Airy Shaw Jiayu Zhang

Potential Anti-Proliferative Natural Products from Bidens biternata (Lour.) Merr. & Sherff and Austrobuxus sanii (Beuzev. & C.T.White) Airy Shaw

Author Zhang, Jiayu

Published 2018

Thesis Type Thesis (Masters)

School School of Environment and Sc

DOI https://doi.org/10.25904/1912/2090

Downloaded from http://hdl.handle.net/10072/380290

Griffith Research Online https://research-repository.griffith.edu.au

Potential Anti-Proliferative Natural Products from Bidens biternata (Lour.) Merr. & Sherff and Austrobuxus sanii (Beuzev. & C.T.White) Airy Shaw

Jiayu Zhang

B.Biomol. Sci.

School of Natural Sciences

Griffith Sciences

Griffith University

Submitted in fulfilment of the requirement of the degree of

Master of Science

February 2018

Abstract

In this project, Bidens biternata (Lour.) Merr.& Sherff collected in Guangxi, China, and Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw from Queensland, Australia were chosen for NMR and MTS assay-guided chemical investigation. B. biternata, a traditional Chinese medicine (TCM), has been used to treat appendicitis, sore throat, diarrhea, dysentery, and stomach ache for hundreds of years. However, there are few of studies on the chemical constituents. Previous biological activity study indicated that the ethanol extract of B. biternata inhibited cell growth against several cancer cell lines. This warranted further research into the bioactive compounds from the TCM.

The plant genus Austrobuxus belongs to one of the endemic taxa in Australia. A. swanii is a rare subtropical rainforest tree. There is also insufficient information of this species reported. A recent paper from our group reported four new picrotoxane terpenoids from A. swanii; the

LC-MS analysis showed potential new picrotoxane terpenoids in the crude extract, which led to further chemical investigation of the plant species.

Three phenyltriynes were isolated from the first biota, B. biternata, by using C18 flash column chromatography, diol silica gel chromatography and semi-preparative HPLC with the guidance of LC-MS and NMR data. One new picrotoxane terpenoid, austrabuxusin E, along with four know austrabuxusins (A-D) were isolated from the second biota, A. swanii by using solvent partitioning and several HPLC purification with the guidance of LC-MS and NMR data. The chemical structures of the pure compounds were elucidated by 1D and 2D NMR (COSY, HMBC, HSQC, ROSEY) and MS spectroscopic data. The anti-proliferative activity of the extracts and pure compounds were evaluated against Caco-2 and SKOV3 cell lines by the MTS assay.

Statement of Originality

This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

Jiayu Zhang

Table of content

Abstract ...... I

Table of content ...... III

List of Tables ...... VI

List of Figures ...... VII

List of schemes ...... X

Acknowledgments...... XI

Conferences attended ...... XII

Abbreviations ...... XII

Chapter 1: Introduction ...... 1

1.1 Natural products in drug discovery ...... 1

1.1.1 Natural products in folklore ...... 1

1.1.2 Primary and secondary metabolites ...... 1

1.1.3 Drug discovery from natural products ...... 2

1.2 Traditional Chinese medicine ...... 4

1.3 Cancer ...... 6

1.3.1 Chemotherapy ...... 6

1.4 Objectives ...... 7

1.5 Reference ...... 8

Chapter 2: Chemical investigations of a traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff………...... 11 III

2.1 Introduction ...... 11

2.1.1 Bioactive compounds from Biden spp...... 11

2.1.2 Bidens biternata (Lour.) Merr. & Sherff ...... 14

2.2 Analysis of Bidens biternata (Lour.) Merr. & Sherff extracts ...... 15

2.2.1 Purification strategies for the active fractions ...... 17

2.3 Large-scale Extraction and Chemical Investigations of Bidens biternata (Lour.) Merr. & Sherff ...... 22

2.3.1 Structure determination of phenylheptatriyne (2.1) ...... 23

2.3.2 Structure determination of 7-phenyl-4,6-heptadiyn-2-ol (2.2) ...... 27

2.3.3 Structure determination of 5-Phenyl-2,4-pentadiyn-1-ol (2.3) ...... 30

2.4 Anti-proliferation activity of pure compounds from Bidens biternata (Lour.) Merr. & Sherff ...... 32

2.5 References ...... 32

Chapter 3: Chemical investigations of Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw…………...... 36

3.1 Introduction ...... 36

3.2 Chemical analysis of Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw extracts…...... 36

3.3 Large-scale chemical investigation of Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw ...... 38

3.4 Structure elucidation of austrobuxusin E (3.1) ...... 40

3.5 Anti-proliferation activity of pure compounds from Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw ...... 45 IV

3.6 References ...... 48

Chapter 4: Experimental Procedures ...... 50

4.1 General experimental procedure ...... 50

4.2 Extraction and Isolation of B.biternata ...... 50

4.2.1 Plant material ...... 50

4.2.2 C18 flash chromatography...... 51

4.2.3 Normal silica gel fractionation...... 51

4.2.4 Diol column chromatography ...... 51

4.2.5 C18 reverse phase HPLC fractionation ...... 51

4.3 Chemical investigation of Austrobuxus swanii ...... 52

4.3.1 Plant material ...... 52

4.3.2 Liquid-phase extraction ...... 52

4.3.3 C18 reverse phase HPLC fractionation ...... 52

4.4 Biology experimental ...... 53

4.4.1 Chemical and reagents ...... 53

4.4.2 Cell lines and cell culture ...... 53

4.4.3 Statistical analysis ...... 54

Appendices ...... 55

List of Tables

Table 1.1 Selected TCM-derived products used in cancer therapy.27 ...... 7

Table 2.1 Vernacular names of B. biternata ...... 14

Table 2.2 Traditional applications of B. biternata...... 15

Table 2.3 NMR spectroscopic data of phenylheptatriyne (2.1) ...... 26

Table 2.4 NMR spectroscopic data of 7-phenyl-4,6-heptadiyn-2-ol (2.2) ...... 29

Table 2.5 NMR spectroscopic data of 5-Phenyl-2,4-pentadiyn-1-ol (2.3) ...... 31

Table 3.1 NMR spectroscopic data of austrobuxusin E (3.1) ...... 44

List of Figures

Figure 1.1 Paclitaxel (Taxol ®) (1.1) and baccatin III (1.2) ...... 3

Figure 1.2 Grandisine A (1.3) and Grandisine B (1.4) ...... 3

Figure 1.3 quinine (1.5), Artemisinin (1.6) and Arteether (1.7) ...... 4

Figure 2.1 The chemical structures of okanin (2.1), friedelan (2.2), friedelanol (2.3),

cymaroside (2.4), luteolin (2.5), apigenin (2.6), apigenin 7-O-glucoside (2.7),

ctopiloyne (2.8), phenylheptatriyne 1 (2.9), phenylheptatriyne 2 (2.10),

phenylheptatriyne 3 (2.11)...... 13

Figure 2.2 Chemical structure of flavonoids or flavonoid glycosides isolated from

Bidens spp...... 14

1 Figure 2.3 H NMR spectrum (800 MHz, DMSO-d6) of six fractions after C18 silica flash

column...... 16

Figure 2.4 Effects of B. biternata fractionations on cell viabilities of different cancer

cell lines as determined by MTS assay for 24h...... 17

Figure 2.5 HPLC UV trace for (Fraction 5) 90% MeOH/ 10% H2O fraction...... 18

Figure 2.6 1H NMR spectrum (800 MHz, DMSO-d6) of Sephadex gel fractionation

fraction 2 to 5...... 19

1 Figure 2.7 H NMR spectrum (800 MHz, DMSO-d6) of normal phase silica gel

chromatography...... 20

VII

Figure 2.8 HPLC UV trace for (Fraction 3) 50% EtOAc/ 50% hexane fraction...... 21

Figure 2.9 HPLC UV trace for (Fraction 4) 70% EtOAc/ 30% hexane fraction...... 21

Figure 2.10 Structure of phenylheptatriyne (2.1), 7-phenyl-4,6-heptadiyn-2-ol (2.2), 5-

Phenyl-2,4-pentadiyn-1-ol (2.3)...... 23

Figure 2.11 HPLC UV trace for (Fraction 3) 20% EtOAc/80% hexane fraction...... 23

Figure 2.12 HPLC UV trace for (Fraction 4) 30% EtOAc/70% hexane fraction...... 23

1 Figure 2.13 H NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1) ...... 24

13 Figure 2.14 C NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1) ..... 25

Figure 2.15 COSY (—) and HMBC (→) correlations for phenylheptatriyne (2.1) ...... 25

Figure 2.16 HMBC spectrum for phenylheptatriyne (2.1) ...... 26

1 Figure 2.17 H NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol

(2.2) ...... 28

Figure 2.18 COSY (—) and HMBC (→) correlations for 7-phenyl-4,6-heptadiyn-2-ol

(2.2) ...... 28

Figure 2.19 HMBC spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol

(2.2) ...... 29

1 Figure 2.20 H NMR spectrum (800 MHz, DMSO-d6) for 5-phenyl-2,4-pentadiyn-1-ol

(2.3) ...... 30

VIII

Figure 2.21 COSY (—) and HMBC (→) correlations for 5-phenyl-2,4-pentadiyn-1-ol

(2.3) ...... 31

Figure 2.22 HMBC spectrum for 5-phenyl-2,4-pentadiyn-1-ol (2.3) ...... 31

1 Figure 3.1 H NMR spectrum (800 MHz, DMSO-d6) of DCM and MeOH extract...... 37

Figure 3.2 Anti-proliferative activity of plant extracts and untreated controls against

Caco-2 and SKOV3 cancer cell lines as determined by MTS assay for 24 h...... 38

Figure 3.3 HPLC UV trace for MeOH extract of A. swanii...... 39

Figure 3.4 The structure of the known compound 3.1, 3.2, 3.3, 3.4, 3.5 ...... 40

1 Figure 3.5 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) ...... 41

13 Figure 3.6 C NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) ...... 42

Figure 3.7 COSY (—) and HMBC (→) correlations for austrobuxusin E (3.1) ...... 43

Figure 3.8 HMBC spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) ...... 44

Figure 3.9 Effects of austrobuxusins A to E on cell mortality of Caco-2 cell line as

determined by MTS assay for 24 h...... 48

List of schemes

Scheme 2.1 Small-scale extraction of B. biternata by C18 flash chromatograph and

normal silica chromatography...... 20

Scheme 2.2 Large-scale extraction of B. biternata ...... 22

Scheme 3.1 Large-scale extraction of A. swanii ...... 39

Acknowledgments

It is FINALLY the day that I’ve waited so long for! I would like to say “Thank you” again and again with my deepest appreciation to those who have offered me invaluable help!

Firstly, I would like to express my heartfelt gratitude to my principal supervisor, Associate Professor Yunjiang Feng for the opportunity she offered me to be her Master student and her support in my graduate study. She is a very nice supervisor with great patience, considerations, careful guidance and suggestions.

I gratefully acknowledge and thank Dr Ian Cock. Thank him very much for his careful instructions, continuous support for my biological research, and valuable insights throughout my project and thesis writing.

Besides, I would like to sincerely thank Prof Ronald J Quinn for his patience and kind help and understanding and valuable advice on my thesis and oral presentation.

I’d also like to extend my gratitude to all my friend and mentors. Thank you to Dr Wendy Loa-Kum-Cheung for training me in LC-MS, NMR and HRESIMS analysis, to Dr Dongdong Wang for training me in chromatography and HPLC, Dr Joseph Sirdaarta for training me in cell culture and MTS assay for anti-proliferative testing. Thank you also to Chao Wang, Miaomiao Liu, Mario Wibowo and Kah Yean Lum for sharing the incredible experience with me.

Conferences attended

GRIDD/SIMM Symposium 2016

Talk: Potential Anti-Proliferative Natural Products from Bidens biternata

Abbreviations

1D one dimensional

2D two dimensional br broad

C18 octadecyl bonded silica calcd calculated

CDCl3 deuterated chloroform

COSY correlation spectrospectroscopy

DCM dichloromethane

DIOL diol-bonded silica

D doublet

DMSO dimethylsulfoxide

DMSO-d6 deuterated DMSO

HPLC high-pressure liquid chromatography

HRESIMS high-resolution electron spray ionization mass spectrometry or spectrum

HSQC gradient heteronuclear single-quantum coherence spectroscopy

Hz Hertz

XII

IC50 half maximal inhibitory concentration

IR infrared

J coupling constant

LC-MS liquid chromatography/mass spectrometry m multiplet

MeOH methanol mg milligram min minute(s) mL milliliter nM nanomolar concentration

NMR nuclear magnetic resonance

PDA photo diode array ppm parts per million

ROESY rotational nuclear Overhauser effect spectroscopy rpm revolutions per minute s singlet

SD standard deviation sp species t triplet

TFA trifluoracetic acid or trifluoroacetate

TCM traditional Chinese medicine XIII

UV ultraviolet wt weight

δ chemical shift (ppm)

℃ degrees Celsius

[α]D specific rotation

µM micromolar

µg microgram

µL microlitre

XIV

Introduction

Chapter 1: Introduction

1.1 Natural products in drug discovery

1.1.1 Natural products in folklore

Natural products have played a central role in human health for a long time. Historically, the applications of natural products are in the forms of traditional medicines, potions and oils. These formulations can be used to prevent, diagnose and treat many diseases and illnesses. The knowledge of the traditional usage of natural products has been accumulated from experience in palatability trials and from observing untimely deaths over hundreds of centuries, and some bioactive natural products have still not been identified.

