM

MIC (µg/mL)

Compound BW25113 ∆TolCa MG1655b USA 300c MSSA 476d

Majusanic acid B (16) >200 >200 >200 >200

Majusanic acid C (5) 25 >200 >200 >200

Angustanoic acid E (4) 6.25 >200 12.5 12.5

Angustanoic acid F (7) 50 >200 >200 >200

Angustanoic acid G (8) 25 >200 200 200

Angustanol (13) >200 >200 >200 >200 a E. coli BW25113 ∆TolC, b E. coli MG1655, c S. aureus USA 300, d S. aureus MSSA.

Angustanoic acid E showed also good activity against Bacillus anthracis sterne and

Bacillus cereus 14579 with MICs of 6.25 µg/mL (Table 4-4). However, it was not active against Acinetobacter calcoaceticus. For the comparison, ofloxacin has MIC of 0.8 µg/mL against B. anthracis sterne (214) and doxycycline has MIC of 0.37 µg/mL against B. cereus

14579. (215)

TABLE 4-4: ANTIMICROBIAL ACTIVITY OF ANGUSTANOIC ACID E (4)

MIC (µg/mL)

B. anthracis sterne B. cereus 14579 E. faecalis V583 A. calcoaceticus

6.25 6.25 200 >200

While angustanoic acid E (4) was active against both Gram-positive and Gram- negative bacteria, majusanic acid C (5), angustanoic acids G (8) and F (7) were active

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against Gram-negative bacteria. These differences in activity spectrum might be associated with differences in compounds structures, specifically different substituents at C-15, of the abietane diterpene skeleton characteristic for all active compounds.

Interestingly, abietane diterpenes have been previously recognized for their potent antibacterial activities. Totarol, isolated from Podocarpus nagi (216) (Podocarpaceae) (217), is an example of a potent antibacterial abietane diterpene which was found to be active against Gram-positive bacteria such as Propionibacterium acnes, Streptococcus mutans,

Bacillus subtilis, Brevibacterium ammoniagenes and Staphylococcus aureus with MICs of

0.39, 0.78, 0.78, and 1.56 µg/mL, respectively. (218)

4.3.4 Conclusion

Abietane diterpenes, angustanoic acid E (4), F (7) and G (8) and majusanic acid C

(5), isolated from I. angustisepalum exhibited antibacterial activities against Gram-positive and Gram-negative bacteria. I. angustisepalum can serve as a potential source of antibacterial compounds.

4.4 NEURAL CELL PROTECTION ASSAY

Previous literature reports indicated the neuroprotective potential of various compounds isolated from Illicium, including macranthol (Figure 1-8c) isolated from I. simonsii and also aniselactone B (Figure 1-14a) and erythro form of anethole glycol (Figure 1-14b), isolated from I. parvifolium subsp. oligandrum. The following subsections discuss evaluation of the neural cell protection of extracts and compounds derived from I. angustisepalum.

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4.4.1 Cell cultures

In the neural cell protection assay two types of cells were used: pheochromocytoma

(PC12) and primary rat cortical neurons.

4.4.2 Experimental procedures

PC12 cells were received from the American Type Culture Collection (Rockville, MD,

USA) and seeded onto 96-well culture plate at 2×104 cells/well, according to published procedures. (219) The cells were incubated at 37°C for 48 hours. Then the cells were cultured in serum-free media and incubated with respective drugs for another 24 hours. The cell survival was determined with CellTiter 96® AQueous One Solution cell proliferation kit

(Promega, Madison, WI, USA), based on the cleavage of MTS into a formazan. In brief, the media was removed at the end of assay. Then the cells were washed with D-Hanks and suspended in 100 μL of serum-free media. Subsequently, 20 μL of CellTiter 96® AQueous

One Solution was added into each well of the 96-well plate and incubated for 1 hour in a humid atmosphere (95% air with 5% CO2) at 37 ºC. The absorbance was measured at 490 nm. The cell survival was expressed as a percentage of the non-treated control.

Primary hippocampal neurons were prepared from E18 Sprague–Dawley (SD) rat embryos and cultured on 96-well plates. The dissociated neurons were fed with Neurobasal medium (Life Technologies) supplemented with 2% B27 (Life Technologies). The seeding densities were 1 X 105 cells/well. On the third day in vitro, MPP+ alone, compound alone, or

MPP+ together with different compounds were added to the neurons. After 24h neuronal cells viability was determined in MTT assay. (220) In brief, MTT (Sigma–Aldrich; 5 mg/ml in DPBS) was added to each well for 4 h to be converted into a purple formazan dye by viable cells.

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DMSO was added to dissolve the formazan, and the absorbance of the solution was detected at 595 nm by DTX880 multimode detector (Beckman Coulter, Brea, CA, USA). Each compound, at each concentration was tested in triplicate. Cell viability was expressed as a percentage of non-treated control. Brain derived neurotrophic factor (BDNF) and antioxidant protein (AOP-6A) were used as positive controls.

4.4.3 Results and discussion

Fractions (Pe, Ea, Bu, Wa) and compounds (1-15) from I. angustisepalum were evaluated for neuroprotective activity using two types of cells, pheochromocytoma (PC12) and primary cortical neurons. In the first step it was determined if fractions and compounds have an effect on the basal cells viability (cytotoxicity assay). In the second step it was determined if fractions and compounds have protective effect against cells damage induced

+ by toxic agents such as hydrogen peroxide (H2O2) and 1-methyl-4-phenylpyridinium (MPP ).

The cytotoxic (Figure 4-1) and protective (Figure 4-2) effects of the four main fractions were evaluated in MTT assay with PC12 cells. The results show that fractions are not toxic within the concentration range of less than 50 µg/mL and that fractions do not show protective effects against H2O2 induced damage in PC12 cells, at the doses of 1-10 µg/mL.

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140

120

100 1μg/mL 80 10μg/mL

60 50μg/mL Cell Cell Survival(%) 40 100μg/mL

0 DMSO Pe Ea Bu Wa

FIGURE 4-1: The cytotoxic effect of fractions from I. angustisepalum on PC12 cells

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100

1μg/mL 60 5μg/mL 10μg/mL Cell Cell Survival(%) 40

0 DMSO H2O2 H2O2+Pe H2O2+Ea H2O2+Bu H2O2+Wa

FIGURE 4-2: The protective effects of fractions from I. angustisepalum against H202 induced oxidative damage in PC12 cells.

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The cytotoxic (Figure 4-3) and protective (Figure 4-4) effects of selected compounds isolated from I. angustisepalum were determined in MTT assay with primary culture of cortical neurons. Based on the cytotoxic data, compounds are not cytotoxic, at the tested range of concentrations 0.4–10 µg/mL. Angustisepalin (11) and angustanoic acid F (7) increased basal cell viability, at similar level as BDNF which served as a positive control.

The protective effect against MPP+ was observed for angustanoic acids E (4) and G

(8), 2,6-dimethoxychavicol (6), majusanic acid C (5) and (-)-T-muurolol (2). While compounds

5 and 4 showed similar protective activity to BDNF at 2 µg/mL, compound 6 was slightly weaker. The most significant protection from the MPP+ damage was observed in compound

8 at 0.4 µg/mL and in compound 2 at 10 µg/mL which was close to the protective effect of

AOP-6A, which served as second positive control.

160 140 120 100 80 0.4 μg/mL 60 2 μg/mL

Cell Cell Survival(%) 40 20 10 μg/mL 0

FIGURE 4-3: The cytotoxic effects of compounds isolated from I. angustisepalum on the primary cortical neurons

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120

100

60 0.4ug/mL 40

2ug/mL Cell Cell Survival(%) 20 10ug/mL 0

FIGURE 4-4: The protective effects of compounds isolated from I. angustisepalum against MPP+ induced oxidative damage in the primary cortical neurons

4.4.4 Conclusions

In the search for neuroprotective agents, fractions of petroleum ether (Pe), ethyl acetate (Ea), butanol (Bu) and water (Wa) and isolates (1-15) were tested. The two step analysis determined if the fractions and compounds have an effect on the basal cell viability

(cytotoxicity assay) and if the fractions and compounds have protective effect against cells damage induced by toxic agents such as hydrogen peroxide (H2O2) and 1-methyl-4- phenylpyridinium (MPP+).

