Cane Toad Skin Extracts

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Compound Identification and Functional Characterisation of Cane Toad Skin Extracts

Author Zulfiker, Abu Hasanat Md

Published 2016

Thesis Type Thesis (PhD Doctorate)

School School of Medical Science

DOI https://doi.org/10.25904/1912/61

Downloaded from http://hdl.handle.net/10072/365733

Griffith Research Online https://research-repository.griffith.edu.au Compound identification and functional characterisation of cane toad skin extracts

Abu Hasanat Md. Zulfiker Master of Science (Pharmaceutical Sciences)

School of Medical Science

Menzies Health Institute Queensland

Griffith University

Submitted in fulfilment of the requirements of the degree of

Doctor of Philosophy

June 2016

Dedication

This thesis is dedicated to my parents Md. Abdul Zalil Sarkar and Mrs. Nurmahal

Zalil who brought me to this beautiful world. Without you, I would not be here today.

Thank you very much for your great support and endless care.

Acknowledgements

I have made great efforts in this project, which would not have been possible without the kind support and help of many individuals and organizations. I would like to extend my sincere thanks to all of them.

First of all, I am highly indebted to my principal supervisor, Professor Ming Wei for his guidance, encouragement and patience over the last few years of my PhD journey.

Thank you so much for giving me the opportunity to work in freedom, to look at research in different ways and on the whole to grow as an independent researcher.

My sincere gratitude to my co-supervisor, Dr. I Darren Grice for his indispensable advice, support, constant supervision and direction regarding the project. I also greatly appreciate his immense support for proof reading of documents, which has helped me to improve my writing ability.

I would like to acknowledge the valuable support of Dr. Saeed Hashimi who contributed to my lab work, protocol design, manuscript proof reading and many discussions that helped me to shape this project. Much of my experimental work would have not been completed without his kind co-operation and help.

I would like to thank Ben Matthews, Laboratory Manager, SmartWater Research

Centre, Griffith University for his wonderful support for my analytical work and proof reading of the analytical part of the thesis.

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My appreciation also goes to my laboratory colleagues especially Mohsen Sohrabi who gave me much support during my PhD journey. It was a great pleasure to work with them. I am thankful to Dr. Deepak Samuel Ipe for his great mental support from the very beginning of my PhD. His friendship was vital for me to keep up with my work.

Thanks also to Md. Atiqur Rahman, S. M. Riajul Wahab and Md. Forhadul Islam for their support and help during my PhD.

I would like to express my gratitude towards my parents and family members, my wife, mother-in-law, father-in-law and their family for the endless support, inspiration and care throughout my life.

I greatly acknowledge the School of Medical Science colleagues and members of the

Institute for Glycomics as well as the Smart Water Research Centre for their kind co- operation and encouragement, which has helped me to complete this project.

I would like to recognize Griffith University, Griffith Graduate Research School, Griffith

Health and Menzies Health Institute Queensland (MIHQ) for awarding me the scholarship as well as the grant support to finish my PhD.

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.

(Signed) ------Abu Hasanat Md. Zulfiker

v Abstract

Amphibians are storehouse of bioactive compounds. Among them, the skin of toad species is rich in biologically active compounds such as peptides, proteins, steroids, alkaloids and opioids. Some of these compounds have found significant therapeutic applications, for example as antibacterials, antifungals, antiprotozoals, antidiabetics, antineoplastics, analgesics and sleep inducing agents. In Traditional Chinese

Medicine (TCM) aqueous extracts of Chinese toad skins have been used for centuries to treat pain, swelling, heart failure and several types of cancer with minimal to no side effects, generating a 10 billion USD market in China. Numerous compounds have been identified from these Chinese toad skin extracts, which have reported therapeutic activities in various disease conditions, either as a single compound or as a group of compounds. In Traditional Korean Medicine, toad extracts have also been reported to show potential activity against anxiety and depression.

In Australia and America cane toad skins have a history of recreational use for euphoric purposes. This information coupled with knowledge of its use in China and

Korea enabled us to hypothesise that Queensland cane toad skin extracts would likely contain similar ‘biologically-active’ compounds in selective extracts. This thesis reports on research work carried out to identify such ‘biologically-active’ extracts and/or compounds, then functionally characterise these in cultured cells for investigation of their therapeutic relevance to neuropsychiatric disorders, especially in obsessive compulsive disorder (OCD) - potentially identifying potent therapeutics for future development.

To test this hypothesis, the research work presented in chapter 3 initially focussed on designing and optimising several aqueous-based extraction systems of Australian cane toad skins. These were based on various previously reported methods used for compound identification in Chinese toad skin extracts. Extracts were generated in three different solvent systems, including aqueous, organic (60% ethanol) and acidic

(acetic acid at two different pHs: 1.78 & 2.17) to attempt to obtain maximum number and amounts of constituents. Thus four extracts were prepared from collected cane toad skins for HPLC-mass spectrometry (HPLC-ESI-Q-TOF-MS/MS) analysis, producing a list of constituents in each extract. The identification of compounds was based on accurate mass measurements for molecular ions and MS/MS analysis, whereby accurate mass pseudo-molecular ions and characteristic fragment ions were compared to published reference data, including mass bank and NIST (National

Institute of Standards and Technology). Analysis identified 42 compounds across the four extracts from a number of chemical classes, including alkaloids, amino acids, bufadienolides, fatty acids, nucleobases, nucleosides and vitamins. Of the 42 compounds identified in this study, 29 were identified in the aqueous extract (AE), 35 in the 60% ethanol extract (EE), 10 in the pH 1.78 acetic acid extract and 11 in the pH

2.17 acetic acid extract. Interestingly, the acetic acid extracts contained only one constituent not present in either the AE or EE extract. Therefore, the AE and EE extracts provided the greatest potential for further functional assays in cultured cells.

To the best of my knowledge, this is the first comprehensive report on the identification of constituents in Australian cane toad skins.

In chapter 4 functional characterisation assays investigating in vitro molecular mechanisms were developed. These assays were utilized to assess the potential of

vii the cane toad skin extracts as potential therapeutics against disease conditions such as OCD. Based on recent evidence, the hypothesis was investigated whereby agonistic activation of the serotonin (5-HT)-2 receptor (likely 5- HT2A) in certain areas of brain such as the pre-frontal cortex (PFC) would improve OCD symptoms. OCD is also associated with abnormalities of dopamine neurotransmission, suggesting lower dopamine (D2/3) receptor availability, primarily in the striatum. Additionally, OCD is reportedly related to dysfunction of 5-HT2A-D2 receptor complex (heteromer) regulation. So the primary objective of the assays was to determine whether AE and

EE could upregulate 5-HT2A or D2 and increase the interaction of 5-HT2A-D2 receptor heteromer formation. Thus it was demonstrated that AE (50 µg/mL), but not EE (50

µg/mL) significantly (p<0.05) upregulated both 5-HT2A and D2 receptor expression and increased the interaction of 5-HT2A-D2 receptor complex (heteromer) formation upon treatment of CLU213 cells with AE. The CLU213 cells were previously validated as expressing both 5-HT2A and D2 receptors. The classical signalling pathway (Gq11-

PLCβ) was also investigated in connection with 5-HT2A mediated agonist activation.

The results indicate that AE (50 µg/mL) upregulates 5-HT2A receptor expression by a mechanism that seems to involve activation of the Gq/11 G protein PLCβ signalling pathway and c-FOS transcription factor. Taken together, this molecular mechanism could provide a basis to progress further development of cane toad skin extracts as a novel therapeutic agent for OCD.

In chapter 5, functional characterisation studies were extended by conducting anti- inflammatory assays on AE, EE and BT (Bufotenine-a known marker reported to be present commonly in toad skins). BT was identified in both AE and EE by HPLC-ESI-

Q-TOF-MS/MS in this research. Inflammation is reported as a common condition in

viii numerous diseases including OCD. This experiment was performed with AE (0.1-10

µg/mL), EE (0.1-10 µg/mL) and BT (0.1-10 nM) in a model of human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13- acetate (PMA) for the release of tumor necrosis factor (TNF)-α and interleukin (IL)-6.

It was found that AE, EE and BT significantly suppressed the release and expression of TNF-α and IL-6 in a dose dependant manner when cells were pre-treated at non- cytotoxic concentrations. Further investigation showed that this inhibition of TNF-α and

IL-6 release and expression was related to a mechanism which triggered nuclear factor (NF)-kappa (κ)B activation and translocation from cytoplasm to nucleus. Results obtained suggest that AE, EE and BT are potential inflammatory inhibitors and good candidates for further development as anti-inflammatory therapeutic agents.

The Australian Cane toad is a devastating pest in Queensland and Australia in general. Therefore, an attractive outcome of this research would be to find a therapeutic application of its skin that could lead to the development of a beneficial cane toad management plan throughout Australia - a goal that this research provides sound scientific basis for progressing.

Contents Page Title------i

Dedication------ii

Acknowledgement------iii

Statement of originality------v

Abstract------vi

Content------x

List of Tables in Chapter 1------xiv

List of Figures in Chapter 1------xiv

List of Abbreviations in Chapter 1------xv

Publication lists------xvii

Chapter 1: Introduction------1

1.1 Cane toads------2

1.1.1 History of cane toad------4

1.1.2 Bio-compounds identified from various toad species------10

1.1.3 Bio-compounds identified from cane toads------14

1.1.4 The use of toad products in medicine------16

1.2 Anxiety Disorder------17

1.2.1 Prevalence------18

1.2.2 Assessment, diagnosis and/or symptoms of specific anxiety disorders------19

1.2.3 Management of anxiety disorders------21

1.2.3.1 Psychoeducation ------22

1.2.3.2 Psychological treatments ------22

1.2.3.3 Pharmacological management------23

1.2.3.4 Pharmacological, non-pharmacological or both----- 23

1.2.4 Anxiety disorder and inflammation------30

Contents Page

1.2.5 Obsessive Compulsive Disorder (OCD)------30

1.2.5.1 Prevalence------31

1.2.5.2 Symptoms------32

1.2.5.3 Pathogenesis------33

1.2.5.3.1 Neuroanatomy------33

1.2.5.3.2 Neurochemistry------35

1.2.5.3.3 Neurogenetics------36

1.2.5.3.4 Neuroimmunology------37

1.2.5.3.5 Neuroethology------37

1.2.5.4 Management of OCD------38

1.2.5.4.1 Pharmacotherapy------38

1.2.5.4.1.1 Tricyclic antidepressants (TCA)- 38

1.2.5.4.1.2 Selective Serotonin Reuptake Inhibitors (SSIRs)------39

1.2.5.4.2 Other pharmacological strategies------39

1.2.5.4.3 Psychotherapy------41

1.2.5.4.4 Combination of pharmacological treatment and cognitive behavioural therapy (CBT)------41

1.2.5.5 OCD and inflammation------42

1.3 Serotonin 2A receptor (5-HT2AR)------43

1.3.1 Significance of 5-HT2AR in OCD------45

1.3.2 Bufotenin as 5-HT2AR agonist and its significance in OCD--- 46

1.3.3 Significance of 5-HT2AR agonists in inflammation ------50

1.3.4 Coupling of 5-HT2A and D2 receptors and its significance in OCD as well as other cognitive and mood disorders------50

Contents Page 1.4 Bioassays to study OCD and inflammation------52

1.4.1 Bioassays to study OCD------52

1.1.4.1 In vitro studies------52

1.4.1.2 In vivo studies------53

1.4.2 Bioassays to study inflammation------57

1.5 Significance of the project------59

1.6 Aims and Objectives------61

Chapter 2: Methodology------63

2.1: Extraction of Australian cane toad skins------64

2.1.1: Collection of cane toad skins------64

2.1.2: Extraction of Australian cane toad skins 64

2.1.2.1: Extraction in aqueous and 60% ethanol solvents- 64

2.1.2.2: Aqueous extraction at acidic pH------65

2.1.3: HPLC-Q-TOF/MS analysis of Australian cane toad skin extracts------66

2.1.4: Cell culture------66

2.1.4.1: Protocols used for cell culture------67

2.1.5: Sample preparation------68

2.1.6: Cytotoxicity assays------69

2.1.7: Western blot------69 2.1.7.1: Protein extractions------69

2.1.7.2: Protein assay------70

2.1.7.3: Western blotting------71

2.1.8: Co-immunoprecipitation------72

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Contents Page

2.1.9: Quantitative Real Time PCR (qPCR)------72

2.1.9.1: RNA extraction------72

2.1.9.2: cDNA preparation from total RNA------73

2.1.9.3: qPCR------73

2.1.9.3.1: Method 1------73

2.1.9.3.2: Method 2------74

2.1.10: Immunofluorescence microscopy------74

2.1.11: ELISA------75

Chapter 3: Compound identification in Australian cane toad skin extract

(Research paper 1)------76

Chapter 4: Functional characterisation of Australian cane toad skin extract

(Research paper 2)------97

Chapter 5: Functional characterisation of Australian cane toad skin extract

(Continued) (Research paper 3)------141

Chapter 6: Overall discussion------157

Chapter 7: Conclusion and future directions------165

Appendix------170

References------185

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List of Tables in Chapter 1 Contents Page

Table 1.1: Identification of various biocompounds to date in the skin of various toad species ------10

Table 1.2: Short description of anxiety disorders as defined by ICD-10 and DSM-IV-TR------20

Table 1.3: Approved US Food and Drug Administration (FDA) drugs for anxiety disorders------25

Table 1.4: Commonly used drugs for anxiety disorders in primary care (Non- FDA)------29

Table 1.5: Obsessions and compulsions in OCD------31

Table 1.6: Drugs used in the treatment of OCD------40

Table 1.7: Animal models of OCD------54

List of Figures in Chapter 1 Contents Page

Figure 1.1.: Cane toad (Bufo marinus)------3

Figure 1.2: Structure of indolealkylamines identified in cane toad skins so far------15

Figure 1.3: Increased activity in orbitofrontal cortex and caudate in patients with OCD------34

Figure 1.4: Normalisation of cortico-striatal-thalamic-cortical circuits by either pharmacotherapy or psychotherapy in OCD------36

Figure 1.5: Structure of serotonin, psilocybin and psilocin------47

Figure 1.6.: Structure of Acetyl ester of bufotenin------49

Figure 1.7: Graphical representation of significance of abnormal dopamine neurotransmission and 5HT2A-D2 heteromer formation in OCD ------52

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List of Abbreviations in Chapter 1 AE Aqueous extracts of cane toad skin

BBB Blood brain barrier

BT Bufotenine

CBT Cognitive behavioural therapy

CNS Central nervous system

CSF Cerebrospinal fluid

DAG Diacyl glycerol

DOI 2,5-Dimethoxy-4-iodoamphetamine

DMT Dimethyl tryptamine

EE 60% Ethanol extract of cane toad skin

ELISA Enzyme-linked immunosorbent assay

FDA Food and Drug Administration

GAD Generalised anxiety disorder

GP General physician

GPCR G-protein coupled receptor

HC Hallucinogenic compound

HPLC High performance liquid chromatography

5-HT 5-Hydroxytryptamine

IL Interleukin

IP Inositol phosphate

LPS Lipopolysaccharide

LSD Lysergic acid diethylamide

MAOI Monoamine oxidase inhibitor

5-MeO- 5-Methoxy-N,N-dimethyltryptamine DMT

List of Abbreviations in Chapter 1 (Continued)

NASSA Noradrenergic and specific serotoninergic antidepressant

NFkB Nuclear factor kappa B

OCD Obsessive compulsive disorder

OFC Orbitofrontal cortex

5-OH-DMT 5-Hydroxy-N,N-dimethyltryptamine

PD Panic disorder

PKC Protein kinase C

PLC Phospholipase C

PMA Phorbol-12-myristate-13-acetate

PTSD Post-traumatic stress disorder

PTX Pertussis toxin

SAD Social anxiety disorder

SNRI Selective norepinephrine reuptake inhibitor

SSRI Selective serotonin reuptake inhibitor

TCA Tricyclic antidepressants

TNF-α Tumour necrosis factor alpha

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Publication list

List of journal articles and conference abstracts published during my PhD candidature:

First Author Publications:

1. Abu Hasanat Md. Zulfiker, Mohsen Sohrabi, Ji Qi, Ben Matthews, Ming Q. Wei, I.

Darren Grice. Multi-constituent identification in Australian cane toad skin extracts using high-performance liquid chromatography high-resolution tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2016; 129 (10):

260-272, DOI: 10.1016/j.jpba.2016.06.031 (IF: 3.16).

Publication link:http://www.sciencedirect.com/science/article/pii/S0731708516303508

2. Abu Hasanat Md. Zulfiker, Saeed M. Hashimi, I. Darren Grice, and Ming Q. Wei.

Cane toad skin extract - induced upregulation of serotonin 2A and Dopamine D2 receptors via Gq/11-PLCβ signalling pathway. Journal of Cellular Biochemistry. 2016

(Accepted Author Manuscript. doi:10.1002/jcb.25627) (IF: 3.44).

Publication link: http://onlinelibrary.wiley.com/doi/10.1002/jcb.25627/full

3. Abu Hasanat Md. Zulfiker, Saeed M. Hashimi, Ji Qi, I. Darren Grice, and Ming Q.

Wei. Aqueous and Ethanol Extracts of Australian Cane Toad Skins Suppress Pro-

Inflammatory Cytokine Secretion in U937 Cells. Journal of Cellular Biochemistry. 2016

(Accepted Author Manuscript. doi:10.1002/jcb.25577) (IF: 3.44).

Publication link: http://onlinelibrary.wiley.com/doi/10.1002/jcb.25577/full

xvii

4. Abu Hasanat Md. Zulfiker, Gian Luigi Mariottini, Ji Qi, I. Darren Grice and Ming Q.

Wei. Indolealkylamines from toad vertebrates and sea invertebrates - their identification and potential activities on the central nervous system. Central Nervous

System Agents in Medicinal Chemistry. 2015; 15 (2):1, DOI:

10.2174/1871524915666150724100245.

Publication link: http://benthamscience.com/journals/central-nervous-system-agents- in-medicinal-chemistry/article/133441/

Co-Author Publications

5. Mohsen Sohrabi, Raj G. Nair, Lakshman P. Samaranayake, Li Zhang, Abu Hasanat

Md. Zulfiker, Adnan Ahmetagic, David Good, Ming Q. Wei . The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology. Journal of Microbiological Methods. 2016 Mar;

122:64-72. doi: 10.1016/j.mimet.2016.01.013. Epub 2016 Jan 23 (IF: 2.02).

Publication link: http://www.sciencedirect.com/science/article/pii/S0167701216300136

6. Ji Qi, C K Tan, Saeed M Hashimi, Abu Hasanat Md. Zulfiker, David Good, Ming Q

Wei. Toad glandular secretions and skin extractions as anti-inflammatory and anticancer agents. Evidence-based Complementary and Alternative Medicine.

Volume 2014, Article ID 312684, 9 pages, http://dx.doi.org/10.1155/2014/312684

2014 (IF: 1.93).

Publication link: http://www.hindawi.com/journals/ecam/2014/312684/

xviii

Conference Presentations:

1. Abu Hasanat Md. Zulfiker, Saeed M. Hashimi, I. Darren Grice, Ming Q. Wei.

Potential of Australian Cane Toad Skin Extracts in the Treatment of Obsessive

Compulsive Disorder. Presented poster in “ANXIETY AND DEPRESSION

CONFERENCE 2016” at Philadelphia Marriott Downtown Philadelphia,

Pennsylvania, USA, March 31–April 3, 2016.

Publication link:

https://www.aievolution.com/ada1601/index.cfm?do=abs.viewAbs&abs=1544

2. Abu Hasanat Md. Zulfiker, Saeed M. Hashimi, Ji Qi, Mohsen Sohrabi, I.

Darren Grice, Ming Q. Wei. Bufotenine inhibits production of pro-inflammatory

cytokines in lipopolysaccharide-induced macrophage U937 cells via NF-κB

signalling pathway. Presented poster in “Gold Coast Health and Medical

Research Conference 2015” at Mercure Gold Coast Resort, Gold Coast,

Australia, 3-4 December, 2015.

3. Qi Ji, Chun Li, Abu Hasanat Md. Zulfiker, Mohsen Sohrabi. Ming Q. Wei. Cane

toad skin aqueous extracts inhibit TNF-α induced activation of NF-kB pathways

in A549 cells. Presented poster in “Gold Coast Health and Medical Research

Conference 2015” at Mercure Gold Coast Resort, Gold Coast, Australia, 3-4

December, 2015.

4. Abu Hasanat Md. Zulfiker, I. Darren Grice and Ming Q Wei. Potential anti-

inflammatory activity of cytotoxic aqueous and ethanolic extracts of Australian

cane toad skins. Presented 3 min flash presentation at “Royal Australian

xix

Chemical Institute Medicinal Chemistry and Chemical Biology” One Day

Symposium, South Bank, Brisbane, Australia, 25 September, 2015.

5. M. Sohrabi, R. Nair, L. Zhang, L. Samaranayake, A. Zulfiker, A. Ahmetagic, D.

Good, M. Wei. Potential Utility of Microbial Metabolites in Cancer Diagnostics.

Presented poster at “International Journal of Dental Research Conference

2015” Boston, Massachusetts, USA, 11-15 March, 2015.

Publication link:

https://live.blueskybroadcast.com/bsb/client/_new_default.asp?action=SEARC

H&Client=404900

6. Abu Hasanat Md. Zulfiker, Mohsen Sohrabi, I. Darren Grice, Ming Q. Wei.

Identification and assessment of function of bioactive extracts from Australian

cane toad skins. Presented poster in “Gold Coast Health and Medical

Research Conference 2014” at Mercure Gold Coast Resort, Gold Coast,

Australia, 4-5 December, 2014.

7. Mohsen Sohrabi, Abu Hasanat Md. Zulfiker, Li Zhang, Raj G Nair, Lakshman

P Samaranayake, Adnan Ahmetagic, David Andrew Good, and Ming Q Wei.

Developing a microbiota signature from gut microbiota’s metabolites.

Presented poster at “Gold Coast Health and Medical Research Conference

2014” Mercure Gold Coast Resort, Gold Coast, Australia, 4-5 December, 2014.

Chapter 1: Introduction

1.1 Cane toad (Bufo marinus)

The scientific name of Cane toad is Bufo marinus which has come from Central and

South America. They are included in the Bufonidae (toad) family of the order Anuran and class amphibian. The more common names are the giant, marine toad. They are now present in Hawaii, Florida, the Philippines and most remarkably Australia. B. marinus was first imported to Australia in the 1920s from Puerto Rico (1). Later, in

1935 a larger batch was brought from Hawaii to Queensland, Australia with the hope that they would control the destructive grey-backed cane beetles that were infesting sugar cane crops. Therefore, the toad is now known as the ‘cane toad’ in Australia.