The earliest formal record of natural products was recorded on clay tablets in cuneiform from Mesopotamia in 2600 B. C, although San cave paintings in Southern Africa are dating from the tens of thousands of years prior to this, also depict medicinal plant use. Many ancient medicinal are still in common usage. The oil from Cupressus sempervirens L. (cypress) and Commiphora species (myrrh) is still used to treat coughs, colds, and inflammation in many societies1. There are also many other well-known examples of documents recording medicinal plant use. The Ebers Papyrus (2900 B.C.) is an Egyptian pharmaceutical treatise which includes 700 plants and their uses. The Chinese Materia Medica (1100 B.C.) (Wu Shi Er Bing Fang, contains 52 prescriptions), Shennong Herbal (~100 B.C, 356 drugs) and the Tang Herbal (659 A.D., 850) are further Chinese pharmaceutical documents describing applications of natural products.1 The Greek physician, Dioscorides, (100 A.D.), recorded the different methods of collection, storage, and application of natural products.

1.1.2 Primary and secondary metabolites

The medicinal properties of plants are due to their individual compliments of different natural products produced by each plant. The term natural product refers to any substances produced by a living organism, but in the area of drug discovery, natural products are defined as secondary metabolites. According to Albrecht Kossel,2 naturally 1

Introduction occurring compounds can be divided into three major classes: primary, and secondary metabolites, and high-molecular-weight polymeric materials.3-4 Primary metabolites including the nucleic acids, the standard amino acids, and sugars are necessary for the metabolism and reproduction of all cells. Secondary metabolites are the small molecules that act as a defense against competitors, herbivores and pathogens or as signal compounds. In contrast to primary metabolites, secondary metabolites are not essential to the growth and development of the producing organism but may provide advantages for the plants survival and give it competitive advantages.5 Base on the structural characteristics, secondary metabolites can be classified into following six categories: fatty acid-derived substances and polyketides, terpenoids and steroids, phenylpropanoids, alkaloids, nonribosomal polypeptides and enzyme cofactors.6-7 Within the field of medicinal chemistry, many secondary metabolites of plants, fungi, and animals exert pharmacological features because of their ability to modulate the signal transduction pathways.

1.1.3 Drug discovery from natural products

Plants have been used as a primary source of medicines for thousands of years.8-9 Over millions of years of evolution, chemically diverse secondary metabolites have been produced by the plants to withstand bacteria, insects, and fungal infections. Many medicinal plants were documented in traditional medicine. 80% of 122 plant-derived were related to their original ethnopharmacological purpose.10 Thus, an understanding of ethnobotany is an invaluable tool in the field of natural product discovery.

Furthermore, there are numerous examples of allopathic drugs that originated from plants. Paclitaxel (Taxol ®) (1.1), a common chemotherapeutic drug used to treat the breast, ovarian and lung cancer therapy,11-12 was first isolated from the bark of the Taxus brevifolia Nutt.(Pacific Yew) by the United States Department of Agriculture (USDA) in 1962. In 1992, taxol (1.1) was approved by American Food and Drug Administration (FDA) for clinical usage.11 Because of the short supply from the natural sources, the total synthesis of the drug was designed to increase the quantities. Furthermore, Baccatin III (1.2), a structural analog of taxol, was used as a precursor to produce paclitaxel. Baccatin III (1.2) can be isolated from the needles of the tree and had a higher yield than that of Paclitaxel (Figure 1.1). 2

Introduction

Figure 1.1 Paclitaxel (Taxol ®) (1.1) and baccatin III (1.2)

Grandisines A and B are two indole alkaloids. Both of those two compounds were isolated from the leaves of Elaeocarpus grandis (Mueller), rainforest trees in Australia. Grandisine A and B have the ability to bind the human δ-opioid receptor and are considered as potential lead compounds for analgesic agents.13

Figure 1.2 Grandisine A (1.3) and Grandisine B (1.4)

The anti-malarial drug quinine (1.5) was first isolated from the bark of Cinchona succirubra Pav. Ex Klotzsch. It has a long history of usage as a treatment of malaria, fever, indigestion, mouth and throat diseases and cancer. Another potent antimalarial drug, Artemisinin, was also isolated from the plant Artemisia annua L. in 1987.14 Subsequent modifications of artemisinin (1.6) resulted in artemisinin and artemether (1.7), which have both been approved as antimalarial drugs.15 A. annua was initially used in traditional Chinese medicine as a treatment for chills and fevers.

Introduction

Figure 1.3 quinine (1.5), Artemisinin (1.6) and Arteether (1.7)

1.2 Traditional Chinese medicine

Side effects, low efficiency for several chronic diseases, high cost of new drugs development, microbial resistance and unclear mechanism of some diseases are some reasons for re-emergence of medicinal herbs in the public and scientific communities.16 Compare with Western medicine currently using pure compounds, Traditional Chinese Medicine (TCM) commonly combines different medical material, such as plant, part of animals, insect and mineral to treat and relieve the symptoms of different diseases.17 TCMs are believed to have fewer side effects than pure drugs and may be a better choice for long-term treatment. They are particularly useful to provide dietary supplement or develop as new drugs for patients who have low resistance to the side effects of modern drugs, such as children and the older people.18-19

Using TCM to treat diseases or keep healthy is contentious and is not yet widely accepted in Western countries. The activities of TCMs on record cannot be completely translated into modern medicinal concepts. The theory of TCM is an integral part of the Chinese culture. In TCM, the whole biological system is described based on the traditional philosophical beliefs such as Yin-yang theory and five elements (metal, wood, water, fire, earth).20 Furthermore, the whole human body is seen as a holistic system. To keep the human body healthy, the yin, and yang, and the five elements have to be all in balance. Different symptoms of the patients are described by either too much yin (cold, slow, wet and chronic) or too much yang (hot, rapid, dry and acute). Furthermore, herbal materials are classified in terms of five tastes (acrid, sweet, bitter, sour, salty), four properties (hot, warm, cool and neutral), the meridians, the compatibility, the contraindication, the

Introduction toxicity, the processing etc.20 Herbs with various properties are combined to increase the therapeutic effect and reduce toxicity and side effects.

Another difference between TCM and modern medication is that TCM using multidrug to multi targets while modern medicine using a single drug to single target. A typical TCM prescription is made up of many different herbs, some are known as the principal drug, others as the associated, the adjuvant or messenger drugs. These are described as emperor, minister, adjuvant, and courier respectively in ancient TCM books. The principal herbs target the main symptoms while the associates assist the principal drug and target other symptoms, the adjuvants enhance the activity and reduce the toxicity, the messengers guide the ingredient into right pattern.20 In contrast, modern western drugs build on our understanding of pharmacology and physiology. The explosion of modern technology helps us to understand the principles of both the disease and drugs in cellular and molecular levels. Then the active compound can work on the drug target can be a potential drug. The understanding of specific drug target has helped scientists to screen active compounds from plant extracts against certain diseases. This has led to the identification of bioactive molecules from TCM. However, the pharmacology of TCM multi-herb prescriptions as yet to fully understand.

Many research works suggest that TCM can be a useful resource for drug discovery. A comprehensive analysis of the Traditional Chinese Medicine Database (TCMD) and the Comprehensive Medicinal Chemistry Database (CMCD) was carried out by Kong and Li in 2008.17 Their study suggested that >85% compounds are structurally similar (similarity >85%).17 A total of 327 TCMD compounds, belonging to 1186 TCMs, were found in the CMCD database. This study showed that TCM, in general, has far better selectivity than normal plants.

The researchers also compared the activity of 327 compounds with that of 1186 TCMs recorded in the ancient books. More than 100 TCM have the same pharmacological effects to those of isolated compounds. For instance, the laxative drugs the stems and roots of Polygonum cuspidatum Siebold & Zucc and the leaflets of Senna alexandrina Mill. contain laxative agents (danthron, emodin, sennosides A+B, etc.); the antispasmodic drugs Datura metel L. contain antispasmodic agents (anisodamine, scopolamine,

Introduction hyoscyamine). These examples suggested that traditional applications of TCM, at least in part, indicated bioactive compounds to the disease on herb book records.

There is great potential to discover new drug candidates from bioactive TCMs. It is interesting to note that, only part of the activity categories (such as analgesic, anthelmintic, antihemorrhagic, etc.) are recorded in the TCM medicinal conceptual system, and only a small number of compounds are recorded in both TCMD and CMCD.

Despite advances in technology and the knowledge of the biological system, modern drug discovery is hindered by expense, difficulty, and inefficiency. In 2010, the expenditure of research and development for new molecular entity was about US $1.8 billion.21 In addition, many drugs failed Phase I, II or III clinical trials. The records of different TCM can be used as big library to save the money and time for drug discovery.

There are thousands of records of plants, animal parts, insect and minerals in TCM books. Chemical investigation of these TCM products has led to the isolation of small molecule drugs. Potential drug candidate could be developed based on a large number of TCM therapies, and also this will help a better understanding of the TCM theory.

1.3 Cancer

Cancer is a global non-infectious disease arises from mutated cells. The mutation in the genes induced the cell proliferates at high speed. During the proliferative progression, the defective gene is passed down to daughter cell. In time, the structure of the descendent cells changes to the tumor (neoplasm).22 The cancer statistics data from the World Health Organization (WHO) show that about 14.1 million cancer cases were diagnosed and 8.2 million cancer deaths occurred in 2012 worldwide.23 To date, there are over 200 different types of cancers. Lung cancer is the most prevalent and most fatal cancer. There are 1,825,000 cases of lung cancer every year, and the number of deaths caused by the lung cancer is 1,590,000. This number is over double times than the number of deaths caused by liver cancer (746,000).23

1.3.1 Chemotherapy

Introduction

Many cancer therapy modalities have been developed including chemotherapy, radiotherapy, surgery, hormonal therapy, biological therapy, hematopoietic stem cell or bone marrow transplants. Chemotherapy is one of the most widely used methods, which using drugs to kill cancer cells or inhibit proliferation. Nowadays, chemotherapy is mainly used to cure cancer, restrict cancer metastases, limit cancer cell growth and relieve the symptoms of cancer. The anticancer drugs/chemotherapeutic drugs are introduced into the bloodstream and circulated throughout the whole body to fight cancer cells.

Natural products and their extracts make a significant contribution to our antitumor drug options. According to the data updated by Newman,24 from the 1940s to 2014, 49% of anticancer drugs were developed from natural products or their derivatives. Some plants have particularly good abilities to block cancer cell proliferation. Vinblastine, which was isolated from Vinc. rosea, taxol from T. brevifolia, and matrine from Sophora flavescens Aiton and camptothecin.24 Plants have an almost unlimited capacity to attract researchers 25,26 for new and novel chemotherapeutics . TCM as part of natural product also has great future in cancer therapy. Table 1.1 presents some applications of TCM-derived products in cancer therapy.

Table 1.1 Selected TCM-derived products used in cancer therapy.27

TCM product Target Plant name Camptothecin Topoisomerase Ⅰ Camptotheca acuminata Podophyllotoxin Topoisomerase Ⅱ Podophyllum emodi var. chinesnse Vindesine and Mitotic-spindle proteins Catharanthus roseus vinorelbine microtubules of the cytoskeleton Paclitaxel microtubules Taxus chinensis Emodin Kinase-2 Rheum palmatum Verbasco telomerase Pedicularis striata Gambogic acid telomerase Garcinia hanburyi Capsaicin angiogenesis capsicum Sinomemine angiogensis Sinomenium acutum Homoharringtoine Receptor on the 60S ribosome Cephalotaxus species subunit Nrocantharidin and Protein phosphatase 1 and 2A Mylabris Cantharidin

1.4 Objectives

Introduction

In this project, two different biotas (Bidens biternata and Austrobuxus swanii) were selected for the NMR and bio-activity guided chemical investigations. Techniques employed in the project included different chromatography, HPLC, LC-MS, and NMR. The anti-proliferation activities were tested by MTS assay. B. biternata is a TCM that has been traditionally used as a treatment of influenza, swollen and sore throat, enteritis, dysentery, jaundice, intestinal carbuncle, epilepsy in children, malnutrition in infants and hemorrhoids.28 Although many applications were recorded by some Chinese Medicinal Herbs book, the mechanism, and active constituents have not been fully reported. So, the first objective of this project was pre-fractionation B. biternata and targeted the fraction which has good anti-proliferative activity by using MTS assay and NMR data. Then the second objective was isolated pure compounds from the target fraction. At last, the anti- proliferative activity of pure compounds was evaluated again by MTS assay.

The second plant Austrobuxus swanii, an Australian endemic plant, is a rare subtropical rainforest tree in New South Wales and south-eastern Queensland, Australia. There is insufficient information on its chemical composition. However, one recent paper from our group reported the isolation of austrobuxusin A-D, structurally complex picrotoxane terpenoids from A. swainii.29 Further study indicated that more austrobuxusin analogues in this plant and therefore, it was chosen for the NMR and bio-active guided chemical investigation. Overall, the purpose of this project was to use MTS assay and NMR- guided isolation to identify compounds from the two biotas.

1.5 Reference

1. Cragg, G. M.; Newman, D. J., Biodiversity: A continuing source of novel drug leads. Pure and Applied Chemistry 2005, 77 (1), 7-24.