The results show that fractions are not toxic within the concentration range of less than 50 µg/mL and that fractions do not show protective effects against H2O2 induced damage in PC12 cells, at the doses of 1-10 µg/mL. It was also found that compounds are not cytotoxic, at the tested range of concentrations 0.4–10 µg/mL. Moreover, angustisepalin (11) and angustanoic acid F (7) increased basal cell viability, at similar level as BDNF which served as a positive control. The protective effect against MPP+ was observed for angustanoic acids E (4) and G (8), 2,6-dimethoxychavicol (6), majusanic acid C (5) and (-)-

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T-muurolol (2). While compounds 5 and 4 showed similar protective activity to BDNF at 2

µg/mL, compound 6 protected cells slightly weaker. The most significant protection from the

MPP+ damage was exerted by compound 8 at 0.4 µg/mL and by compound 2 at 10 µg/mL which was close to protective effect of AOP-6A which served as second positive control.

These findings indicate that angustanoic acids E (4) and G (8), majusanic acid C (5) and (-)-T-muurolol (2) have a neuroprotective potential.

In the previous studies, thymol was shown to have in vitro acetylcholinesterase inhibitor activity and also neuroprotective effects. (221) (222) (223) Further findings showed that thymol has antidepressant and neuroprotective (through attenuation of amyloid β or scopolamine induced cognitive impairment in rats) activities in vivo. (223) (224)

Compounds with the exception of thymol were evaluated for neuroprotective activity for the first time.

4.5 ACETYLCHOLINESTERASE INHIBITION ASSAYS

Acetylcholinesterase (AChE) inhibitors have been previously reported from I. simonsii and I. verum. Based on these findings, the evaluations of I. angustisepalum for the AChE were undertaken.

4.5.1 TLC-bioautographic assay

TLC-bioautography assay is a suitable tool for the simultaneous separation of extracts and detection of active principles. The following subsections present application of TLC- bioautography for identification of acetylcholinesterase inhibitors in extracts and fractions of

I. angustisepalum.

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4.5.2 Experimental procedures

The assay was carried out according to the modified Marston’s method (225) (226).

Fractions (Eo, Pe, Ea, Bu, Wa) and compounds (1-14) were prepared for TLC at concentration of 1 mg/mL and 5 μL of each sample was spotted on the TLC plate. Plates were tested with and without migration. AChE (500 U) was dissolved in 75 mL of 0.05 M

Tris–hydrochloric acid buffer at pH 7.8; bovine serum albumin (75 mg) was added to the solution in order to stabilize the enzyme during the bioassay. Final concentration of enzyme stock solution was (6.7 U/mL). The stock solution was kept at 4°C. 1-naphtyl acetate (75 mg) was dissolved in ethanol (20 mL) and diluted with 30 mL of water. The plate was then sprayed with enzyme stock solution and dried with cool air, to avoid deactivation of the AChE.

Afterwards, the plate was sprayed with solution of 1-naphtyl acetate, and dried with cold air.

Subsequently, the enzyme was subjected to incubation process. In brief, the plate was placed inside a plastic tank containing a small amount of water, but the water was not in contact with the TLC plate. Then the tank was covered and incubation was carried out at

37°C for 20 min. The enzyme was stable under these conditions.

The enzyme was detected with solution consisting of Fast Blue B salt (25 mg) and distilled water (50 mL). After incubation was completed, Fast Blue B salt solution was sprayed onto the plate to produce a purple coloration. AChE inhibitors produced white spots on the purple background, according to chemical reaction presented in Figure 4-5.

Physostigmine positive control solutions were prepared in concentrations: 0.1 µg,

0.01 µg, 0.001 µg µg, 0.0001 µg and 0.00001 µg and controls were run in chloroform- methanol-water (85:15:1) as depicted on Figure 4-6.

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FIGURE 4-5: Reaction of enzyme AChE with 1-naphtyl acetate and formation of the purple dye in the TLC-bioautographic assay (225)

4.5.3 Results

Physostigmine, an alkaloid of Physostigma venenosum Balf. (Leguminosae), is a reversible inhibitor of the acetylcholinesterase that prevents breakdown of acetylcholine and therefore it is applied as a cholinergic agent. (227) To determine the limits of detection for the

TLC bioassay, the physostigmine solutions were prepared and spotted on the TLC plate.

This way the lowest detectable amount of active compound was determined. The lowest amount of physostigmine that inhibited the enzyme was observed at 10-4 µg (Figure 4-6) which was comparable to published values. (225) (226)

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FIGURE 4-6: Determination of detection limits. Lanes 1-6: physostigmine, 10-1µg, 10-2µg, -3 -4 -5 -6 10 µg, 10 µg, 10 µg and 10 µg. Samples were applied to a silica gel G60 F254 plate, the TLC plate was eluted in chloroform–methanol–water (85:15:1 v/v/v) and the TLC-bioautography was carried out.

In the preliminary TLC bioautographic screening of fractions from I. angustisepalum, the volatile fraction (Eo) and ethyl acetate fraction were found to be active. Bioautographic analysis of the Eo led to identification of one active principle with Rf of 0.9 (Figure 4-7) which by means of GC-MS analysis and TLC co-chromatography was identified as thymol (1).

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FIGURE 4-7: Analysis of essential oil. Lane 1: volatile fraction (Eo), 10 µL; Lane 2: -2 physostigmine, 10 µg. Samples were applied to silica gel G60 F254 plate and the TLC plate was eluted in petroleum ether–ethyl acetate (6:4 v/v).

A. Chromatogram-Detection: 1mL 50% ethanolic H2SO4 + 10 mL 2% methanolic p- benzaldehyde, then heating.

B. Bioautograph-Detection: AChE + 1-naphtyl acetate, incubation in 37°C, then Fast Blue B Salt.

The sixteen combined subfractions generated from the separation of ethyl acetate fraction were subjected to TLC bioautography as shown on Figure 4-8.

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FIGURE 4-8: Identification of active subfractions. Lanes 1-16: Ea subfractions, 5 µL; Lane 17: Ea, 5µL; Lanes 18 and 19: Physostigmine 10-2 µg and 10-3 µg. Samples were applied to a silica gel G60 F254 plate and the TLC plate was eluted by petroleum ether–ethyl acetate (6:4 v/v).

A. Chromatogram- Detection: 1mL 50% ethanolic H2SO4+10mL 2% methanolic p- benzaldehyde, then heating.

B. Bioautograph- Detection: AChE+1-naphtyl acetate, incubation in 37°C, then Fast Blue B Salt.

As described earlier, the ethyl acetate fraction afforded sixteen subfractions, out of which three were active in the TLC bioautography, including 9–10, 22 and 23-27.

Subsequently, subfractions were phytochemically explored which resulted in the isolation of

3,5-dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin

(11), clovane-2,9-diol (12), angustanol (13), majucin (14), 2-Hydroxy-7-methyl-hexan-1,5- olide (15) and majusanic acid B (16).

All compounds were tested for acetylcholinesterase inhibition using TLC bioautography. TLC bioautographic detection identified six active principles including 3,5-

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dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-Hydroxy-7-methyl-hexan-1,5-olide (15).

4.5.4 Discussion

The thin-layer chromatography (TLC) is an effective method for analyzing complex matrices such as plant extracts. When TLC is used in combination with the biological detection, it is called TLC bioautography, and has been established in 1946. (228) Many authors have applied this technique in their phytochemical studies. (229) (230) (225)

Bioautography carried out on thin-layer chromatographic (TLC) plate allows for the detection of the biologically active compounds directly on the TLC plate. Sample can be separated in a suitable solvent system or just applied on TLC and tested without migration. (231) It requires a small amount of the analytical sample and also it is perfect for the evaluations of complex plant extracts. Another advantage of this method is that, in contrast to HPLC, a few samples can be tested simultaneously on TLC. Organic solvents used for developing the plate, which cause inactivation of enzymes and hinder biological detection, can be completely evaporated before the enzymatic reaction.

Alzheimer’s disease (AD) is the most common type of dementia and a leading neurodegenerative disorder. According to the cholinergic hypothesis, memory impairment in

AD patients results from the dysfunctions in the central neurotransmission of acetylcholine.

Therefore inhibitors of acetylcholinesterase (AChE) currently form the basis of pharmacotherapy of AD. In the course of a search for natural AChE inhibitors,

I. angustisepalum was found to be active by using a bioautographic assay. Since former studies have identified the Illicium genus to be a potential source of AChE inhibitors, this

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under-studied member of Illicium was further investigated. (43) (40) In the present study, a volatile fraction and four major fractions (i.e., ethyl acetate, butanol, petroleum ether and water extracts) of I. angustisepalum were tested for AChE-inhibiting activity, and positive results were observed in the volatile fraction and in the ethyl acetate fraction. Bioautographic detection led to identification of six active principles including thymol (1), 3,5- dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-Hydroxy-7-methyl-hexan-1,5-olide (15).