They failed to control the beetles, but were remarkably successful at reproducing and spreading due to a lack of natural predators, good breeding conditions including abundant food. They now number well into the millions, and are still increasing across thousands of square kilometres of north eastern Australia (2). At present Bufo marinus is regarded as a major environmental and ecological threat that has destroyed and displaced a considerable number of native fauna and insects (3). Cane toads are highly malleable to all but extremely cold conditions and are usually found in temperate to sub-tropical forested areas near a water source (where breeding takes place). Cane toads can grow up to 25 cm and weigh up to 1.8 kg for a large female, although males are smaller. They are nocturnal, being active at night, sleeping and hiding themselves at daytime. They have good appetites and generally eat anything they can catch and fit in their mouths. They are recognized to live up to 40 years in imprisonment, but the life span in the wild is usually much shorter due to predation (1). They are regarded as pests and the government eradication efforts include asking residents to aid in their collection and disposal.

A B

Figure 1.1: Cane toad (Bufo marinus).

Toads are characterized by warty skin, large eyes with horizontal pupils, unwebbed front hands, large parotid glands and bony ridges on the head. Cane toads are reportedly the most venomous of all toads. The venom is secreted by numerous wart- like glands on the dorsal skin surface of the toad, and most venom is deposited in the parotid glands (swollen masses behind each ear). The venom carries powerful toxins that make them unpleasant to most predators. Each toad can generate about 600 mg of freeze dried venom. Under extreme stressful situations such as being bitten by a predator, they can discharge enough toxin (as a milky white sticky fluid) to quickly kill a large dog (1).

There are examples of finding snakes or domestic animals dead with a cane toad in their mouth or gut. Cane toads have also been found to affect the population of monitor lizards and various other frogs or toads (4). That is why, some animals have developed strategies to avoid these venomous creatures e.g., two snake species have been reported to grow smaller heads so that they can no longer eat venomous cane toad in

Australia (5, 6). Humans have also been reported to die after consumption of a mixture

of toad skin-secretion components (7). The toxin's strength is so intense that hedgehogs (Insectivora, Erinaceidae) utilise it to improve their mechanical anti- predatory adaptations (8). Hedgehogs hold these secretions in their mouths and lick it onto their spines, as the toxin in fresh or dried form on their spines likely enhances the pain or potential of infection in its predators.

1.1.1 History of the cane toad

The venomous toad B. marinus is native to the new world, where it is a common occupant of low swampy habitats ranging from Florida west along the Gulf Coast to

Mexico, then south to Panama and northern South America. In post Columbian times, it spread rapidly throughout the Antilles and south along the Pacific coast of South

America and inland into the Amazon basin (9).

Cane toad came to Europe immediately after the voyages of Columbus, and exterminators quickly discovered that when the toad was placed in boiling olive oil, the secretions of the glands could be easily skimmed off the surface. In early sixteenth- century Italy, exterminators developed sophisticated procedures to extract toad toxins into a salt which could then be scattered on the intended victim’s food (10). As a matter of fact, in the early eighteenth century it was actually added to explosive shells and mixed with the saltpetre used to make gunpowder, as the toad venom was considered to be so highly toxic (11, 12). Probably, commanders thought that if the cannon did not kill their enemies, the toad toxins would (13).

Not surprisingly, at the very beginning, European physicians included toad venom into their material medica. Dried and powdered toad justified a noticeable place for the treatment of dropsy, fever, and various ailments in many important pharmacopoeia, including Thesaurus Pharmakologicus, written by Johannes Schroder and published in Leyden in 1672, Pharmacologia, by Samuel Dale (London 1692) and The London

Dispensatory, by William Salmon, published in 1702. Toads continued to be an important therapeutic agent throughout the eighteenth century, appearing in

Pharmacopoeia Universalis by R. James (London 1747) and The English

Dispensatory by John Quincy (London 1749). In late 1833, powdered toad was annotated in an important medical compendium, Pharmacologia, written by J. A. Paris.

The Chinese were well ahead of their Western colleagues in their knowledge of the properties of toad venom along with many other things. For centuries they had been making the toxic secretions into smooth disks which they termed Ch’an Su or “toad venom” in Mandarin (13).

Cane toad skin has a long history of use for various purposes, including euphoric uses.

The large parotid glands present in the back of the toad are known as “veritable chemical factories” (14); they generate and discharge at least 26 compounds, all of which are biologically active. Some of these, for example the phenylethylamine bases and derivatives such as the catecholamines dopamine, epinephrine, and norepinephrine and a number of indole derivatives such as serotonin are benign and occur naturally in human tissue. These chemicals act as chemical messengers between the synapses and also as neurotransmitters which connect individual neurons. The indole derivatives and catecholamines are present in numerous species and are not poisonous in animals (13). The parotid glands also discharge other

compounds in significant amounts, including bufotenin (BT), a purported hallucinogenic agent (15), and two very poisonous cardioactive steroids, bufogenin and bufotoxin. These compounds have been identified in the skin or glands of various toads and it is their unique toxic properties that have gained the animal a notorious place in a range of exterminators and black magicians all over the world (13).

Dispute over the use of toads as a hallucinogen has continued until now, although historical evidence does exist to support this. First, all over Central America the toad was widely used as a symbol, especially in Mayan iconography (16). Many discovered

Mayan artefacts including small ceramic serving bowls, have apparent illustrations of the toad and particularly graphic portrayals of its distinctive venom glands (14, 16, 17).

Second, at one post-classic Mayan site on Cozumel Island, Mexico, an archaeologist observed that almost all amphibian remains were B. marinus (18). This report was consistent with an earlier, similar discovery at San Lorenzo, Mexico, that led an eminent archaeologist to imply that the Olmec civilization used the toad as a narcotic

(19). Third, one of the components discharged by the toad is BT, a compound that is present in a hallucinogenic snuff prepared today by South American Indians from the seeds of Anadenanthera peregrine (20). A report from Western medical literature has indicated that pure BT upon intravenous administration into human subjects causes hallucinations (15). Fourth, the followers of the hallucinogen hypothesis all cite an unpublished report of the existing use of B. marinus in a hallucinogenic preparation in

Vera Cruz, Mexico (14, 16).

Those who consider the toad as a hallucinogen also draw attention to the spreading of B. marinus remains in various archaeological sites (19, 21). Michael Coe (19)

commented on the use of toads at the San Lorenzo site: “These toads are a puzzle, as they can not be skinned without an extremely dangerous poison getting into the meat. We are now looking into the possibility that the Olmecs used them for a hallucinogenic substance called BT which is one of the active ingredients (19). A survey of the archaeological literature demonstrates that a substantial number of B. marinus are present in middens throughout Central America, leading other archaeologists to propose that pre-Columbian Indians used the toad, not as a hallucinogen, but as food, after carefully removing the skin and parotid glands (22, 23).

In 1979 Richard Cooke (23) butchered and cooked toads and described their taste as pleasant as smoked chicken. He suggested that the toad was not employed as a drug, but as a survival food based on the temporal and spatial distribution of Bufo marinus which was supported by the fact that it is today used for that purpose by the Campa

Indians of the lower Apurimac River in Peru (13).

There has been a central myth of the toad hallucinogen hypothesis that no one has been able to determine how the toad preparations could have been used safely. It is true that the glands secrete BT, a known component of South American hallucinogenic snuffs (20) which is only slightly lipid soluble and cannot therefore pass the blood brain barrier, but has good psychotropic properties (13). However in toad venom, very potent cardioactive steroids such as bufotoxin and bufogenin are also present (12, 24).

It is obvious that ingesting a straight maceration of the parotid glands would induce cardiac failure long before the recipient would feel the putative hallucinogenic properties of BT (21). It is speculated that the Maya would have been interested in poisoning many of their priesthood, who probably would have been the ones taking the drug. Only if procedures had been devised that specifically reduced the effect of

toxic constituents could B. marinus have been made into a hallucinogen. Sometimes folk healers have showed a sophisticated biological and chemical knowledge, to improve particular hallucinogenic portions with numerous admixtures (20). However, the knowledge of chemistry and differential solubilities necessary to exclude both bufotoxin and bufogenin in an orally ingested preparation would represent an impressive achievement (13).

Toads have also been used to make so called ‘love drinks’ and other witches’ brews and ointments (25, 26). Scientists have regarded these as fictional over time. Although in China and Mesoamerica, toads are documented as being used in magical brews.

Chinese toad secretions (ch’an su) are now known to contain significant quantities of

BT (24). In China and Japan, BT-containing preparations are sold as aphrodisiacs

(27). In the late Classic period, B. marinus skeleton enclosed in a cylindrical ceramic container was found in Seibal, a Mayan ceremonial center which may have been used as a vessel for drinking belche. Various cane toad skeletons were also discovered in post Classic Mayan ritual depots on the Caribbean island of Cozumel (28, 29) for use in rituals. B. marinus skin is also an ingredient of toad-based arrow poisons, which have the same chemical substances of the strongest African vegetable arrow poisons

(30). All of these findings demonstrate that cane toad was used in rituals or had a cosmological importance. Recently cumulative incidents of ‘toad lickin’ have been reported by the press (31). ‘Toad lickin’ is performed by licking the skin of the cane toad, whereby the skin secretions induced cause a rapid furry sensation on the lips and tongue. According to reports, abusers are likely to lick even twice daily. Five to 10

(up to 30) minutes later, nausea is common, and sometimes hallucinations occurring

20 to 30 minutes after ingestion may occur, but the effect is not as long-lasting as LSD.

The thickened juice of boiled animals is a drink in Australia (although not common)

(32), while cane toad skin tea (a decoction of the dried skin) is also known to be a preparation.

1.1.2 Biocompounds identified from various toad species.

Table 1.1: Identification of various biocompounds to date in the skin of various toad species.

Compound Class Identified compounds Species References

Alkaloid Morphine Bufo marinus (33) (non-peptide opioid) Alkaloid (pyrrolizidines) Allopumiliotoxins, decahydroquinolines, dehydro-5,8-disubst, Melanophryniscus stelznerii, (34, 35) homopumiliotoxins, 3,5-disubstituted indolizidines; 5,8-disubstituted Melanophryniscus rubriventris indolizidines, 3,5-disubstituted pyrrolizidines, 1,4-disubstituted quinolizidine, indolizidines , izidines, pumiliotoxins, 5,6,8-trisubstituted indolizidines, spiropyrrolizidines

Bufadienolide related Marinoic acid Bufo marinus (36)

Fatty acids Suberic acid, succinic acid Bufo bufo gargarizans cantor (37) Hydroxyphenylalkylamines Epinephrine and norepinephrine Bufo marinus, Bufo bufo (38) Non-protein BM-ANF1 Bufo melanostictus Schneider (39) Nucleobases Thymine, uracil Bufo bufo gargarizans cantor (40) Peptides Alyteserin-1c, Alytes obstetricans (41) Bombinin-like-peptides (BLP1, BLP2, BLP3) Bombina orientalis (42) Bombinin related peptides Bombina variegate (43) Cyclo(Ala-Ala), cyclo(Pro-Gly), Bufo bufo gargarizans cantor (40, 44)

Maximins & maximin H, PR-bombesin Bombina maxima (45, 46)

Compound Class Identified compounds Species References Proteins BAS-AH, baserpin Bufo andrewsi (47, 48) Bm-TFF2 Bombina maxima (49) Steroids Arenobufagin, hellebrigenin, marinobufagin, telocinobufagin Ansonia inthanon (50) (Bufadienolides) Marinosin Bufo marinus (51)

Argentinogenin, bufalin, bufotalidin, gambufotalidin, hellebrigenol, Bufo marinus (1) jamacobufagin, marinobufagin, resibufogenin, telocinobufagin,

marinobufotoxin, telecinobufotoxin Arenobufagin, arenobufagin-3-suberoylarginine, bufalin, bufalin-3- Bufo bufo gargarizans cantor (44, 52-56)

succinoylarginine ester, bufarenogin, ψ-bufarenogin, ψ-bufarenogin-3- suberoylarginine bufotalin, bufotalin-3-succinoylarginine ester, bufotalin-3- suberoylarginine ester (vulgarobufotoxin), bufotalinin, bufotoxin, cinobufagin, cinobufagin-3-succinoylarginine ester, cinobufagin-3-glutaroylarginine ester, cinobufagin-3-adipoylarginine ester, cinobufagin-3-pimeloylarginine ester, cinobufagin-3-suberoylarginine ester, cinobufagin-3-sulphate, cinobufotalin, cinobufotalitoxin, 3-dehydrobufalin, de-O-acetylcinobufotalin, desacetylbufotalin, desacetylcinobufagin, desacetylcinobufotalin, desacetylcinobufotalin-3-suberoylarginine, desacetylcinobufotalin-3- succinoylarginine ester, gamabufotalin, gamabufotalin-3-suberoylarginine ester (gamabufotalitoxin), gamabufotalin-3-succinoylarginine ester, gamabufotalin-3-adipoylarginine ester, gamabufotalin-3-pimeloylarginine ester, hellebrigenol, hellebrigenin and its stereoisomer, hellebrigenin-3- hemisuberate, 11β-hydroxylbufalin, 12β-hydroxylbufalin, 19-hydroxylbufalin resibufogenin-3-succinoylarginine ester, marinobufagin, 11β- hydroxyresibufogenin,12β-hydroxyresibufogenin,

Compound Class Identified compounds Species References

Steroids N-[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-N-methylurea, 3-oxo-D4- Bufo bufo gargarizans cantor (44, 52-56) (Bufadienolides) resibufogenin, 19-oxo-cinobufotalin, 19-oxocinobufotalin-3-adipoylarginine ester, resibufogenine-3-sulphate, resibufogenin, telocinobufagin, telocinobufagin-3-hemisuberate1β-hydroxylbufalin, telocinobufagin-3- suberoylarginine Bufalin, bufalitoxin, bufotalin, cinobufagin, cinobufotalin, desacetylcinobufagin, Bufo asper (50, 57) marinobufagin, resibufogenin, resibufotoxin, telocinobufagin, vulgarobufotoxin,

Arenobufagin, bufalin, bufotalin, gamabufotalin, marinobufagin, marinobufagin- Bufo bankorensis BORBOU (58) 3-succinyl-L-arginine ester, marinobufagin-3-glutaryl-L-arginine ester, marinobufagin-3-adipoyl-L-arginine ester, marinobufagin-3-pimeloyl-L-arginine ester, marinobufagin-3-suberoyl-L-arginine ester, resibufogenin , telocinobufagin, telocinobufagin-3-succinyl-L-arginine ester, telocinobufagin - 3-glutaryl-L-arginine ester, telocinobufagin-3-adipoyl-L-arginine ester, telocinobufagin-3-pimeloyl-L-arginine ester, telocinobufagin-3-suberoyl-L- arginine ester Arenobufotoxin, bufalin-3-adipoylarginine ester, bufalin-3-pimeloylarginine Bufo vulgaris formosus Boulenger (59-62) ester, bufalin-3-succinoylarginine ester, bufalin-3-sulphate, bufalitoxin, bufogenin-3-sulfates, cardenobufotoxins,cinobufagin-3-pimeloylarginine ester, cinobufagin-3-adipoylarginine ester, cinobufotalitoxin, cinobufotoxin, gamabufotalin-3-pimeloylarginine ester,

Compound Class Identified compounds Species References

Steroids gamabufotalin-3-adipoylarginine ester, gamabufotalitoxin, resibufogenin-3- Bufo vulgaris formosus Boulenger (59-62) (Bufadienolides) succinoylarginine ester, sarrmentogenin-3-sulfate Marinobufagin-3-suberoyl-L-glutamine ester, telocinobufagin-3-suberoyl-L- Bufo americanus (63) glutamine ester Marinobufagin, telocinobufagin Bufo divergens (50)

Arenobufagin, arenobufotoxin, bufotalinin, hellebrigenin, hellebritoxin, Bufo viridis Laur (64, 65) marinobufagin, telocinobufagin, marinobufotoxin, telocinobufotoxin, resibufogenin Bufotalin-3-suberoyl-histidine, bufotalin-3-methylhistidine, cinobufagin Bufo melanostictus schneider (66, 67) Steroids (Cholesterols) Cholesterol, cholestane-3β,5α,6β-triol, cholestenone, palmitatic acid Bufo bufo gargarizans cantor (44)

cholesteryl ester Miscellaneous Sleep inducing factor (SIF) Bufo melanostictus Schneider (68) Bufogargarizanine B, bufogargarizanine C, bufogargarizanine D, 4-amido-3- Bufo bufo gargarizans cantor (37, 40) hydroxymethyl-coctylamidezotetra-α-furanone

1.1.3 Biocompounds identified from cane toad

Various classes of compounds such as indolealkylamines (IAAs), bufadienolides, peptides, proteins, fatty acids and hydroxyphenylalkylamines have been identified in various toad skins including cane toad (B. marinus) (Table 1.1). Among them, bufadienolides and IAAs were found as the major class of compounds. Compounds from the bufadienolide class identified to date in the skin of various toad species are presented in Table 1.1. Compounds of the IAA class have been recently reviewed by

Zulfiker at al (Appendix). In summary, there have been 15 IAAs, namely: serotonin (5-

HT), BT, bufotenidine, bufotenine-O-sulphate, bufothionine, bufoviridine, dehydrobufotenin, dehydrobufothionine, indole-3-acetic acid, 5-HT-O-sulphate, 5- methoxytryptamine, N-methyl-5-HT, N-methyl-5-methoxytryptamine, O- methylbufotenine and O-methylbufoviridine isolated to date from different toad species

(69). Cane toad secretions are also reported to contain catecholamines (dopamine, N- methyldopamine, adrenaline, noradrenaline), tryptamines (5-HT, N-methyl-5-HT, BT, bufotenidine and dehydrobufotenine), along with glycoside-like toad toxins (31, 70).

Pharmacologically active substances in B. marinus skin extracts have been found to contain BT, bufotenidine, dehydrobufotenine and bufothionine (71). It was found that

B. marinus presents dehydrobufotenin, 5-HT and traces of BT in the parotoid gland secretion and bufotenidine, dehydrobufotenine, 5-HT and bufotenin in the skin secretion. Variations of different IAAs were also observed in the skin and parotoid gland secretions of similar species (72). The skin has also been reported to contain morphine.

The toad toxins (bufotoxine, bufogenine, and bufadienolides) are cardiotoxic and have digitalis like effects: including nausea, vomiting, increase in blood pressure, confusion, and psychotic states (32). Smoking is presumably the protective way to take-in cane toad secretions, as the boiling method reportedly destroys the poisonous constituents while leaving the BT intact. The ethnobotanist Brett Blosser smoked dried cane toad

secretions (~ 1 mg every few minutes) and stated experiencing tryptamine-like hallucinations. The small number of reports on the effects of smoked toad skin state that they are hallucinogenic. One Australian user stated, "I am seeing the world through the consciousness of a toad" (27).

H2N (H3C)2N (H3C)2N HO HO O

N H N N H 5-HT H Bufotenine Dehydrobufotenine

H CHN (H C) N 3 (H3C)2N 3 2 H CO HO 3 O3SO

N N N H H H N-Methyl-5-HT O-Methylbufotenine Bufothionine

(H C) N H2N (H3C)2HN 3 2 H3CO O3SO HO

N N N H H SO3H 5-Methoxytryptamine Bufotenine-O-sulphate Bufoviridine

(H C) N H3CHN (H3C)3N 3 2 O HO H3CO

N N N H H SO3H N-Methyl-5-methoxytryptamine Bufotenidine O-Methylbufoviridine

HOOC H2N HO

N N H H 5-HT-O-sulphate Indole-3-acetic acid

Figure 1.2: Structure of indolealkylamines (IAAs) identified in cane toad skins so far.

1.1.4 The use of toad products in medicine

Toad skin and venom secretions have been used in traditional Chinese and Japanese medicine for more than 3000 years (19). Chinese traditional medicines (TCMs), have also earned popularity in western countries. More than one million prescriptions for these medicines were dispensed in Britain in 1999 and more than 600 TCM clinics exist now in that country alone. Toad skin and venom contain a range of complex secondary metabolites. It is assumed that these may have been developed through evolution, presumably due to survival pressures and subsequently to be synthesized to act as poisons to potential predators or to act upon endogenous mechanisms. This rich supply of natural chemicals demonstrates an exceptional source which may lead towards potential pharmaceuticals (1).

Dried and powdered toad preparations known as Chan Su or Senso, reached Europe from China in the 17th century (22) and were listed as “bufones exciccati” in the official pharmacopoeias at that time. They were indicated as diuretics in the management of dropsy (oedema) as well as other diseases before being substituted by digitalis, introduced by William Withering in 1785. In China and Japan, the dried venom of

Chinese toad is still indicated for numerous medicinal purposes including oedema, fever, pain relief, infections and tonsillitis. Toad products are also used in cosmetics that have been FDA approved in USA, particularly skin lotions and creams, to treat skin disorders such as dermatitis (1). Parotid gland secretions of Chinese toad, Chansu in native Mandarin, have a long history of clinical use in China (23). Over the last decade

Chansu-related toad medicines have also earned great favor in USA and other western countries. The venom is used topically in the treatment of sores, toothache and local

inflammation. In recent years topical preparations containing toad venom are extensively being used to successfully treat different cancers and heart failure (73).

Due to current agriculture herbicide contamination in China and the continuing relaxation of economic trade barriers, TCM associations are importing this animal product, with Australia being a viable source. An Australian company based in

Queensland has exported cane toad venom and skin to the China markets to use in traditional medicinal preparations. Thus research that might add value to and improve the prospect of cane toad export is welcomed as an opportunity to make this pest commercially useful (1).

A water extract of Chinese toad skin is used in China, is administered as an intravenous injection for anti-cancer therapy and generates reportedly a 10 billion USD market.

While some researchers have tested the bilogical effects of B. marinus parotid secretions (74), investigations on the skin extracts are still lacking. Only cardiac glycoside-like activity using human or animal isolated organs in vitro as bioassay tissues are reported for aqueous and chloroform extracts of cane toad skins (1). To the best of our knowledge, investigation of aqueous cane toad skin extracts and any of its constituents in the treatment of obsessive compulsive disorder (OCD) is still unexplored. According to the Mark Report in 2007, cane toad numbers are increasing every year throughout eastern and northern Australia. So if the cane toad pest could be turned into a medicinal resource, then a new direction for the control of this biohazard could result.

1.2 Anxiety disorders

Anxiety disorders, being a group, are among the most common mental health disorders and often cause major functional impairment (75). Historically, anxiety disorders have received comparatively little research attention. In recent times epidemiological studies have described anxiety disorders as the commonest class of mental illness (76), which are frequently comorbid with other conditions including both medical and psychiatric.

As a consequence there is a need to develop effective treatments, both pharmacologic and psychological to provide symptomatic relief (75).