2. Kossel, A., Ueber die chemische Zusammensetzung der Zelle. Archiv für Physiologie 1891, 4, 181-186.

3. Kliebenstein, D., Secondary metabolites and plant/environment interactions: a view through Arabidopsis thaliana tinged glasses. Plant, Cell & Environment 2004, 27 (6), 675-684.

4. Karlovsky, P., Secondary metabolites in soil ecology. Secondary Metabolites in Soil Ecology 2008, 1-19.

5. Maplestone, R. A.; Stone, M. J.; Williams, D. H., The evolutionary role of secondary metabolites—a review. Gene 1992, 115 (1), 151-157. 8

Introduction

6. Hanson, J. R., Natural products: the secondary metabolites. Royal Society of Chemistry: 2003; Vol. 17.

7. McMurry, J. E., Organic Chemistry with Biological Applications. Cengage Learning: 2014.

8. McRae, J.; Yang, Q.; Crawford, R.; Palombo, E., Review of the methods used for isolating pharmaceutical lead compounds from traditional medicinal plants. The Environmentalist 2007, 27 (1), 165-174.

9. Swanson, T., Intellectual property rights and biodiversity conservation: an interdisciplinary analysis of the values of medicinal plants. Cambridge University Press: 1998.

10. Fabricant, D. S.; Farnsworth, N. R., The value of plants used in traditional medicine for drug discovery. Environmental Health Perspectives 2001, 109 (Suppl 1), 69.

11. Cragg, G. M., Paclitaxel (Taxol®): a success story with valuable lessons for natural product drug discovery and development. Medicinal Research Reviews 1998, 18 (5), 315-331.

12. Wall, M. E., Camptothecin and taxol: discovery to clinic. Medicinal Research Reviews 1998, 18 (5), 299-314.

13. Carroll, A. R.; Arumugan, G.; Quinn, R. J.; Redburn, J.; Guymer, G.; Grimshaw, P., Grandisine A and B, Novel Indolizidine Alkaloids with Human δ-Opioid Receptor Binding Affinity from the Leaves of the Australian Rainforest Tree Elaeocarpus g randis. The Journal of Organic Chemistry 2005, 70 (5), 1889-1892.

14. Dias, D. A.; Urban, S.; Roessner, U., A historical overview of natural products in drug discovery. Metabolites 2012, 2 (2), 303-36.

15. Newman, D. J.; Cragg, G. M., Natural products as sources of new drugs over the last 25 years. Journal of Natural Products 2007, 70 (3), 461-477.

16. Patwardhan, B.; Warude, D.; Pushpangadan, P.; Bhatt, N., Ayurveda and traditional Chinese medicine: a comparative overview. Evidence-Based Complementary and Alternative Medicine 2005, 2 (4), 465-473.

17. Kong, D. X.; Li, X. J.; Tang, G. Y.; Zhang, H. Y., How many traditional Chinese medicine components have been recognized by modern Western medicine? A chemoinformatic analysis and implications for finding multicomponent drugs. ChemMedChem 2008, 3 (2), 233-6.

18. Lee, K.-H., Research and future trends in the pharmaceutical development of medicinal herbs from Chinese medicine. Public Health Nutrition 2000, 3 (4a), 515-522.

19. Konkimalla, V. B.; Efferth, T., Evidence-based Chinese medicine for cancer therapy. Journal of Ethnopharmacology 2008, 116 (2), 207-210. 9

Introduction

20. Teng, L., Traditional Chinese herbal medicine. Pharmaceutical Journal (Vol 276) 2006.

21. Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L., How to improve R&D productivity: the pharmaceutical industry's grand challenge. Nature Reviews Drug Discovery 2010, 9 (3), 203-214.

22. Kintzios, S. E.; Barberaki, M. G., Plants that Fight Cancer. Crc Press: 2004.

23. Torre, L. A.; Siegel, R. L.; Ward, E. M.; Jemal, A., Global cancer incidence and mortality rates and trends—an update. Cancer Epidemiology and Prevention Biomarkers 2016, 25 (1), 16-27.

24. Newman, D. J.; Cragg, G. M., Natural Products as Sources of New Drugs from 1981 to 2014. J Nat Prod 2016, 79 (3), 629-61.

25. Reed, J. C.; Pellecchia, M., Apoptosis-based therapies for hematologic malignancies. Blood 2005, 106 (2), 408-418.

26. Talib, W. H.; Mahasneh, A. M., Antiproliferative activity of plant extracts used against cancer in traditional medicine. Scientia Pharmaceutica 2010, 78 (1), 33.

27. Efferth, T.; Li, P. C.; Konkimalla, V. S. B.; Kaina, B., From traditional Chinese medicine to rational cancer therapy. Trends in Molecular Medicine 2007, 13 (8), 353- 361.

28. Dharmananda, S., BIDENS.

29. Demirkiran, O.; Campitelli, M.; Wang, C.; Feng, Y., New picrotoxane terpenoids, austrobuxusin AD, from the Australian endemic plant Austrobuxus swanii. Tetrahedron 2016, 72 (51), 8400-8405.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

Chapter 2: Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

2.1 Introduction

The genus Bidens (Asteraceae: Heliantheae) has about 240 different species and is widely distributed.1 Previous chemical investigation studies of related Bidens spp. identified a variety of compounds, including polyacetylenes, polyacetylenic glycosides, okaninglycoside, acetylenes, sesquiterpene lactones, quercetin, phenolic compounds and flavonoids.2-3 In China, Bidens spp. is used to treat diabetes, inflammation, enteritis, bacillary dysentery and pharyngitis.4 In Brazil, it is used to treat pain, fever, angina, diabetes, edema, infections and inflammation.5 Moreover, further biological studies have proved it has anti-hyperglycemic,6-7 antihypertensive,8-9 antiulcerogenic antiulcerogenic,10 hepatoprotective,11 antipyretic,12 immunosuppressive and anti-inflammatory,13-15 anti-leukemic,16-17 anti-malarial,5 antibacterial,18 and antitumor19 effects.

2.1.1 Bioactive compounds from Bidens spp.

Many species have been investigated for their medicinal properties, because of biological activities of some extracts and fractions from Bidens spp. Bidens pilosa S.F. Blake and Biden bipinnata L. have been particularly well studied and many compounds have been isolated and bioactivity data reported. Okanin (2.1) (Figure 2.1) one of the most abundant chalcones isolated from the genus Bidens, has been reported to treat inflammation. The excess production of nitric oxide (NO) leads to the development of various inflammatory diseases and some inducible pro-inflammatory enzymes such as inducible nitric oxide synthase are the key to generate a large amount of NO in activated macrophages for a long period.20-21 Okanin (2.1) could inhibit the expression of nitric oxide production and inducible nitric oxide synthase (iNOS) via reduced expression of heme oxygenase-1.22

In addition to flavonoid, many triterpenoids (Figure 2.2) from Bidens spp. also showed anti- inflammatory activity. The friedelin (2.2) and friedelanol (2.3), cymaroside (2.4) and luteolin (2.5), apienin (2.6) and apigenin 7-O-glucoside (2.7) .23 Some alkynes have been isolated from the Bidens species. The polyacetylene 2-O-b-D-glucosyltrideca-11E-en-3,5,7,9-tetrayn-1,2- diol (2.7) from B. camphylotheca can inhibit lymphocyte proliferation.14 Ctopiloyne (2.8) has 11

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff been reported to be a potential anti-hyperglycemic agent by inhibiting Th1 differentiation and promoting Th2 differentiation.4

Phenylheptatriynes (2.9, 2.10, 2.11) inhibit the growth of Gram-positive bacteria when irradiated with ultraviolet light at wavelengths between 360-370 nm. Polyacetylenes 2 (2.12) and 3 (2.13) are bacteriostatic and have fungistatic activity, even in the dark.23 Phenylheptatriyne (2.11) showed potent anticancer activity against Human oral (KB), liver 24 (HepG), colon (Caco-2) and breast (MCF-7) cancer cell lines with an IC50 values of 8.0, 0.49, 0.70, 10.0 µg/mL, respectively.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

O OH H H H H H H HO OH

O HO HO OH 2.1 2.2 2.3

OH OH O OH O OH O HO OH

HO OH OH O O O HO O HO O

2.4 OH 2.5 OH 2.6 OH

HO OH HO OH OH O O HO OH HO HO O O O O OH OH 2.7 2.8

2.9 2.10

2.11

Figure 2.1 The chemical structures of okanin (2.1), friedelan (2.2), friedelanol (2.3), cymaroside (2.4), luteolin (2.5), apigenin (2.6), apigenin 7-O-glucoside (2.7), ctopiloyne (2.8), phenylheptatriyne 1 (2.9), phenylheptatriyne 2 (2.10), phenylheptatriyne 3 (2.11).

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

OH OH OH O OH OH HO HO O O OH O OH OH O OH HO O O O OH O OH OH HO O HO HO O OH OH OH OH 2.12 2.13 2.14

O OH OH OH OH O O O O HO O O O O O OH OH O OH O 2.15 2.16 2.17

Figure 2.2 Chemical structure of flavonoids or flavonoid glycosides isolated from Bidens spp. Isoquercitrin (2.12), Vitexin (2.13), Astragalin (2.14), 5,6,7,4’-tetramethoxylflavone (2.15), 5,3’,4’-trihydroxy-3,7- dimethoxyflavone (2.16), Quercetin (2.17).

2.1.2 Bidens biternata (Lour.) Merr. & Sherff

Bidens biternata (Lour.) Merr. & Sherff is a widespread weed and distributed all over the world. Table 2.1 shows some vernacular names of B. biternata. It is commonly known as Spanish needles because of its seeds were easy to stick into hair and clothing.25 The florets of B. biternata are composed of the white petal and yellow disc florets, so Chinese call it gold center silver plate. The stem of B. biternata is quadrangular, grooved and hairy and leaves are opposite with dentate margins.

Table 2.1 Vernacular names of B. biternata

Names Regions Jinzhanyinpan China Phutjom, Kerrai, Kathori, Kuro India Agedi, Ketul Indonesia Konchem Thailand Dipmal, Phutium Pakistan

As a TCM, B. biternata is used for detoxification, respiratory tract infections, blood stasis effect, acute appendicitis, sore throat, acute jaundice, hepatitis, gastroenteritis, malaria, 14

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff rheumatoid joint pain, topical cure boils, traumatic swellings, snakebite and pain.26 Similar to Bidens Pilosa L. and other Bidens spp., many traditional applications of B. biternata in difference area were reported (Table 2.2), including toothache, leprosy, fever, cough, asthma, liver infection, diabetes, toothache, cutaneous infections and many others. However, chemical constituent of B. biternata has not been well investigated as the B. Pilosa L. The literature displayed many compounds with different medicinal activities were isolated from the same genus. Besides, in the previous screen by our group, the ethanol extract of B. biternata present anti-proliferative activity. In this project, B. biternata was chosen for further chemical investigation by using NMR and MTS assay as a guide.

Table 2.2 Traditional applications of B. biternata.

Ailments Mode of applications reference Cutaneous infections Poultice of leaf is applied 27 Liver infections Decoction of leaves is used 28 Headache Bruised leaves are applied on forehead 27 Cold Decoction of the whole plant is given 29 Eye and ear complaints Juice of fresh leaves is used as eye and ear drops 30 Toothache Roots are chewed 31 Cough Infusion is given 32 Snakebite Fresh roots are made into a paste and given to drink 33 Pimples Leaves paste is applied 34 Wounds Leaves are rubbed as a hemostatic 27 Diabetes Decoction of leaves and root is given 27 Chronic dysentery Eczema Decoction of the whole plant is used 35

2.2 Analysis of Bidens biternata (Lour.) Merr. & Sherff extracts

The dried ethanol extract (5g) was pre-adsorbed onto C18 bonded silica gel using methanol

(MeOH) followed by evaporation. The crude extract was purified by C18 silica flash column, eluting with a 20% stepwise gradient from 10% MeOH/ 90% H2O to 100% MeOH (including 0.1% TFA). Each fraction was dried and analyzed by LC-MS and 1H NMR (Figure 2.3) to obtain crucial information about the chemistry of fractions.36,37

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

1 Figure 2.3 H NMR spectrum (800 MHz, DMSO-d6) of six fractions after C18 silica flash column. (Fraction 1) 10% MeOH/ 90% H2O fraction, (Fraction 2) 30% MeOH/ 70% H2O fraction, (Fraction 3) 50% MeOH/ 50% H2O fraction, (Fraction 4) 70% MeOH/ 30% H2O, (Fraction 5) 90% MeOH/ 10% H2O fraction, (Fraction 6) 100% MeOH fraction.

NMR fingerprints of the six fractions (Figure 2.3) implied that different types of compounds were enriched in each different fraction. 1H NMR spectra of fraction 1,2,3 and 4 displayed typical signals for sugar moieties between δH 2.80 and δH 5.50 indicative of carbohydrates.