According to literature, thymol was shown to have good in vitro acetylcholinesterase inhibitor activity. (221) (222) On the other hand, 3,5-dimethoxychavicol (6), (2R, 3R)-

3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2- hydroxy-7-methyl-hexan-1,5-olide (15) have never been evaluated for the acetylcholinesterase inhibition. The present study shows the acetylcholinesterase inhibiting potential of these compounds, for the first time. Interestingly, sesquiterpene-sesquineolignan, clovanedunnianol (Figure 1-6a) moderately inhibited acetylcholinesterase with IC50 value of

4.58 µM. (39)

4.5.5 Conclusion

In the course of search for natural acetylcholinesterase inhibitors I. angustisepalum was investigated using TLC assay based on enzymatic reaction. Six active principles were identified in essential oil and ethyl acetate extract from I. angustisepalum. Bioautographic detection led to identification of six active principles including thymol (1), 3,5- dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-hydroxy-7-methyl-hexan-1,5-olide (15). It is the first time that

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AChE inhibitors are reported from I. angustisepalum. These findings suggest that genus

Illicium is a promising source for natural AChE-inhibitors.

4.5.6 Ellman’s assay

Ellman’s assay, based on the spectrophotometric detection, was applied for determination of the IC50 values of six compounds identified as active in the TLC- bioautography. The following subsections explain the experimental procedures as well as results of the Ellman’s.

4.5.7 Experimental procedures

The microplate assay was carried out according to the modified Ellman’s method.

(229) In a 96‐well 25 μL of 7.5 mM ACTI (in buffer B: 50 mM Tris–HCl, pH 7.8 containing 0.1

M NaCl and 0.02 M MgCl2.6H2O), 125 μL of 1.5 mM DTNB (in buffer B), 50 μL of buffer A

(50 mM Tris–HCl, pH 7.8 containing 0.1% BSA) and 25 μL selected compounds (at concentrations: 1 mg/mL to 7.8 μg/mL made in 10% DMSO, in buffer A) were mixed and the absorbance was measured using a microplate spectrophotometer at 405 nm every 15 seconds for 75 seconds. Then 25 μL of AChE (0.11 U/mL in buffer A) was added and the plate was incubated at 25 °C for 10 min. The final assay volume was 250 µL. Absorbance was measured again every 15 seconds for 120 seconds. Assay was performed in triplicate..

10% DMSO solutions in buffer A were used as a negative control (blank). Physostigmine solutions at final concentrations (15–0.11 μg/mL made in 10% DMSO, in buffer A) served as a positive control. The final concentration of DMSO in each well was 1%. AChE inhibitors prevented production of thiocholine and also yellow ion, according to chemical reaction presented in Figure 4-9.

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Enzyme inhibition was expressed as IC50 which was calculated by curve fitting according to classical sigmoidal dose-response equation (variable slope) obtained by plotting percentage of inhibition versus concentrations (eight concentrations were used). The percent of inhibition was calculated relative to a blank (due to absence of inhibitors is considered as

100% enzyme activity).

FIGURE 4-9: Detection of AChE inhibition with the Ellman’s reagent (225)

4.5.8 Results

The TLC bioautographic detection led to identification of six active principles including thymol (1), 3,5-dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-Hydroxy-7-methyl-hexan-1,5-olide (15). As the TLC bioautography is a semi-quantitative method the enzyme based spectrophotometric assay was used to supplement the results of the TLC bioautography assay. IC50 values of pure compounds, found to be active in TLC based assay, are summarized in Table 4-5.

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TABLE 4-5: AChE INHIBITION OF THE ACTIVE COMPOUNDS FROM I. ANGUSTISEPALUM

Compound IC50 (µg/mL)

Thymol (1) 13.47

2,6-dimethoxyxhavicol (6) 28.86

(2R, 3R)-3,5,7,3’4’-Pentahydroxyflavonone (10) 16.10

Angustisepalin (11) 14.49

Clovane-2,9-diol (12) 11.02

Majucin (14) 45.72

2-Hydroxy-7-methyl-hexan-1,5-olide(15) 19.41

Positive control IC50 (µg/mL)

Physostigmine 0.09649 a The values shown are the means obtained from three independent experiments

4.5.9 Discussion

According to the literature acetylcholinesterase inhibiting activity was so far identified with I. simonsii and I. verum. The acetylcholinesterase inhibiting activity of the ethanol extract from the aerial parts of I. simonsii was tested in vitro using Ellman’s assay. Isolated terpene- sesquineolignans, clovanedunnianol (Figure 1-6a), p-menthadunnianol (Figure 1-6b), mixture of sesquineolignans simonsol E (Figure 1-6c) and simonsin A (Figure 1-6d), and phenylpropane isodunnianol (Figure 1-6e) moderately inhibited acetylcholinesterase with

IC50 values of 4.58 µM, 6.55 µM, and 10.34 µM, 13.0 µM respectively. (39) (40) In another study, the standardized fruit extract of I. verum was tested in vitro using TLC-bioautography and Ellman’s assay. In the TLC-bioautography the extract showed weak AChE inhibiting activity at 10mg/mL, and the IC50 was 58.67 μg/mL. (41) Also, anethole derived from an essential oil of I. verum has been reported to inhibit AChE at 39.89 μg/mL.

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Compounds isolated in this study from I. angustisepalum, thymol (1), 3,5- dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-Hydroxy-7-methyl-hexan-1,5-olide (15), showed moderate AChE inhibiting activity. Out of six active compounds only thymol was previously evaluated for

AChE inhibition. In one study, thymol showed weak AChE activity at EC50 of 0.74 mg/mL.

(221) Up to date a number of monoterpene alcohols, including carvacrol, thymol and anethole were found to have good AChE inhibiting activity, which suggest that this particular class of compounds, including thymol and similar compounds might find potential application as

AChE inhibitors. (221)

4.5.10 Conclusion

In the course of search for natural acetylcholinesterase inhibitors, thymol (1), 3,5- dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-Hydroxy-7-methyl-hexan-1,5-olide (15), were identified.

IC50 were established using modified Ellman’s method. Compounds moderately inhibited AChE, and clovane-2,9-diol (12) and thymol (1) showed the best activity among tested compounds, with IC50 of 11.02 and 13.47 µg/mL, respectively.

4.6 PROMOTION OF NEURAL GROWTH ASSAY

Promotion of neural growth activity has been widely described in Illicium plants.

Activity was found in sesquiterpenes and phenylpropanes in several Illicium species such as

I. lanceolatum, I. jiadifengpi, I. anisatum, I. simonsii and I. merrillianum. The following subsections describe experimental procedures, results obtained and discussion of results compared to the literature information.

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4.6.1 Isolation and culture of neurons

Primary cortical neurons were prepared from E18 Sprague Dawley (SD) rat embryos and cultured on poly-L-lysine (0.1 mg/mL; Sigma-Aldrich) coated 96-well plates. (220)

Dissociated neurons were fed with Neurobasal medium (Life Technologies) supplemented with 2% B27 (Life Technologies). The seeding densities were 0.25 × 105 per well.

4.6.2 Experimental procedures

The neurite outgrowth induced by the compounds isolated from I. angustisepalum was analyzed using the mean length of the neurite measured under a microscope as previously described in the literature.(89) Briefly, isolated cortical neurons were treated with various concentrations of test samples (1, 5 or 20 μg/mL). After 24h incubation, neurons were fixed with 4% paraformaldehyde in PBS for 20 min, and then permeated with 0.1% Triton X-

100 in PBS for 20 min to block activity of the endogenous peroxidase with freshly prepared

0.3% H2O2 for 20 min. For anti-MAP2 staining, the neurons were incubated overnight at 4 °C with primary antibody [anti-MAP2 (1:1000)] and then treated with horse peroxidase- conjugated second antibody [Simple Stain PO (1:2)] for 1h. Subsequently, the peroxidase was developed with 200 μL substrate Simple Stain DAB solution. The neurite outgrowth induced by compounds was evaluated under microscope as an average of neurite length.

These neural cells that made network-formation of less than three cells were stained with anti-MAP2 and selected for further measurements. The length of the longest axon extended from the body of the cell was measured and calculated by high content technology using

ImageXpress Micro XLS Widefield High-Content System (Molecular Devices, USA). Brain derived neurotrophic factor (BDNF) was used in this study as positive control at 500 nM.