1.2.1 Prevalence

Twelve month prevalence rates in Australia in 2007 indicate that anxiety disorders are the most common mental health problem, affecting 14.4% of the population (although some people experienced more than one type of anxiety disorder). Post-traumatic stress disorder (PTSD) is the most widespread affecting 6.4% of the population, followed by social phobia (4.7%), agoraphobia (2.8%), generalised anxiety disorder

(GAD, 2.7%), panic disorder (2.6%), and obsessive compulsive disorder (OCD, 1.9%)

(77). The 2007 UK Adult Psychiatric Morbidity Survey reported a 9% prevalence of mixed anxiety and depression in their doorstep community survey, while generalised anxiety disorder (GAD) affected 4.4% of the population, other anxiety disorders 3.6%, and only 2.3% reported depressive disorders (78). Women experienced higher rates than men (18% and 11%, respectively), and the highest rate of anxiety disorders was in the 35–44 year old age group (18%). One in 5 women and 1 in 10 men report a specific phobia (79). General practice is often the first port-of-call for patients with anxiety disorders; 1 in 10 people experiencing an anxiety disorder within the past 12 months visited a GP for their mental health problems, but did not receive care from any other provider (80). The Bettering the Evaluation and Care of Health program showed

that GPs treat psychological problems at a rate of 11.5 per 100 encounters and anxiety is the second commonest psychological problem managed after depression (81).

1.2.2 Assessment, diagnosis and/or symptoms of specific anxiety disorder

There has been increasing focus to encourage GPs to improve their skills in the diagnosis and management of depression, although anxiety disorders have been comparatively neglected. In the community, the symptoms of anxiety disorders are more common than those of depression and are frequently accompanied by significant morbidity (78). Yet there has been little research to evaluate outcomes in anxiety other than depressive disorders, and the majority of research has come from the USA or

Europe (82). Medical conditions that may be related to anxiety include (83):

•hypoglycaemia

• hyper- or hypo-thyroidism

• cardiac disorders

• chronic respiratory disease

• vitamin B deficiency

• inner ear infections

• acute reactions to aspartame

• withdrawal from benzodiazepines.

If there is no sign of a medical cause for the patient’s symptoms, assessment should change to look for the characteristics of specific anxiety disorders and other coexisting psychiatric disorders and assessing the degree of distress (84).

Table 1.2: Short description of anxiety disorders as defined by ICD-10 (85) and DSM- IV-TR (83).

Anxiety Definition disorders Panic disorder Panic disorder is characterized by recurrent panic attacks. Panic attacks are discrete

(PD) periods of intense fear or discomfort, accompanied by at least four somatic and psychic

symptoms (palpitations, sweating, trembling, dyspnoea, choking sensations, chest

pain, nausea, abdominal distress, dizziness, feeling of unreality, fear of dying, etc.). Panic disorder A panic attack reaches a peak within 10 min and lasts 30 – 45 min on average. Usually,

(PD) the patient is afraid that he has a serious medical condition such as myocardial

infarction.

Agoraphobia About two-thirds of all patients with panic disorder suffer from agoraphobia, which is

defined as fear in places or situations from which escape might be difficult or in which

help may not be available in the event of having an unexpected panic attack. These

situations include being in a crowd or standing in a line, being outside the home alone,

or traveling in a bus, train or automobile. These situations are avoided or endured with

marked distress.

Generalized The main features of generalized anxiety disorder are excessive anxiety and worry.

anxiety disorder The patients suffer from somatic anxiety symptoms as well as from restlessness,

(GAD) irritability, difficulty concentrating, muscle tension, sleep disturbances and being easily

fatigued. Patient may express constant worry that the patient or a relative will shortly

become ill or have an accident.

Specific phobia Specific phobia is characterized by excessive or unreasonable fear of single objects or

situations (e.g., flying, heights, animals, seeing blood).

Social phobia SAD is characterized by marked, persistent, and unreasonable fear of being observed

(social anxiety or evaluated negatively by others in social performance or interaction situations and is

disorder; SAD) associated with somatic and cognitive symptoms. The feared situations are avoided or

else are endured with intense anxiety or distress. These situations include fear of

speaking in public, speaking to unfamiliar people or being exposed to possible scrutiny

by others.

Anxiety Definition disorders

Obsessive- OCD is characterized by recurrent obsessions or compulsions, or both, that cause

compulsive impairment in terms of distress, time, or interference with functioning. Concerns

disorder (OCD) involving contamination, harm, hoarding, and sexual, somatic and religious

preoccupations are the most common obsessions. Compulsions include washing,

checking, repeating, ordering, counting, hoarding and touching (rare).

Post-traumatic PTSD develops after a terrifying ordeal that involved physical harm or the threat of

stress disorder physical harm. The person who develops PTSD may have been the one who was

(PTSD) harmed, the harm may have happened to a loved one, or the person may have

witnessed a harmful event that happened to loved ones or strangers. The condition is

characterized by recurrent and intrusive distressing recollections of the event,

nightmares, a sense of reliving the experience with illusions, hallucinations, or

dissociative flashback episodes, intense psychological or physiological distress at

exposure to cues that resemble the traumatic event, avoidance of stimuli associated

with the trauma, inability to recall important aspects of the trauma, loss of interest,

estrangement from others, sleep disturbances, irritability, difficulty concentrating,

hypervigilance, and exaggerated startle response. The full symptom picture must be

present for more than 1 month.

1.2.3 Management of anxiety disorders

Anxiety is a common human physiological mechanism intended to help the body respond to a threat. The autonomic changes that happen in anxiety are important to avoid danger and moderate anxiety can, in fact, improve performance. However, when anxiety is accompanied by very high levels of autonomic arousal, erroneous cognitions including exaggerated threat perceptions and dysfunctional coping strategies, it can result in significant distress and impairment in work, school, family, relationships, and/or activities of daily living. Patients showing anxiety symptoms in a general practice setting do not always fit the characteristics of a particular anxiety disorder. However, it 21

is important for the general practitioner to know how to assess patients for particular anxiety disorders and the basic principles of management of these disorders (86).

1.2.3.1 Psychoeducation

Psychoeducation is important when dealing with someone with any anxiety disorder.

Most people with anxiety symptoms also suffer a secondary ‘worry about worry’ (e.g., that anxiety is dangerous, that they are going crazy or ‘losing it’), which clearly aggravates the anxiety. Psychoeducation is also important in some treatment options

(e.g., relaxation, cognitive behaviour therapy (CBT), exercise, yoga). GPs are in a good position to deliver such information as they are a trusted and authoritative source of information about health and physiology (84).

1.2.3.2 Psychological treatments

Psychological treatments have been presented as an effective medication for anxiety disorders, with CBT having the greatest evidence for efficacy (11, 13). Some GPs may have extraordinary skills in CBT, but in most cases the patient may need to be referred to a psychologist.

CBT for anxiety disorders starts with psychoeducation about anxiety, as outlined above, and training patients in relaxation techniques (such as progressive muscular relaxation or guided imagery) or increasingly, mindfulness based on meditation.

Preliminary evidence indicates that mindfulness techniques may be of particular benefit

(87). Exposure techniques are helpful if there is a specific feared stimulus. These involve exposing the patient to feared stimuli in a graded fashion until anxiety reduces.

The diagnosis will indicate the stimuli to which the individual will require exposure, for 22

example, bodily sensations in panic disorder or social situations in social phobia.

Usually exposure techniques are coupled with behavioural strategies to address avoidance behaviour and cognitive strategies that challenge specific maladaptive beliefs (e.g., ‘I must control my horrible thoughts’ in OCD). Other aspects of CBT for anxiety include training in problem solving to help patients develop adaptive coping and interpersonal skills and relapse prevention strategies. In addition to in-session exposure practice, home based tasks and monitoring sheets to help the patient report on their experiences between consultations have been shown to be helpful (88-90).

1.2.3.3 Pharmacological management

SSRIs and SNRIs are the first line of drugs indicated for anxiety disorders (75). Various other drugs are also used to treat anxiety disorders, which are listed in Tables 2 and 3.

1.2.3.4 Pharmacological, non-pharmacological management or both

Anxiety disorder management with concurrent CBT and pharmacotherapy has not been found effective in long term treatment, in spite of its regular use in practice (91,

92). Some researchers think that the concurrent use of CBT and anxiolytic medications may be detrimental to the elimination phase of exposure based therapies, depending on the effects of glucocorticoid activity on learning of emotional material (93). Patients appraise both CBT and pharmacotherapy, but prefer CBT to medication for anxiety disorders treatment, and reportedly find CBT to be more effective in the long term (94,

95). A general guideline is to begin with CBT but pharmacotherapy may be considered if patients are unresponsive or depression levels are high. In severe cases, initiating evidence based medication before starting CBT is considered. In clinical decision making, both patient preferences and characteristics must be taken into account. Daily 23

assessment is important to observe the clinical improvement. But the disorders demanding more specialised psychological interventions (e.g., OCD, PTSD, GAD) or even more complex situations (e.g., severe, comorbid) may require referral to a professional and skilled psychologist or psychiatrist (84).

Table 1.3: Approved US Food and Drug Administration (FDA) drugs for anxiety disorders.

Group Mechanism of Drugs Indication Dosage Clinical Considerations action Selective Inhibition of reuptake Fluoxetine GAD,s Preliminary dose 5 mg/day in am. Enhance by 5 Weight neutral; no need to taper off (96).

Serotonin at the presynaptic OCD, PD mg/wk up to 20 mg. Wait a month to assess drug

Reuptake serotonin (5-HT) effects before enhancing dose, enhancing by 20

Inhibitors transporter pump, mg/mo. Maximum dose generally 80 mg/day (96).

resulting in increased (SSRIs) Paroxetine GAD, PD, Panic disorder: preliminary 10 mg/day (12.5 Most sedating SSRI; most potential for overall levels of brain PTSD, mg/day CR); generally wait a few wk to assess weight gain; difficult to discontinue; 5-HT OCD, drug effects before enhancing dose, but can favourite first choice for GAD, PTSD (96).

SAD enhance by 10 mg/day (12.5 mg/day CR) once a

wk; maximum dose generally 60 mg/day (75

mg/day CR); single dose.

Escitalopram GAD PreliminaryOther anxiety 10 mg/day;disorders: increase initial 20 to 20 mg/day mg/day (25 if Comparatively expensive; less GI

needed;mg/day CR); single generally dose any wait time a few of wk day. to 10assess mg symptoms than citalopram (96).

escitalopramdrug effects equivalent before enhancing to 40 mg dose,citalopram but with can

fewerenhance side by effects. 10 mg/day Give an (12.5mg/day adequate trial CR) of once 10 mg a

beforewk; maximum giving 20 dose mg. Some 60 mg/day patients (75 require mg/day dosing CR);

withsingle 30 dose or 40 (96) mg. (96).

Group Mechanism of Drugs Indications Dosage Clinical Considerations action

Selective Inhibition of Sertraline OCD, PD, Preliminary 25 mg/day; increase to 50 mg/day Good first choice SSRI; in pregnancy,

Serotonin reuptake at the PTSD, SAD after 1 wk, usually wait a few wks to assess drug “zombie” effect common (96).

Reuptake presynaptic effects before increasing dose; maximum does

Inhibitors serotonin (5-HT) generally 200 mg/day; single dose (96).

transporter pump, (SSRIs) resulting in

increased overall Tricyclic Inhibition both Clomipramin OCD Preliminary 25 mg/day; enhance over 2 wk to 100 Take with meals; more side effects than levels of brain 5-HT Antidepressants serotonin and e mg/day; maximum dose usually 250 mg/day at SSRIs (96).

(TCAs) norepinephrine night. OCD may need high doses (e.g., 200-250

reuptake from the mg/day) (96).

synaptic cleft

synaptic cleft Doxepin Anxiety Preliminary 25 mg/day at bedtime; enhance by Useful for anxiety with skin, hair, scalp,

25 mg every 3-7 days. 75 mg/day; enhance nail issues (scratchers/pickers); recent

progressively until desired efficacy; can be dosed FDA approval as a sleep agent (96).

daily at bedtime or in divided doses; maximum

dose 300 mg/day (96).

Group Mechanism of Drugs Indications Dosage Clinical Considerations action

Serotonin Inhibition reuptake Venlafaxine GAD, PD, Preliminary dose 37.5 mg daily (XR) or 25-50 mg Difficult discontinuation; monitor for

Norepinephrine of norepinephrine XR SAD in 2-3 doses (immediate release) for 1 wk, if hypertension; useful for pain syndromes

Reuptake and serotonin from tolerated; enhance daily dose no faster than 75 (96).

Inhibitors (SNRIs) the synaptic cleft mg every 4 days until desired efficacy; maximum

dose 375 mg/day. Try doses at 75 mg increments

for a few wk before enhancing by 75 mg (96).

Duloxetine GAD For generalized anxiety, preliminary 60 mg daily; Weight neutral; fewer sexual side effects;

maximum dose 120 mg/day(96). hepatotoxicity—watch alcohol intake

(96). Benzodiazepines Binding to a Lorazepam Occasional 0.5 mg orally Short acting, rapid tolerance; acute,

specific site on the situational short-term use agent; contraindicated for

GABA-A receptor, anxiety those with acute narrow-angle glaucoma, Clonazepam Short term 0.5 mg orally daily, may split dose (96). Potential for dependence; avoid use with resulting in an sleep apnea, or myasthenia gravis; avoid use while history of chemical dependency; not for enhanced effect of use with history of chemical dependency starting those with acute narrow-angle glaucoma the inhibitory (97) SSRI (2-4 or hepatic failure; good choice for first 2- neurotransmitter Alprazolam Occasional 0.25 mg orally (96). Multiple drug-drug interactions ; high risk week) 4 wk when starting SSRI for panic GABA situational of tolerance, abuse, dependence (97); anxiety usesymptoms only in (97)very. limited situations such as

air travel or needle phobia (96); use in

27 collaboration with mental health

specialist

Group Mechanism of Drugs Indications Dosage Clinical Considerations action

Other Non- Buspirone Anxiety Preliminary 15 mg twice a day; enhance by 5 Generally not as effective for mono-

Benzodiazepines mg/day every 2-3 days until desired efficacy; therapy as other agents; may augment

maximum dose 60 mg/day (96). SSRIs, which is recommended to reduce

sexual dysfunction (96). Hydroxyzine GAD 50-100 mg 4 times a day (96). Can be agitating (97); driving

precautions; good augmentation with

SSRIs (96).

Table 1.4: Commonly used drugs for anxiety disorders in primary care (Non-FDA).

Drugs Classification Clinical Use Clinical considerations

Mirtazapine α2 antagonist; dual GAD, PTSD, PD Side effects worse at lower doses, gets better as dose increases (96);

serotonin and breaking a 15-mg tablet in half and giving 7.5 mg may increase sedation (96,

norepinephrine agent; 97); some patients require more than 45 mg daily, including up to 90 mg in

antidepressant (96) patients who tolerate these doses (96).

Propranolol Βeta-blocker Performance anxiety; prevention Caution in individuals with asthma, comorbid depression (96) and diabetes

of PTSD in those with symptoms of (97).

acute stress disorder

Prazosin Centrally active α1 Nightmares in PTSD Caution with other antihypertensives (98)

adrenergic antagonist,

Clonidine antihypertensiveCentrally active α1 Performance anxiety, Contraindicated with other antihypertensives; avoid abrupt discontinuation; adrenergic antagonist, hyperarousal PTSD taper dose gradually in hypertensive patients (98).

antihypertensive

Trazodone 5-HT2 antagonist; reuptake Insomnia in PTSD, PD, GAD Falls are common in elderly (98).

Gabapentin inhibitorAnticonvulsant Anxiety Useful for anxiety syndromes in those with bipolar disorder; good choice with

anxiety with pain syndromes; relatively few drug-drug interactions (99)

Guanfacine Centrally active α2A Nightmares in PTSD If guanfacine is terminated abruptly, rebound hypertension may occur within

agonist; antihypertensive 2-4 days (100).

1.2.4 Anxiety disorders and inflammation

The role of inflammation in various disorders such as cardiovascular disease, diabetes, and cancer has been considered as a common mechanism of disease for decades (101-103). This important insight to neuropsychiatric disorders is now expanding to have an effect in the disciplines of psychiatry and neurology.

Likewise researchers and clinicians are recognizing the significance of immune and inflammatory processes in neurodegenerative disorders, mood and anxiety disorders as well as schizophrenia (101, 104, 105).

With regard to anxiety disorders, very little attention has been paid to these diseases, though it is apparent that many patients with anxiety disorders also show an inflammatory phenotype like depression. PTSD is the most studied so far, where several studies have revealed the enhancement of inflammatory cytokines and their signalling pathways, including nuclear factor kappa B (NF-

κB), which is a lynchpin molecule to start the inflammatory cascade and has been displayed to be exquisitely stress responsive (106, 107). Inflammatory markers have also been reported to increase in other anxiety-related conditions including panic disorder, OCD, anxiety-related personality dimensions, as well as diagnoses such as neuroticism and borderline personality disorder (108-111).

1.2.5 Obsessive compulsive disorder (OCD)

OCD is a chronic, disabling anxiety disorder characterized by obsessions and/or compulsions that are associated with significant distress and/or interference in functioning of daily life (19). It is described as an anxiety disorder in DSM-IV

(American Psychiatric Association 1994). 30

1.2.5.1 Prevalence

Studies suggest that OCD affects up to 1 in 50 people worldwide (112) which is manifested across development (113), and is related to substantial dysfunction and psychiatric comorbidity(114, 115).

Table 1.5: Obsessions and compulsions in OCD.

Obsessions Compulsions Contamination concerns Washing, bathing, showering

Harm to self/others Checking, praying, asking for reassurance Sexual/religious concerns

Symmetry, precision concerns Arranging, ordering

Saving concerns Hoarding

OCD has also estimated to be the fourth most prevalent life time psychiatric disorder in the general population (116, 117). Some researchers estimate that

OCD patients constitute up to 10% of the population in outpatient psychiatric clinics that makes OCD the fourth most common diagnosis after phobias, substance abuse and major depression. OCD can initiate in childhood and adolescence, sometimes as early as age two. Males are affected at an earlier age compared to females, but both genders have the same prevalence in adulthood. Mean age of onset is about 20 years. Most OCD patients develop the disorder by age 25 with 15% showing symptoms after the age of 35 (118).

About 1% of children and adolescents are found with clinically significant obsessive-compulsive symptoms (79, 80) along with as many as 4% of youths showing subclinical symptoms (81). Approximately 80% of adult OCD cases have 31

preliminary onset in childhood (83, 84), which typically runs a prolonged course without starting proper treatment. The proportion of childhood OCD between boys and girls is about 3:2, which is due in part to a higher frequency of tic disorders in boys (119). However, this ratio is mostly balanced by adolescence/early adulthood (23, 113).

1.2.5.2 Symptoms

Obsessions are repetitive, disturbing invasive thoughts or images that cause disabling distress. Common obsessions include fears of contamination, sexual behaviour (e.g., fear of doing something sexually inappropriate), aggressiveness

(e.g., fear of harming another person or self), or religious taboos (e.g., concerns about committing blasphemy or offending a religious figure), and symmetry/exactness and hoarding/saving obsessions (22, 23). The distressing nature of obsessions motivates compulsions, which are repetitive, overt or covert behaviours performed to temporarily relieve the anxiety associated with obsessions. Common compulsions include cleaning, checking, repeating behaviours, reassurance seeking, covert rituals (e.g., counting, praying), and hoarding/collecting (22, 73, 120). Unlike adults with OCD, children do not need to have insight into their obsessive-compulsive symptoms (19), though the majority of children have some degree of insight (77, 86).

The outcome of this disorder is extremely severe in the life of children/adolescents, interfering in their personal, social, school and family life

(114, 121). OCD is frequently comorbid with depression (70%) (120). Moreover, various studies have reported higher comorbidity rates, indicating that up to 80%

of children have certain related disorders. These comorbid disorders result in an incremental increase in the degree of interference, worsening the condition of the disorder and narrow treatment response (122).

1.2.5.3 Pathogenesis

1.2.5.3.1 Neuroanatomy

Preliminary manifestations of OCD are initiated by specific neuronal circuits which presumably came from an association between post encephalitis parkinsonian and obsessive-compulsive symptoms together with striatal lesions (123). OCD

Symptoms have also been reported in several neurological disorders with striatal involvement, including Tourette’s syndrome, Sydenham’s chorea, Huntington’s disorder, and Parkinson’s disorder (124).

On the contrary, OCD patients having abnormalities of various measures and paradigms can be used in neuropsychiatric (e.g., neurological soft signs, olfactory identification, evoked potentials, prepulse inhibition, intracortical inhibition) and neuropsychological (e.g., executive function, visual memory function) studies

(124, 125). These abnormalities are congruent with cortico-striatal-thalamic cortical dysfunction and impaired inhibition, and studies suggest that they are specific to OCD (126). Structural imaging studies have elucidated the significant decreased volume and increased grey matter density of the corticostriatal- thalamic-cortical circuits in OCD patients. Similarly, functional imaging studies

(127, 128) steadily represent enhanced baseline activity in the orbitofrontal cortex, cingulate gyrus, and striatum in OCD patients along with significantly increased activity when their obsessions are exacerbated (Figure 1.3). Besides 33

the regional cerebral glucose metabolic rates in OCD patients determined by positron emission tomography demonstrate the involvement of a neuronal circuitry in the orbitofrontal cortex (OFC), the head of the caudate nucleus and the thalamus (129-132). In fact, OCD patients are found to have an increased metabolic activity in this circuitry, and both effective pharmacological and behavioural treatments are required to normalize this metabolic activity (133-

136).

Other areas of the brain might also underlie a part in OCD. For instance, temporal dysfunction has been related to OCD (128, 137), and the connection of the amygdala with OCD is also found in some reports (Figure 1.3) (138). Imaging research in children with OCD supports the involvement of cortico-striatal- thalamic-cortical circuits and could determine the evolution of various abnormal brain areas over time (127). Functional imaging studies (135) elucidate the normalization of brain activity in the corticostriatal-thalamic-cortical circuitry in patients following pharmacotherapy and behavioural therapy.

Figure 1.3: Increased activity in orbitofrontal cortex and caudate in patients with OCD [Modified from (139)].

1.2.5.3.2 Neurochemistry

Serotonin (5-HT) seems to be a significant factor in the pathogenesis of OCD.

Studies (140-144) suggest that the key 5-HT sub-receptors in OCD seem to be the 5-HT2C and 5-HT1D receptors. Studies (145) indicate that high dose and long duration of administration of SSRIs are required to desensitize the 5-HT1D receptor, which is associated with what is clinically monitored to reduce the symptoms of OCD. In animals, administration of SSRIs for 8 weeks (but not 3 weeks) resulted in the enhanced release of 5-HT in the OFC, due to the terminal

5-HT auto-receptors becoming desensitized. It was also demonstrated that that the activation of normosensitive postsynaptic 5-HT2-like receptors may underlie the enhanced 5-HT release in the OFC (146) because increased release of 5-HT by the serotonergic neurons of the OFC would decrease the symptoms clinically

(Figure 1.4) (147).

Dopamine is another neurotransmitter of the cortico-striatal-thalamic-cortical system that offers promise in the pathology of OCD. Recently decreased striatal

D2/3 receptor availability in OCD has been documented (148). On the other hand, administration of dopamine agonists in preclinical studies has been reported to worsen the symptoms and tics of OCD (149). Dopamine blockers, such as atypical antipsychotics, are the FDA-indicated treatment for Tourette’s syndrome, which is thought to be part of the OCD spectrum (147).