Moreover, for fraction 4, 5 and 6 the strong proton signal at δH 1.24 represented aliphatic fatty compounds, interesting aromatic signals between δH 5.5 and δH 8.5 were also observed from these fractions.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

Figure 2.4 Effects of B. biternata fractionations on cell viabilities of different cancer cell lines as determined by MTS assay for 24h.(Fraction 1) 10% MeOH/ 0% H2O fraction, (Fraction 2) 30% MeOH/70% H2O fraction, (Fraction 3) 50% MeOH/50% H2O fraction, (Fraction 4) 70% MeOH/30% H2O, (Fraction 5) 90% MeOH/10% H2O fraction, (Fraction 6) 100% MeOH fraction. The cancer cells were exposed to B. biternata fractionations for 24h. The experiment was performed in triplicate in at least three independent experiments. * indicates results that are significantly different to the untreated control (p<0.01). ** indicates results that are significantly different to the untreated control (p<0.001)

The anti-proliferation activity of six fractions determined by MTS assay against to Caco-2 colorectal and SKOV3 ovarian cancer cell lines at a concentration of 0.3 mg/mL. The earlier fractions (Fr 1-3) showed minimal anti-proliferative activity against the two cell lines. Indeed, fraction 3 induced proliferation of Caco-2 cells above that of the untreated cells at that concentration. Later eluting fractions (Fr 4-6) showed good antiproliferative activity towards at least one of the tested cell lines. Fraction 4 showed significant inhibition to SKOV3 cell line although it induced an apparent (but not significant) promotion to Caco-2. Fraction 5 showed good antiproliferative activity against both Caco-2 and SKOV3 cell. Indeed, for the SKOV3, almost 100% inhibition was achieved in the MTS assay. Fraction 6 had a little inhibitory effect on the SKOV3 cell and the good inhibitory effect on Caco-2. Given its good activity and interesting 1H-NMR signals, fraction 5 was a target for further purification.

2.2.1 Purification strategies for the active fractions

The 1H-NMR spectrum of fraction 5 (Figure 2.3) indicated that the fraction contained a mixture of fatty acids and their derivatives as the major compounds. As fatty acids/derivatives are often regarded as a group of nuisance compounds with a variety of non-selective activities, 17

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff different chromatographic techniques were used to remove the fatty compounds and concentrate the minor components in the active fraction.

I. C18 reverse phase HPLC

The reverse phase C18 HPLC was firstly carried out for the purification. Isocratic HPLC conditions of 90% H2O/10% MeOH was eluted with a linear gradient from 10% MeOH/90%

H2O to 100% MeOH over 60 mins, all at a flow rate of 9 mL/min. Sixty fractions (60 X 1min) were collected (Figure 2.5), followed by 1H-NMR analysis fractions.

Figure 2.5 HPLC UV trace for (Fraction 5) 90% MeOH/ 10% H2O fraction. Timed fractions (1 min x 60) were collected from 1-60 min.

According to HPLC result and NMR fingerprint, the C18 column provided poor separation with the major fatty compounds co-eluting with the minor compounds of interest between 20 min to 28 min. In addition, the 1H NMR spectra of fractions 20-30 showed that fatty acids and their 1 derivatives (a characteristic CH2 signal at δH 1.24 in the H-NMR spectrum) were spread over all fractions. A large number of fatty acids and their derivatives cannot be removed and would cause problems for further purification. Therefore, a further chromatographic method was required to remove the fatty acids and their derivatives.

II. Sephadex LH-20 gel filtration

From our experience, Sephadex LH-20 gel filtration works well to remove fatty compounds.

Fractions 20-30 from the C18 reverse phase HPLC chromatogram were combined and further separated on Sephadex LH-20 eluting with MeOH and dichloromethane as the mobile phase (v/v, 1:1). Fourteen fractions were collected (50 mL per fraction) from the chromatogram.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

Fraction 5

Fraction 4

Fraction 3

Fraction 2

Figure 2.6 1H NMR spectrum (800 MHz, DMSO-d6) of Sephadex gel fractionation fraction 2 to 5. Isometric fractions (50 ml x 14) were collected.

1H NMR spectra of fractions 2 to 5 (Figure 2.6), indicated some separation of the components. However, fatty acids and their derivatives still co-eluted with other minor compounds. Fractions 2 and 5 displayed several broad aliphatic signals characteristic of fatty acid compounds, whilst fractions 3 and 4 contained broad aliphatic signals as well as some smallar signal at δH 5.0 – 8.0.

III. Normal phase silica gel chromatography and diol silica gel chromatography

Normal silica gel chromatography was also trialed in an attempt to remove fatty compounds from the active fractionations. As shown in Scheme 2.1, the dried sample (1.2039 g) was pre- adsorbed onto silica gel, then further purified by silica gel flash chromatography, eluting with a 20% stepwise gradient from 100% hexane to 100% EtOAc. Each fraction was dried and analyzed by 1H NMR to obtain important information about the effectiveness of the purification process.36,37

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

Crude

C18 flash chromatography

10%(MeOH) 30%(MeOH) 50%(MeOH) 70%(MeOH) 90%(MeOH) 100%(MeOH)

Silica 1.2039g chromatography

Hexane 30% EtOAc 50% EtOAc 70% EtOAc 100% EtOAc 0 g 0.2591g 0.0835g 0.1406g 0.1458g

Scheme 2.1 Small-scale extraction of B. biternata by C18 flash chromatograph and normal silica chromatography.

1 Figure 2.7 H NMR spectrum (800 MHz, DMSO-d6) of normal phase silica gel chromatography. (Fraction 1) 100% hexane fraction, (Fraction 2) 30% EtOAc/ 70% hexane fraction, (Fraction 3) 50% EtOAc/ 50% hexane fraction, (Fraction 4) 70% EtOAc/ 30% hexane, (Fraction 5) 100% EtOAc fraction.

The NMR data analysis (Figure 2.7) indicated that normal phase silica gel chromatography provided better separation of the components in comparison with previous techniques (Figure 2.5). Fractions 1 and 2 mainly contained fatty compounds with the 1H-NMR signals at the

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff upfield region, while fraction 3, 4 and 5 comprised compounds with downfield 1H-NMR signals in addition to upfield signals.

Figure 2.8 HPLC UV trace for (Fraction 3) 50% EtOAc/ 50% hexane fraction. Timed fractions (1 min x 60) were collected from 1-60 min.

The fraction from 20 to 25 min may contain minor compounds which had aromatic structures, whilst the fraction from 45 to 60 min contained lipophilic compounds. Furthermore, the 1H NMR proved that the fraction 45 to 60 min predominated with several aliphatic signals in the upfield region. In contrast, the fraction from 20 to 25 min contained a mixture of minor compounds with the fatty acid ester.

Figure 2.9 HPLC UV trace for (Fraction 4) 70% EtOAc/ 30% hexane fraction. Timed fractions (1 min x 60) were collected from 1-60 min.

The UV trace of fraction 3 (Figure 2.8) and the UV trace of fraction 4 (Figure 2.9) demonstrated that lipophilic materials could be separated by C18 column after normal silica gel fractionation. However, NMR fingerprint of fraction 4 after HPLC indicated the fatty acid ester mixed with minor compounds. The normal diol silica gel chromatography was applied to achieve better separation.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

In summary, the normal diol silica gel fractionation provided a better separation strategy to remove lipophilic compounds from the minor components in the active fraction. A purification strategy was therefore established which employs reverse phase C18 chromatography followed by diol silica gel chromatography, this will allow the removal of the abundant fatty compounds from the active fractions and targets minor compounds for isolation work.

2.3 Large-scale Extraction and Chemical Investigations of Bidens biternata (Lour.) Merr. & Sherff

The outline of the large-scale isolation of compounds from B. biternata was presented in in Scheme 2.2.

B. biternata (20 g)

C18 flash Chromatography MeOH/H2O/1% TFA

10% MeOH 30% MeOH 50% MeOH 70 % MeOH 90% MeOH 100% MeOH Fraction Fraction Fraction Fraction Fraction Fraction MTS (4.9 g) (3.3 g) (1.4 g) (1.3 g) (0.9 g) (0.3 g) Assay

DIOL Chromatography Hexane/EtOAc

Hexane 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% MTS EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc Assay HPLC HPLC

Compound 1 Compounds 2, 3, 4

Scheme 2.2 Large-scale extraction of B. biternata

The dried ethanol extract (20g in methanol) was pre-adsorbed onto C18 bonded silica gel. The crude extract then was purified by C18 silica flash column, eluting with a 20% stepwise gradient from 10% MeOH/ 90% H2O to 100% MeOH (including 0.1% TFA). Each fraction was dried and analyzed by 1H NMR. 90% fraction was selected for further purification based on their similar 1H-NMR spectra with that of the active fraction.

The dried 90% MeOH fraction (0.9g) was further purified by diol silica gel chromatography, eluting with a 10% stepwise gradient from pure hexane to 100% EtOAc, to afford 11 fractions. 1H-NMR analysis indicated that fractions 1-3 from diol column contained compounds of 22

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

interest. Further purification of fraction 1 and 3 by C18 reverse phase HPLC (eluting with linear gradient from 90% H2O/10% MeOH to 100% MeOH over 60 min, all at a flow rate of 4 mL/min) (Figure 2.11 & Figure 2.12) led to the isolation of compounds phenylheptatriyne (2.1, 4.4mg, 0.000088% dry wt), 7-phenyl-4,6-heptadiyn-2-ol (2.2, 0.6mg, 0.000012% dry wt), 5-Phenyl-2,4-pentadiyn-1-ol (2.3, 0.5mg, 0.00001%).

Figure 2.10 Structure of phenylheptatriyne (2.1), 7-phenyl-4,6-heptadiyn-2-ol (2.2), 5-Phenyl-2,4-pentadiyn-1- ol (2.3)

Figure 2.11 HPLC UV trace for (Fraction 3) 20% EtOAc/80% hexane fraction. Timed fractions (1 min x 60) were collected from 1-60 min.

Figure 2.12 HPLC UV trace for (Fraction 4) 30% EtOAc/70% hexane fraction. Timed fractions (1 min x 60) were collected from 1-60 min.

2.3.1 Structure determination of phenylheptatriyne (2.1)

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

The 1H NMR spectrum (Figure 2.13) for phenylheptatriyne (2.1) showed 4 signals (Table 2.1), one methyl singlet (H 2.08), three aromatic methine multiples (H 7.61, 7.50 and 7.43). The 13C NMR spectrum (Figure 2.14) and edited HSQC spectra revealed the presence of 13 carbons corresponding to one methyl and seven quaternary carbons. Analysis of the COSY and HSQC spectra established a mono-substituted benzene ring. The remaining carbon signals (c 58.3, 64.0, 67.5, 73.9, 75.7 and 81.1) were assigned to 3 sets of triple bonds. The assignment was confirmed by the HMBC correlation (Figure 2.16) from H-2’, H-6’ (H 7.61) to the triple bond carbon C-7 (c 75.7), and from H3-1 (H 2.08) to the triple bond carbons C-2, C-3, C-4, C-5, C-6 (c 73.9, 64.0, 67.5, 58.3, 81.1) respectively. Thus, the structure of phenylheptatriyne (2.1) was assigned as 2.1

1 Figure 2.13 H NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1)

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

13 Figure 2.14 C NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1)

2.1

Figure 2.15 COSY (—) and HMBC (→) correlations for phenylheptatriyne (2.1)

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

Figure 2.16 HMBC spectrum for phenylheptatriyne (2.1)

Table 2.3 NMR spectroscopic data of phenylheptatriyne (2.1) in DMSO-d6 (800MHz) at 25 ℃

Position 13C, type 1H, mult. (J in Hz) COSY HMBC 1 4.2, CH3 2.08, s 5, 3, 4, 2, 6 2 73.9, C 3 64.0, C 4 67.5, C 5 58.3, C 6 81.1, C 7 75.7, C 1’ 119.6, C 2’ 133.0, CH 7.61, m 3’, 5’ 7, 129.0 (3’, 5’), 4’, 6’ 3’ 129.0, CH 7.43, m 2’, 6’ 1’, 5’, 133.0 (2’, 6’) 4’ 130.5, CH 7.50, m 2’, 6’ 5’ 129.0, CH 7.43, m 2’, 6’ 1’, 3’, 133.0 (2’, 6’) 6’ 133.0, CH 7.61, m 3’, 5’ 7, 129.0 (3’, 5’), 2’, 4’ 13C data recorded by 1D 13C NMR

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

2.3.2 Structure determination of 7-phenyl-4,6-heptadiyn-2-ol (2.2)

7-phenyl-4,6-heptadiyn-2-ol (2.2) had similar 1H NMR spectrum to that of 2.1. The 1H NMR spectrum (Figure 2.17) for 7-phenyl-4,6-heptadiyn-2-ol (2.2) showed 7 signals (Table 2.2), including one methyl (H 1.14), one methylene (H 2.48), one aliphatic methine multiplet (H

3.82), and tree aromatic methine (H 7.53, 7.44 and 7.40) signals. HSQC data analysis linked carbon atoms to their directly attached protons. It also showed an exchangeable proton signal at H 4.95. Analysis of the COSY spectrum established two spin systems: a mono-substituted aromatic ring and 2-propanol. Three carbons (c 84.2, 65.8 and 74.6) were assigned to two sets of triple bonds between the aromatic ring and isopropanol functional groups. It was confirmed by the HMBC correlation (Figure 2.19) from H-2’/ H-6’ (H 7.53) to the triple bond carbon C-

7 (c 74.6), and from H-3 (H 2.48) to the triple bond carbons C-5 (c 65.8), also LRESIMS data showed as m/z 185.4 [M + H] +. Thus, the structure of 7-phenyl-4,6-heptadiyn-2-ol (2.2) was assigned as 2.2