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4.6.3 Results and discussion

Primary cortical neurons were treated with compounds (1-15) isolated from

I. angustisepalum for 24h, and the length of all neurites of each differentiated cell was analyzed by high content image acquisition and analysis. BDNF, which is classified as the member of the nerve growth factor (NGF) family, was used as a positive control. BDNF is released from the cortical neurons, and also it is essential for the survival of striatal neuron cells in the brain. Noteworthy, BDNF expression was found to be reduced in patients suffering from the Alzheimer's and Huntington disease. (196)

It was observed that at 20 μg/mL three [thymol (1), majucin (14) and majusanic acid

C (5)] out of fifteen tested compounds increased neurite length more than BDNF. Angustanol

(13) was slightly less effective than BDNF. At doses of 5 μg/mL and 1 μg/mL, angustanol

(13), clovane-2,9-diol (12) and thymol (1) showed moderate outgrowth promotion but all were less effective than BDNF. Figure 4-10 presents outgrowth effects of tested compounds.

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100 m)

μ 80 60 40 1µg/mL

Length ( Length 20 5µg/mL 0 20µg/mL

FIGURE 4-10: Neurite outgrowth promoting effects of selected compounds from I. angustisepalum on the primary cortical neurons

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Adult neurogenesis, also called a birth of neurons, plays a key role in cognitive functions and in the control of mood. Studies show that it is an important therapeutic target in neurodegenerative and mental disorders.

In the present study fourteen compounds (1-15) isolated from I. angustisepalum extract were tested for their potential in promoting differentiation and neurite outgrowth in hippocampal neurons. After 24h of incubation, thymol (1), majucin (14) and majusanic acid

C (5), at dose of 20 μg/mL caused an increase in neurite length (thymol>majucin>majusanic acid C>BDNF>angustanol) greater than that of BDNF. It was also found that angustanol (13) was slightly less effective than BDNF. At doses of 5 μg/mL and 1 μg/mL, angustanol (13), clovane-2,9-diol (12) and thymol (1) showed some neurite outgrowth promotion but all were less effective than BDNF.

According to the literature, thymol (1) was shown to have in vitro acetylcholinesterase inhibitor activity and also neuroprotective effects. (221) (223) Further findings showed that thymol has antidepressant and neuroprotective (attenuation of amyloid β or scopolamine induced cognitive impairment in rats) activities in vivo. (223) (224) Despite of number of studies and actives attributed to thymol, the monoterpene was not evaluated for neurite outgrowth promoting activity until this study. In addition, as a small molecule thymol is capable of crossing blood-brain-barrier (BBB) which is a filtering mechanism that protects neural tissues from toxins. The effectiveness of drugs that target disorders of the central nervous system highly depend on their ability to cross BBB.

According to previous reports, seco-prezizaane-type sesquiterpenes isolated from

Illicium have neurotrophic potential. Examples of isolates with neurotrophic activity include

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(2R)-hydroxy-norneomajucin (Figure 1-15c), jiadifenolide (Figure 1-15e), jiadifenoxolane A

(Figure 1-15f), jiadifenin (Figure 1-15i) and (2S)-hydroxy-3,4-dehydroneomajucin (Figure 1-

15j), which exhibited a significant neurotrophic activity promoting neurite outgrowth in the primary cultures of fetal rat cortical neurons at concentrations ranging from 1 to 10 μM/L, 0.01 to 10 μM and 0.1 to 10 μM, respectively. (126) (127) (94) Interestingly, all of these neurotrophic compounds were isolated from I. jiadifengpi. Majucin (14), is the first majucin- type seco-prezizaane sesquiterpene which was isolated from I. majus. Numerous studies focused on exploration of Illicium plants in the search for neurotrophic compounds. Most of the discovered neurotrophic compounds are seco-prezizaane sesquiterpenes. In consideration of previous reports, majucin-type sesquiterpene scaffold is be responsible for promoting neurite outgrowth in the primary cultures of fetal rat cortical neurons. (94) (127)

(126) (232) Interestingly, the majucin-type scaffold responsible for the neurotrophic activity, is characterized with a complex cage-like architecture including six to eight chiral centers and two quaternary carbons.

Literature search found no previous reports of neurotrophic evaluation of majucin (14).

Angustisepalin (11), which is neomajucin-type sesquiterpene, had no significant activity on neurite outgrowth, as expected.

Based on the literature information, majusanic acid C (5), angustanol (13) and clovane-2,9-diol (12) have never been evaluated for the neurotrophic potential. The present study shows the neurotrophic potential of these terpene compounds, for the first time.

Interestingly, clovanemagnolol (Figure 4-11), isolated from the bark of Magnolia obovata

Thunb. (Magnoliaceae), promoted neurite growth at 0.1 µM and ChAT induction at 1 µM.

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(202) (203) Chemically clovanemagnolol is a combination of a sesquiterpene and neolignan, in which sesquiterpene moiety is same as that of clovane-2,9-diol (12).

FIGURE 4-11: Clovanemagnolol with distinguished clovane-2,9-diol moiety (marked in green) (202) (203)

4.6.4 Conclusion

In the pursuit of non-peptide neurotrophic compounds in plants, compounds isolated from I. angustisepalum were tested for neurotrophic activity, by promoting neurite outgrowth in the primary cultures of fetal rat cortical neurons.

From fourteen tested compounds, three terpene compounds, thymol (1), majucin (14) and majusanic acid C (5), caused an increase of neurite length greater than that of BDNF. It was also found that compound angustanol (13) was slightly less effective than BDNF. At the dose of 20 μg/mL compounds showed neurotrophic activity in the following order, thymol>majucin>majusanic acid C>BDNF>angustanol>BDNF>angustanol. At doses of 5

μg/mL and 1 μg/mL, compounds angustanol, clovane-2,9-diol and thymol showed some neurite outgrowth promotion but all were less effective than BDNF.

These findings indicate that thymol has a potential as a drug lead for in the therapy of neurodegenerative disorders. In addition to that, these findings support the hypothesis that the rare seco-prezizaane-type sesquiterpenoids found in the Illicium plants produce

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compounds with potential as leads in development of the non-peptide neurotrophic agents useful in the treatment of neurodegenerative disorders such as Alzheimer’s disease.

The abietane-type compounds, majusanic acid C (5) and angustanol (13) and sesquiterpene cloavane-2,9-diol (12) were evaluated for neurotrophic activity for the first time.

CHAPTER 5

CONCLUSIONS

5.1 GENERAL CONCLUSIONS

According to the modern definition, the pharmacognosy explores naturally occurring substances for their medicinal, ecological, or other functional qualities. It embraces a wide range of disciplines including organic, medicinal and analytical chemistry, ethnobotany, taxonomy and many more. As I. angustisepalum is an understudied species of Illicium, its further pharmacognostic analysis was carried out. In addition, I. angustisepalum was subjected to series of in vitro assays.

I. angustisepalum is a sparingly studied plant species, whose classification and identification as a member of the genus Illicium has been based on the morphological characteristics of its flowers, fruits, leaves, pollen and seeds. (150) In order to substantiate the chemical characteristics of this plant species, the metabolite fingerprint by GC-MS was established and compared with those of better-known species, I. verum and I. lanceolatum.

The existing variations of the volatiles within this taxon were also identified. The present study has successfully described the variation of the volatile secondary metabolites between

I. angustisepalum, I. lanceolatum and I. verum using metabolite fingerprinting. The volatile compounds shared by the analyzed Illicium species, (E)-2-hexenal (organic aldehyde), a- cadinol (sesquiterpene), a-muurolol (sesquiterpene), cis-carveol (monoterpene), cubenol

(sesquiterpene), n-hexanol (organic alcohol) and β-caryophyllene (sesquiterpene), can be considered as chemotaxonomic markers of Illicium. Terpenes, including mono- and sesquiterpenes may have the potential to produce new insights into the historical debate

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concerning systematic position of genus Illicium and its incorporation into Schisandraceae family.

In order to obtain an insight of the chemical composition of the plant species and to search for novel and/or biologically active metabolites, the twig part of I. angustisepalum was explored. The structures of isolated secondary metabolites were elucidated using a combination of spectroscopic techniques, such as 1D and 2D NMR and HRESIMS, and also by comparisons with the published data. The phytochemical analysis led to isolation of a total of 16 compounds. 2-Hydroxy-2-methyl-6-methyleneoct-7-en-3-yl benzoate (3) and 2- hydroxy-2-methyl-hexan-1,5-olide (15) were isolated as natural products for the first time.

Compounds 1, 2, 5, 9, 10, 12, 14 and 16 were identified in I. angustisepalum for the first time.

Compounds 4, 6, 7, 8, 11 and 13 were reported from I. angustisepalum previously. Among all isolated compounds few major chemical classes were distinguished which is a testament to chemical diversity characteristic for Illicium plants.