Figure 1.4: Normalisation of cortico-striatal-thalamic-cortical circuits by either pharmacotherapy or psychotherapy in OCD [Modified from (139)].

Indeed, many other systems, including glutamate neurotransmission (150), some neuropeptides (151), and gonadal steroids (152, 153) also contribute a part.

Eventually, the role of second and third messenger pathways in OCD will need to be defined (154, 155).

1.2.5.3.3 Neurogenetics

Studies suggest (156) that there is a genetic relation with OCD and that functional genetic polymorphisms may have a role in the pathogenesis (157). The polymorphisms of 5-HT system genes such as the 5-HT transporter has also been implicated, but not proven consistent so far (158). Based on an earlier report on 5-HT1D polymorphism (143) it is thought that the terminal auto-receptor is important to mediate OCD, but remains inconclusive. Recently dopaminergic polymorphisms have been reported to be responsible, indicating that alleles were distributed differently in OCD patients with and without tics (159).

1.2.5.3.4 Neuroimmunology

Early reports on correlation between OCD and Sydenham’s chorea were confirmed in a systematic investigation (160) which led to the consideration that perhaps some cases of OCD resulted from autoimmune processes that disrupted cortico-striatal-thalamic-cortical circuits. Interestingly, it is reported that autoimmune neuropsychiatric disorder is associated with streptococcal infections

(161). It was found (162) to increase the expression of D8/17, a B lymphocyte antigen and marker of susceptibility to development of sequelae after streptococcal infection, in OCD patients. Moreover several immune dysfunctions in OCD such as abnormal autoantibodies have also been documented (163).

Thus the putative relationship between immune dysfunctions and OCD requires additional study to ascertain its specificity (versus other disorders) (164), its frequency (compared with other possible striatal insults), and its association to other psychobiological factors (such as genetic variables) (165).

1.2.5.3.5 Neuroethology

Development of animal models for OCD remains an important goal for the future to help search for new pharmacotherapeutic agents (139). In veterinary behavioural practice, a fascinating set of animal models is found (166). Acral lick dermatitis in dogs, for instance, is designated by repetitive licking of the paws, which is an indication in some cases of OCD where the hands are licked instead of washing. The syndrome is more common in some canine families than others, and its pharmacotherapy response profile appears to be like that of OCD (167).

Other findings (168) demonstrate the contribution of environmental factors in stereotypic promotion. For instance, the behaviours relating to stereotypies can

be produced by confinement or by emotional deprivation. Fluoxetine (SSRI) has been found to be more effective than placebo in the pharmacotherapy of stereotypies in emotionally deprived primates (169). Yet, further work is required to identify the exact origins and nature of such dysfunction; such research necessitates incorporation of a broad range of data comprising neuroanatomical, neurochemical, neurogenetic, neuroimmuno-logical, and neuroethological variables.

1.2.5.4 Management of OCD

For symptomatic control of OCD pharmacologic and behavioural therapy is indicated, but long-term management is needed. SSRIs are the first-line of treatment, but usually higher dosages and longer duration are required for effectiveness over SSRI treatment for depression (170).

1.2.5.4.1 Pharmacotherapy

1.2.5.4.1.1 Tricyclic antidepressants (TCA)

Clomipramine was first considered for use in this disorder in 1967 (171). This drug has been investigated to verify its supremacy in prepost test designs (Table

1.5) (172), with a placebo control condition (173, 174), with CBT (175) and with another TCA, desipramine (176-179). Later it was possible to establish clomipramine as the only agent of this group that has enough criteria for the treatment of OCD. By comparing the differential efficacy of this substance in children versus adults, it was evident that there were considerably better benefits in adults. Moreover, follow-up, substitution or crossed design studies have

revealed that there is a higher frequency of recurrence of the subjects after the drug is discontinued (180).

1.2.5.4.1.2 Selective serotonin reuptake inhibitors (SSIRs)

Introduction of SSRIs is considered effective not only for OCD, but because they also have a better safety and tolerability profile than does clomipramine. In fact, all available SSRIs are found to be effective and better tolerated in randomised controlled studies of OCD (Table 1.5) (181), and a few are also regarded as potential candidates in paediatric OCD (182).

1.2.5.4.2 Other pharmacological strategies

Other pharmacological strategies are considered useful after failure of first line treatments. These strategies may be an extension of SSRIs and third line monotherapies (183). Low doses of dopamine blockers is the best evidenced treatment for augmentation of SSRIs. Traditional neuroleptics was also employed

(184), but recently the use of better tolerated new generation antipsychotic agents such as risperidone (185-187) and aripiprazole (188) in adults has been utilized.

In some studies of adults (controlled) and children (uncontrolled), combinations of antidepressants have been considered useful. Various other drugs such as lithium (189, 190), buspirone (191, 192), pindolol (193) have been used as augmenting strategies, theoretically, to enhance 5-HT neurotransmission but, to date, with little success.

Table 1.6: Drugs used in the treatment of OCD.

Group Drugs Dose/duration Results References

TCA Clomipramine 50-225 mg/day Significant pretest-posttest (172) 8 weeks improvement SSRI Fluoxetine 10-20 mg/day Significant pretest-posttest (194) 8 weeks improvement Sertraline 50-200 mg/day Significant reduction of 49% in (195) 24 weeks patients Paroxetine 20 mg/day Significant pretest-posttest (196) 12 weeks improvement. Mean reduction of 50% Fluvoxamine 25-200 mg/day Mean reduction 25% (197) 10 weeks Citalopram 10-30 mg/day Significant pretest-posttest (198) 8 weeks improvement. Reduction superior to 50% in 80% of the sample Escitalopram 10-20 mg/day Improved functioning, and better (199) 16 weeks tolerability MAOI Phenelzine 45-90 mg/day Improved significantly, effective (200) 12 weeks as clomipramine NASSA Mirtazapine 30-60 mg/day May be an effective (201) 12 weeks pharmacotherapy for OCD

In cases of SSRI-resistant OCD patients, addition of the amino acid precursor tryptophan with pindolol is the only recognised therapeutic potential as a pro- serotonergic strategy (193).

Third line monotherapy is indicated when both first choice treatments as well as augmentation strategies are found to be ineffective. An atypical antipsychotic drug, ziprasidone (202) has been used, although other therapies such as plasma exchange (203, 204) or the administration of glutamate antagonists (205) have also been investigated.

Pharmacotherapy in OCD should be maintained for at least one year (206) as it is possible that some patients have a slower response to treatment (207). 40

Furthermore, it is reasonable to discontinue the drugs gradually after the decision is made to do so (e.g., decreasing dose by 25% every few months) (139).

1.2.5.4.3 Psychotherapy

Psychotherapy was thought to be an effective approach for OCD management for a long time. Behavioural therapy was the first psychotherapy (208), and is useful in OCD in adults and children. However, the precise way in which exposure results in normalisation of corticostriatal- thalamic-cortical circuitry needs to be fully characterised (139).

Cognitive interventions might also have a role in OCD treatment (209). In practice, a cognitive-behavioural approach is frequently used, indicated individually or in groups (210). Because OCD symptoms may have a great impact on the patient’s family and assessment of such an effect as well as inclusion of the patient’s partner or family in development of a treatment strategy would appear suitable in some cases (211).

1.2.5.4.4 Combination of pharmacological treatment and cognitive behavioural therapy (CBT)

There are very few reports on combined psychological therapy and drug treatment studies. Attempts have however been made to introduce CBT along with SSRI or clomipramine to observe whether the child/adolescent improved

(212-217). One of the best combinations reported is CBT together with sertraline, which is considered superior to drug therapy and CBT alone (218). In a recent

study, comparing the results on adding CBT to drug treatment (SSRI) with the previous one (drug treatment only) concludes that children treated with complete

CBT and medication obtained greater diminutions in their obsessive symptoms

(214). In a study reported in 2010 involving addition of D-cycloserine to CBT (219) found very promising results.

1.2.5.5 OCD and inflammation

Clinical studies of OCD patients observed higher tumour necrosis factor alpha

(TNF-α) and Interleukin-6 (IL-6) plasma levels in comparison with the healthy controls. This finding gives indication that altered levels of proinflamatory cytokines exist in these patients, clearly demonstrating the involvement of the immune system in the pathophysiology of OCD (110). Patients with early onset

OCD compared with control subjects were found to have high IL-12 and TNF-α concentrations. This is the first report of elevated IL-12 and TNF-α in paediatric patients (220). Moreover elevated levels of TNF-α have also been found in CSF samples drawn from patients with OCD, attention-deficit hyperactivity disorder and schizophrenia (221). Furthermore, lower concentrations of TNF-α in peripheral blood has been reported in previous studies of adult OCD patients

(222-224). These reports indicating elevated blood levels of two major proinflammatory cytokines, namely TNF-α and IL-6 provides evidence of immune activation in OCD and that ongoing immune activation may underlie the pathogenesis of OCD. Interestingly, most studies have reported a normal or increase in proinflammatory cytokine levels, in contrast to other psychiatric disorders such as major depression and schizophrenia, where a decrease has been reported (225-227).

1.3 Serotonin 2A receptor (5-HT2AR)

The mammalian 5-HT2A receptor is a subtype of the 5-HT2 receptor. It belongs to the 5-HT receptor family and is a postsynaptic G protein-coupled receptor

(GPCR) (228) which is widely expressed in the CNS (229). 5-HT2ARs are highly expressed in pyramidal neurons in the middle layers of the medial prefrontal cortex (230). They are also present in the frontal cortex, cerebral cortex, basal forebrain, hippocampus, amygdala, dorsal thalamus, hypothalamus, superior colliculus, substantia nigra, pedunculopontine nucleus, legmental area, and myelencephalon (231). 5-HT2ARs are involved in mediation of normal and psychotic states, working memory, regulation of GABAergic and cholinergic neuronal cells, sleep, peripheral pain, and cardiovascular functions. In addition,

5-HT2ARs are associated in several psychiatric disorders, such as schizophrenia, depression, and anxiety disorders i.e., OCD (232, 233). Thus, selective ligands are required to investigate the pharmacological role of 5-HT2A receptors (234).

5-HT2ARs are primarily known to couple to the Gαq signal transduction pathway.

When agonists bind with the receptor and stimulate it, then Gαq and β-γ subunits dissociate to trigger downstream effector pathways. Gαq stimulates phospholipase C (PLC) activity, which afterwards promotes the release of diacylglycerol (DAG) and inositol triphosphate (IP3) leading to the stimulation of protein kinase C (PKC) activity and Ca2+ release (235). So agonist activation of the 5-HT2A receptor (5-HT2AR) triggers multiple signal transduction pathways.

Signal transduction cascades are known to ultimately regulate gene transcription which leads to cellular responses (236). The G protein subtypes activated by 5-

HT2AR are mainly Gq/11. Agonists acting at 5-HT2A receptors located on the apical

dendrites of pyramidal cells within regions of the prefrontal cortex are believed to induce hallucinogenic activity (237). It was found that the classical Gq/11-PLC-β 5-

HT2AR signalling pathway is needed to generate a response to hallucinogenic compounds (HCs), but is not unique and specific to HCs (238). The psychedelic effects of LSD are characterised by its strong partial agonist effects at 5-HT2A receptors. It is known that specific 5-HT2A agonists are psychedelics and most 5-

HT2A specific antagonists block the psychedelic activity of LSD (239).

Some reports have demonstrated pertussis toxin (PTX) sensitive Gi/o proteins are involved in cellular responses mediated by 5-HT2AR activation (240, 241). Earlier it was found that the signalling produced by hallucinogen and non-hallucinogen

5-HT2AR agonists causes induction of c-fos and requires Gq/11-dependent signalling in mouse cortical primary neurons. On the other hand, the signalling of hallucinogenic 5-HT2AR agonists also induces egr-2, which is Gi/o-dependent.

That is to say, expression of c-fos is induced by all 5-HT2AR agonists (238) and expression of egr-2 is hallucinogen-specific (237). In a 2007 study it was reported that all HCs investigated induced egr-1 expression (238). Moreover, it is reported that in several cell types, Gi/o protein-regulated phosphorylation networks involve

Gβγ subunit-mediated activation of Src (242-245). It was suggested that both HCs and nonhallucinogenic compounds (NHCs), while acting as agonists at the same

5-HT2AR expressed by cortical neurons, induce different patterns of signalling that are responsible for their different behavioural activities (238). In vitro studies also demonstrated that different agonists acting at the same receptor may exhibit distinct patterns of signal transduction (246).

1.3.1 Significance of 5-HT2AR in OCD

Clinical and preclinical experimental data demonstrate that 5-HT2 receptor activation (most likely 5-HT2A subtype) may be therapeutic to improve OCD symptoms. Most psychedelic drugs are potent agonists at 5-HT2A and 5-HT2C receptors and their binding potency to these receptors is strongly relevant to their human potency as hallucinogens. So the activation of 5-HT2 receptors by hallucinogens may lead to acute reduction of, and possible longer-lasting beneficial effects on, the symptoms of OCD. Probably the biological mechanism of these effects involves agonist activation of 5-HT2 receptors, but whether it is the acute physiological response to this activation, or the adaptive changes following 5-HT2 receptor activation, remains unclear (247). The desensitization of

5-HT2 receptors may be a consideration because the activity of 5-HT2 antagonists lead to a desensitization of 5-HT2 mediated responses (248), though very few 5-

HT2 antagonists have been studied in this respect. In addition, we now know that there are at least 14 subtypes of 5-HT receptor in the brain, although again only a limited number of potential drugs that are in clinical use (including hallucinogens) have been investigated for their effects on these different receptor subtypes. Many questions still remain unanswered. However, if hallucinogens really show the way to ameliorate the symptoms of OCD, then attempts to understand the specific mechanisms underlying such improvement may lead to development of faster-acting and more effective treatments (247).

So the anti-OCD effect could be mediated by an increased 5-HT neurotransmission in the OFC as a result of an increased 5-HT release, where 5-

HT exerts its effect mainly via 5-HT2-like receptors. Since postsynaptic 5-HT2 receptors induce the effect of increased 5-HT release in OFC, a 5-HT2 agonist having capability to selectively activate these cortical receptors should produce a rapid anti-OCD effect (249, 250). In support of this possibility to obtain a rapid therapeutic response, the beneficial effect of some hallucinogens with 5-HT2 agonistic properties, such as psilocybin, would offer potential (251). Following on from this there are several reports regarding the beneficial effects of psilocybin as well as other hallucinogenic drugs such as lysergic acid diethylamide (LSD) in patients with OCD and related disorders (247, 252, 253). Recently nine human subjects with DSM-IV defined OCD were administered 100-300 µg/kg psilocybin and marked decreases in OCD symptoms of variable degrees were observed in all subjects (254).

Thus potential drug development strategies is possible with regard to OCD and/or various others anxiety disorders for 5HT2A agonists. The first step is to develop non-hallucinogenic 5HT2A agonists. As different groups of drugs bind selectively to 5HT2A receptors, it should, at least in theory, be possible to design new nonhallucinogenic 5HT2A agonist. The important underlying postulation of this strategy is that 5HT2A agonism is responsible for the robust anti-OCD effect seen with hallucinogen (255).

1.3.2 Bufotenin as a 5-HT2AR agonist and its significance in OCD

Bufotenin (BT), also known as 5-hydroxy-N,N-dimethyltryptamine (5-OH-DMT), mappin, N,N-dimethylserotonin, 3[2-(dimethylamino)ethyl]-IH-indol-5-01, is a hallucinogenic methylated indoleamine (tryptamine derivative) present in human

body fluids (256-258) and is closely related to N,N-DMT, 5-MeO-DMT, psilocybin and psilocin.

H N HO PO NH(CH ) HO 2 3 3 2 N(CH3)2 HO

N N N H H H 5-HT (Serotonin) Psilocybin Psilocin

Figure 1.5: Structure of 5-HT, psilocybin and psilocin.

BT binds to 5-HT receptor (mainly 5-HT2A agonist) (259). The effective hallucinogenic dose in humans is 50-100 mg and the effects last for only 45-60 minutes. As a result, BT-induced hallucinations are often called a “businessman trip” (260). About 16 mg is indicated as an effective dosage. Very few pharmacological studies are available of this substance. Because of better water solubility, BT is superior to DMT or 5-MeO-DMT as a urinary marker (261). At 8 mg IV (as BT oxalate) the subject (human volunteer) reported intense emotional and perceptual changes involving extreme anxiety as well as a sense that death was immanent, although hallucinations were not present. Even up to 16 mg intranasal dose produced no other effects except profound local irritation. BT is reported as a psychotomimetic agent under certain circumstances (262). Rapid

IV (3 min or less) or intramuscular injections of 4-16 mg of BT have been found to have strong psychological and/or physiological reactions (15, 262, 263). To date BT is not believed to be a true psychedelic drug (264) though this study is not based on any personal experiments. In its pure form, BT has not obtained any cultural significance as a psychoactive substance. Reportedly IV

administration of BT over 3 minutes at doses of 0.06 to 0.25 mg/kg caused tingling in the head and neck to the extremities, constriction of the chest, visual disturbances, colour hallucinations, mental confusion, mild disorientation, nausea, retching, vomiting and nystagmus (15). Within a few minutes after stopping the IV push there was a gradual relaxation, lassitude and withdrawal, followed by a slow return to mental awareness and significant euphoria lasting for several hours. In another report, it was observed that lower doses and slower administration of BT was devoid of the unpleasant initial symptoms, but the euphoric state developed directly and its effects are reminiscent of LSD-25 and

Mescaline (265). In a study from 1959 BT extracted from the seeds of Piptadenia peregrine or the revised Anadenanthera peregrine and tested on humans, whereby snuff (560 mg) was taken intranasally which contained 6 mg of bufotenine producing no intoxication. Neither subjective nor objective effects were observed after inhalation of as much as 1 g of the snuff powder, repeated at 30 minute intervals- patients merely sneezed and coughed. Pure BT (6 mg as the salt-creatinine sulphate) was administered intranasally, and the first symptoms appeared. At 10 mg intranasally symptoms of fear, associated with a flushed face, lacrimation, tachycardia and tachypnea were evident. At 40 mg intranasal spraying, there were no further subjective or objective effects beyond the initial 10 mg dose (263). Interestingly, when mice were injected intraperitoneally with 0.5 mg, they died apparently from respiratory failure (266).

BT is reported as an endogenous substance in humans, reportedly present in human cerebrospinal fluid (CSF), urine and/or blood. Using the most common methods of reporting, studies have identified BT concentrations ranging from 1 to

62.8 µg/24 h, and from 0.48 to 218 ng/mL (267). In blood, concentrations of BT 48

reportedly ranged from 22 pg/mL (HPLC-radioimmunoassay) (268) to 40 ng/mL

(direct fluorescence assay of extracts) (269). There are no reports on the presence of BT in CSF (267).

BT is reported to be behaviourally inactive or only very weakly active in humans and animals; this may be due to its low partition coefficient and resultant inability to penetrate the blood-brain barrier (BBB) (270). Attempted administration of larger doses of BT to human subjects creates peripheral effects which may obscure any central effects (263). Indeed, BT is poorly lipid soluble, as reflected by its partition coefficient (0.06) (271). The inability of BT to penetrate the BBB has been demonstrated experimentally by measurements in animal brain levels, at various time intervals, following the administration of BT (272).

To overcome this limitation of BT-esters may be prepared by direct acylation.

O N(CH3)2 H3CCO

N H Acetyl bufotenine

Figure 1.6: Structure of acetyl-BT

BT itself possesses a high affinity for 5-HT receptors. In order to determine whether the ester function serves simply as a protecting group or whether the BT- esters themselves might possess some affinity, 5-HT receptor-binding affinities were determined and found that all BT-esters studied possess a receptor affinity similar to that of BT itself. The low lipid solubility of BT limits its ability to cross

BBB and has impeded direct investigation of its central activity. To avoid these limitations mice were administered IV 30 µmoles/kg of the acetyl-BT which is 49

more lipid soluble and were killed by immersion in liquid nitrogen at either 60 or

120 minutes post injection. Assay of brain tissue displayed presence of BT and absence of acetyl-BT at both times. Measuring LSD-like activity as a function of brain concentration, the order of potency was found to be BT>5-MeO-DMT>DMT

(270).

1.3.3 Significance of 5-HT2AR agonist in inflammation

It has been reported that upon stimulation of 5-HT2A receptors in peripheral tissues rapidly inhibit a variety of TNF-α mediated proinflammatory markers, including intracellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and IL-6 gene expression, nitric-oxide synthase activity, and nuclear translocation of nuclear factor kB (273).

1.3.4 Coupling of 5-HT2A & D2 receptor and its significance in OCD as well as other cognitive and mood disorders

OCD is associated with the abnormalities of dopamine neurotransmission.

Studies reveal that neurotransmitter systems such as dopamine are also involved in the pathophysiology of OCD, in addition to 5-HT. Preclinical, neuroimaging and neurochemical studies demonstrate that the dopaminergic system is associated with induction or exacerbation of the symptoms that are indicative of OCD. (274).

Studies, though debatable, suggest that OCD patients show lower dopamine receptor availability primarily in the striatum secondary to increased dopamine drive (275). It has also been reported that decreased striatal D2/3 receptor availability occurs in OCD (148). Another studies suggest that 5-HT2A and D2 receptors located post-synaptically can accumulate into functionally interacting 50

heteromers in PFCx (276, 277). Although the molecular mechanisms that control this 5-HT2A and D2 receptor interaction are still unclear (278), it is assumed that

OCD is associated with dysfunctional 5HT2-D2 receptor regulation (279).

Generally the stimulation of 5HT2 receptors causes the reduced synthesis and release of dopamine (280). This upregulation of 5HT2 receptor activity causes decreased synaptic levels of striatal dopamine resulting in upregulation of postsynaptic striatopallidal D2 receptors which may be indicative of the therapeutic activity of OCD (281). Myoinositol was assumed to have its effect in the treatment of OCD through this mechanism (279). It is also of note that the activity of neuroleptic drugs such as pimozide act through the 5HT2-D2 receptor complex in SSRI resistant OCD patients (282).

The 5-HT2A–D2 receptor complex might also have a significant implication in understanding the pathophysiology of some other neuropsychiatric disorders and the mechanism of action of agents used to treat them. In fact, atypical antipsychotics target this heteromer, reducing its formation (278). Although the clinical significance of this 5-HT2A–D2 receptor complex formation in PFCx is not well documented. However, dimerization of 5-HT2A and D2 receptors may indicate a mechanism by which these receptors might control each other’s activity.

Recently it has been suggested that activation of D2 receptors would increase 5-

HT2A receptor affinity to specific agonists and could modify the signalling of 5-

HT2A receptors (277). In other recent reports it has been suggested that the activity of 5-HT2A receptors in PFCx would be synergistically increased by the 5-

HT2A–D2 receptor complex formation (276, 283). Thus drug treatments that modify the expression of either 5-HT2A or D2 receptors are likely to modify the formation of this 5-HT2A–D2 receptor complex. So if drug exposure modifies the 51

expression of cortical 5-HT2A receptors in PFCx, it could also modify the 5-HT2A–

D2 heteromer formation in this brain region.