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

1 Figure 2.17 H NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol (2.2)

2.2

Figure 2.18 COSY (—) and HMBC (→) correlations for 7-phenyl-4,6-heptadiyn-2-ol (2.2)

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

Figure 2.19 HMBC spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol (2.2)

o Table 2.4 NMR spectroscopic data of 7-phenyl-4,6-heptadiyn-2-ol (2.2) in DMSO-d6 (800MHz) at 25 C

Position 13C, type 1H, mult. (J in Hz) COSY HMBC 1 22.6, CH3 1.14, d (6.2) 2 2, 3 2 64.5, CH 3.82, m 1, 3 2-OH 4.95, d (4.7) 2 3 29.1, CH2 2.48a 2 1, 5 4 84.2, C 5 65.8, C 6 N 7 74.6, C 1’ 121.0, C 2’ 131.8, CH 7.53, m 3‘, 5‘ 7, 4’, 2‘, 6’ 3’ 128.9, CH 7.40, m 2‘, 6‘ 1’, 3‘, 5’ 4’ 128.9, CH 7.44, m 2’, 6’ 5’ 128.9, CH 7.40, m 2‘, 6‘ 1’, 3’, 5‘ 6’ 131.8, CH 7.53, m 3‘, 5‘ 7, 2’, 4', 6‘ 13C data recorded by 1D 13C NMR. N signal was not observed. a Signals were overlapping

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

2.3.3 Structure determination of 5-Phenyl-2,4-pentadiyn-1-ol (2.3)

5-Phenyl-2,4-pentadiyn-1-ol (2.3) had similar 1H NMR spectrums to 2.1. The 1H NMR spectrum for 5-Phenyl-2,4-pentadiyn-1-ol (2.3) showed 4 signals (Table 2.3) belonging to one methylene (H 4.26), tree aromatic methine (H 7.64, 7.52 and 7.44) protons. Analysis of the COSY and HSQC spectrum established two spin systems: mono-substituted aromatic ring H- 2’/H-3’/H-5’/H-6’ and an oxygenated methylene. In addition, an exchangeable proton signal was observed at H 5.57. MS and carbon NMR analysis were not carried out due to a small amount of the sample. By comparing the NMR data with literature,38 2.3 was determined as 5- Phenyl-2,4-pentadiyn-1-ol (2.3)

1 Figure 2.20 H NMR spectrum (800 MHz, DMSO-d6) for 5-phenyl-2,4-pentadiyn-1-ol (2.3)

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

Figure 2.21 COSY (—) and HMBC (→) correlations for 5-phenyl-2,4-pentadiyn-1-ol (2.3)

Figure 2.22 HMBC spectrum for 5-phenyl-2,4-pentadiyn-1-ol (2.3)

o Table 2.5 NMR spectroscopic data of 5-Phenyl-2,4-pentadiyn-1-ol (2.3) in DMSO-d6 (800MHz) at 25 C

Position 13C, type 1H, mult. (J in Hz) COSY HMBC 1 49.3, CH2 4.26, s 1-OH 2, 3 1-OH - 5.57, b 1 2 82.8, C - 3 69.0, C - 4 N - 5 77.6, C - 1’ 119.7, C - 2’ 132.5, CH 7.64, m 3‘, 5‘ 7, 6’ 3’ 128.8, CH 7.44, m 2‘, 6‘ 1’, 4’ 4’ 129.8, CH 7.52, m 2‘, 6‘ 5’ 128.8, CH 7.44, m 2‘, 6‘ 1’, 4’ 6’ 132.5, CH 7.64, m 3‘, 5‘ 7’, 2’ 31

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

13C data recorded by 2D spectrum. N signal was not observed. a Signals were overlapping. b broad peak

2.4 Anti-proliferation activity of pure compounds from Bidens biternata (Lour.) Merr. & Sherff

Three different pure compounds 2.1-2.3 were tested in vitro for growth inhibitory activity against Caco-2 and SKOV3 cell lines. However, only small quantity of compounds was isolated. Therefore, instead of determining IC50 values by screening across a range of concentrations, spot testing was performed to examine the anti-proliferation activity. The resulted suggested that all three compounds showed anticancer activity against Caco-2 cells with phenylheptatriyne (2.1) displaying 39% inhibition at a concentration of 0.171 mg/mL; 7- phenyl-4,6-heptadiyn-2-ol (2.2) displaying 77% inhibition at 0.067 mg/mL; 5-Phenyl-2,4- pentadiyn-1-ol (2.3) displaying 42% inhibition at 0.167 mg/mL.

In conclusion, the anti-proliferative activity of B. biternata was mainly concentrated on non- polar fraction after C18 flash chromatography. Three phenyltriynes type of compounds were isolated and structure were identified by 2D NMR. All three compounds possessed anticancer activity against the Caco-2 cell line and the SKOV3 cell line. Given limited quantity of samples isolated, anti-proliferative activity was evaluated at a single dose. For the further study, compound 2.1, 2.2 and 2.3 can be obtained by synthetic way. So, the better anticancer activity profile can be established. Moreover, the fraction 4 (70% MeOH/30% H2O) showed activity to only the SKOV3 cancer cell line. and fraction 3 (50% MeOH/50% H2O) showed good activity to proliferate the Caco-2 cancer line. No chemical investigation on these two fractions in this project, because of the limit time and out of the objective. However, the chemical investigation of this two fractions in future may achieve more novel compounds.

2.5 References

1. Karis, P.; Ryding, O.; Bremer, K., Asteraceae Cladistics and Classification. K. Bremer, Ed 1994, 559-569.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

2. Christensen, L. P.; Lam, J., Acetylenes and related compounds in Heliantheae. Phytochemistry 1991, 30 (1), 11-49.

3. Jensen, S. L.; Sﬁrensen, A., Studies Related to Naturally Occurring Acetylene. Acta Chemica Scandinavica 1961, 15 (9), 1885-1891.

4. Chiang, Y.-M.; Chang, C. L.-T.; Chang, S.-L.; Yang, W.-C.; Shyur, L.-F., Cytopiloyne, a novel polyacetylenic glucoside from Bidens pilosa, functions as a T helper cell modulator. Journal of Ethnopharmacology 2007, 110 (3), 532-538.

5. Brandão, M.; Krettli, A.; Soares, L.; Nery, C.; Marinuzzi, H., Antimalarial activity of extracts and fractions from Bidens pilosa and other Bidens species (Asteraceae) correlated with the presence of acetylene and flavonoid compounds. Journal of Ethnopharmacology 1997, 57 (2), 131-138.

6. Ubillas, R. P.; Mendez, C. D.; Jolad, S. D.; Luo, J.; King, S. R.; Carlson, T. J.; Fort, D. M., Antihyperglycemic acetylenic glucosides from Bidens pilosa. Planta Medica 2000, 66 (01), 82-83.

7. Alarcon‐Aguilar, F.; Roman‐Ramos, R.; Flores‐Saenz, J.; Aguirre‐Garcia, F., Investigation on the hypoglycaemic effects of extracts of four Mexican medicinal plants in normal and Alloxan‐diabetic mice. Phytotherapy Research 2002, 16 (4), 383-386.

8. Dimo, T.; Azay, J.; Tan, P. V.; Pellecuer, J.; Cros, G.; Bopelet, M.; Serrano, J. J., Effects of the aqueous and methylene chloride extracts of Bidens pilosa leaf on fructose- hypertensive rats. Journal of Ethnopharmacology 2001, 76 (3), 215-221.

9. Leandre, K. K.; Claude, J.; Kouakou, A.; Jacques, D. Y.; Flavien, T.; Etienne, E. E., β- Adrenomimetic actions in the hypotension and vasodilatation induced by a chromatographic active fraction from Bidens pilosa L.(Asteraceae) in mammals. Current Bioactive Compounds 2008, 4 (1), 1-5.

10. Alvarez, A.; Pomar, F.; Sevilla, M.; Montero, M., Gastric antisecretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiata Schult. Bip. Journal of Ethnopharmacology 1999, 67 (3), 333-340.

11. Yuan, L.-P.; Chen, F.-H.; Ling, L.; Dou, P.-F.; Bo, H.; Zhong, M.-M.; Xia, L.-J., Protective effects of total flavonoids of Bidens pilosa L.(TFB) on animal liver injury and liver fibrosis. Journal of Ethnopharmacology 2008, 116 (3), 539-546.

12. Sundararajan, P.; Dey, A.; Smith, A.; Doss, A. G.; Rajappan, M.; Natarajan, S., Studies of anticancer and antipyretic activity of Bidens pilosa whole plant. African Health Sciences 2006, 6 (1), 27-30.

13. Arıdogan, B. C., Immunomodulatory effects of phytocompounds. Modern Phytomedicine: Turning Medicinal Plants into Drugs 2006.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

14. Pereira, R. L.; Ibrahim, T.; Lucchetti, L.; da Silva, A. J. R.; de Moraes, V. L. G., Immunosuppressive and anti-inflammatory effects of methanolic extract and the polyacetylene isolated from Bidens pilosa L. Immunopharmacology 1999, 43 (1), 31-37.

15. Horiuchi, M.; Seyama, Y., Improvement of the antiinflammatory and antiallergic activity of Bidens pilosa L. var. radiata SCHERFF treated with enzyme (Cellulosine). Journal of Health Science 2008, 54 (3), 294-301.

16. Chang, J.-S.; Chiang, L.-C.; Chen, C.-C.; Liu, L.-T.; Wang, K.-C.; Lin, C.-C., Antileukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb. The American Journal of Chinese Medicine 2001, 29 (02), 303-312.

17. Rojas, J. J.; Ochoa, V. J.; Ocampo, S. A.; Muñoz, J. F., Screening for antimicrobial activity of ten medicinal plants used in Colombian folkloric medicine: A possible alternative in the treatment of non-nosocomial infections. BMC Complementary and Alternative Medicine 2006, 6 (1), 2.

18. Rabe, T.; Van Staden, J., Antibacterial activity of South African plants used for medicinal purposes. Journal of Ethnopharmacology 1997, 56 (1), 81-87.

19. Kviecinski, M. R.; Felipe, K. B.; Schoenfelder, T.; de Lemos Wiese, L. P.; Rossi, M. H.; Gonçalez, E.; Felicio, J. D. a.; Wilhelm Filho, D.; Pedrosa, R. C., Study of the antitumor potential of Bidens pilosa (Asteraceae) used in Brazilian folk medicine. Journal of Ethnopharmacology 2008, 117 (1), 69-75.

20. Fujiwara, N.; Kobayashi, K., Macrophages in inflammation. Current Drug Targets- Inflammation & Allergy 2005, 4 (3), 281-286.

21. Korhonen, R.; Lahti, A.; Kankaanranta, H.; Moilanen, E., Nitric oxide production and signaling in inflammation. Current Drug Targets-Inflammation & Allergy 2005, 4 (4), 471- 479.

22. Kil, J.-S.; Son, Y.; Cheong, Y.-K.; Kim, N.-H.; Jeong, H. J.; Kwon, J.-W.; Lee, E.-J.; Kwon, T.-O.; Chung, H.-T.; Pae, H.-O., Okanin, a chalcone found in the genus Bidens, and 3- penten-2-one inhibit inducible nitric oxide synthase expression via heme oxygenase-1 induction in RAW264. 7 macrophages activated with lipopolysaccharide. Journal of Clinical Biochemistry and Nutrition 2011, 50 (1), 53-58.

23. Geissberger, P.; Séquin, U., Constituents of Bidens pilosa L.: do the components found so far explain the use of this plant in traditional medicine? Acta Tropica 1991, 48 (4), 251-261.

24. Kumari, P.; Misra, K.; Sisodia, B. S.; Faridi, U.; Srivastava, S.; Luqman, S.; Darokar, M. P.; Negi, A. S.; Gupta, M. M.; Singh, S. C., A promising anticancer and antimalarial component from the leaves of Bidens pilosa. Planta Medica 2009, 75 (01), 59-61.

25. Bhatt, J.; Singh, J.; Singh, S.; Tripathi, R.; Kohli, R., Invasive Alien Plants An Ecological Appraisal for the Indian Subcontinent. CABI: 2011; Vol. 1.

Chemical investigations of traditional Chinese medicine (TCM) Bidens. biternata (Lour.) Merr. & Sherff

26. Zhu, S.; Yilin, C.; Yousheng, C.; Yourun, L.; Shangwu, L.; Xuejun, G.; Tiangang, G.; Shixin, Z.; Ying, L.; Qiner, Y., Asteraceae (Compositae). 2011.

27. Panda, H., Medicinal plants cultivation & their uses. Asia Pacific Business Press Inc.: 2002.

28. Nanda, Y.; Singson, N.; Rao, A. N., Ethnomedicinal plants of Thadou tribe of Manipur (India)-1. Pleione 2013, 7 (1), 138-145.

29. Shah, S.; Ram, J.; Pala, N.; Tripathi, P.; Kumar, M., Medicinal plant wealth of oak dominated forests in Nainital catchment area of Uttarakhand. Academia Journal of Medicinal Plants 2014, 2 (1), 6-13.

30. Ghosh, G.; Ghosh, D.; Melkania, U.; Majumdar, U., Traditional medicinal plants used by the'Adi, Idu and'Khamba tribes of Dehang-Debang biosphere reserve in Arunachal Pradesh. International Journal of Agriculture, Environment & Biotechnology 2014, 7 (1), 165.