Despite of a number of pharmacological studies of Illicium plants, I. angustisepalum has not been subjected to biological evaluations yet. The present study successfully created the first in vitro biological profile of I. angustisepalum. The fractions and extracts from

I. angustisepalum were evaluated for their biological potentials in a battery of in vitro bioassay systems for cytotoxic, antimicrobial, neuroprotection, acetylcholinesterase inhibiting and promotion of neural growth activities. Compounds (1-15) were tested for cytotoxic activity against MDA-MB-435, MDA-MB-231 and OVCAR3 but none of the isolated showed significant cytotoxic activity. In the anti-microbial assay, abietane diterpenes, angustanoic acid E (4), F (7) and G (8) and majusanic acid C (5), isolated from I. angustisepalum had showed good antibacterial activities against Gram-positive and Gram-negative bacteria

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which suggests that I. angustisepalum is a potential source of antibacterial compounds. In the search for neuroprotective agents, fractions petroleum ether (Pe), ethyl acetate (Ea), butanol (Bu) and water (Wa) and isolates (1-15) were tested. In the two step analysis, it was determined if fractions and compounds have an effect on the basal cells viability (cytotoxicity assay) and if fractions and compounds have protective effect against cells damage induced

+ by toxic agents such as hydrogen peroxide (H2O2) and 1-methyl-4-phenylpyridinium (MPP ).

The results showed that fractions are not toxic within the concentration range of less than 50

µg/mL and that fractions do not show protective effects against H2O2 induced damage in

PC12 cells, at the doses of 1-10 µg/mL. It was also found that compounds are not cytotoxic, at the tested range of concentrations 0.4–10 µg/mL. Moreover, angustisepalin (11) and angustanoic acid F (7) increased basal cell viability, at similar level as BDNF which served as a positive control. The protective effect against MPP+ was observed for angustanoic acids

E (4) and G (8), 2,6-dimethoxychavicol (6), majusanic acid C (5) and (-)-T-muurolol (2). While compounds (5) and (4) showed similar protective activity to BDNF at 2 µg/mL, compound (6) protected cells slightly weaker. The most significant protection from the MPP+ damage was exerted by compound (8) at 0.4 µg/mL and by compound (2) at 10 µg/mL which was close to protective effect of AOP-6A which served as second positive control. These findings indicate that angustanoic acids E (4) and G (8), majusanic acid C (5) and (-)-T-muurolol (2) have a neuroprotective potential. All compounds with the exception of thymol were evaluated for neuroprotective activity for the first time. In the course of search for natural acetylcholinesterase inhibitors I. angustisepalum was investigated using TLC bioautography and also enzymatic assays. In the TLC bioautography, six active principles were identified in essential oil and ethyl acetate extract from I. angustisepalum. Including thymol (1), 3,5-

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dimethoxychavicol (6), (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavonone (10), angustisepalin (11), clovane-2,9-diol (12) and 2-hydroxy-2-methyl-hexan-1,5-olide (15). IC50 were established using modified Ellman’s method. Compounds moderately inhibited AChE, and clovane-2,9- diol (12) and thymol (1) showed the best activity among tested compounds, with IC50 of 11.02 and 13.47 µg/mL, respectively. It is the first time that AChE inhibitors are reported from

I. angustisepalum. These findings suggest that genus Illicium is a promising source for natural AChE-inhibitors. In the promotion of neural growth assay three terpene compounds, thymol (1), majucin (14) and majusanic acid C (5), caused an increase of neurite length greater than that of BDNF. It was also found that compound angustanol (13) was slightly less effective than BDNF. At the dose of 20 μg/mL compounds showed neurotrophic activity in the following order:

Thymol > majucin > majusanicacid > BDNF > angustanol > BDNF > angustanol.

5.2 PERSPECTIVE

5.2.1 Metabolite fingerprinting of I. angustisepalum

The comparison of the metabolite fingerprints of I. angustisepalum, I. lanceolatum and

I. verum described the variation of the volatile secondary metabolites found in these three plant species. Although, study presented interesting patterns of volatile metabolites, the extracts used in the study were prepared from the samples collected on the same day and in the same region. Therefore, it is recommended that subsequent research works should be carried out with samples collected in at least two regions. It is also advised that samples from each of the regions should be collected during the warm season with higher outdoor temperatures and greater amount of sunlight and also during the cold season with lower

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outdoor temperatures and less amount of sunlight. Such comparison would introduce more insights into the chemotaxonomic differences between these three Illicium species.

5.2.2 Chemical and biological characterization of isolated compounds

Both the present study and previous literature reports suggest that plants from genus

Illicium produce a majority of bioactive metabolites. Sixteen compounds were isolated from

I. angustisepalum. The chemical classes of these isolates are quite different although all were derived from the same plant species. In addition to the abietane diterpenes previously obtained from I. angustisepalum, several other chemical scaffolds were found, including rarely occurring in nature seco-prezizaane sesquiterpenes. The battery of in vitro assays was used to create the first biological profile of I. angustisepalum. The results of the neural cell protection, AChE inhibition and promotion of neural cell growth assays showed that a number of compounds obtained from I. angustisepalum have interesting biological potentials.

Angustanoic acids G (8) and (-)-T-muurolol (2) protected neural cells from the MPP+ damage at 0.4 µg/mL and at 10 µg/mL, respectively. Clovane-2,9-diol (12) and thymol (1) moderately inhibited AChE. And thymol (1), majucin (14) and majusanic acid C (5), caused an increase of the neurite length greater than that of BDNF, in the promotion of neural growth assay. All these findings, suggest that phytochemical and biological explorations of this genus should continue.

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APPENDICES

APPENDIX A: TLC - SPECTRA - CRYSTALLOGRAPHIC DATA

Figure A-1: Co-chromatography of volatile fraction with thymol standard

Figure A-2: Subfractions (1-15) obtained from the separation of ethyl acetate fraction. Plate was eluted by petroleum ether-ethyl acetate (6:4 v/v)

162

163

APPENDIX A (continued)

13 Figure A-3: C NMR spectrum (100 MHz, methanol-d4) of 2-hydroxy-7-methyl-hexan-1,5-olide (15)

164

APPENDIX A (continued)

Figure A-4: HSQC NMR spectrum (400 MHz, methanol-d4) of 2-hydroxy-7-methyl-hexan-1,5-olide (15)

165

APPENDIX A (continued)

Figure A-5: HMBC NMR spectrum (600 MHz, methanol-d4) of 2-hydroxy-7-methyl- hexan-1,5-olide (15)

166

APPENDIX A (continued)

13 Figure A-6: C NMR spectrum (100 MHz, methanol-d4) of 2-Hydroxy-2-methyl-6-methyleneoct- 7-en-3-yl benzoate (3)

167

APPENDIX A (continued)

Figure A-7: HSQC NMR spectrum (400 MHz, methanol-d4) of 2-Hydroxy-2-methyl-6- methyleneoct-7-en-3-yl benzoate (3)

168

APPENDIX A (continued)

Figure A-8: HMBC NMR spectrum (400 MHz, methanol-d4) of 2-Hydroxy-2-methyl-6- methyleneoct-7-en-3-yl benzoate (3)

169

APPENDIX A (continued)

1 Figure A-9: Comparison of H NMR (400 MHz, red spectrum was recorded in methanol-d4, blue spectrum was recorded in DMSO-d6) spectra of (2R, 3R)-3,5,7,3’,4’-pentahydroxyflavanone (10)

170

APPENDIX A (CONTINUED)

TABLE A-1: ATOMIC POSITIONAL AND EQUIVALENT ISOTROPIC THERMAL PARAMETERS FOR 6Β-HYDROXY-4-STIGMASTEN-3-ONE (9) WITH ESTIMATED STANDARD DEVIATIONS IN PARENTHESES Atom x Y z U (eq) [Å2]