Figure 1.7: Graphical representation of significance of abnormal dopamine neurotransmission and 5HT2A-D2 heteromer formation in OCD. Lower D2 receptor availability in striatum is reported to discharge lower amounts of the D2 receptor, which further causes OCD. On the other hand, dysfunctional 5HT2A-D2 receptor regulation in pre-frontal cortex might cause OCD. Stimulation of 5-HT2A receptor by an agonist in the prefrontal cortex could improve these abnormalities and ameliorate OCD symptoms.

1.4 Bioassays to study OCD and inflammation

1.4.1 Bioassays to study OCD

1.4.1.1 In vitro studies

Very few cell-based screening methods have been used as the major approaches to study OCD. Recently some investigators conducted in vitro cellular studies

using CLU213 cells as well as animal experiments to investigate the activity of cortical 5-HT2A receptors. Specifically they investigated the 5-HT2A receptor mediated neurotransmission and/or upregulation to determine if it was associated with several physiological functions as well as neuropsychiatric disorders such as stress response, anxiety, depression and schizophrenia. The study also reported agonist-induced enhanced coupling of 5-HT2A-D2 receptor, 5-HT2A up-regulation and D2 receptor down-regulation (CLU213 cell line) which was hypothesized to contribute to the pathophysiology of some cognitive and mood disorders (284-

287). Cellular studies regarding OCD are still unexplored.

1.4.1.2 In vivo studies

OCD involves behavioural, cognitive and ‘metacognitive symptoms, most of which cannot be reflected with confidence in an animal model. For example, though the behaviour of a laboratory animal might be similar to a compulsion, we can never know whether mental experiences equivalent to obsessions are the underlying motivation. However, some important characteristics of OCD can be investigated in animals (288) and various animal models that have been used to date are outlined in Table 1.6.

Table 1.7: Animal models of OCD used to date (289-307).

Model Animal Putative compounds/ Basic principle used mechanism Naturally-occurring Mice SSRIs (fluoxetine) This behavioural test is based on the development of stereotypy i.e., repetitive, purposeless

stereotypy behaviour which is similar to compulsive behaviour. Stereotypy develops spontaneously in the

captive deer mouse (Peromyscus maniculatus) and bank vole (Clethrionomys glareolus), and

includes behaviours such as patterned running, repetitive jumping and backward somersaulting.

Quinopirole-induced Rat mCPP/Serotonergic This model is based on behaviour induced by chronic treatment with quinpirole, a dopamine D2

compulsive checking agonist (prominent 5- receptor agonist. In open field, such ‘quinpirole-sensitized’ rats exhibit an enhanced locomotory

HT2A/2C receptors agonist) response to this drug and repeatedly visit one or two select places or objects much more

frequently than do control rats, a behaviour similar to compulsive checking.

Spontaneous Rat 5-HT1A agonist/ 8-hydroxy- When a rat is placed in a T-maze with equally rewarded arms, it normally does not enter the

alteration behaviour 2(di-N-propylamin)-tetralin same arm on successive trials. Repeated entry into one of the arms is considered a measure of

(8-OH-DPAT) compulsive-like behaviour.

Compulsive lever Rat SSRIs, 5-HT2C or D1 In this model, the rat is first trained to press one of two levers in order to activate a light/tone

pressing receptor antagonists stimulus that signals the delivery of a food pellet into the food tray, which the animal then

approaches to receive the reward. After this training, ‘signal attenuation’ trials are administered,

where the levers are absent and the light/tone signal is presented but the food pellet is not compulsive-like behaviour. delivered. Thus, the signal attenuation phase essentially removes the feedback signal (delivery

of the reward) that would normally terminate lever pressing behaviour. Then in the test phase-

Model Animal Putative compounds/ Basic principle used mechanism

Compulsive Rat SSRIs, 5-HT2C or D1 the levers are reintroduced into the apparatus, and pressing the lever activates the light/tone stimulus, but lever pressing receptor antagonists no food is delivered.

Genetic models Rodent D1 receptor agonists This model is based on stereotyped grooming. Thus, a ‘grooming chain’ comprises four sequentially

& Mice performed phases of distinct, repeated actions. Striatal lesions decrease the proportion of grooming chains

that are carried to completion.

Pathogenic and Mice 5-HT2C and D2 In this model, mice immunized with a GABHS (Group A β-hemolytic streptococcus) homogenate displayed neurodevelopm antagonists increased rearing behaviour in open field and produced brain reactive antibodies. GABHS-immunized ental model mice were subsequently found to exhibit deficits in motor coordination, social interaction and olfactory

discrimination, along with increased rearing behaviour and altered performance in a learning and memory

task. Consistent with the presence of a pathogenic IgG, intravenous infusion of GABHS-immune serum

increased rearing behaviour in naive mice, an effect not observed in mice infused with IgG-depleted

GABHS-immune serum. Normal Rabbit SSRIs This model is based on three behaviours: grooming, hoarding and nest building. Each of these behaviours behaviour with is goal-directed, persists for a prolonged period of time, and has repetitive and stereotyped components. compulsive like features

Model Animal Putative compounds/ Basic principle used mechanism Schedule- Rat SSRIs, 5-HT2A/2C In this model, excessive drinking is induced in adult rats through the use of a fixed-time operant Induced Polydipsia receptor agonists, 5- schedule. The rats are drug experienced and are food deprived overnight prior to each test session. The

HT1A (8-OH-DPAT), 5- test apparatus consists of a sound-attenuated chamber surrounding a Plexiglas test box (30x25x30 cm)

HT6 receptor agonists, which is equipped with a stainless-steel grid floor and a mechanism to permit the automatic delivery of

Diazepam, buspirone, one 45 mg food pellet each min into a food cup located within the apparatus. A bottle containing tap

haloperidol, water attached to the test apparatus is kept available during test sessions with intake measured to the

chlorpromazine, nearest 1 g. This schedule of food delivery causes a repetitive drinking behaviour that results in the

amphetamine, etc. consumption of a large volume of water, generally 5- to 10-fold greater than baseline, during the 120

minute test session. On test days, drugs are administered acutely to investigate its effect on the

adjunctive drinking behaviour. Evaluation is done to compare the effect of each dose to that obtained for

the vehicle condition. The lowest dose tested (MED, minimum effective dose) is determined. Wheel running Rats SSRIs (fluoxetine) This model is also known as food restriction-induced hyperactivity (FRIH). Drugs are treated to rats to

measure the ability to attenuate development of FRIH syndrome. Food restriction is begun the day prior

to placement in the running wheel cage. After placement in the running wheel cages, rats with restricted

food access are fed for 90 min in the middle of the light period. During the 90 min feeding period access

to the wheels is closed for all rats, including the control group that have free access to food during the

rest of the day. At the end of the 90 min feeding period rats are weighed and injected with drug or saline.

All rats are sacrificed on day 10 after placement in the running wheel, 1 hr after the end of the feeding

period. All rats have free access to water throughout the study.

1.4.2 Bioassays to study inflammation

Cytokines are well known today as biomarkers for inflammatory studies. They are considered as “immune-transmitters” which act as messengers between the immune system and the brain (308). Cytokines are produced from various types of cells including cells of the CNS (astrocytes, microglia and even neurons). For example, activated macrophages secrete various pro-inflammatory cytokines such as TNF-α, IL-

6 and IL-1 (309).

Most of the cytokine release studies from monocytes/macrophages involved the use of transformed cell lines. That is to say, bioassays to study inflammation involves in vitro cytokine release studies and for this purpose THP-1 and U937, human leukaemia- derived monocytic cells, are routinely used. These cells can be easily differentiated from macrophage phenotypes when induced by stimuli such as phorbol esters

(phorbol-12-myristate-13-acetate, PMA). Probably the most suitable method for cytokine release inhibition studies is based on the murine macrophage-like cell line,

RAW 264.7 cells. These adherent cells can easily be maintained and activated by various inflammatory stimuli including bacterial LPS or proinflammatory cytokines.

Upon treatment the cultured macrophages stimulate the release of IL-1β, IL-6 and TNF-

α that can be quantified by ELISA or other bioassay methods (310).

There are various mechanisms involved for cellular inflammatory cytokine release studies. Specific genes are involved in each of the mechanisms that are activated in macrophages and regulate cytokine expression at various cellular levels. There are specific binding sites of the promotor genes for these cytokines (e.g., TNF-α) for transcription factors such as NF-κB, AP-1 (activator protein-1), CRE, and

CCAAT/enhancer binding protein β (C/EBPβ, also called NF-IL6) (311). Among them, one of the most widely investigated transcriptional controls of pro-inflammatory cytokines is mediated by NF-κB. In macrophages, NF-κB exists in a heterodimeric inactive form by binding with an inhibitory IκB protein subunit in the cytoplasm. NF-κB is activated after phosphorylation and subsequent proteolytic degradation of IκB via the action of the IκB kinase (IKK) (312). Then the liberated NF-κB is bound to the κB motif of the promoter region of target genes after translocation into the nucleus, which induces the release of inflammatory cytokine (TNF-α, IL-1β and IL-6) genes (310).

Cytokine production from macrophages includes several other mechanisms such as the phosphatidylinositol-3 protein kinase/Akt, and the mitogen-activated protein kinases (MAPKs) other than the NF-κB system (313-315). The MAPKs comprise of a group of serine–threonine protein kinases which transmit signal transduction from the cell surface to the nucleus. The MAPK signalling cascade is important in cell survival and proliferation, differentiation, and apoptosis. Three well-defined MAPK cascades in macrophages responsible for proinflammatory cytokines expression are ERK

(extracellular signal-related kinase), p38 MAPK, and JNK/SAPK (C-Jun N-terminal kinase), ERK1/2, JNK, and p38 MAP kinase (316). Numerous inflammatory stimuli such as mitogens, proinflammatory cytokines and reactive oxygen species, UV radiation and physical stress activate the MAPK system (316, 317). MAPK signalling is also reported as a prerequisite to transcription factor activation, such as NF-κB. Non- receptor type protein tyrosine kinases such as Syk and Src have been found to be necessary in monocyte-derived proinflammatory cytokines release (318). As all of these mechanisms induce the expression of inflammatory cytokine genes in macrophages, they are important for anti-inflammatory targets of new drugs (319).

1.5 Significance of the project

Toad skins have been used for centuries as a medicinal product in the treatment of various diseases, such as fever, edema, pain relief, infections, tonsillitis, sores, toothache, inflammation and various forms of cancers. Toad skins have also been reported as a rich source of various bioactive compounds such as IAAs, bufadienolides, peptides, proteins, fatty acids and hydroxyphenylalkylamines (Table 1.1). Most of these compounds have been found to have potential therapeutic activity in the treatment of various disease conditions either as a single entity or as a group (320-323). For example, a water extract of Chinese toad skin is used in China, administered as an intravenous injection for anti-cancer therapy, has generated reportedly a 10 billion USD market. The use of cane toad skin has a long history of use for euphoric reasons as well. In recent decades, cumulative incidents of toad lickin’ have been reported by the press (31) where it was reported to cause a rapid furry sensation on the lips and tongue after inducing the secretions. While some researchers have examined the biological effects of cane toad parotid venom secretions (74), investigations on the skin extracts are still lacking. Only cardiac glycoside-like activity using human or animal isolated organs in vitro as bioassay tissues of aqueous and chloroform extracts of cane toad skin are reported (1).

On the other hand, anxiety disorders, as a group, are among the most common mental health disorders and often cause major functional impairment (75). Historically, anxiety disorders have received comparatively little research attention. In recent times though epidemiological studies have described anxiety disorders as the most common class of mental illness, which are frequently comorbid with other conditions, both medical and psychiatric (76). As such, there has been great emphasis on the requirement of

developing effective treatments, both pharmacologic and psychological, to provide symptom relief (75). Twelve month prevalence rates in Australia in 2007 clearly show that anxiety disorders are the most common mental health problem, affecting 14.4% of the population (although some people experience more than one type of anxiety disorder). Among them, OCD is a major chronic, disabling anxiety disorder characterized by obsessions and/or compulsions that are associated with significant distress and/or interference in functioning of daily life (19). The outcome of this disorder is extremely severe in the life of children/adolescents, interfering in their personal, social, school and family life (114, 121). Moreover, various studies have revealed higher comorbidity rates, indicating that up to 80% of children have certain related disorders. These comorbid disorders result in an incremental increase in the degree of interference, worsening the condition of the disorder and narrowing the treatment response (122). Studies suggest that OCD affects up to 1 in 50 people in Australia

(112), which is estimated to be the fourth most prevalent life time psychiatric disorder in the general population (116, 117). Potent SSRIs are the only antidepressant agents which have proved useful to date, in the treatment of OCD (250). But higher doses and longer duration of treatment has made this group of drugs ineffective. Even in clinical trials, about 50% of patients have not found significant improvement (185). Other drugs such as lithium (189, 190), buspirone (191, 192), pindolol (193) have been tried as augmenting strategies to treat SSRI-resistant patients, theoretically, intended to increase 5-HT neurotransmission but so far, with little success. Other treatment strategies such as CBT, combination of SSRIs and CBT, and the use of polypharmacy have not been effective. It is assumed that almost 40% to 60% of patients would fail to respond to standard therapies after adequate treatment (324). For these reasons, identifying new options for treatments has become a high priority (254). To the best of our knowledge, there are no investigations of aqueous cane toad skin extracts nor any

of its components in the treatment of OCD. Moreover, like numerous other disorders such as cardiovascular disease, diabetes and cancer (101), recently inflammation has also been reported as a common condition in neuropsychiatric disorders such as depression, anxiety and OCD as well (104).

This research project aimed to initially assess various methods of extraction of

Queensland cane toad skins to obtain the optimum number of constituents in the maximum amounts in aqueous-based extracts. The extracts (cocktail of constituents) were then assayed to establish the mechanisms of function of each extract in order to assess their therapeutic potential to develop as drugs in the treatment of OCD or other mood and cognitive disorders along with inflammation. An additional environmental aspect to this research is that the number of cane toads are continuing to increase every year throughout the eastern and northern regions of Australia (325), so if the cane toad regarded as a serious pest could be turned into a medicinal resource, then a new direction for the control of this biohazard could result.

1.6 Aims and objectives

Aim 1: Preparation and compound identification of cane toad skin extracts (Chapter 3)

Objective 1: After collection of cane toad skins, generate extracts in three different solvents including aqueous, 60% ethanol and acetic acid (at two different pH: 1.78 and

2.17) based on the commonly used methods for the extraction of Chinese toad skins.

Thus four extracts were generated and a simple, easy and reliable HPLC-ESI-Q-TOF

MS/MS method developed to identify compounds in these extracts.

Aim 2: Functional characterisation of the aqueous and 60% ethanol extracts to develop a molecular model for potential drug development in the treatment of OCD (chapter 4)

Objective 2: For functional characterisation, an in vitro cellular model has been chosen to develop a molecular mechanism based on previous evidence and linked to a disease condition such as OCD based on that molecular mechanism. The aqueous extract could be promising for development of a therapeutic agent against OCD since aqueous extracts of Chinese toad skins are known to have potential against various disease conditions.

Aim 3: Further functional characterisation to assess the activity of aqueous and 60% ethanol extracts and their principal constituent, BT against inflammation (chapter 5)

Objective 3: Extension of functional characterisation assays to assess the activity of the aqueous and 60% ethanol extracts and their known active constituent, BT against inflammation. An in vitro cellular model was used for this experiment to evaluate anti- inflammatory activity.

Chapter 2: Methodology

2.1 Extraction of Australian cane toad skins

2.1.1 Collection of Australian cane toad skins

The dried skins of Australian cane toad (Bufo marinus) were kindly donated by Jing

Jing from the University of Queensland, Australia. Skins used were without the glandular glands and were stored at room temperature in a sealed container. Before extraction, the dried skins were washed with water to remove dirt and cut into small pieces (around 2 cm2).

2.1.2 Extraction of Australian cane toad skins

The extraction method used in this study is based on the commonly used method for the preparation of Chinese medicine from toad skins (33, 326). The extraction procedure used is as follows:

2.1.2.1 Extraction in aqueous and 60% ethanol solvents

Aqueous

 Cane toad skins (about 10.095 g) were dried, chopped, then immersed in 200

mL MilliQ water in a flask with a water-jacketed condenser

 The mixture was brought to reflux

 Then gently refluxed for 8 hours and allowed to cool

 Skins were removed via filtration (filter paper)

 Steps one to three were repeated and then aqueous extracts were combined

 The combined extracts were again filtered

 The solution was divided into two parts: Lot 1 and Lot 2 64

Lot 1:

 The extract was centrifuged at 2000g x 10 minutes

 The supernatant was then filtered (filter paper)

 The extract was then freeze-dried to give Lot 1 (aqueous extract) for analysis

Lot 2:

 The solution was freeze dried while the solid residue was kept for further

extraction with 60% ethanol

 Ethanol 60% (40% aqueous) (100 mL) (approximately 10 times the volume of

the sample) was added then the solution was stirred for 4 hours

 The supernatant was then centrifuged at 2000g x 10 minutes

 Supernatant was decanted off, then filtered

 Solvent was then removed from the supernatant solution (rotary evaporation),

followed by freeze-drying to give Lot 2 as the 60% ethanol extract for analysis

2.1.2.2 Aqueous extraction at acidic pH

Cane toad skins were extracted under acidic conditions (glacial acetic acid) at two different pH (2.17 and 1.78) as 2.5 M and 15.1 M, respectively. The procedure is as follows:

 Cane toad skins (about 10.018 g) were dried, chopped then immersed into 28.4

mL MilliQ water and 171.6 mL glacial acetic acid to obtain pH 1.78 solution in a

250 mL flask with a water-jacketed condenser. For 2.17 pH, about 10.070 g

skins were immersed into 28.4 mL glacial acetic acid and 171.6 mL MilliQ water

 The mixtures were brought to the boil

 Then gently refluxed for 8 hours and allowed to cool 65

 Skins were removed by filtration (filter paper)

 The extracts were centrifuged at 2000g x 10 minutes

 Glacial acetic acid was removed using Rotary evaporator (Buchi Rotavapor R-

200, Australia)

 The solid residues were dissolved in 10 mL MilliQ water repeatedly to solublise

all solid mass

 The extract was then freeze-dried to give the acidic acid extracts (pH 1.78 and

2.17) for analysis

2.1.3 HPLC-Q-TOF/MS analysis of Australian cane toad skin extracts

All four extracts (aqueous, 60% ethanol and acetic acid in both pH) were analysed to identify the constituents by HPLC-Q-TOF-MS/MS. This is discussed in detail in the experimental section (chapter 3). BT, which was identified to be present in the aqueous and 60% ethanol extracts (described in chapter 3 and discussed in detail in chapter 5) by comparison to a commercial standard. Initial chromatographic separation was performed on an Agilent 1290 HPLC, using an Agilent Eclipse plus C18 column (2.1 x

100 mm, 1.8 µm particle size). MS was carried out on an Agilent 6530 Q-TOF mass spectrometer equipped with a Jet Stream ESI source. All analyses were performed using the Agilent MassHunter Qualitative analysis and the Agilent Personal Compound

Database/Library (PCBL) software platforms.

2.1.4 Cell culture

CLU213 (Rat Hypothalamic Cell Line) cell line was purchased from Cedarlane

Laboratories (CL) (Burlington, NC, USA). Human pro-monocytic U937 cell line was

obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). To culture and preserve of each cell line, CL and ATCC suggested strictly following the recommended protocol and recommended the use of DMEM or RPMI media with FBS respectively.

2.1.4.1 Protocols used for cell culture

 Cryo-vials containing cell lines were collected from liquid nitrogen storage tank

and immediately placed on ice

 Frozen cryo-vials were placed in a water bath at 37oC. After thawing for 1 minute

with constant agitation, the vials containing the cells were wiped with 80%

ethanol before transferring to the cell culture hood

 Cells were diluted with 5 mL of pre-warmed culture medium in a 50 mL centrifuge

tube. Then the tube was centrifuged for 5 minutes at 1500 rpm, collected the

cell pallet and resuspended in 10 mL culture medium. Cell suspended culture

medium was then transferred in a 75 cm2 tissue culture flasks (TC75 flasks)

o  The flasks were incubated at 37 C with a constant supply of 5% CO2 in an

incubator

 Cells were examined microscopically every day during continuation of culture

 Cells were viewed to check confluence and confirm the absence of bacterial and

fungal contaminants using an inverted microscope

 Old culture medium was replaced with fresh culture medium every three days or

as necessary

 For sub-culture after confluence, the cell monolayer was washed with PBS twice

 For adherent cells (CLU213), 3 mL 0.25% trypsin/EDTA was added to the

washed cell monolayer in 75 cm2 tissue culture flask. The flask was rotated to

cover the entire surface with trypsin while the flask was kept in the incubator,

then incubated for a further 3 to 5 minutes

 The cells were examined using an inverted microscope to ensure cells

detachment of adherent cells (CLU213)

 Fresh serum-containing medium (7 mL) was added into the flask to inactivate

the trypsin, cell suspension was then transferred to a 50 mL centrifuge tube

 For suspension cells (U937), the cell suspension was taken out from the flask

using pipette and place into a 50 mL falcon tube.

 The tube was then centrifuged for 5 minutes at 1500 rpm. The cell pellet was

then collected and resuspended in 30 mL culture medium. Cell suspended

culture medium was then transferred into three 75 cm2 tissue culture flasks.

Flasks were then returned to the cell culture incubator

 For cell preservation, cell pellet was suspended in preserving medium

containing culture medium, 10% FBS and 10% DMSO. Cell suspension was

prepared in a concentration of 1 million cells/mL. Cell suspension was then

transferred into cryo-vials (1 mL per cryo-vial). Cryo-vials were then transferred

into a Mr Frosty freezing container (Thermo Scientific) and kept overnight at -

80oC for slow freezing. The following day, cryo-vials were transferred to a liquid

nitrogen storage tank

2.1.5 Sample preparation

Only aqueous and 60% ethanol extract sample preparation were used for the remaining experimental work as no additional compounds were identified by HPLC-ESI-Q-TOF-

MS in the acetic acid extracts (both pH 2.17 and 1.78). The sample preparation for experiments is discussed in the relevant sections (chapters 4 and 5).

2.1.6 Cytotoxicity Assays

All cytotoxicity assays included in this thesis were conducted using the WST-8 assay

(cell counting kit, Sigma Aldrich, Australia), following the protocol below:

 100 µL of cell suspension (around 1x104 cells/well) were dispensed into a 96-

well plate

 Cells were incubated overnight and then treated with various concentrations of

the indicated drugs

 After incubation for an appropriate length of time (24 to 72 hours), 10 µL of

WST-8 reagent solution was added

 Plate was incubated at 37°C for 4 hours in a humidified, 5% CO2 incubator

 The optical density (OD) value was measured at 450 nm

 The percentage of “inhibition of proliferation” was calculated as follows:

Percentage of inhibition = (mean control OD-mean experimental OD/mean

control OD x 100) %

2.1.7 Western Blot

For all western blot experiments included in this thesis, the following protocols were used.