31. Priyadi, H.; Takao, G.; Rahmawati, I.; Supriyanto, B.; Nursal, W. I.; Rahman, I., Five Hundred Plant Species in Gunung Halimun Salak National Park, West Java: A Checklist Including Sundanese Names, Distribution, and Use. Cifor: 2010.

32. Bhat, J. A.; Kumar, M.; Bussmann, R. W., Ecological status and traditional knowledge of medicinal plants in Kedarnath Wildlife Sanctuary of Garhwal Himalaya, India. Journal of Ethnobiology and Ethnomedicine 2013, 9 (1), 1.

33. Kadel, C.; Jain, A. K., Folklore claims on snakebite among some tribal communities of Central India. 2008.

34. Kala, C. P., Current status of medicinal plants used by traditional Vaidyas in Uttaranchal state of India. 2005.

35. Zahara, K.; Bibi, Y.; Tabassum, S.; Bashir, T.; Haider, S.; Araa, A.; Ajmal, M., A review on pharmacological properties of Bidens biternata: A potential nutraceutical. Asian Pacific Journal of Tropical Disease 2015, 5 (8), 595-599.

36. Khokhar, S.; Pierens, G. K.; Hooper, J. N.; Ekins, M. G.; Feng, Y.; Davis, R. A., Rhodocomatulin-Type Anthraquinones from the Australian Marine Invertebrates Clathria hirsuta and Comatula rotalaria. Journal of Natural Products 2016, 79(4), 946-953

37. Feng, Y.; Davis, R. A.; Sykes, M.; Avery, V. M.; Camp, D.; Quinn, R. J., Antitrypanosomal cyclic polyketide peroxides from the Australian marine sponge Plakortis sp. Journal of Natural Products 2010, 73 (4), 716-719.

38. Bowling, N. P.; Burrmann, N. J.; Halter, R. J.; Hodges, J. A.; McMahon, R. J., Synthesis of Simple Diynals, Diynones, Their Hydrazones, and Diazo Compounds: Precursors to a Family of Dialkynyl Carbenes ( R1 C C C C C R2 ). The Journal of Organic Chemistry 2010, 75 (19), 6382-6390.

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

Chapter 3: Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

3.1 Introduction

Austrobuxusin spp. belong to the plant family of Picrodendraceae. More than 36 genera are described worldwide for this particular family. The genus Austrobuxus is endemic to Australia. Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw is a rare subtropical rainforest tree commonly known as pink cherry.1 This species has been poorly investigated for any therapeutic properties, with little scientific reports related to its chemical components and associated biological activity. One recent paper from our group reported the isolation of austrobuxusins A-D, structurally complex picrotoxane terpenoids from A. swainii.2

Picrotoxane and related natural products have been reported from plant genera Picrodendron.3- 6 Dendrobium,7-14 Baccaurea,15 and Coriaria.16-17 Picrotoxane terpenoids possess a complex chemical structure with a spiro γ-butyrolactone skeleton. A literature review suggested that the terpenoid components have potent inhibitory activity against GABA receptors of mammals and insects,18-19 anti-microbial activity against Colletotrichum gloeosporioides15 and neurotoxicity.20

Given the chemical complexity and biological activities and cytotoxicities of the picrotoxanes, I was attracted to further research on Austrobuxus swanii and its potential for anti-cancer activity. My objective was to 1) identify and purify potential new/novel austrobuxusin analogue. 2) isolate large quantity of known austrobuxusins for anticancer activity testing.

3.2 Chemical analysis of Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw extracts

The air-dried and ground leaves of Austrobuxus swanii (5g) were extracted with n-hexane followed by DCM (dichloromethane) and MeOH. The hexane extract (0.025g) mainly contained fatty acids and was not chosen for further investigation. The NMR analysis (Figure 3.1) of the DCM (0.134g) and MeOH (1.202g) extraction showed that MeOH extract contained characteristic NMR signals of austrobuxusins at H 2.99, 3.17, 3.20, 3.63, 4.04, 4.72. Furthermore, the MeOH extract appeared to contain a higher concentration of the

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw austrobuxusins than DCM extract. The anti-proliferation activity of the three extracts were determined by MTS assay anti-proliferative against Caco-2 and SKOV3 cell lines at a concentration of 0.3 mg/mL. The result (Figure 3.2) indicated that the three extracts all showed good anti-proliferation activity against SKOV 3 cell line, but a little inhibition effect on Caco- 2 cell line. The MeOH extract was chosen for further isolation work.

Fraction 2

Fraction 1

1 Figure 3.1 H NMR spectrum (800 MHz, DMSO-d6) of DCM and MeOH extract. Fraction 1: DCM extract, Fraction 2: MeOH extract.

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

Figure 3.2 Anti-proliferative activity of plant extracts and untreated controls against Caco-2 and SKOV3 cancer cell lines as determined by MTS assay for 24 h. NC: negative control, Fraction 1: hexane extract, Fraction 2: DCM extract Fraction 3: MeOH extract. Results were expressed as mean ± SD as a percentage of vehicle control. The experiment was performed in triplicate in at least three independent experiments. * indicates results that are significantly different to the untreated control (p<0.01). ** indicates results that are significantly different to the untreated control (p<0.001)

3.3 Large-scale chemical investigation of Austrobuxus swanii (Beuzev. & C.T.White) Airy Shaw extracts

Austrobuxus swanii was collected from Queensland, Australia. The air-dried and ground leaves (20.56g) were extracted with n-hexane, followed by DCM and MeOH (Scheme 3.1). All extracts were dark brown colored, and a portion of three extracts were subjected to in vitro anti-proliferation assay. According to the 1H NMR spectrum and anti-proliferation profile, the

MeOH extract was subjected to NMR-guided fractionation using semi-preparative C18 HPLC with Betasil C18 column. Isocratic HPLC conditions of MeOH/H2O/TFA (10:90:0.1) were used to eluted with a linear gradient to MeOH/H2O/TFA (100:0:0.1) over 60 mins, all at a flow rate of 9 mL/min. Sixty fractions (60 x 1min) were collected (Figure 3.3) and NMR fingerprint was used to check the purity of these fractions. According to the NMR fingerprint, fractions 29 and 30 were purified by Luna C18 column at flow rate of 4 mL/min eluting using a linear 1 gradient from MeOH/H2O/TFA (75:45:0.1) to MeOH/H2O/TFA (45:55:0.1). H-NMR analysis of the fractions led to the isolation of a new compound, austrobuxusin E (3.1) and a known compound, austrobuxusin A (3.2). This is the first time that austrobuxusin E has been reported in A. swanii extracts. Further purification of fraction 26 by Luna C18 column at flow rate of 4 mL/min eluting using a linear gradient from MeOH/H2O/TFA (30:70:1) to MeOH/H2O/TFA 38

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

(40:60:1) yielded two more known compounds, austrobuxusin B (3.3) and austrobuxusin C

(3.4). Further purification of fraction 32 by Luna C18 column at a flow rate of 4 mL/min eluted with a linear gradient from MeOH/H2O/TFA (45:55:1) to MeOH/H2O/TFA (45:55:1) yielded one more known compound, austrobuxusin D (3.5).

Scheme 3.1 Large-scale extraction of A. swanii

Figure 3.3 HPLC UV trace for MeOH extract of A. swanii. Timed fractions (1 min x 60) were collected from 1- 60 min.

Five austrobuxusins were isolated, including the new compound austrobuxusin E (3.1, 17.1 mg, 0.52% dry wt), along with four known compounds, namely austrobuxusin A (3.2, 3.9 mg, 0.12% dry wt), austrobuxusin B (3.3, 12.1 mg, 0.37% dry wt), austrobuxusin C (3.4, 12.9 mg, 0.39% dry wt), austrobuxusin D (3.5, 3.9 mg, 0.12% dry wt).

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

Figure 3.4 The structure of the known compound 3.1, 3.2, 3.3, 3.4, 3.5

3.4 Structure elucidation of austrobuxusin E (3.1)

The newly detected compound, austrobuxusin E (3.1), was isolated as a colorless powder with an [α]D value of + 21.6 (MeOH, c 2.5). The molecular formula of the compound was + determined as C25H32O13 based on the (+)- HRESIMS data (m/z 563.1727 [M + Na] ). IR absorptions at 3370 (br) and 1766 cm-1 indicated the presence of hydroxyl and carbonyl functionalities. The 1H NMR (Figure 3.5) and HSQC spectrum of austrobuxusin E (3.1) showed signals due to three methyl protons (H 1.82, 1.41 and 0.91), two methylenes (H 4.84 and 3.64/3.41) and 16 methines. In addition, signals at H 5.01, 4.58, 4.27, 4.26, 4.21(three signals overlap) indicated the existence of seven oxygenated methines, characteristic of a sugar moiety. The 13C NMR spectra (Figure 3.6) and HSQC spectra revealed the presence of 25

3 carbons corresponding to 16 methines, 3 methyls, 2 methylenes (including one sp CH2 at c

2 61.0 and one sp CH2 at c 112.3) and six quaternary carbons. Six quaternary carbons included two carbonyl carbons (c 174.0 and 174.2), one sp2 carbon (c 140.0) and four aliphatic sp3 carbons (c 54.5, 74.1 and 101.1). A series of sugar signals were displayed in the 1H NMR

13 spectrum (Figure 3.5) between H 2.85 -4.26. The C NMR spectrum (Figure 3.6) indicated

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw the presence of a glucose unit (a) due to a set of carbon signals at c 103.0, 77.4, 77.1, 73.7, 69.9 and 61.0.

1 Figure 3.5 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

13 Figure 3.6 C NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

In addition to the glucose moiety (a), detailed analysis of the COSY spectrum established the

O O following spin systems: H3C CH CH (b), CH CH CH CH (c), H2C CH CH3 (d),

CH CH (e). The connectivity between substructures a and b through an oxygen atom was established by the HMBC correlation (Figure 3.7) from oxygenated methine proton (H 4.26) in a to carbon at c 76.5 in b. Additional HMBC correlations were observed from the methine proton (H 4.21) in b to a carbonyl carbon at c 174.2 to form big fragment f. The correlations from methylene proton (H 4.84) and methyl proton (H 1.82) in d to carbon at c 47.1 in c indicated the link between d and c. Moreover, the HMBC correlations between oxygenated methine proton (H 5.01) and methine proton (H 3.09) in c and carbon at c 174.0 indicated the attachment of -O-CO- groups to substructure c. The connectivity between c and e through an oxygenated quaternary carbon (c 74.1) were established by the HMBC correlations from oxygenated methine protons (H 3.76, 4.21) in e and the methine proton (H 3.09) in c to the same quaternary carbon (Figure 3.8). The formation of fragment g was determined by the 42

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

HMBC correlations from a methyl group (H 0.91), the oxygenated methine proton (H 4.27) in b, oxygenated methine protons (H 3.76, 4.21) in e to quaternary carbon (c 54.5). Finally, the connection between the substructures g and f through the formation of two five-membered ring systems were established by the HMBC correlations: from the methyl proton (H 0.91) and the oxygenated methine proton (H 4.27, 4.21) in g to quaternary carbon at c 101.1, and from the oxygenated methine proton (H 4.21) in g to the quaternary carbon at c 84.9. The structure of austrobuxin E was therefore assigned as 3.1 in figure 3.4.

HO OH HO OH O HO O O O HO O O O O HO a O b HO f

O e O O O O O O O c O O c O O O

g d

HO OH HO OH 2' 3' O O 1' HO O 17 HO O 4' 16 O O O 12 O 5' 18 14 HO 13 O HO 6' 19 O O O 7 6 11 f O O 1 2 O OH O 15 O OH 3 5 4

9 8 10 g 3.1

Figure 3.7 COSY (—) and HMBC (→) correlations for austrobuxusin E (3.1)

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

Figure 3.8 HMBC spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Table 3.1 NMR spectroscopic data of austrobuxusin E (3.1) in DMSO-d6 (800MHz) at 25 ℃

Position 13C, type 1H, mult. (J in Hz) COSY HMBC ROESY 1 54.5, C - - - - 2 84.3, CH 4.27, d (3.9) 3 1, 3, 7, 13, 14 3, 7, 9 3 80.3, CH 5.01, dd (4.4) 2, 4 15 2 4 47.1, CH 3.36, brs 3, 5 a 5 49.1, CH 3.09, m 4 1, 3, 4, 6, 11, 15 2w, 10, 11, 6 74.1, C - - - -

7 21.7, CH3 0.91, s 1, 2, 6, 13 2, 9 8 140.0, C - - - -

9 112.3, CH2 4.84, d (8.9) 10 4, 10 2, 7, 10 10 22.8, CH3 1.82, s 9 4, 8, 9 4, 5, 9 11 63.3, CH 3.76, d (2.6) 12 1, 6, 13W 5, 12 12 57.0, CH 4.21a 11 1, 6, 13, 14 11, 14, 16, 19 13 101.1, C - - - - 14 84.9, CH 4.58, brs - 12, 13, 17, 18 16, 19w 15 174.0, C - - - - 16 54.5, CH 2.78, d (2.3) 18 13, 17, 18, 19W 14, 18, 19 17 174.2, C - - - - 18 76.5, CH 4.21a 19 14, 16, 17 11, 14, 16, 19

19 19.5, CH3 1.41, d (6.4) 18 16, 18 14, 16 44

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

1’ 103.0, CH 4.26 d (7.9) 2’ 18 - 2’ 73.7, CH 2.85, td (8.4, 5.8) 3’, 1’, 3’, 1’ - 2’-OH 4.77, (5.9) 2’ -OH - - 3’ 77.4, CH 3.14, m 2’ - - 3’-OH 4.97, d (4.9) 3’ - - 4’ 69.9, CH 3.01, m 4’-OH 5’ - 4’-OH 4.93, d (5.4) 4’ - - 5’ 77.1, CH 4.21a - - -

6’a 61.0, CH2 3.41 6’b 11 - 6’b 3.64, m 6'a - - 6’-OH 6’a,6’b - - N signal was not observed. a Signals were overlapping, w signal was weak

ROESY experimental data (Table 3.1) indicated that the relative configuration of austrobuxusin was the same as those of the known compounds austrobuxusin A-D2. The

ROESY correlations from H-2 to CH3-7 and H-9 indicated that the H-3, the methyl group at C-1 and the isopropenyl group at C-4 were on the same side. The ROESY correlations between H-5 and H-11 suggested that the bicycle [4, 3, 0] nonane ring system was cis-fused. All this showed that the configuration at C-2 and C-14 were as same as the austrobuxusins A (3.2). The relative configuration of C-14 has been included in the structure 3.1 in Figure 3.4. It is to be noted that the absolute configuration of the sugar moiety is yet to be determined.