C1 0.1849(5) 0.5180(5) 0.28197(14) 0.0195(11) C2 0.1054(5) 0.5325(5) 0.31870(14) 0.0198(13) C3 0.1262(4) 0.3784(5) 0.34943(13) 0.0116(10) C4 0.0432(4) 0.3953(5) 0.38586(13) 0.0123(10) C5 -.1031(4) 0.3909(7) 0.37037(15) 0.0214(11) C6 0.0740(5) 0.5732(6) 0.40850(14) 0.0187(11) C7 0.0319(5) 0.5803(6) 0.45193(15) 0.0227(12) C8 0.0898(5) 0.4317(7) 0.47824(16) 0.0254(12) O9 0.1173(6) 0.4471(7) 0.51551(13) 0.0504(16) C10 0.1051(4) 0.2618(6) 0.45654(15) 0.0218(12) C11 0.0778(4) 0.2408(5) 0.41548(14) 0.0152(11) C12 0.0788(4) 0.0563(6) 0.39702(16) 0.0201(11) O13 -.0506(3) -.0039(5) 0.38448(14) 0.0309(13) C14 0.1511(4) 0.0486(5) 0.35896(16) 0.0191(13) C15 0.1103(4) 0.1957(5) 0.32782(13) 0.0121(10) C16 0.1901(4) 0.1919(5) 0.29155(14) 0.0140(10) C17 0.1917(5) 0.0224(6) 0.26570(15) 0.0205(11) C18 0.2416(4) 0.0863(6) 0.22550(14) 0.0178(11) C19 0.2531(4) 0.2916(6) 0.22724(14) 0.0157(11) C20 0.1582(4) 0.3413(5) 0.25900(13) 0.0142(10) C21 0.0184(4) 0.3329(7) 0.23833(14) 0.0224(13) C22 0.2367(4) 0.3792(6) 0.18408(14) 0.0183(11) C23 0.2440(5) 0.5838(7) 0.18640(15) 0.0231(14) C24 0.3395(4) 0.3046(7) 0.15846(14) 0.0216(12) C25 0.3330(5) 0.3803(8) 0.11433(14) 0.0260(14) C26 0.4135(5) 0.2704(7) 0.08662(14) 0.0210(12) C27 0.4688(5) 0.3865(7) 0.05434(15) 0.0276(16) C28 0.5746(6) 0.5109(9) 0.07277(18) 0.0357(17) C29 0.3369(5) 0.1099(7) 0.06806(17) 0.0284(16) C30 0.2338(5) 0.1629(9) 0.03154(17) 0.0336(16) C31 0.4227(7) -.0370(9) 0.0539(2) 0.0419(19) [Å]: Angstrom

171

APPENDIX A (CONTINUED)

TABLE A-2: BOND LENGTHS (Å) FOR 6Β-HYDROXY-4-STIGMASTEN-3-ONE (9) WITH ESTIMATED STANDARD DEVIATIONS IN PARENTHESES Atoms Bond Length (Å) Atoms Bond Length (Å) C1 - C2 1.535(7) C27 - C28 1.514(8) C1 - C20 1.535(6) C29 - C30 1.555(8) C2 - C3 1.530(6) C29 - C31 1.527(9) C3 - C4 1.553(6) C3 - C15 1.545(5) C4 - C5 1.546(6) C4 - C6 1.544(6) C4 - C11 1.526(6) C6 - C7 1.529(7) C7 - C8 1.492(7) C8 - C10 1.479(7) O9 - C8 1.217(7) C10 - C11 1.341(7) C11 - C12 1.514(6) C12 - C14 1.524(7) O13 - C12 1.434(6) C14 - C15 1.527(6) C15 - C16 1.522(6) C16 - C17 1.530(6) C16 - C20 1.553(6) C17 - C18 1.540(7) C18 - C19 1.551(6) C19 - C20 1.558(6) C19 - C22 1.542(6) C20 - C21 1.530(6) C22 - C23 1.544(7) C22 - C24 1.539(6) C24 - C25 1.538(7) C25 - C26 1.543(7) C26 - C27 1.530(7) C26 - C29 1.532(7) [Å]: Angstrom

172

APPENDIX A (CONTINUED)

TABLE A-3: BOND ANGLES (º) FOR 6Β-HYDROXY-4-STIGMASTEN-3-ONE (9) WITH ESTIMATED STANDARD DEVIATIONS IN PARENTHESES Atoms Angle ° (e.s.d) Atoms Angle ° (e.s.d)

C2 - C1 - C20 110.9(4) C17 - C18 – C19 108.2(4) C1 - C2 - C3 114.0(4) C18 – C19 – C20 102.2(3) C2 - C3 - C4 112.8(3) C18 – C19 – C22 113.2(4) C2 - C3 - C15 112.3(3) C20 – C19 – C22 119.4(4) C4 - C3 - C15 112.4(3) C1 – C20 - C16 106.5(3) C3 - C4 - C5 111.5(3) C1 – C20 – C19 116.0(4) C3 - C4 - C6 109.4(3) C1 – C20 – C21 110.9(4) C5 - C4 - C6 108.3(4) C16 – C20 – C19 100.2(3) C5 - C4 - C11 109.8(3) C16 – C20 – C21 112.6(3) C6 - C4 - C11 109.9(3) C19 – C20 – C21 110.1(3) C3 - C4 - C11 108.0(3) C19 – C22 – C23 112.5(4) C4 - C6 - C7 113.9(4) C19 – C22 – C24 109.2(4) C6 - C7 - C8 111.3(4) C23 – C22 – C24 110.9(4) O9 - C8 - C7 122.1(5) C22 – C24 – C25 114.5(4) O9 – C8 - C10 122.1(5) C24 – C25 – C26 112.3(4) C7 – C8 - C10 115.8(4) C25 – C26 – C27 111.7(4) C8 – C10 - C11 123.7(4) C25 – C26 – C29 111.1(4) C4 – C11 - C10 123.0(4) C27 – C26 – C29 113.7(4) C4 – C11 - C12 117.5(4) C26 – C27 – C28 113.4(4) C10 – C11 - C12 119.5(4) C26 – C29 – C30 112.2(4) O13 – C12 - C11 110.6(4) C26 – C29 – C31 113.3(5) O13 – C12 - C14 107.2(4) C30 – 29 – C31 109.3(5) C11 – C12 - C14 112.6(4) C12 –C14 - C15 112.7(3) C3 –C15 - C16 109.3(3) C14 – C15 - C16 111.5(3) C3 - C15 - C14 109.8(3) C15 – C16 - C17 119.0(3) C15 – C16 - C20 115.0(3) C17 – C16 - C20 104.2(4) C16 – C17 - C18 103.5(4)

173

APPENDIX A (continued)

Table A-4: PLANT NAME INDEX

Latin binomial Ref. Family Ref. Pages Bidens graveolens (233) Asteraceae (234) 96 Cassia alexandrina (235) Fabaceae (236) 2 Catharanthus roseus (237) Apocynaceae (238) 3 Centella asiatica (239) Apiaceae (236) 2 Coptis chinensis (240) Ranunculaceae (241) 3 Curcuma longa (242) Zingiberaceae (243) 2 Ephedra sinica (244) Ephedraceae (245) 3 Glycyrrhiza glabra (246) Fabaceae (236) 2 Illicium anisatum (16) Schisandraceae (247) 4, 13-16, 21, 25, 29-30, 53, 97-98, 125 Illicium arborescens (248) Schisandraceae (247) 10, 25, 27-28 Illicium brevistylum (249) Schisandraceae (247) 8 Illicium burmanicum (250) Schisandraceae (247) 10, 17 Illicium difengpi (251) Schisandraceae (247) 7-8, 19, 22 Illicium dunnianum (252) Schisandraceae (247) 8, 53 Illicium floridanum (253) Schisandraceae (247) 13, 18 Illicium griffithii (100) Schisandraceae (247) 13-16, 20-21, 25, 99, 101, 104-105 Illicium henryi (254) Schisandraceae (247) 8, 22 Illicium jiadifengpi (255) Schisandraceae (247) 8, 24, 30, 96, 98 Illicium jiadifengpi (256) Schisandraceae (247) 96 f. minwanense Illicium lanceolatum (257) Schisandraceae (247) 10, 13, 15-16, 29, 33, 38, 40-42, 44-45, 47, 50-51, 53-54, 125, 132, 135 Illicium macranthum (258) Schisandraceae (247) 8 Illicium merrillianum (36) Schisandraceae (247) 10 Illicium majus (259) Schisandraceae (247) 8, 24, 96 Illicium micranthum (260) Schisandraceae (247) 8, 10, 97 Illicium micranthum (37) Schisandraceae (247) 10 subsp. tsangii Illicium pachyphyllum (261) Schisandraceae (247) 8, 34 Illicium parvifolium (262) Schisandraceae (247) subsp. oligandrum 8, 13-15, 18, 20-22, 24-25, 27, 103 Illicium simonsii (263) Schisandraceae (247) 8, 10, 12, 20, 26, 29, 99, 101-102, 105, 108, 114, 124-125 Illicium tashiroi (38) Schisandraceae (247) 10, 25 Illicium (264) Schisandraceae (247) 8 ternstroemioides Illicium verum Schisandraceae (247) 38, 40-42, 44, 48, 50, 51-54, 96-97, 104, 114, 132, 135 Magnolia obovata (265) Magnoliaceae (20) 97, 130 Melia azedarach (242) Meliaceae (266) 3 Papaver somniferum (267) Papaveraceae (268) 2 Pinus sylvestris (217) Pinaceae (269) 97 Rauvolfia serpentina (270) Apocynaceae (238) 2 Ricinus communis (271) Euphorbiaceae (272) 2