2.1.7.1 Protein Extractions

Protein extractions were conducted using RIPA buffer (Thermo Scientific, Rockford, IL

61105, USA) according to manufacturer’s instructions. The working RIPA buffer was prepared by adding Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL

61105, USA) at a concentration of 10 µL/mL. The whole protein extraction was

performed at 4ºC with the lysates after finishing the protein extraction stored at -80ºC until used in the assay.

Nuclear and cytoplasmic proteins were extracted using NE-PER® Nuclear and

Cytoplasmic Extraction Reagents (Thermo Scientific, IL, USA) following manufacturer’s protocol. Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL 61105, USA) was used as a concentration of 10 µL/mL and added immediately before the experiment. The extraction of nuclear and cytoplasmic proteins were performed at 4ºC and the extracted proteins were stored at -80ºC until used in the assay.

2.1.7.2 Protein Assay

DC protein Assay (Biorad, USA) kit was used for the quantification of proteins. The protocol for protein quantification (microplate assay protocol) was as follows:

 For the assay, 96 well plate was used

 The working reagent was prepared by adding 20 µL of reagent S to each mL of

reagent A

 Protein standard were prepared 3-5 dilutions (such as 0.2 to 1.5 mg/mL) for the

standard curve each time the assay was performed. The standards were

prepared in the same buffer as the sample

 Standards and samples (5 µL each) were pipetted into a clean, dry microtiter

plate

 Reagent A (25 µL) containing reagent S was added into each well

 Reagent B was added into each well. Care was taken to avoid cross-

contamination and formation of bubbles

 After 15 minutes, absorbance was read at 750 nm in a microplate reader

(POLARstar Omega, BMG Labtech, Ortenberg, Germany)

 Protein concentration of samples was calculated using the standard curve

2.1.7.3 Western blotting

For the western blot, the following working procedure was used:

 Mini-PROTEAN® TGX Stain-Free™ Protein Gels (4–20%) or TGX Stain-Free™

FastCast™ Acrylamide Starter Kit (10% or 12%) (BIO-RAD, Hercules, CA, USA)

was used to assemble the electrophoresis cell. Inner and outer buffer chambers

were filled with running buffer. The upper (inner) buffer chamber of each core

was filled with 200 mL and the lower (outer) buffer chamber was filled to the

indicator mark for 2 gels (550 mL) with 1x running buffer

 Protein samples were mixed with 1X/4X SDS sample buffer plus β-

mercaptoethanol

 Samples were heated at 95 °C for 5 min and equal amounts of proteins were

loaded (20-50 μg) onto the SDS-PAGE gels

 The electrophoresis cell was connected to the power supply and electrophoresis

carried out using 100 to 200 V with a run time of 30 to 60 minutes in Mini Trans-

Bolt module (Bio-Rad)

 After gel electrophoresis, proteins were transferred to PVDF membranes (0.2

µM, BIO-RAD, Hercules, CA, USA) using the transblot turbo (Biorad). The

transfer condition was set as 1.5 A, 25 V, 10 min (at constant voltage)

 After transfer, the membrane was blocked in 5% skim milk for 1 hour at room

temperature

 Primary antibody was diluted (the details of primary antibody such as name,

dilution, etc., is mentioned in individual experiments (chapters 4 and 5) in 71

antibody dilution or blocking solution. The membrane was then incubated with

the diluted antibody overnight at 4°C with gentle agitation

 After overnight incubation, the membrane was washed at least 3 times with

TBST, for 5 minutes each wash

 The membrane was then incubated for 1 hour at room temperature with

secondary antibody after dilution in TBST (the details of secondary antibody

presented later is recorded in each individual experiment in chapters 4 and 5)

 The membrane was then washed at least 3 times with TBST, for 5 minutes each

wash

 Immun-Star WesternC Chemiluminescence substrate solution (Bio-Rad) was

added (0.1 mL per cm2 of membrane) and the chemiluminescence was

visualised using a CCD imager ChemiDoc system (Bio-Rad)

2.1.8 Co-immunoprecipitation

Co-immunoprecipitation was performed using the Thermo Scientific Pierce co-IP kit

(Rockford, IL, USA) according to the manufacturer’s protocol. This is discussed in detail in the experimental section of chapter 4.

2.1.9 Quantitative Real Time PCR (qPCR)

All of the qPCR experiments included in this thesis, the following working procedures are used.

2.1.9.1 RNA extraction

Total RNA was isolated using Aurum Total RNA Mini Kit (Bio-rad Laboratories, USA) and RNeasy plus Mini Kit (Qiagen GmbH, Hilden, Germany) from CLU213 and U937 cells respectively according to manufacturer’s instructions. The extracted RNA was stored at -80ºC.

2.1.9.2 cDNA preparation from total RNA

The concentration of RNA samples was measured by NanoDrop spectrophotometer

ND-1000 (Thermo Fisher Scientific, DE, USA). Then cDNA was synthesized from total

RNA (1 µg) using iScript Reverse Transcription Supermix (Bio-rad Laboratories, USA) for CLU213 cells and RT2 First Strand Kit (Qiagen, Frederick, MD, USA) for U937 cells according to manufacturer’s instructions.

2.1.9.3 qPCR qPCR was performed using iQ™ SYBR® Green Supermix kit (Bio-rad Laboratories,

USA) for CLU213 cells (Method 1) and RT² SYBR Green ROX qPCR Mastermix

(QIAGEN, Frederick, MD, USA) for U937 cells (Method 2). All of the primers used in this thesis are mentioned in each individual experiment later.

2.1.9.3.1 Method 1

In this method, iQ™ SYBR® Green Supermix kit (Bio-rad Laboratories, USA) was used for the quantification of mRNA levels according to the manufacturer’s instruction using a Rotor gene Q cycler (QIAZEN, Frederick, MD, USA). The primers (both forward and reverse) were used at a final concentration of 500 nM in a final 10 µL reaction volume.

A negative control without cDNA or any known DNA template was included for each 73

primer pair. Quantitative gene expression levels were measured using a two-step cycling protocol. The reaction parameters were as follows: 95ºC for 3 min, (95ºC for 15 sec, and 57ºC for 1 min) for 40 cycles. Melting curves were included to ensure that only a single product was performed. Relative gene expression levels were calculated using the 2[-∆∆C(T)] method. For each sample, the amount of targeted mRNA was normalized to that of the reference transcript GAPDH mRNA.

2.1.9.3.2 Method 2

In this method, RT² SYBR Green ROX qPCR Mastermix (QIAGEN, Frederick, MD,

USA) was used for U937 cells for quantitative determination of gene expression levels according to the manufacturer’s instruction. The experiment was performed in a Rotor

Gene Q cycler (QIAZEN, Frederick, MD, USA) using a two-step cycling protocol in a final 25 µL reaction volume. A negative control without cDNA or any known DNA template was included for each primer pair. The reaction parameters were as follows:

95ºC for 10 min, (95ºC for 15 sec, 60ºC for 1 min) for 40 cycles. Relative gene expression levels were calculated using 2[-∆∆C(T)] method. The test sample target levels were normalized to human HPRT1 expression.

2.1.10 Immunofluorescence microscopy

Immunofluorescence microscopy was carried out on both CLU213 and U937 cells. The experiments were performed on CLU213 cells (0.2 x 105/ well) after seeding onto sterile

13 mm tissue culture cover slips (Sarstedt Inc., Newton, NC, USA) within 24 well plate.

For U937 cells (1 x 106/ dish), the experiment was conducted on cells that were seeded onto a 35 mm sterile dish (Sarstedt Inc., Newton, NC, USA). In both cases, the cells were fixed using 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X- 74

100 in PBS for 5 min, blocked for 1 hour with 5% bovine serum albumin (BSA) in PBST and incubated overnight at 4ºC with the respective primary antibody (Santa Cruz

Biotechnology, USA) in 1% BSA/PBST. The name and concentration of each primary antibody is discussed in the experimental section of chapters 4 and 5. Following primary antibody incubation, cells were further incubated with anti-rabbit Alexa-fluor

488-conjugated secondary antibody (Invitrogen) at a concentration of 1:1000 in 1%

BSA/PBS for 1 hour at room temperature in the dark. Cells were then stained with

Hoechst 33342, trihydrochloride, trihydrate (Thermo Fisher Scientific, Australia) for 5 min at room temperature in the dark. A minimum of three washes was performed between each of the above mentioned steps. A Nikon A1R+ confocal microscope was used with 60x objective lens, then Images were acquired with a Nikon digital camera and NIS-Elements acquisition software (version 4.13). Images taken were processed with Adobe Photoshop CS8 to reduce file size.

2.1.11 ELISA

ELISA (elisakit.com; Caribbean Park, Scoresby, VIC 3179, Australia) was performed to evaluate the secreted TNF-α and IL-6 in U937 cells (5 x 105 cells/mL) according to the manufacturer’s instruction. This is discussed in detail in the experimental section of chapter 5.

Chapter 3: Compound identification in

Australian cane toad skin extracts

This chapter represents a co-authored paper, with the bibliographic details as follows:

Title: Multi-constituent identification in Australian cane toad skin extracts using high- performance liquid chromatography high-resolution tandem mass spectrometry

Authors: Abu Hasanat Md. Zulfiker, Mohsen Sohrabi, Ji Qi, Ben Matthews, Ming Q. Wei, I. Darren Grice

Journal: Journal of Pharmaceutical and Biomedical Analysis

Article type: Research paper

Publication status: Published

Publication link:http://www.sciencedirect.com/science/article/pii/S0731708516303508

I am the first author of this paper and my significant contribution to this paper involved: research design, execution of all laboratory experiments, including extraction of toad skins, sample preparation, interpretation of MS results, manuscript writing.

14/06/2016

Signed------(Date) ------(Abu Hasanat Md. Zulfiker) 14/06/2016

Signed------(Date) ------(Associate supervisor and corresponding author: Dr. I. Darren Grice) 14/06/2016

Signed------(Date) ------(Co-corresponding author and Principal supervisor: Dr. Ming Q. Wei)

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License Number 3913140632080 License date Jul 20, 2016 Licensed Content Publisher Elsevier Licensed Content Publication Journal of Pharmaceutical and Biomedical Analysis Licensed Content Title Multi-constituent identification in Australian cane toad skin extracts using high-performance liquid chromatography high-resolution tandem mass spectrometry Licensed Content Author Abu Hasanat Md. Zulfiker,Mohsen Sohrabi,Ji Qi,Ben Matthews,Ming Q. Wei,I. Darren Grice Licensed Content Date 10 September 2016 Licensed Content Volume 129 Number Licensed Content Issue n/a Number Licensed Content Pages 13 Start Page 260 End Page 272 Type of Use reuse in a thesis/dissertation Portion full article Format electronic Are you the author of this Yes Elsevier article? Will you be translating? No Order reference number Title of your Compound identification and functional characterisation of cane toad thesis/dissertation skin extracts Expected completion date Jun 2016 Estimated size (number of 268 pages) Elsevier VAT number GB 494 6272 12 Requestor Location Abu Hasanat Md. Zulfiker Griffith Health Centre (G40) Level 9 (9.12)

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Journal of Pharmaceutical and Biomedical Analysis

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Multi-constituent identiﬁcation in Australian cane toad skin extracts using high-performance liquid chromatography high-resolution tandem mass spectrometry

Abu Hasanat Md. Zulfiker a, Mohsen Sohrabi a, Ji Qi a, Ben Matthews b, Ming Q. Wei a,∗∗, I. Darren Grice c,∗ a School of Medical Science & Menzies Health Institute Queensland, Gold Coast campus, Griffith University, Australia b Smart Water Research Centre, Gold Coast Campus, Griffith University, Australia c Institute for Glycomics and School of Medical Science, Gold Coast Campus, Griffith University, Australia article info a b s t r a c t

Article history: Toad skins and venom glandular secretions have been widely used for centuries in traditional Chinese and Received 16 February 2016 Japanese medicine for the treatment of various ailments such as cancer, sores, toothache, local inflamma- Received in revised form 13 June 2016 tion and pain. The active chemical constituents from traditional oriental medicines have demonstrated Accepted 19 June 2016 potential in the development of effective therapeutic pharmaceuticals. Our primary focus in this research Available online 1 July 2016 was to identify and characterise ‘active’ compounds or groups of compounds for their potential as neuropsychiatric disorder therapeutics. For this aim, we utilised a variety of solvents, i.e., the aqueous, 60% Keywords: ethanol (aqueous) and acetic acid (aq) (at two different pHs) for extractions of Australian cane toad Australian cane toad skin Constituents skins to identify chemical constituents. The identification of compounds was carried out using HPLC- Aqueous extract ESI-Q-TOF-MS/MS based on the accurate mass measurement for molecular ions and MS/MS analysis, Ethanolic extract whereby accurate mass pseudo-molecular ions and characteristic fragment ions were compared to pub- Q-TOF-MS lished reference data, including mass bank and NIST. As a result, we have to date identified 42 major constituents including alkaloids, amino acids, bufadienolides, fatty acids, nucleobases, nucleosides and vitamins mostly from the aqueous and 60% ethanol extracts. Of the 42 constituents identified, 29 were found in the aqueous extract, 35 were found in the ethanol (aq) extract and only 10 in the pH 1.78 acetic acid extract and 11 in the pH 2.17 acetic acid extract of the cane toad skins. Therefore, the aqueous and 60% ethanolic extracts present the greatest potential for ongoing development in our assays. There have been no previous reports on the identification of many of the constituents we have here identified in Australian cane toad skins. These findings, while somewhat consistent with findings in toad skins in other countries, identifies the presence of potential bioactive constituents. Our results showed that HPLC-ESI-Q-TOF-MS/MS is an effective method to characterise and identify components in Australian cane toad skin extracts. Chemical profiling is an essential initial step in the identification and therapeutic exploitation of bioactive agents present in Australian cane toad skin extracts. Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.