Austrobuxusin A (3.2, 3.9 mg, 0.12% dry wt), austrobuxusin B (3.3, 12.1 mg, 0.37% dry wt), austrobuxusin C (3.4, 12.9 mg, 0.39% dry wt), austrobuxusin D (3.5, 3.9 mg, 0.12% dry wt) were also isolated. All four compounds were identified on the basis on the analysis of spectroscopic and spectrometric and by comparison with literature values.2

3.5 Anti-proliferation activity of pure compounds from Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw extracts

Compounds 3.1-3.5 were all tested in vitro for growth inhibitory activity against Caco-2 cell line and SKOV 3 cell line at a concentration of 166 M. Anti-proliferative activity was evaluated by the MTS assay. All five pure compounds showed inhibitory effect on Caco-2 cells only and did not significantly affect SKOV3 proliferation. Austrobuxusin C (3.5) showed the greatest inhibitory effect, inhibiting proliferation by approximately 89% inhibition on the Caco-2 cell growth (compared to the untreated control). Austrobuxusin B (3.3) displayed the lowest inhibitory effects with inhibition of approximately 53% of the Caco-2 cell growth

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw recorded. The other three pure had variable inhibition of Caco-2 cells proliferation, with 71% (3.1), 60% (3.4), 68% inhibition to Caco-2 cells respectively.

Interestingly, A. swanii extracts were good inhibitors of SKOV3, but the pure compound only presented low inhibition effect on Caco-2. There were two hypotheses based on this result. 1) There were other compounds in MeOH extraction responsible for the anti-proliferative activity of three extracts against SKOV3 cells. According to the process presented in 3.3, all five austrobuxusin compounds isolated from four fractions (26, 29, 30 and 32) after HPLC fractionation. However, the UV trace of HPLC (Figure 3.3) indicated that more compounds could be isolated from other fractions. Because of the limit time of this project, not all fractions were fully investigated. So, further study could be focus on other fractions for more compound, which potentially inhibited the SKOV3 cancer cell. 2) There were other compounds which may have cooperated with compound 3.1-3.5 to enhance the anti-proliferative effects of these compounds on SKOV3. Combinational therapies often enhanced efficacy of the cancer treatment. In combinations, the individual components, which targeted different pathways, may act in synergistic or additive manner. For instance, The high antioxidant agent could induced the upregulation of Nrf2 and nucleus translocation21. So, drugs with a high anti-oxidant activity and cancer drug(s) often be combined together to achieve a better cancer treatment. Same action may be showed in the extract caused the activity increasing of extract. For further study, more compound can be isolated and the anti-proliferative activity can be evaluated by MTS assay. If any compounds showed significant inhibition, many technologies such as cyclin kinase inhibition assays, caspase assays, microscope studies can be used to determine whether the compound had cytostatic activity or cytotoxic activity.

A new austrobuxusin E (3.1) was isolated in the project. The absolute configuration of this compound was not determined because of the limited time. GC-MS had been tried to identify the sugar enantiomers after conversion of D and L sugars to chiral derivatives. The derivatives of standard D-gluscose had earlier retention time then L-gluscose. However, GC-MS data of the D-glucose product and L-glucose product did not show significant difference. It was highly possible that the reaction was not successful. In the future, three different methods can be used to determine the configuration of the new austrobuxusin. Firstly, the reaction of the sugar chiral derivatives can be repeated to achieve the right product. Secondly, more austrobuxusin E can be isolated. Then the [α]D value of the sugar moiety isoalted from the acid hydrolysis can be

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw measured to determine the full stereochemical configuarion. At last, the crystal of the new austrobuxusin was achieved during the isolation work. However, the quality of the austrobuxusin E was not good enough to achieve crystal structrue. Different conditions can be used for the crystallization to produce high quality crystals.

In conclusion, the main purpose of this plant was achieved, five picrotaxane type of compounds, austrobuxusins A-E (3.1-3.5) were isolated from the Australian endemic plant Austrobuxus swanii. The chemical structure of the new austrobuxusin analogue 3.1 was elucidated by the comprehensive analysis of NMR and MS spectroscopic data. Austrobuxusins A-D showed moderate activity against Caco-2 cell line. It is speculated that more compounds are present in the MeOH extract which may be responsible or assistant for its activity against SKOV3 and possibly Caco-2 cell lines.

Log concentration of Austrobuxusin D

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

Log concentration of Austrobuxusin D

Figure 3.9 Effects of austrobuxusins A to E on cell mortality of Caco-2 cell line as determined by MTS assay for 24 h. A: austrobuxusin A (3.2), B (3.3) and C (3.4); B: austrobuxusin D (3.5); C: austrobuxusin E (3.1). Results were expressed as mean ± SD as a percentage of vehicle control. The experiment was performed in triplicate in at least three independent experiments. * indicates results that are significantly different to the untreated control (p<0.01). ** indicates results that are significantly different to the untreated control (p<0.001)

3.6 References

1. Floyd, A., Rainforest trees of mainland south-eastern Australia. 2008.

2. Demirkiran, O.; Campitelli, M.; Wang, C.; Feng, Y., New picrotoxane terpenoids, austrobuxusin AD, from the Australian endemic plant Austrobuxus swanii. Tetrahedron 2016, 72 (51), 8400-8405.

3. Koike, K.; Suzuki, Y.; Ohmoto, T., Picrotoxane terpenoids, picrodendrins S and T, from Picrodendron baccatum. Phytochemistry 1994, 35 (3), 701-704.

4. Suzuki, Y.; Koike, K.; Ohmoto, T., Eight picrotoxane terpenoids, picrodendrins KR, from Picrodendron baccatum. Phytochemistry 1992, 31 (6), 2059-2064.

5. Nagahisa, M.; Koike, K.; Narita, M.; Ohmoto, T., Novel picrotoxene norditerpene lactones from Picrodendron baccatum. Tetrahedron 1994, 50 (37), 10859-10866.

6. Koike, K.; Ohmoto, T.; Kawai, T.; Sato, T., Picrotoxane terpenoids from Picrodendron baccatum. Phytochemistry 1991, 30 (10), 3353-3356.

7. Zhang, X.; Tu, F.-J.; Yu, H.-Y.; Wang, N.-L.; Wang, Z.; Yao, X.-S., Copacamphane, picrotoxane and cyclocopacamphane sesquiterpenes from Dendrobium nobile. Chemical and Pharmaceutical Bulletin 2008, 56 (6), 854-857.

8. Zhao, C.; Liu, Q.; Halaweish, F.; Shao, B.; Ye, Y.; Zhao, W., Copacamphane, Picrotoxane, and Alloaromadendrane Sesquiterpene Glycosides and Phenolic Glycosides from Dendrobium moniliforme. Journal of Natural Products 2003, 66 (8), 1140-1143. 48

Chemical investigations of Austrobuxus swanii (Beuzev. & C.T. White) Airy Shaw

9. Qin, X.-D.; Qu, Y.; Ning, L.; Liu, J.-K.; Fan, S.-K., A new picrotoxane-type sesquiterpene from Dendrobium findlayanum. Journal of Asian Natural Products Research 2011, 13 (11), 1047-1050.

10. Zhang, X.; Liu, H. W.; Gao, H.; Han, H. Y.; Wang, N. L.; Wu, H. M.; Yao, X. S.; Wang, Z., Nine new sesquiterpenes from Dendrobium nobile. Helvetica Chimica Acta 2007, 90 (12), 2386-2394.

11. Shengjie, Y.; Lam, Y.; Chai, J., Evolution of liquid-bond strength in powder injection moulding compact during thermal debinding: numerical simulation. Modelling and Simulation in Materials Science and Engineering 2004, 12 (2), 311.

12. Zhao, W.; Ye, Q.; Dai, J.; Martin, M.-T.; Zhu, J., Allo-aromadendrane-and picrotoxane-type sesquiterpenes from Dendrobium moniliforme. Planta Medica 2003, 69 (12), 1136-1140.

13. Ye, Q.; Qin, G.; Zhao, W., Immunomodulatory sesquiterpene glycosides from Dendrobium nobile. Phytochemistry 2002, 61 (8), 885-890.

14. Yang, M.; Chen, L.-J.; Zhang, Y.; Chen, Y.-G., Two new picrotoxane-type sesquiterpenoid lactones from Dendrobium williamsonii. Journal of Asian Natural Products Research 2017, 1-5.

15. Pan, Z.-H.; Ning, D.-S.; Huang, S.-S.; Wu, Y.-F.; Ding, T.; Luo, L., A new picrotoxane sesquiterpene from the berries of Baccaurea ramiflora with antifungal activity against Colletotrichum gloeosporioides. Natural Product Research 2015, 29 (14), 1323-1327.

16. Shen, Y.-H.; Li, S.-H.; Li, R.-T.; Han, Q.-B.; Zhao, Q.-S.; Liang, L.; Sun, H.-D.; Lu, Y.; Cao, P.; Zheng, Q.-T., Coriatone and Corianlactone, Two Novel Sesquiterpenes from Coriaria nepalensis. Organic Letters 2004, 6 (10), 1593-1595.

17. Wang, Y.-Y.; Tian, J.-M.; Zhang, C.-C.; Luo, B.; Gao, J.-M., Picrotoxane Sesquiterpene Glycosides and a Coumarin Derivative from Coriaria nepalensis and Their Neurotrophic Activity. Molecules 2016, 21 (10), 1344.

18. Ozoe, Y.; Akamatsu, M.; Higata, T.; Ikeda, I.; Mochida, K.; Koike, K.; Ohmoto, T.; Nikaido, T., Picrodendrin and related terpenoid antagonists reveal structural differences between ionotropic GABA receptors of mammals and insects. Bioorganic & Medicinal Chemistry 1998, 6 (4), 481-492.

19. Schmidt, T. J.; Gurrath, M.; Ozoe, Y., Structure–activity relationships of seco- prezizaane and picrotoxane/picrodendrane terpenoids by Quasar receptor-surface modeling. Bioorganic & Medicinal Chemistry 2004, 12 (15), 4159-4167.

20. Larsen, L.; Joyce, N. I.; Sansom, C. E.; Cooney, J. M.; Jensen, D. J.; Perry, N. B., Sweet poisons: honeys contaminated with glycosides of the neurotoxin tutin. Journal of Natural Products 2015, 78 (6), 1363-1369.

21. Mokhtari, R. B.; Homayouni, T. S.; Baluch, N.; Morgatskaya, E.; Kumar, S.; Das, B.; Yeger, H., Combination therapy in combating cancer. Oncotarget 2017, 8 (23), 38022. 49

Experimental Procedures

Chapter 4: Experimental Procedures

4.1 General experimental procedure

UV spectra were recorded on a Jasco V-650 UV/Vis spectrophotometer. Optical rotations were recorded on Jasso P-1020 polarimeter using quart cell with a dimension of 3 x 100 mm. NMR spectra were recorded on either Varian or Bruker NMR instrument. Varian: NMR spectra were recorded at 25 oC on a 500 MHz and 600 MHz Unity INOVA spectrometers, the latter equipped with a triple resonance cold probe. Bruker: spectra were recorded at 25 oC on an 800MHz NMR Ascend AVANCE III HD spectrometer which was also equipped with a triple resonance cold probe. The 1H and 13C NMR chemical shifts were referenced to the solvent peaks for DMSO- d6 at H 2.50 and C 39.5, for CD3Cl3 at H 7.26 and C 77.0, respectively. LRESIMS spectra were recorded on a Waters ZQ mass spectrometer. HRESIMS data were recorded on a Bruker Daltronics Apex III 4.7e Fourier-transform mass spectrometer. A BIOLINE orbital shaker was used for the large-scale extractions of Austrobuxus Swanii. Alltech Davisil 40-60 µm 60 Å C18 bonded silica, Altech Davisil 30-40 µm 60 Å diol-functionalized silica and Altech Davisil 30- 40 µm 60 Å silica was used for column chromatography work. A Waters HPLC system equipped with a 600 pump, a Waters 996 PDA detector, and either a Waters 717 autosampler or Gilson 215 liquid handler and a Thermo Scientific Dionex UltiMate 3000 HPLC system equipped with a WPS-3000 autosampler were used for HPLC. Two Thermo C18 Betasil 5µm column (21.2 mm x 150 mm), a Phenomenex Luna 5 µm C18 column (10 mm x 250 mm) were used for semi-preparative HPLC separations. Sephadex LH-20 packed into different size of open glass columns (e.g., 25 mm x 500 mm) was used for gel permeation chromatography. All solvents used for chromatography, [α]D, UV, and MS were Lab Scan HPLC grade, and the H2O was Millipore Milli-Q PF filtered. All other reagents, standards, and dry solvent were high- purity products as indicated in the text.