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APPENDIX A (continued)

Table A-4: PLANT NAME INDEX

Latin binomial Ref. Family Ref. Pages Tanacetum annuum (273) Asteraceae (234) 96 Taxus brevifolia (274) Taxaceae (275) 3

CURRICULUM VITA

EDUCATION PhD - Medicinal Chemistry and Pharmacognosy Program UNIVERSITY OF ILLINOIS AT CHICAGO, WHO Collaborating Center for Traditional Medicine, College of Pharmacy, Chicago, IL 2015

PharmD - 6 Year Accredited Pharmacy Program JAGIELLONIAN UNIVERSITY, Medical College, Faculty of Pharmacy, Krakow, Poland 2003-2009 Thesis Title: “Comparative phytochemical analysis of lichens of Cladonia genus” (written in Polish and English). In collaboration with University of Iceland, School of Health Sciences, Faculty of Pharmaceutical Sciences, Reykjavik, Iceland (ERASMUS Program Scholarship). Mentors: Sesselja Omarsdottir, PhD, Associate Professor of Pharmaceutical and Natural Products Chemistry, University of Iceland; Irma Podolak, PhD, Associate Professor of Pharmacy, Jagiellonian University.

Visiting Scholar - ERASMUS Program Scholarship Winter-Spring 2008 UNIVERSITY OF ICELAND, School of Health Sciences, Faculty of Pharmaceutical Sciences, Reykjavik, Iceland

RESEARCH EXPERIENCE Principal Investigator - Research Assistant May 2012-Present UNIVERSITY OF ILLINOIS AT CHICAGO, WHO Collaborating Center for Traditional Medicine, College of Pharmacy, Chicago, IL Mentor: Chun-Tao Che, PhD  Dissertation: Pharmacognostic investigations and in vitro profile of Illicium angustisepalum.  The metabolite fingerprint of I. angustisepalum by GC-MS was established and compared with those of better-known species, I. verum and I. lanceolatum. Existing variations of the volatiles within this taxon were identified.  The extract of I. angustisepalum was subjected to phytochemical and biological evaluations: o As a result of the phytochemical work, a total of sixteen compounds were isolated and identified, including two novel structures. o The first in vitro biological profile for this plant was established.

Innovation and Development PhD Intern Jun 2013-Sep 2013 FRESENIUS KABI U.S.A, Skokie, Illinois Manager: Anton Stetsenko, MD, MBA Project Title: The Component Optimization of a FLIPR Assay  Assisted in design of inter-disciplinary research project structured to 1) isolate, identify and evaluate biological activity of impurities observed in the pharmaceutical product and to 2) accurately calculate product’s potency.  Optimized one of the main components of calcium-flux assay (FLIPR), essential for determination of biological activity of isolated impurities.  Experimentally determined conditions optimal for full factorial Design of the Experiment (DOE), and identified factors crucial for cell seeding uniformity and method robustness.  Created experimental protocols, and contributed to development of ‘cell seeding fishbone’ for DOE.

Research Assistant - Co-Investigator Jan 2012-Dec 2012 UNIVERSITY OF ILLINOIS AT CHICAGO, College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, Chicago, IL Mentor: Animesh Barua, PhD (RUSH UNIVERSITY, College of Medicine, Chicago, IL) Project Title: Protective effects of curcuminoids (Curcuma longa L.) in the hen model of human ovarian cancer  Used LC-PDA to qualitatively and quantitatively assess curcuminoids content in the hens’ serum.

Research Assistant - Co-Investigator July 2011-Nov 2011 UNIVERSITY OF ILLINOIS AT CHICAGO, College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, Chicago, IL Mentor: Ruxana T. Sadikot, MD, MRCP (UNIVERSITY OF ILLINOIS, College of Medicine, Chicago, IL) Project Title: Measurement of curcumin in commercially available curcumin preparations

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 Determined curcumin content in commercial products using combination of NMR and LC-PDA techniques.  Evaluated curcumin bioavailability in the plasma of healthy volunteers.

Research Rotation Program Jan 2011-May 2011 UNIVERSITY OF ILLINOIS AT CHICAGO, College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy Chicago, IL Mentor: Brian T. Murphy, PhD Project Title: Biogeography of actinomycetes from the Massachusetts coastline  Applied 16S rRNA analysis to evaluate: a) the distribution of Actinobacteria from different SCUBA collection sites, b) the effectiveness of pre-treatment methods that select for typical antibiotic-producing genera, and c) the effects of media composition on actinomycete diversity.

ERASMUS Program Feb 2008-May 2008 UNIVERSITY OF ICELAND, School of Health Sciences, Faculty of Pharmaceutical Sciences, Reykjavik, Iceland Mentors: Sesselja Omarsdottir, PhD, PharmD (EU); Irma Podolak, PhD, PharmD (EU) Project Title: Comparative phytochemical analysis of lichens of Cladonia genus  Phytochemically compared Polish and Icelandic lichens species.  Isolated and purified two lichenic acids: usnic and fumaroprotocetraric acids.

PharmD Student Research Project Oct 2006-Apr 2007 JAGIELLONIAN UNIVERSITY, Medical College, Faculty of Pharmacy, Krakow, Poland Mentor: Agnieszka Galanty, PhD Project Title: Phytochemical analysis and biological activity of species from Agavaceae  Fractionated plant extract using chromatographic (HPTLC, CC) protocols.  Evaluated cytotoxic and antimicrobial potentials of extracts and subfractions.

CLINICAL PHARMACY EXPERIENCE PharmD Summer Intern - JOHN H. STROGER, JR., HOSPITAL OF COOK COUNTY, Chicago, IL Summer 2007 Manager: Pang Chong, PharmD Inpatient Hospital Pharmacy:  Assisted with medication ordering and verifications, medication stocking, compounding bulk medications and pediatric syringes.  Learned medication delivery and automation systems, purchasing and ordering procedures, controlled substances policies, pharmacy policy & procedures, pharmacy administration guidelines, and role of pharmacy/pharmacist in the hospital.

Outpatient Hospital Pharmacy:  Assisted with prescription filling and checking, and medication pick up for patients.  Learned the roles of staff pharmacists and technicians as well as clinical pharmacy and their role in providing patient care throughout the hospital.

RETAIL PHARMACY EXPERIENCE Licensed Retail Pharmacist - GALEN, Private Pharmacy, Alwernia, Poland Dec 2009-Jul 2010  Overseen technicians’ and intern’s activities.  Supervised medications’ and medical preparations dispensing, medication’s ordering, and medication stocking.  Provided patients with information about treatment procedures, medication risks, special diets, and physician instructions thus implemented pharmaceutical care guidelines.  Compounded ointments, creams, suspensions and solutions for patients in compliance with the Polish Pharmacopeia Edition 9 (FP IX).  Analyzed pharmacy sales, developed strategy for procuring products yielding high margin, and assisted in negotiating competitive pricing.

Postgraduate Intern - BATOREGO, Private Pharmacy, Krakow, Poland Mar 2009-Sep 2009 Manager: Jozefa Krol, PharmD (EU)

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 Mastered medications’ and medical preparations dispensing, medication’s ordering, and medication stocking and in the pharmaceutical care.  Learned ethical and administrative aspects of the pharmacist’s profession.

Pharmacy Summer Intern - PHARMED, Private Pharmacy, Alwernia, Poland Summer 2006 Manager: Ewa Turzynska, PharmD (EU)  Familiarized with the layout and use of outpatient’s pharmacy rooms and with medications’ composition with regard to A, B, N registers and their synonymic names and Latin nomenclature.  Learned medicinal preparations and pharmaceutical raw materials proper storage procedures as well as pharmaceutical preparations expiration and withdrawal procedures.  Learned pharmacist’s responsibilities including prescriptions fulfillment procedures, merchandise ordering and receiving, narcotics receipts and expenditure supervision (keeping the narcotic book).

TEACHING/MENTORING EXPERIENCE Teaching Assistant Aug 2010-May 2012 UNIVERSITY OF ILLINOIS AT CHICAGO, College of Pharmacy, Chicago, Illinois Mentors: Bradley Cannon, PharmD; Sandra Cuellar, PharmD; Janet Engle, PhD; Birgit Jaki, PhD; Louise Parent-Stevens, PharmD; Latha Radhakrishnan, PharmD  Coordinated between course lecturers and students for four semesters.  Proctored and graded quizzes and exams.  Attended lectures and answered students’ questions.  Supervised blackboard system: made announcements, posted course documents and grades.