1. Introduction Florida and the Philippines. It is included in the Bufonidae (toad) family, of the order Anuran, class amphibian. More common names The Australian cane toad, Bufo marinus, is an introduced species. are the giant, marine toad. It was first introduced to Queensland, Originally the toad was found in Central and South America, though Australia in 1935 from Hawaii as a biological control agent to limit today it has quite a ubiquitous distribution including Hawaii, populations of the destructive grey-backed cane beetle and it’s larvae that were infesting sugar cane crops, thus gaining the moniker in Australia of the ‘cane’ toad. The cane toad failed to control the beetles, but was remarkably successful at reproducing and spread- ∗ Corresponding author at: Institute for Glycomics and School of Medical Science, ing due to the lack of natural predators, a favourable breeding Gold Coast Campus, Griffith University, QLD 4222, Australia. ∗∗ environment and abundant food. They now number well into the Corresponding author. millions, and are still spreading into thousands of square kilome- E-mail addresses: m.wei@griffith.edu.au (M.Q. Wei), d.grice@griffith.edu.au (I.D. Grice). tres of north eastern Australia [1]. At present Bufo marinus is a http://dx.doi.org/10.1016/j.jpba.2016.06.031 0731-7085/Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved. A.H.Md. Zulfiker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 261 major environmental and ecological threat that is out-competing a For the two separate aqueous acetic acid extracts, cane toad considerable number of native fauna. skin (10 g) were immersed in 200 mL of 2.5 M (pH 2.17) and 15 M Toad skin and venom secretions have been used in traditional (pH 1.78) acetic acid, respectively in a flask with a water-jacketed Chinese and Japanese medicine for more than 3000 years [2] and condenser. The mixture was brought to gentle reflux for 8 h then reportedly contain a range of complex secondary metabolites. allowed to cool overnight. The skins were removed by filtration This rich supply of natural chemicals demonstrates an exceptional (filter paper). The filtrate was then centrifuged at 2000g for 10 min resource that may provide potential pharmaceuticals. Characterisa- and the supernatant was reduced under reduced pressure (Buchi tion and identification of chemical constituents in cane toad skin is Rotavapor R-200). The solid mass was then dissolved with MilliQ essential for scientifically robust investigation of the pharmacolog- water and freeze-dried to give the pH 1.78 acetic acid (28.8% w/w) ical activity of skin extracts and targeted development of potential and pH 2.17 acetic acid (68.9% w/w) extract. therapies. Several methods including paper chromatography [3] and high performance liquid chromatography (HPLC) [4] have been 2.4. Sample preparation used to isolate constituents from toad skins. These techniques have drawbacks, with relatively low sensitivity and little capac- The freeze dried extracts (aqueous, 60% ethanol (aqueous) and ity to identify isolated chemicals without further analysis. Recently the two acetic acid extracts) were stored at 2–8 ◦C. Prior to HPLC- HPLC-diode array detection (DAD) and HPLC-quadrupole-time-of- Q-TOF-MS/MS analysis the samples were dissolved in methanol (to flight mass spectrometry (HPLC-Q-TOF-MS) have been used to 0.5 g/mL), then centrifuged (13,000g, 10 min) followed by filtration characterise a small number of polar compounds in the aque- of the supernatants (0.22 ␮m membrane filter). Filtrates were then ous toad skin extracts Cinobufacini or Huachansu [5]. In addition, transferred to an auto sampler vial for analysis. HPLC-Q-TOF-MS has also been used to identify a limited number of constituents in parotid gland secretions of the Australian cane toad 2.5. HPLC-Q-TOF-MS/MS system and conditions [6]. The present study utilised HPLC in tandem with accurate mass- Initial chromatographic separation was performed on an Agi- MS, utilising electrospray ionisation (ESI) to separate and analyse lent 1290 HPLC, using an Agilent Eclipse plus C18 column polar compounds in aqueous, 60% ethanol (aqueous) and acidic (2.1 × 100 mm, 1.8 ␮m particle size). In brief, the chromatographic aqueous (acetic acid pH 1.78 and 2.17) extracts of cane toad skins conditions consisted of using an injection volume of 5 ␮L with a collected in Queensland, Australia. flow rate of 0.6 mL/min. Mobile phases consisted of ultrapure water (A) and 95/5 acetonitrile/water (B). For MS analysis in positive-ion 2. Experimental mode both mobile phases where modified with 0.1% v/v formic acid, while for negative-ion mode analysis mobile phases were modified 2.1. Chemicals and reagents with 5 mM ammonium formate. The chromatographic conditions consisted of an initial isocratic phase at 5% B for 5 min, followed by For extraction of cane toad skins, ethanol (AR grade) and acetic a gradient from 5% to 30% B between 5 and 13 min before a sharper acid were purchased from Sigma Aldrich, Castle Hill NSW, Australia. gradient from 30% to 100% B at 13 to 20 min. Column washing was Chemical analysis utilised LCMS grade acetonitrile, methanol and performed by holding 100% B between 20 to 24 min followed by formic acid (Thermo Fisher Scientific, Scoresby Victoria, Australia) re-equilibration to 5% B between 24 to 29 min. and AR grade ammonium formate (Chemsupply, GILLMAN, South MS was carried out on an Agilent 6530 Q-TOF mass spectrom- Australia, Australia), Ultra-pure water was prepared “in-house” eter equipped with a Jet Stream ESI source. Spectra were acquired in both the positive-ion and negative-ion modes. Source ionisation using a laboratory-grade Type-1 water purification system (Sar- ◦ torius, Dandenong South Victoria, Australia). conditions were as follows: drying gas temperature, 300 C; drying gas flow, 10.0 L/min; sheath gas temperature 300 ◦C; capillary voltage, (+) 3000 V and (−) 1500 V; skimmer voltage, 65 V; fragmenting 2.2. Collection of cane toad skins (cone) voltage, 110 V and nebulizer at 25 psi. The mass spectrometer was operated with a range of m/z 100–1700 with an acquisition Dried cane toad skins were kindly donated by Dr. Jing Jing (Uni- rate of 1.4 spectra/second. versity of Queensland), Australia. Skins used were without glands and stored at room temperature, sealed. Before extraction, the dried 2.5.1. MS/MS analysis skins were washed with Ultra-pure water to remove dirt and were The extracts were re-analysed using collision-induced dissoci- × 2 cut into approximately 2 2 cm pieces with non-metallic imple- ation (CID) MS/MS analysis targeting the compounds identified in ments. the initial accurate mass scan. CID analysis was performed using auto-MS/MS mode which only targeted a list of preferred precur- 2.3. Extraction of Australian cane toad skins sor ions for MS/MS analysis, without the use of active exclusion of previously analysed precursor ions. This allowed MS/MS spec- The extraction method utilised in this study was based on the tra to be collected over the full peak of the targeted precursor-ion commonly used method for the preparation of Chinese medicines while accounting for retention time shifts between the initial MS from toad skins [7]. Cane toad skins (10 g) were dried, chopped, run and later MS/MS fragmentation analysis. Fragmentation spec- then immersed in 200 mL MilliQ water in a flask with a water- tra were collected at 10, 20 and 40 V CID energy for every targeted jacketed condenser. The mixture was brought to gentle reflux for precursor ion within a mass range of 20–1700 m/z at a rate of 2 8 h, then allowed to cool overnight. The skins were removed by fil- spectra/second. tration (filter paper), then the filtrate was divided into two equal parts. One part was centrifuged at 1500g for 10 min and the super- 2.5.2. Data analysis natant freeze-dried to give the aqueous extract (24.4% w/w). The All analyses were performed using the Agilent MassHunter second portion of supernatant was freeze-dried, then treated with Qualitative analysis and the Agilent Personal Compound 60% ethanol (aqueous), centrifuged at 1500g × 10 min, supernatant Database/Library (PCBL) software platforms. In brief, this consisted removed and freeze-dried to give the 60% ethanol (aqueous) extract of generating putative compound lists using the molecular extrac- (4.8% w/w). tion feature function in the qualitative analysis platform. Initially, 262 A.H.Md. Zulfiker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 solvent blanks where analysed to generate a list of potential molecular ions where then compared with ions reported in the contaminating compounds. The putative contaminant compounds literature for the identified constituents. were then excluded when they occurred within sample analyses to decrease the likelihood that the detected compounds where 3.2. HPLC-ESI-Q-TOF-MS/MS analysis due to a background contamination event. Identification of the putative compounds was performed using the formula generation The total ion chromatograms (TIC) of cane toad skin extracts function, based on mass accuracy and isotopic peak spacing and (A–D) obtained by HPLC-ESI-Q-TOF-MS in positive-ion mode are ratio. Further identification was performed using accurate mass shown in Fig. 1. database searches using purpose made databases of compounds of interest. 3.2.1. Identification of alkaloids Alkaloids are cyclic nitrogen containing compounds, once thought to be limited only to the plant kingdom although they are 3. Results and discussion now known to also be distributed in amphibians, including toad skins [8]. Alkaloids are well known for many of their pharmaco- In the present work, various aqueous-based extraction systems logical effects. One of the remarkable characteristics of alkaloids were designed and optimized to isolate known ‘neuro-active’ mark- is their toxicity against numerous human cancer cell lines, which ers identified previously in the skins. Aqueous and ethanol solvents has led to their being utilized in the treatment of various cancers were reported to provide the optimum extraction of known mark- and are known to induce anti-inflammatory, anti-asthmatic and ers [5,6]. Acetic acid (at two different pHs) were also used to anti-anaphylactic affects [9]. generate a further two extracts to investigate if a richer profile A total of ten alkaloids have been identified in this study on the of markers would be obtained in comparison to the aqueous and basis of accurate mass spectra (mass accuracy, isotopic spacing and 60% ethanolic extracts. The four extracts were then analyzed by distribution) and comparison to fragmentation spectra reported HPLC–MS-Q-TOF (ESI and AJSI) to generate a list of identified and in the literature. Seven out of the 10 alkaloids were identified as unidentified (to date) constituents in each extract. indolealkylamines (IAAs) based on their positive-mode TOF-MS/MS spectra (see Figs. 1–3) (peaks 13, 14, 18, 20, 21, 23 and 24). Com- 3.1. Optimization of HPLC-ESI-Q-TOF-MS/MS system and mon fragmentation pathways for the IAAs have been presented in conditions Table 1 indicating diagnostic fragment ions to confirm the identification of these compounds. IAAs are reported to be abundantly Optimization of MS parameters was an essential step to obtain present in the skin of numerous toad species and have also been good chromatographic component separation and limit charge identified in Chansu and Huachansu TCMs [5]. Discovery of these suppression during ionisation using ESI mass spectrometry. For compounds in Australian cane toad skins gives support to the value this reason, mobile phase (methanol-water, acetonitrile-water, of further study into the biological activity and therapeutic utility methanol-water with 0.1% formic acid and acetonitrile-water with of skin preparations of this animal. 0.1% formic acid) and chromatographic gradient conditions were initially optimised. Acetonitrile-water with 0.1% formic acid gave 3.2.1.1. Indolealkylamines. IAAs are characterised by the presence good separation and abundant signal response both in the positive of a central bicylic indole ring, typically with alkyl or alkylamines and negative-ion scan mode and was therefore selected to optimize side chains on the third carbon on the five membered rings and the HPLC–MS experiments. a hydroxyl group on the fifth carbon of the benzene ring. The Owing to the high structural multiplicity of the various chemical hydroxyl group can be further functionalised by ether or ester link- constituents detected in the four extracts, obtaining good responses ages. Collision induced fragmentation during MS/MS analysis of the to all chemical constituents was challenging. So the MS parameters IAAs primarily results in loss of these side groups creating com- (gas flow rate, nebulizer gas pressure, dry gas flow, spray voltage, mon fragment ions between different IAAs. The confirmation ions capillary temperature and entrance voltage potential) were opti- used in this study consisted of the partial fragmentation of the alky- mized to obtain the highest intensity signal for all the analytes. lamine side chain leaving a propene group to give a m/z 160.075 ion, Similarly, the MS/MS parameters (fragmentation amplitude and followed by complete loss of all side chains leaving the indole rings compound stability) were adjusted to produce a range of high and to give a m/z 117.05 ion [10]. low molecular weight product ions to facilitate identification. Peak 13 was identified as 5-methoxytryptamine (13). Accurate Similarly, the conditions used for compound extraction by the mass analysis yielded m/z 191.1180, consistent with an M+H ion data analysis software were optimised. Compound extraction soft- for the formula with C11H14N2O, and produced fragment ions at ware tools used in this study were pivotal, generating automated m/z 174.0909 corresponding to loss of the primary alkylamine and integration of peaks from the extracted ion chromatograms of the m/z 160.0754 IAA diagnostic ion. Formation of the m/z 160.07 the putative compound. Additionally, correct determination of the ion would involve cleavage of the ether-linked methyl group. Due molecular formula based on accurate mass as well as spacing and to the reactivity of the oxide ion from loss of the methyl group ratio of detected isotopic peaks in relation to the primary ion it is likely that this group would be stabilised by the addition of was required. To exclude low abundance compounds from the hydrogen to form a hydroxyl during ion formation. These finding analysis, automated compound detection only included putative are consistent with other reports in the literature for 13 [10–12]. compounds with a peak height in excess of 25,000 abundance units. Peak 14 was identified as 5-HT (also known as serotonin or 5- Detected compounds then underwent further screening based on hydroxytryptamine, note: 5-HT will be used throughout rest of this formula identification, with an acceptability criterion of 20 ppm paper) (14). The identified m/z 177.1022 ion was consistent with the mass error between the calculated formula exact mass and the M+H of the formula C10H12N2O and fragmentation analysis yielded detected mass. The list of putative compounds detected in the sam- the IAA diagnostic m/z 160.0762 and m/z 117.0584 ions. This finding ples was then screened manually and compounds with poor peak is consistent with the literature [10]. integration and unacceptable mass errors excluded. Peak 18 was idenditified at bufotenine (18). The identified Diagnostic fragment ions where identified for each of the puta- m/z 205.1335 ion is consistent with the M+H of the formula tive compounds based on the most abundant fragment ions at C12H16N2O and fragmentation analysis generated the IAA diagnos- 10, 20 and 40 V collision energy. These ions along with pseudo- tic m/z 160.0748 ion [10,13]. A.H.Md. Zulfiker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 263 [10–12] [10,13] [10] [11,16] [5,11,17] [17] [5,11] [11,16,17] [6,27] [5] [5,17] [6,27] [11] [11,14] [11,12] [11,12] [11,12] [15] [15] [11,12] [5,11] [5,11] c c acid c c 5-Methoxy- tryptamine Cytisine Bufotenine N -Methyl- cytisine Serotonin (5-HT) Valine Adenine Nicotinic Xanthine Leucine Pimeloyl arginine Adenosine Ethenzamide Guanosine Suberoyl arginine N -Acetyl-5- hydroxytryptamine Indole-3-acetic acid Mefenorex Identification References Eritadenine Linalyl acetate Guanine Hypoxanthine b 315.1650 (100) 329.1846 Negative-ion mode 110.0349, 92.0504, 80.0259, 58.0659 55.9350 44.0508, 42.0347 53.0144, 43.0303 81.0086, 55.0299, 55.0295, 54.0361 117.0575 117.056, 77.0389, 44.0503 117.0572 96.9597, 117.0584 118.0862, 72.0817, 55.0555, 136.0618, 119.0356, 94.0407, 92.0407, 67.0301, 65.0143 124.0388, 106.0270, 80.0500, 78.0336, 53.0388, 52.0193 153.0396, 136.0140, 111.0380, 82.0405, 53.0141 86.0964, 69.0705, 56.0500, 41.0397 317.1815, 175.1187, 158.0919, 112.0861, 70.0653 268.1016,136.0613, 119.0327 130.9900, 121.0829, 93.0695 152.0563, 135.0298, 110.0353 331.1985, 158.0928, 112.0868 160.0758, 140.987, 132.0811, 130.0629, 117.0586, 115.0547, 77.0391 176.0706, 134.8896, 130.0614, 103.0516, 77.0400 119.0603, 56.9424, Positive-ion mode 110.0347, 55.0298, 110.0343, 94.0396, 82.0400, 5 5 2 4 5 4 O O 174.0909, 160.0754, O O 160.0757, 148.0745, O 160.0748, 115.0543, O 160.0756, 117.0572, O O O 160.0762, 132.0807, O 2 2 2 2 2 O 4 2 4 2 2 2 2 5 2 5 2 2 O 152.0563, 135.0291, O 137.0453, 119.0353, O 5 N N N N N N N ClN 212.1176, 120.0622, N O N N 5 5 4 4 NO N NO NO NO N N NO N N 24 20 14 26 14 16 16 14 18 13 12 13 9 11 5 11 5 5 4 4 13 11 H H H H H H H H H H H H H H H H H H H H H H 5 5 9 5 6 5 5 6 13 10 12 9 11 10 10 14 11 12 12 12 10 12 Formula Observed diagnostic CID fragment ions C C C C C C C C C C C C C C C C C C C C C C a Mass error (ppm) − 4.2341 − 2.2048 − 1.9678 − 3.2882 − 4.8371 − 3.6484 − 3.7849 − 0.9458 − 2.2379 − 5.4188 − 2.0929 − 3.3878 − 2.1119 − 2.6161 − 2.9249 − 2.9249 − 2.0674 − 2.8397 − 8.8578 m / z Measured Calculated 118.0863 118.0868 136.0620 136.0623 254.0884252.0738 254.0889 152.0567 252.0732 152.0572 2.3802 124.0393 124.0399 137.0458 137.0463 153.0400 153.0400 4.8940 132.1019 132.1024 317.1821315.1687 317.1824 268.1040 315.1668 268.1046 6.0285 197.1546166.0859 197.1541 166.0868 2.2824 191.1180 191.1184 177.1022 177.1028 284.0989 284.0995 331.1986329.1855 331.1982191.1179 329.1825 1.2077 191.1184 9.1134 205.1335 205.1341 205.1335 205.1341 219.1129 219.1133 176.0706 176.0711 212.1182 212.1206 − − − + + + + + + + + + + + + + + + + + + + + + + [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M − H] [M − H] [M+H] [M+H] [M+H] [M − H] 176.0955 204.1264 283.0929 135.0550 253.0811 136.0389 131.0951 317.1748 196.1479 330.1903 175.0639 211.1109 165.079 190.1106 204.1264 218.1051 123.0322 152.0334 117.0795 151.0499 267.0980 190.1116 1.29 14 1.48 19 2.44 15 1.55 2 0.40 3 0.41 6 0.64 89 0.96 1.10 11 1.32 16 1.73 21 2.72 22 2.83 12 1.34 13 1.42 18 2.43 20 2.62 5 0.61 7 0.93 Peak Retention time (min) Precusor Ion (amu)1 M Selective ion 0.33 4 0.59 10 17 1.98 Table 1 Constituents identified in cane toad skin extracts by HPLC-Q-TOF-MS. 264 A.H.Md. Zulfiker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 [11,12] [20] [21] [11,12] [6] [21] [6,22] [11,12] [6,23] [21,24,29] [21,24] References [11,12] [11,12] [11,12] [11,12] [13] [11,12] [20] ester c c Linoleamide Marinocino-bufagin-3- adipoyl- l -arginine Marino-bufotoxin Bufalin Riboflavin Adalinine Marino-bufagin 3-pimeloyl- l -arginine Marino-bufagin Telocinobufatoxin Algestone acetonide Bufalin-3-suberoyl- l - arginine (Bufalitoxin) Bufotalinin 19-oxo-Desacetylcino- bufagin Identification Zopfiellamide A Martinellic acid cis-9,cis-15- Octadecadienoic acid Oleic acid or 9-Octadecenoic acid N , -Dibutyl- formamide Acetryptin Dehydro-bufotenin b 697.3772, 315.1676, 173.1052 713.4151, 329.1834, 173.1042 697.4210, 329.1838, 173.1027 Negative-ion mode 278.1512, 81.0705, 67.0544 245.2256, 135.1163, 109.0975, 95.0854, 91.0523, 685.3791, 286.1407 713.4141, 695.4019, 331.1977, 175.1192 387.2526, 369.2422, 351.2344, 305.2186, 255.2105 359.1366, 243.0882, 200.0764, 198.0647, 172.0858, 71.0600, 57.0327 – 699.3952, 681.3869, 317.1820, 264.1326, 175.1188 401.2312, 365.2088, 253.1920 715.4282 387.2538, 369.2437 699.4328 415.2112, 351.1916, 237.1634 415.2117, 255.2326 Positive-ion mode 29.0383 188.0933, 174.0721, 156.0667 m / z . 9 9 9 9 2 6 8 2 6 OO 405.2241, 203.1184 204.1186, 203.1172, O O O O 0 O O 2 2 4 4 4 4 4 7 4 4 5 4 2 2 6 6 N N N N O NO N O N O N NO N NO 280.2609, 263.2359, O O N O O NO 72.9376, 57.0711, 41.0398, 14 14 52 56 34 20 23 54 32 58 34 58 35 30 30 41 33 32 34 19 H H H H H H H H H H H H H H H H H H H H 12 12 36 38 24 17 13 37 24 38 9 24 38 25 24 24 27 18 18 18 C C C C C C C C C C Formula Observed diagnostic CID fragment ions C C C C C C C C C C 106/theoretical a × m / z ) Mass error (ppm) − 1.3257 − 0.8842 0.7149 − 7.3174 − 3.1614 − 0.7746 − 1.4879 − 1.3607 − 0.1974 − 1.5663 -theoretical m / z m / z Measured Calculated 203.1184 203.1184203.1186 203.1184 0 0.9846 685.3815 685.3813713.4113 0.2918 713.4112 0.1401 387.2543 387.2535 2.0658 377.1456 377.1461 226.1805 226.1807 699.3974 699.3969 697.3861 697.3812401.2312 6.9545 401.2341 715.4286 715.4282713.4150 713.4126158.1539 0.5591 158.1544 3.3641 387.2532 387.2535 699.4345 699.4333697.4210 697.4176 1.7150 446.2536 4.8750 446.2542 415.2115 415.2120 415.2125 415.2120496.3399 1.2042 496.3399 280.2636 280.2640 279.2335 279.2324 3.9393 281.2495 281.2481 4.9777 − − − − − + + + + + + + + + + + + + + + + + + [M+H] [M+H] [M+H] [M − H] [M − H] [M − H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M − H] [M+H] [M+H] [M+H] [M+H] [M+H] [M − H] 376.1396 698.3891 714.4204 280.2408 712.4036 386.2469 225.1732 698.4263 414.2042 495.3326 400.2254 157.1467 445.2463 414.2042 282.2567 386.2457 684.3734 279.2564 202.1112 202.1113 error was calculated using the following formula: mass error (ppm) = (measured 17.13 19.51 14.64 Mass Fragmentation ions were observed at 10,Tentative 20 assignment. and 40 V collision energy. c a 41 25.15 27 15.38 28 2930 19.29 19.44 32 35 20.15 38 21.45 39 21.70 37 21.44 42 25.87 34 20.08 25 14.11 40 22.69 Peak Retention time (min) Precusor Ion (amu) M23 Selective ion 24 4.52 6.82 26 31 19.45 33 19.59 36 21.24 b Table 1 ( Continued )

A.H.Md. Zulﬁker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 265

Fig. 1. Total Ion Chromatograms (TIC) in the positive-ion mode (from HPLC-ESI-Q-TOF-MS) of (A) aqueous CTS extract; (B) 60% ethanol (aqueous) CTS extract; (C) pH 2.17

acetic acid CTS extract; and (D) pH 1.78 acetic acid CTS extract. The peak numbering in each TIC relates to numbered compounds listed in Table 1.

Peak 20 was identified as N-acetyl-5-HT (20). The identified Peak 21 was identified as indole-3-acetic acid (21) produc-

m/z 219.1129 ion was consistent with the M+H of the formula ing a m/z 176.0706 ion consistent with the M+H of the formula

C12H14N2O2 and produced the diagnostic 160.0758 m/z ion under C10H9NO2. Fragmentation analysis showed a m/z 130.0614 consis-

fragmentation conditions [11]. tent with a HCOOH neutral loss of the carboxyl group, additionally 266 A.H.Md. Zulﬁker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272

Fig. 2. Structure of compounds identiﬁed in the aqueous extract (A), 60% ethanol (aq) extract (B), 15 M acetic acid (pH 1.78) extract (C), and the 2.5 M acetic acid (pH 2.17) extract (D). Compounds 12, 17, 19, 22, 23, 39 are tentatively assigned. A.H.Md. Zulﬁker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 267

H3COCHN (H3C)2N HO HO

N loss of C2H5NO N H loss of C2H7N H N-Acetyl-5-HT (20) Bufotenine (18) [M + H]+ m/z 219.113 [M + H]+ m/z 205.133 H2C

NH2 loss of CH5N H N H N 2 2 160.075 loss of NH3 m/z HO H3CO (C10H10NO)

N N H H 5-Methoxytryptamine ( ) 5-HT (14) 13 + [M + H]+ m/z 177.102 [M + H] m/z 191.118

NH m/z 117.057 (C8H7N)

Fig. 3. Diagnostic MS/MS fragmentation in four major IAAs: N-acetyl-5-HT (20), bufotenine (18), 5-methoxytryptamine (13) and 5-HT (14) giving m/z 160.075 and 117.057 [30].

H 3.2.1.2. Piperidines. Peak 29 has been putatively identiﬁed as adalinine (29). Though no fragmentation was detected for this COOH compound, in the positive-ion mode the m/z 226.1805 ion was consistent with the M+H for formula C13H23NO2 for adalinine having a mass error of −0.8842 ppm. Numerous literature reports exist on the acceptability of a mass error of 5 ppm or less on the [M+H]+ or N [M−H]− ions to identify constituents without needing to employ H (21) MS/MS [6]. As only accurate mass has been used to identify this compound and it has not previously been reported in toad skin

m/z 77.04 [M + H- C4H5NO2] or secretions, further characterisation of this compound will be required to confirm it’s identity. Fig. 4. Diagnostic m/z 77.04 fragment ion from MS/MS fragmentation of indole-3- acetic acid (21). 3.2.1.3. Quinolizidines. Peaks 17 and 19 were identified at cytosine (17) and N-methylcytisine (19), respectively. In the positive-ion an m/z 77.0400 ion was present, consistent with a benzene group mode spectral analysis displayed m/z 191.1179 for peak 17 (con- of the central indole after double ring cleavage (Fig. 4). This frag- sistent with the M+H ion for formula C11H14N2O) and peak 19 mentation pathway corresponds with other literature reports on exhibited m/z 205.1335 (consistent with the M+H ion for formula 21 [14]. C12H16N2O). Both ions were subjected to CID fragmentation and + Peak 23 was tentatively identified as acetryptin (23). The found to produce fragment ions m/z 160.075 (17, [M+H-CH5N] ; 19, + observed ions m/z 203.1184 and m/z 405.2241, are consistent [M+H-C2H7N] ) consistent with loss of the secondary alkylamine with the M+H and 2M+H respectively of the formula C12H14N2O. (Fig. 5). In addition, both ions proceeded to lose a further 43 amu, to No MS/MS data was obtained for this ion so additional identity give m/z 117.05. Several additional diagnostic fragment ions were conformation via fragmentation analysis could not be performed. observed and compared to the reported literature to identify these Acetryptin has not been reported as isolated from natural sources two constituents as cytisine (17)(m/z 148.0745, 77.0389, 44.0503) previously, nor has any pharmacological activity been reported for and N-methylcytisine (19)(m/z 96.9597, 58.0659), respectively the compound. As this is the first study to report 23 as a natural [15]. product and identification has only been via accurate mass determination, further characterisation of the detected compound will 3.2.2. Identification of amino acids be required to confirm the identification. Amino acids are biologically important organic compounds that Peak 24 was identified as dehydrobufotenione (24). The m/z combine to form proteins. Amino acids encompass the second 203.1186 ion for this peak was consistent for the M+H of the for- largest component (water is the largest) of human muscles, cells mula C12H14N2O. The alkylamine sidechain in 24 cylcised with and other tissues in the form of proteins. Outside proteins, amino carbon 7 on the central indole, created a fragmentation pattern acids play vital roles in processes such as neurotransmitter trans- unlike other IAAs. Fragmentation spectra showed m/z 188.0933 port and biosynthesis. They are also essential elements in the field + + [M+H-CH3] and m/z 174.0721 [M+H-CH3-CH3] consistent with of medicine, food industry, biotechnology and biochemistry. Iden- the single, then double loss of the methyls on the quarternary alkyl tification and isolation of amino acids in toad skins has not been amine. The accurate mass and fragmentation data was consistent reported previously. with reference data for 24 reported in the literature [13]. However, in positive ion-mode MS their fragmentation patterns have been well studied. It is well known that amino acids generally 268 A.H.Md. Zulfiker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272

H H NH H NCH3 loss of CH N loss of C2H7N O 5 O O N N N

Cytisine ( ) -Methylcytisine ( ) 17 m/z 160.076 N 19 [M + H]+ 191.118 [M + H]+ m/z 205.133 m/z (C10H10NO)