4.2 Extraction and Isolation of B. biternata

4.2.1 Plant material

The plant material was collected from Guangxi province, China. The plant species was identified by Dr Jinquan Yuan from Guangxi Medicinal Botanic Gardens, and a voucher sample

Experimental Procedures is deposited at the South-Central University for Nationalities, Wuhan, China. 10 kg of the plant material was extracted exhaustively by ethanol to afford the ethanol crude extract 42.35g.

4.2.2 C18 flash chromatography

The dried ethanol extract (5g) was pre-adsorbed onto C18 bonded silica gel using methanol (MeOH) followed by evaporation. Then the extract coated silica was placed on a bed of pre- equilibrated C18-bonded silica and eluted sequentially with MeOH/H2O/TFA (10:90:0.1, 250 mL), MeOH/H2O/TFA (30:70:0.1, 250 mL), MeOH/H2O/TFA (50:50:0.1, 250 mL)

MeOH/H2O/TFA (70:30:0.1, 250 mL), MeOH/H2O/TFA (90:10:0.1, 250 mL) and

MeOH/H2O/TFA, (100:0:0.1, 500 mL). Each fraction was dried via the rotary evaporation to yield six brown-green gum-like fractions. A portion of each fraction was used to do the MTS assay and 1H NMR analysis respectively.

4.2.3 Normal silica gel fractionation

The dried 90% MeOH fraction and 100% MeOH fraction (1.2039g) was per-adsorbed onto C18 bonded silica gel by methanol then dried in fund hood overnight. Then placed on a bed of pre- equilibrated C18-bonded silica, and flushed with MeOH/H2O/TFA (10:90:0.1, 1 L),

MeOH/H2O/TFA (30:70:0.1, 1 L), MeOH/H2O/TFA, (70:30:0.1, 1 L), MeOH/H2O/TFA

(90:10:0.1, 1 L) and MeOH/H2O/TFA, (100:0:0.1, 1 L) respectively. Each fraction was dried via rotary evaporation to yield brown-green gum.

4.2.4 Diol column chromatography

The dried 90% MeOH/10% H2O fraction (0.9g) was per-adsorbed onto diol silica gel by methanol and dried in fund hood overnight. The sample coated diol was then placed on top of pre-equilibrated diol silica in a glass column, and eluted with hexane/EtOAc (100:0, 1 L), hexane/EtOAc (10:90, 1 L), hexane/EtOAc (20:80, 1 L), hexane/EtOAc (30:70, 1 L), hexane/EtOAc (40:60, 1 L) hexane/EtOAc, (50:50, 1 L), hexane/EtOAc (60:40, 1 L), hexane/EtOAc, (70:30, 1 L), hexane/EtOAc (80:20, 1 L), hexane/EtOAc (90:10, 1 L), and hexane/EtOAc (0:100, 1 L) to give 11 fractions.

4.2.5 C18 reverse phase HPLC fractionation

Experimental Procedures

Hexane/EtOAc (20:80) fraction was further purified by C18-bonded silica semi-preparative

HPLC at a flow rate of 4 mL /min with linear gradient elution from MeOH/H2O/TFA (60:40:1) to MeOH within 40 mins, followed by MeOH for 10 mins. Sixty fractions (1 min per fraction) were collected. 1H-NMR analysis suggested that fraction 18 contained 7-phenyl-4,6-heptadiyn- 2-ol (2.2), fraction 22 contained 7-Phenylhepta-2,4,6-triyn-1-ol (2.3)

Compound 2.1: a yellow gum, 1H and 13C NMR data see Table 2.3

Compound 2.2: a yellow gum, 1H and 13C NMR data see Table 2.4

Compound 2.3: a yellow gum, 1H and 13C NMR data see Table 2.5

4.3 Chemical investigation of Austrobuxus swanii

4.3.1 Plant material

The plant material was collected in Queensland, Australia.

4.3.2 Liquid-phase extraction

The air-dried and ground fruit-bodies of Austrobuxus swanii (20.56 g) were divided into two conical flasks (500 mL) and extracted with n-hexane (250 mL). The mixture was shaken on an orbital shaker at 200 rpm for 2 h. The resulting extract was filtered under gravity through 90 mm filter paper and set aside. DCM (250 mL) was added to the A. swanii residues, and the flask was shaken at 200 rpm for 2 h before filtration. MeOH (250 mL) was added and the mixture was shaken at 200 rpm for 2 h. The second volume of MeOH (250 mL) was then added and the MeOH/ A. swanii mixture was shaken for a further 16 h at 200 rpm before the filtration. Finally, all extractions were dried by the rotary evaporator to yield hexane extract (0.1044 g), DCM extract (0.5369 g) and MeOH extract (4.8063 g).

4.3.3 C18 reverse phase HPLC fractionation

A portion of the MeOH extract (0.3665 g) was pre-adsorbed onto the cotton and packed into a stainless-steel guard cartridge. The cartridge was subsequently attached to a semi-preparative

C18 Betasil HPLC column. Isocratic conditions of 10% MeOH/90% H2O were initially performed for the first 10 min, followed by a linear gradient to MeOH over 40 mins, then

Experimental Procedures

MeOH for 10 min, all at a flow rate of 9 mL/min. Sixty fractions (60 x 1min) were collected. Fractions were analyzed by LRESIMS and 1H NMR spectroscopy. Fractions 29 and 30 contained compounds of interest, and was further purified by C18-bonded silica semi- preparative HPLC at flow rate of 4 mL/min eluting with linear gradient from MeOH/H2O /TFA

(75:45:0.1) to MeOH/H2O /TFA (45:55:0.1) within 50 mins, followed by MeOH for 10 mins

(Figure 2.5). Fraction 29 and 30 were further purified by Luna C18 column at flow rate of 4 mL/min eluting with a linear gradient from MeOH/H2O/TFA (75:45:0.1) to MeOH/H2O/TFA (45:55:0.1) within 50 mins, followed by MeOH for 10 mins. 1H-NMR analysis of the fractions led to the isolation of a new compound, austrobuxusin E (3.1) and a known compound, austrobuxusin A (3.2). Further purification of fraction 26 fractions by Luna C18 column at flow rate of 4 mL/min eluting with linear gradient from MeOH/H2O/TFA (30:70:0.1) to MeOH/H2O /TFA (40:60:0.1) within 50 mins, followed by MeOH for 10 mins yielded two more known compounds, austrobuxusin B (3.3) and austrobuxusin C (3.4). Further purification of fraction

32 fractions by Luna C18 column at flow rate of 4 mL/min eluting with linear gradient from

MeOH/H2O/TFA (45:55:0.1) to MeOH/H2O/TFA (45:55:1) within 50 mins, followed by MeOH for 10 mins yielded one more known austrobuxusin D (3.5)

Compound 3.1: a colorless gum with an[α]D value of + 21.6 (MeOH, c 2.5). The molecular formula of the compound was determined as C25H32O13 based on the (+)- HRESIMS data (m/z 563.1727 [M + Na] +). IR absorptions at 3370 (br) and 1766 cm-1. UV almost no absorption. 1H and 13C NMR data see Table 3.1

4.4 Biology experimental

4.4.1 Chemical and reagents

All samples were dissolved in dimethyl sulfoxide (DMSO) and then adjusted to 0.3% using dionised water. The samples were then stored at -20 ℃ until use. Dulbecco’s modified Eagle’s Medium (DMEM), RPMI-1640 Medium were purchased from Invitrogen (Carlsbad, CA).

4.4.2 Cell lines and cell culture

The Caco2 [HTB-37]and SKOV3 [HTB-77] cell lines used in this study were obtained from American Type Culture Collection (Rockville, USA). The positive control used for in vitro 53

Experimental Procedures anticancer acitivity was taxol from Sigma. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, or Ham’s F-12K Medium (Life Technologies) supplemented with 20 mM HEPES, 10 mM sodium bicarbonate, 50 μg/ mL streptomycin, 50 IU/ mL penicillin, 2 mM glutamine and 10% foetal calf serum (Life Technologies). The cells were maintained as monolayers in 75 mL flasks at 37℃, 5% CO2 in a humidified atmosphere until approximately 80% confluent.

A modified MTS assay was used to test the extract and isolated compounds’ anti-proliferative activity against Caco2 and SKOV3 cancer cell lines. A volume of 70 µL aliquot of cells was added to half of the 96-wells plate, from row one to four, and 70 µL of cell media (for negative control) was added to the other half, from row five to eight, for the preparation of the MTS based cell proliferation assay. A volume of 30 µL of the test extracts or pure compounds was added to each well. The total volume of each individual well was 100 µL and the plate was incubated at 37℃, 5% CO2 in a humidified atmosphere for 24 hours. Afterwards, 20μl of Cell Titre 96 Aqueous One solution(Promega) was added to each well and the plates were incubated in the same condition for a further 3 hours. The absorbance was subsequently recorded by a Molecular Devices, Spectra Max M3 plate reader. The antiproliferative activity of each extract was calculated as a percentage of untreated control using the formula:

Proliferation (% untreated control) = (Act/Acc) ×100 Act: corrected absorbance of the test extract Acc corrected absorbance of the untreated control

4.4.3 Statistical analysis

Data are expressed as the mean ± SEM of at least three independent experiments. One-way ANOVA was used to calculate statistical significance between control and treated groups with a P value < 0.01 considered to be statistically significant, using GraphPad Prism 5.0.

Appendices

Table of appendices

1 Figure S.1 H NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1) ...... 58

13 Figure S.2 C NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1) ...... 59

Figure S.3 HSQC NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1) . 60

Figure S.4 HMBC NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1) 61

1 1 Figure S.5 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne

(2.1) ...... 62

1 Figure S.6 H NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol

(2.2) ...... 63

Figure S.7 HSQC NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-

ol (2.2) ...... 64

Figure S.8 HMBC NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-

ol (2.2) ...... 65

1 1 Figure S.9 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-

heptadiyn-2-ol (2.2) ...... 66

1 Figure S.10 H NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-pentadiyn-1-ol

(2.3) ...... 67

Appendices

Figure S.11 HSQC NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-pentadiyn-

1-ol (2.3) ...... 68

Figure S.12 HMBC NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-pentadiyn-

1-ol (2.3) ...... 69

1 1 Figure S.13 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-

pentadiyn-1-ol (2.3) ...... 70

1 Figure S.14 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) ...... 71

13 Figure S.15 C NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) ...... 72

Figure S.16 HSQC NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) .. 73

Figure S.17 HMBC NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) . 73

1 1 Figure S.18 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E

(3.1) ...... 74

Figure S.19 ROESY NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1) 75

1 Figure S.20 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin A (3.2) ...... 76

1 Figure S.21 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin B (3.3)...... 77

1 Figure S.22 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin C (3.4)...... 78

1 Figure S.23 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin D (3.5) ...... 79

Figure S.24 High resolution mass spectrum for austrobuxusin E (3.1) ...... 80

Appendices

Figure S.25 Infrared spectrum for austrobuxusin E (3.1) ...... 80

Figure S.26 UV spectrum for austrobuxusin E (3.1) ...... 81

Appendices

1 Figure S.1 H NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1)

Appendices

13 Figure S.2 C NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1)

Appendices

Figure S.3 HSQC NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1)

Appendices

Figure S.4 HMBC NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1)

Appendices

1 1 Figure S.5 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for phenylheptatriyne (2.1)

Appendices

1 Figure S.6 H NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol (2.2)

Appendices

Figure S.7 HSQC NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol (2.2)

Appendices

Figure S.8 HMBC NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol (2.2)

Appendices

1 1 Figure S.9 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for 7-phenyl-4,6-heptadiyn-2-ol (2.2)

Appendices

1 Figure S.10 H NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-pentadiyn-1-ol (2.3)

Appendices

Figure S.11 HSQC NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-pentadiyn-1-ol (2.3)

Appendices

Figure S.12 HMBC NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-pentadiyn-1-ol (2.3)

Appendices

1 1 Figure S.13 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for 5-Phenyl-2,4-pentadiyn-1-ol (2.3)

Appendices

1 Figure S.14 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Appendices

13 Figure S.15 C NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Appendices

Figure S.16 HSQC NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Figure S.17 HMBC NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Appendices

1 1 Figure S.18 H- H COSY NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Appendices

Figure S.19 ROESY NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin E (3.1)

Appendices

1 Figure S.20 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin A (3.2)

Appendices

1 Figure S.21 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin B (3.3)

Appendices

1 Figure S.22 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin C (3.4)

Appendices

1 Figure S.23 H NMR spectrum (800 MHz, DMSO-d6) for austrobuxusin D (3.5)

Appendices

Figure S.24 High resolution mass spectrum for austrobuxusin E (3.1)

Figure S.25 Infrared spectrum for austrobuxusin E (3.1)

Appendices

Figure S.26 UV spectrum for austrobuxusin E (3.1)