Mentoring Aug 2013-Present UNIVERSITY OF ILLINOIS AT CHICAGO, College of Pharmacy, Chicago, Illinois  Guided three first year graduate students through research rotations: helped to design experiments and trained with research techniques applicable to projects.

AWARDS AND HONORS  The US President’s Volunteer Service Award (Nominated) 2015  University of Illinois at Chicago (UIC) Chancellor’s Student Service and Leadership Award 2015  University of Illinois at Chicago (UIC) Student Travel Presenter’s Award 2015  UIC Graduate Student Council Travel Award 2015  AAPS Nutraceutical and Natural Products Focus Group Travelship 2014  2nd place at UIC College of Pharmacy Image of Research Annual Competition for “Frozen” 2014  American Society of Pharmacognosy (ASP) Student Travel Award 2014  NIH National Center for Complementary and Alternative Medicine Grant 2013  Honorable Mention at UIC Image of Research Annual Competition for “Colors of Curcuma” 2012  Teaching and Research Assistantships and Board of Trustee Tuition Waivers 2010-Present  ERASMUS PROGRAM Scholarship 2008

LEADERSHIP EXPERIENCES Chairperson, Expanding Your Horizons (EYH) Chicago 2014-Present  Orchestrated a 1-day symposium for 200 middle-school girls to show career options in the fields of STEM.  Lobbied AAPS K-12 Foundation Grant to sponsor 50 scholarships for middle-school girls from underprivileged families.  Story was featured in AAPS Newsmagazine.  Recognized and complemented by UIC Dean and members from UIC executive leadership team. Elected Chairperson, AAPS-UIC Student Chapter 2014-2015  Organized UIC College of Pharmacy Research Day Poster Competition.  Secured funding (1.5K USD) for hosting events that foster students’ career development.  Promoted awareness of career advancement.

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Chairperson for Poster Session, 52nd MIKI Annual Meeting, Chicago, IL 2013-2014  Organized scientific poster session for 200 graduate students and post-doctoral candidates.  Helped fundraise 55K USD to host the meeting. Elected Chair, Student Scientific Society of Pharmacognosy, Jagiellonian University, Poland 2006-2007  Pioneered fresh ideas and spearheaded interactive science seminars to promote research among junior pharmacy students.  Secured funding to conduct research presented at International Students’ Conference of Medical Sciences, Poland.

Elected Class President, P1-P5, Jagiellonian University, Poland 2003-2008  Ensured adequate pharmacy student representation at university level committees.  Revitalized student advocacy by acting as a liaison between faculty and students. VOLUNTEER SERVICES (SELECTED) Asians with Disabilities Outreach Project Think-Tank (ADOPT) 2014-Present Role: Translated and recorded an Employment Services fact sheet, to make the Illinois Vocational Rehabilitation system accessible (in 10 different languages) to Chicago-based Asians with disabilities.

Chicago Department of Transportation Complete Streets Program 2012-Present Role: Assisted with conducting seasonally Quarterly Downtown Cordon Bike Count to help increase bicycle use and create a network that serves all Chicago residents.

“Garden Walk and Continuing Education Symposium”, Chicago, IL 2011-2013 Role: Assisted in guiding/lecturing 300-600 visitors about 140 species of medicinal plants medicinal plants and health products (drugs and dietary supplements).

“Annual Members Night at Field Museum”/“Medicinal Plants of the Field Museum”, Chicago, IL 2011&2015 Role: Managed 300-400 visitors through exhibits displaying 100 species of medicinal plant specimens in the form of herbarium specimens and pharmaceutical products. Led guided tours to elementary-middle school kids and public.

LICENSES AND CERTIFICATIONS  FPGEC Certificate and Pharmacy Technician License in the State of Illinois.  Licensed to work as a registered pharmacist, authorizing to performing independent professional duties in retail pharmacy (Polish/EU Pharmacist License).  Good Documentation Practices (GDP) and current Good Manufacturing Practice (cGMP) Training; Learning Management System (LMS) and Product Complaint and Adverse Reporting Training.  Heartsaver First Aid CPR AED - American Heart Association©.  Statement of Accomplishment with Distinction for SciWrite Writing in the Sciences, Stanford University.

PROFESSIONAL MEMBERSHIPS  Member of Northwestern University Advanced Degree Consulting Alliance (NUADCA) 2014-Present  Member of American Association for Pharmaceutical Scientists (AAPS) 2014-Present  Member of American Society of Pharmacognosy (ASP) 2013-Present  Member of Polish American Pharmacists Association (PAPA) 2013-Present  Member of the District Pharmacy Council in Krakow, Poland 2009-2012

PUBLICATIONS AND PRESENTATIONS Zhao, M., Onakpa, M. M., Santarsiero, B. D., Chen, W. L., Karina M. Szymulanska-Ramamurthy, K. M., Swanson, S. M., Burdette, J. E., & Che, C. T. 16,17-Dinorpimarane, 17-Norpimaranes, and (9βH)-Pimaranes from the tuber of Icacina trichantha. Original research article was submitted for the peer review to Journal of Natural Products.

Szymulanska-Ramamurthy, K. M., Zhao, M. & Che, C. T. (2014, November). Chemical diversity and identification of acetylcholinesterase inhibitors from Illicium angustisepalum. Oral presentation presented at the Baxter-UIC NMR Exchange Meeting, Chicago, IL.

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Szymulanska-Ramamurthy, K. M., Zhao, M. & Che, C. T. (2014, November). Acetylcholinesterase inhibitors from the extract of Illicium angustisepalum. Poster presented at the American Association of Pharmaceutical Scientists (AAPS), San Diego, CA.

Szymulanska-Ramamurthy, K. M., Zhao, M. & Che, C. T. (2014, August). Detection of acetylcholinesterase-inhibiting natural products from Illicium angustisepalum by bioautographic screening. Poster presented at the American Society of Pharmacognosy Annual Meeting and the 14th Annual International Conference on the Science of Botanicals (ASP/ICSB), Oxford, MS.

Zhao, M., Zhang, X., Wang, Y., Huang, M., Duan, J. A., Gödecke, T., Szymulanska-Ramamurthy, K. M., Yin Z. & Che, C. T. (2014, April). Germacranes and m-Menthane from Illicium lanceolatum. Molecules. 4; 19 (4): 4326-37.

Szymulanska-Ramamurthy, K. M. (2013, September). Ph.D. Student Summer Internship Project: Development and Optimization of the FLIPR Assay. Oral presentation presented to the Innovation and Development Team, Fresenius Kabi U.S.A, Skokie, IL.

Zhao, M., Zhang, X., Wang, Y., Huang, M., Duan, J. A., Gödecke, T., Szymulanska-Ramamurthy, K. M., Yin Z. & Che, C. T. (2013, July). Germacranes and m-Menthane from Illicium lanceolatum. Poster presented at the American Society of Pharmacognosy Annual Meeting (ASP), St. Louis, MO.

Szymulanska-Ramamurthy, K. M., Zhao, M. & Che, C. T. (2012, April). Literature review of the genus Illicium with focus on phytochemistry and pharmacology of Illicium angustisepalum. Poster presented at the Medicinal Chemistry Meeting-in-Miniature (MIKI) Annual Meeting, Iowa City, IA.

Szymulanska-Ramamurthy, K. M., Phansalkar, R., Godecke, T., Zhao, M. & Che, C. T. (2012, February). Measurement of curcumin in commercially available curcumin preparations. Poster presented at the UIC College of Pharmacy Research Day, Chicago, IL.

Szymulanska-Ramamurthy, K. M., Tanouye, U. & Murphy, B. T. (2011, April). Biogeography of actinomycetes from the Massachusetts coastline. Oral presentation presented at the UIC Graduate Student Research Rotation Talks, Chicago, IL and poster presented at the UIC Student Research Forum, Chicago, IL.

Szymulanska, K. M., Omarsdottir, S. & Podolak, I. (2008, July). Comparative phytochemical analysis of lichens of Cladonia genus. Master Thesis.

Burakowska, D., Litwin, A. & Szymulanska, K. M. (2007, April). Analiza fitochemiczna i aktywnosc biologiczna gatunkow z rodziny Agavaceae. Poster presented at the International Students Conference of Medical Sciences, Krakow, Poland.

Burakowska, D., Litwin, A. & Szymulanska, K. M. (2007, April). Analiza fitochemiczna i aktywnosc biologiczna gatunkow z rodziny Agavaceae. Przeglad Lekarski. 64; 1: 95.