Fig. 5. Diagnostic fragmentation of cytisine (17) and N-methylcytisine (19) to give m/z 160.076. fragment by producing diagnostic [M+H-HCOOH]+, [M+H-HCOOH- (Fig. 6b). Accurate mass and fragment ions were consistent with + + NH3] ions along with the parent molecular ion [M+H] . In our study the reference data for marinobufotoxin (marinocinobufagin-3- we identified two amino acids in the 60% ethanolic (aq) extract (B). suberoyl-l-arginine) (26) [20]. Peak 1 was identified as valine (1). The detected ion in the Peak 27 was identified as bufalin (27). The identified positive- positive-ion mode m/z 118.0863 was consistent for M+H of the ion mode m/z 387.2543 ion is consistent with the M+H for formula + formula C5H11NO2 and generated fragment ions at m/z 72.0817 C24H34O4. Diagnostic fragment ions at m/z 369.2422 [M+H-H2O] , + + + + [M+H-HCOOH] and m/z 55.0555 [M+H-HCOOH-NH3] . We have 351.2344 [M+H-2H2O] and m/z 305.2186 [M+H-3H2O-CO] were identified this constituent as valine (1) based on comparison of also evident. In addition, a neutral loss of 96 amu from m/z 351.2344 accurate mass and fragmentation data to the relevant reported (elimination of ␣-pyrone ring at C-17 to give m/z 255.2105) was literature [16]. observed and is also regarded as diagnostic in the fragmentation of Peak 8 was identified as leucine (8). The positive-ion mode bufadienolides (Fig. 6c) [21]. spectral data gave m/z 132.1019 (consistent with the M+H for for- Peak 30 was identified as marinobufagin-3-pimeloylarginine mula C6H13NO2) and displayed a similar fragmentation pattern to ester (30). In the positive-ion mode the m/z 699.3974 ion valine (1). Whereby fragment ions were seen associated with loss observed was consistent with M+H for C37H54N4O9. It evi- of the carboxyl groups (m/z 86.0964) [M+H-HCOOH]+ and both the denced a similar fragmentation pattern to 26, indicating that 30 primary amine and carboxyl groups (m/z 69.0705) [M+H-HCOOH- was related in structure to compound 26. Diagnostic fragment + + NH3] . Comparison of accurate mass and fragmentation data with ions were evident at m/z 681.3869 [M+H-H2O] ,m/z 317.1820 + the literature led us to identify the constituent as leucine (8). This [M+H-C24H30O4] (consistent with loss of marinocinobufagin), + data corresponds with the literature reports for the identification m/z 264.1326 [M+H-C24H29O4-NH3-2H2O] and m/z 175.1188 + of leucine [17]. [M+H-C24H29O4-C7H12NO2] (consistent with loss of NH2 and the pimeloyl moieties) (see Fig. 6d). Moreover, in the negative-ion mode, peak 30 showed an [M−H]− at m/z 697.3861 consistent with 3.2.3. Identification of bufadienolides C37H52N4O9. It generated fragment ions at m/z 315.1676 [M−H- Bufadienolides are steroids containing a typical ␣-pyrone ring. − (C13H23N4O4)] (consistent with loss of 3-pimeloyl-l-arginine) They are also known as cardenolides due to exhibiting similar which underwent further fragmentation through the amide bond biological activity to cardiac glycosides. Bufadienoldies have been to give m/z 173.1052. The fragmentation patterns of 26 and 30 have found in various toad species and are reported to have numerous been studied previously with their presence reported in the skin of therapeutic activities including anaesthetic, antimicrobial, antitu- Bufo marinus [20]. mor and blood pressure stimulating effects along with cardiotonic Peak 31 was identified as marinobufagin (31). The m/z 401.2312 activity [18]. They are also the major bioactive constituents of ion observed was consistent with the M+H for formula C24H32O5. chansu and huachansu in TCMs, used in the treatment of heart + Positive-ion fragmentation gave m/z 365.2088 [M+H-2H2O] and failure and various cancers [19]. In the present study, we have m/z 253.1920. The pseudo-molecular ion is outside our ‘acceptable’ identified a total of nine bufadienoldies in the four cane toad skin error limit, however, close comparison of the accurate masses of extracts. All nine bufadienolides were detected in the aqueous the fragment ion data with the literature allowed us to tentatively extract (A), with seven detected in the 60% ethanolic (aq) extract, identify this peak [21]. two detected in the pH 1.78 and three in pH 2.17 acetic acid extracts. Peak 32 was identified as telocinobufagin-3-suberoyl-l- l Peak 25 was identified as marinocinobufagin-3-adipoyl- - arginine (telocinobufatoxin) (32). The m/z 715.4286 ion was the arginine ester (25), showing m/z 685.3815 (consistent with the only ion observed in the positive-ion mode and was consistent with M+H for a formula C36H52N4O9) in positive-ion mode. A frag- M+H for C H N O . In the negative-ion mode the m/z 713.4126 + 38 58 4 9 ment ion was also evident at m/z 286.1407 [M+H-C24H31O5] ion was consistent with the M−H for C38H56N4O9. Fragmentation indicative of cleavage of the ester-linkage with the neutral loss in negative-ion mode gave two ions at m/z 329.1834 [M−H- of the steroidal backbone (Fig. 6a). Consideration of the accurate telocinobufagin]− giving the 3-suberoyl-l-arginine fragment ion mass and fragment ion led to the identification of peak 25 as and m/z 173.1042 indicating a further through-amide bond frag- l marinocinobufagin-3-adipoyl- -arginine ester (25). To the best of mentation in the 3-suberoyl-l-arginine (32) [22]. our knowledge, this is the first identification of marinocinobufagin- Peak 35 was identified as bufalin-3-suberoyl-l-arginine ester l 3-adipoyl- -arginine ester from a natural source. (bufalitoxin) (35). The observed m/z 699.4345 ion was consis- Peak 26 was identified as marinobufotoxin (26), displaying m/z tent with C38H58N4O8 and in the negative-ion mode the m/z 713.4113 consistent with the M+H for formula C38H56N4O9. In frag- 697.4210 ion was also consistent with the pseudo-molecular ion. mentation experiments ions were evident at m/z 695.4019 [M+H- Fragmentation in the negative-ion mode gave m/z 329.1838 [M−H- + + H2O] , 331.1977 [M+H-C24H30O4 (loss of marinocinobufagin)] , O-bufalin]− giving the 3-suberoyl-l-arginine fragment ion and m/z with further successive loss of (NH3+H2O) at m/z 278.1512 173.1027, exhibiting similar fragmentation to that seen for 32 + [M+H-C24H30O4-NH3-2H2O] , and m/z 175.1192 [M+H-C24H30O4- [6,23]. + C8H14NO2] . The m/z 331.1977 ion represents essentially the Peaks 37 and 38 were identified as bufotalinin (37) and 19-oxo- l intact suberoyl- -arginine, while the m/z 175.1192 ion represents desacetylcinobufagin (38), respectively. In the positive-ion mode a further loss of NH2 and the suberoyl moiety from this species

A.H.Md. Zulﬁker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 269

Fig. 6. Schematic representation of tentative fragmentation pattern of ﬁve major bufadienolides (25, 26, 27, 30 and 37) [21].

m/z 415.2115 and 415.2125 ions were consistent with M+H for viously been reported as the major components of aqueous TCM

C24H30O6. Peak 37 generated diagnostic bufadenolide fragment extracts [26].

ions at m/z 351.1916 [M+H-2H2O-CO] and m/z 237.1634 [M+H- The most common fragmentation process for nucleic bases is

3H2O-CO-␣-pyrone] consistent with further loss of H2O and the the loss of water and/or ammonia (NH3), followed by the removal of

␣

-pyrone ring (Fig. 6e). Further fragmentation on peak 38 gave the HCN or CO. For the nucleosides, the initial detachment of the neutral

diagnostic m/z 255.2326 ion, while the m/z 237.1634 ion was also ribose moiety is common due to the weak N C bond, followed by

regarded as diagnostic for bufotalinin (37) [24]. Both 37 and 38 have the elimination of ammonia from the base.

been identiﬁed previously in Chansu [21]. Peak 2 was identiﬁed as adenine (2). In the positive-ion mode

m/z 136.0620 was evident and consistent with M+H for C5H5N5.

Diagnostic fragmentation gave m/z 119.0356 [M+H-NH3] consis-

3.2.4. Identiﬁcation of fatty acids +

tent with loss of ammonia and 92.0407 [M+H-NH3-HCN] further

Fatty acids are known to have important therapeutic roles in

loss of HCN [17].

numerous human disease conditions, including cardiovascular dis-

Peak 4 was identiﬁed as guanine (4). The observed m/z 152.0567

ease, inﬂammation, hypertension, allergies, and immune and renal

ion was consistent with M+H for C5H5N5O. Fragmentation gave

disorders. Fatty acids are ubiquitous in plants and animals, most + +

m/z 135.0291 [M+H-NH3] , m/z 110.0347 [M+H-NH2CN] and m/z

often occurring with 16–20 carbon chain backbones. Palmitic and +

80.0259 [M+H-NH3-CO-HCN] ions [5,11].

oleic acid are most commonly found in organisms, along with other

Peak 6 was identiﬁed as hypoxanthine (6). The m/z 137.0458 ion

acids typical of the species [25]. Two 18 carbon (oleic) fatty acids

was consistent with M+H for C5H4N4O. Fragmentation produced

were identiﬁed in the 60% ethanolic (aq) extract (extract B) in the + +

ions at m/z 119.0353 [M+H-H2O] , m/z 110.0343 [M+H-HCN] , and

negative-ion mode. This is not entirely unexpected as these com- +

m/z 82.04 [M+H-HCN-CO] [5,11].

pounds are likely to undergo partitioning away from the aqueous

Peak 7 was identiﬁed as xanthine (7). The observed m/z

extracts. No fatty acids were detected in the positive-ion mode.

153.0400 ion was consistent with M+H for C5H4N4O2. A similar

Peak 41 was identiﬁed as the unsaturated fatty acid octadeca-

fragmentation pattern was seen for peak 7 to that for gua-

dienoic acid (41). The detected m/z 279.2335 ion was consistent

nine (4), whereby diagnostic fragment ions at m/z 136.0140

with the M−H for C18H32O2. No fragmentation of the M−H was + +

[M+H-NH3] , m/z 111.0380 [M+H-NH2CN] and m/z 81.0086 [M+H-

evident at 10, 20 or 40 V collision energy. We have no data to sup- CO-HCN]+ [5,11].

port the speciﬁc oxidation sites, or identify cis/trans conﬁguration

Peak 10 was identiﬁed as adenosine (10). The m/z 268.1040 ion

[11,12].

observed was consistent with the M+H for C10H13N5O4. Diagnostic

Peak 42 was identiﬁed as oleic acid (42). The observed m/z +

fragment ions at m/z 136.0613 [M+H-ribose] consistent with loss

281.2495 ion was consistent with M−H for C18H34O2. No additional +

of the ribose sugar and 119.0327 [M+H-ribose-NH3] indicative of

fragmentation was observed in either the positive or negative-ion

a further loss of ammonia were also observed [5].

modes [11,12].

Peak 15 was identiﬁed as guanosine (15), whereby the m/z

284.0989 ion was consistent with the M+H for C10H13N5O5. Diag-

nostic fragment ions at m/z 152.0563 [M+H-ribose] , m/z 135.0298

3.2.5. Identiﬁcation of nucleic bases and nucleosides + +

[M+H-ribose-NH3] and m/z 110.0353 [M+H-ribose-NH2CN] were

Nucleobases are nitrogen containing biological compounds

also observed and parallel those observed for the other purine-

linked to a sugar within nucleosides making up the basic building

based nucleoside 10 [5,17].

blocks of DNA and RNA. Nucleobases and nucleosides have pre- 270 A.H.Md. Zulﬁker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272

3.2.6. Identification of vitamins Peak 40 was identified as linoleamide (40). The observed m/z Vitamins play an important role in proper human physiological 280.2636 ion was consistent with M+H for C18H33NO. Diagnos- + functioning. Their presence in TCMs is well known and they have tic fragment ions at m/z 263.2359 [M+H-NH3] and m/z 245.2256 + been characterized previously by LC–MS [17]. In our study two vita- [M+H-NH3-H2O] were also evident [11,12]. mins were identified, both being present in the 60% ethanolic (aq) extract and one in the aqueous extract. 3.2.8. Comment on the level of detection Peak 5 was identified as nicotinic acid (5), displaying m/z While our study and discussion has focused on the qualitative 124.0393 consistent with the M+H for C6H5NO2. Diagnostic detection of constituents in the CTS extracts we believe that we can + fragment ions at m/z 106.0270 [M+H-H2O] , m/z 80.0500 [M+H- indicate some rough levels of detection for the various constituent + + H2O-CO2] and m/z 78.0336 [M+H-H2O-CO] , along with two classes based on previous studies using our MS instrument where additional fragment ions (m/z 53.0388 and m/z 52.0193) were we have known detection limits. Essentially here we are indicating observed [17]. Nicotinic acid (niacin or vitamin B3) is considered an approximate lower-limit of detection for each of the classes. an important human nutrient. We have indicated a much broader classification here in regard to Peak 28 was identified as riboflavin (28). The m/z 377.1456 ion these comments, due to the approximate indications suggested. It observed was consistent with the M+H for C17H20N4O6, while frag- is evident that many additional constituents are likely present in + ment ions at m/z 243.0882 [M+H-C5H12O4] consistent with the the CTS extracts, in addition to those that we have detected, but + loss of the ribityl side chain, m/z 200.0764 [M+H-C5H12O4-CO-NH] any such constituents have fallen below our level of detection in + and m/z 198.0647 [M+H-C5H12O4-CO-NH2] were also observed this study. [11,12]. Below are approximate minimum levels of detection relating to a 5 ␮L injection of CTS extract (500 mg/mL) analysed. The list is not comprehensive for the compounds we have identified here, but 3.2.7. Identification of miscellaneous compounds offers some relative approximate indication of the quantities that Eleven other compounds that are outside of the classes listed these constituents were present at in the extracts. above (see Table S1) were also identified in our study. Alkaloids with a primary amine (positive-ion mode): ∼1 ng/mL Peak 9 was identified as pimeloyl arginine (9). The m/z 317.1821 (good ionisation): ion observed in the positive-ion mode was consistent with (1, 2, 3, 4, 8, 9, 10, 13, 14, 15, 16, 23). M+H, while the m/z 315.1687 ion was consistent with M−H for Constituents with secondary-type amines (positive-ion mode): C13H24N4O5. In the positive-ion mode diagnostic fragment ions at ∼10 ng/mL (good ionisation): + m/z 175.1187 [M+H-C7H10O3] were consistent with the loss of a (2, 3, 4, 6, 7, 9, 10, 15, 17, 18, 19, 20, 21, 22, 24, 28, 29, 39). + pimeloyl moiety and m/z 158.0919 [M+H-C7H10O3-NH3] indicat- Steroidal-based constituents (positive-ion mode) with a car- ing further loss of ammonia [6,27]. bonyl on the bottom ring: ∼10 ng/mL (good ionisation): Peak 12 was tentatively identified as ethenzamide (12). The (34). positive-ion mode gave m/z 166.0859 consistent with M + H Steroidal-based constituents with hydroxyl and saturated bot- for C9H11NO2. Fragmentation generated m/z 121.0829 [M+H- tom ring ∼500 ug/mL (poor ionisation, therefore likely present in + OCH2CH3] consistent with loss of an ethoxy moiety. Comparison higher levels): of this data with Mass bank and NIST data allowed for tentative (27, 31, 37, 38). identification. Fatty acids (negative mode): ∼100 ng/mL (poor ionisation, Peak 16 was identified as suberoyl arginine (16). The m/z therefore likely present in higher levels): 331.1986 ion observed was consistent with M+H for C14H26N4O5. (41, 42). + Diagnostic fragment ions at m/z 158.0928 [M+H-C8H13O3-NH2] Fatty amides (positive mode): ∼∼100 ng/mL (poor ionisation, (consistent with loss of the suberoyl and NH2 moieties) and therefore likely present in higher levels): + m/z 112.0868 [M+H-C8H13O3-NH2-H2O-CO] were also evidenced. (33, 40). Peak 16 displayed a similar fragmentation pattern to that of peak 9 identified as pimeloyl arginine (9) [6,27]. 3.2.9. General comments Peak 22 was tentatively identified as mefenorex (22) giving m/z Of the 42 identified compounds, 29 were found in the aqueous 212.1182 consistent with M + H for C12H18CIN. Induced fragmen- extract (A), 35 were found in the 60% ethanolic (aq) extract (B), 10 + tation produced ions at m/z 119.0603 [M+H-C3H7NCl] consistent were found in the pH 1.78 acetic acid extract (C) and 11 constituents with cleavage of the chlorinated alkyl amine moiety through the were found in the pH 2.17 acetic acid extract (D) (Table S1). Forty + C N bond and m/z 92.0504 [M+H-C3H7NCl-C2H3] indicative of one out of the 42 constituents identified were found in either A or B further C C bond cleavage of the alkyl side chain [11,12]. extracts, with 23 constituents found in both A and B. Interestingly, Peak 33 was identified as N,N-dibutylformamide (33). In the only 13 constituents were found only in A and B (i.e., not in C or D). positive-ion mode a m/z 158.1539 ion consistent the M+H for There were only 8 constituents identified in all four extracts (A, B, C9H19NO was observed. A single diagnostic fragment ion was evi- C and D) (3, 4, 11, 13, 22, 29, 31 and 33). Exclusively, there were denced at m/z 57.0711 (C N bond breakage) [11,12]. 5 constituents found in extract A (6, 7, 12, 15 and 38) and 12 in There were several other compounds that did not show signif- extract B (1, 8, 19, 20, 21, 22, 28, 36, 39, 40, 41 and 42) and only icant fragmentation in MS/MS experiments, but were tentatively 1 constituent found in both the acetic acid extracts (C and D) (2). identified according to acceptable confidence levels of reference Although it certainly seems reasonable that 2 would also be present data matching [11,12]. Those identified compounds were peak 3 in extracts A and B, but evidently at levels below detectability in this identified as eritadenine (3)(m/z 254.0884 consistent with M+H for study. There were 12 constituents that were present in either C or D C9H11N5O4), peak 11 identified as linalyl acetate (11)(m/z 197.1546 extracts, but overall it was clearly evident that these acidic extracts consistent with M+H for C12H20O2), peak 34 identified as algestone contained a notably reduced number of ‘recognised actives’ and acetonide (34)(m/z 387.2532 consistent with M+H for C24H34O4), would therefore not be the ongoing focus of our interests. peak 36 identified as zopfiellamide A (36)(m/z 446.2536 consis- Of the constituents identified 10 were alkaloids, 2 amino acids, 9 tent with M+H for C25H35NO6) and peak 39 tentatively identified bufadienolides, 2 fatty acids, 6 nucleic bases or nucleosides, 2 vita- as martinellic acid (39)(m/z 496.3399 consistent with M+H for mins, and 11 were classed as miscellaneous (Table S1). It seems C27H41N7O2). reasonable to propose that our bioactivity of interest will come A.H.Md. Zulfiker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272 271 from either the alkaloid or bufodienolide classes present in the CTS Appendix A. Supplementary data (19 constituents). Extract A contained 16 of the 19 constituents and extract B 17 of the 19 constituents and between extracts A and B Supplementary data associated with this article can be found, in all 19 constituents detected were present. the online version, at http://dx.doi.org/10.1016/j.jpba.2016.06.031. In comparison to our aqueous extract A an analogous LC-QTOF- MS study on the TCM cinobufacini (a boiling water extract prepared from the skin of Bufo bufo gargarizans Cantor) report the identifi- References cation of 19 major hydrophilic compounds [5]. Only 8 constituents identified in our study (two alkaloids (18 and 24)) and 6 nucleic [1] M. Letnic, J.K. Webb, R. Shine, Invasive cane toads (Bufo marinus) cause mass mortality of freshwater crocodiles (Crocodylus johnstoni) in tropical bases or nucleosides (2, 4, 6, 7, 10 and 15) were also identified in Australia, Biol. Conserv. 141 (2008) 1773–1782. the study of Zhao et al. [5]. Interestingly a review on all reported [2] G.G. Habermehl, Venomous Animals and their Toxins, Springer-Verlag, 1981, chemical constituents isolated to 2011 from the skin of B. bufo 2016, pp. 116–120. [3] M. Roseghini, R. Endean, A. Temperilli, New and uncommon indole- and gargarizans indicates 58 constituents are reported in the TCM cinob- imidazole-alkylamines in skins of amphibians from Australia and Papua New ufacini. Classes principally represented were peptides, steroids Guinea, Z. Naturforsch. C 31 (1976) 118–120. (bufadienolides, cholesterols), indole alkaloids, bufogargarizanines [4] H.J. Wang, L.-x. Yang, B. Gao, M. Cheng, J. Yang, B.-l. Bian, Determination of and organic acids [28]. Out of these 58 constituents we identified bufothionine in different species of toad skin by HPLC, Chin. J. Info. Trad. Chin. Med. 19 (2012) 44–45. only 3 in common with this study, namely dehydrobufotenine (24), [5] H. Zhao, X. Wu, H. Wang, B. Gao, J. Yang, N. Si, B. Bian, Qualitative and bufalin (27) and marinobufagin (31). quantitative analysis of cinobufacini injection using rapid separation liquid Roseghini et al. reported the detection of 5-HT, 5- chromatography coupled with quadrupole-time-of-flight mass spectrometry and HPLC-photodiode array detection, a feasible strategy for the quality hydroxy-N-methyltryptamine, dehydrobufotenine, bufotenine, control of Chinese medicine injections, J. Sep. Sci. 36 (2013) 492–502. bufotenidine, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, [6] J. Jing, W.C. Ren, C. Li, U. Bose, H.S. Parekh, M.Q. Wei, Rapid identification of bufotenine-O-sulphate and bufotenidine-O-sulphate from a paper- primary constituents in parotoid gland secretions of the Australian cane toad using HPLC/MS-Q-TOF, Biomed. Chromatogr. 27 (2013) 685–687. chromatography study on the 80% methanolic (aq) extracts of 100 [7] M. Roseghini, V. Erspamer, R. Endean, Indole-, imidazole- and toads skins of differing species from Australia and Papua New phenyl-alkylamines in the skin of one hundred amphibian species from Guinea [7]. Only 5-HT (14) and bufotenine (18) were commonly Australia and Papua New Guinea, Comp. Biochem. Physiol. 54 (1976) 31–43. [8] A.H. Md Zulfiker, G.L. Mariottini, J. Qi, I.D. Grice, M.Q. Wei, Indolealkylamines detected in both their and our studies. It is apparent from the from toad vertebrates and sea invertebrates—their identification and relevant literature that only 12 of our identified 42 compounds potential activities on the central nervous system, Cent. Nerv. Syst. Agents have been identified previously in toad skins. The low percentage Med. Chem. (2015). [9] E. Küpeli, M. Kos¸ar, E. Yes¸ilada, K.H.C. Bas¸er, A comparative study on the of overlap in identified constituents reported in toad skin extracts anti-inflammatory, antinociceptive and antipyretic effects of isoquinoline demonstrates the high level of complexity present in the skins of alkaloids from the roots of Turkish Berberis species, Life Sci. 72 (2002) toads. While in our study we have not quantified constituents iden- 645–657. tified, previous studies indicate that concentrations vary widely [10] S. McClean, R.C. Robinson, C. Shaw, W.F. Smyth, Characterisation and determination of indole alkaloids in frog-skin secretions by electrospray depending upon species, geographic location and numerous other ionisation ion trap mass spectrometry, Rapid Commun. Mass Spectrom. 16 complex features [13] (for example [3]). However, the glands or (2002) 346–354. venom of toads has been studied more extensively than toad skins [11] NIST. NIST/EPA/NIH Mass Spectral Database. Retrieved 29 October, 2015, from http://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:start. and many additional compounds have been identified in various [12] MassBank. MassBank database. Retrieved 29 October, 2015, from http:// toad species, see for example [24]. Numerous constituents that www.massbank.jp/QuickSearch.html. we have identified in this study have also been identified in the [13] N.M. Maciel, C.A. Schwartz, O. Rodrigues Pires Júnior, A. Sebben, M.S. Castro, M.V. Sousa, W. Fontes, E.N. Ferroni Schwartz, Composition of glandular or venom extract studies reported. indolealkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications, Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 134 (2003) 641–649. 4. Conclusion [14] S. Hou, J. Zhu, M. Ding, G. Lv, Simultaneous determination of gibberellic acid, indole-3-acetic acid and abscisic acid in wheat extracts by solid-phase extraction and liquid chromatography-electrospray tandem mass A simple and reliable HPLC-Q-TOF-MS/MS technique has been spectrometry, Talanta 76 (2008) 798–802. established for the separation and analysis of an aqueous, 60% [15] A. Tei, M. Wink, Lupin, an ancient crop for the new millenium, in: E. Santen, ethanolic (aq) and two acetic acid extracts (pH 1.78 and 2.17) of M. van Wink, S. Weissmann, P. Roemer (Eds.), Proc. 9th Int. Lupin Conference, Klink-Müritz, Germany, 1999, pp. 20–24. Australian CTS. Analysis of the four extracts has lead to the identifi- [16] H.-Y. Zhang, P. Hu, G.-A. Luo, Q.-L. Liang, Y.-L. Wang, S.-K. Yan, Y.-M. Wang, cation (several tentatively) of 42 known constituents. The majority Screening and identification of multi-component in Qingkailing injection of constituents identified were present in the ethanolic (35 of 42) using combination of liquid chromatography/time-of-flight mass and aqueous (29 of 42) extracts. The acetic acid extracts presented spectrometry and liquid chromatography/ion trap mass spectrometry, Anal. Chim. Acta 577 (2006) 190–200. a greatly reduced number of constituents at the levels of detec- [17] M.-H. Liu, X. Tong, J.-X. Wang, W. Zou, H. Cao, W.-W. Su, Rapid separation and tion in our study. Many of the constituents we have identified identification of multiple constituents in traditional Chinese medicine have not been reported in toad skin extracts previously. The results formula Shenqi Fuzheng injection by ultra-fast liquid chromatography combined with quadrupole-time-of-flight mass spectrometry, J. Pharm. presented add significantly to our further understanding of the Biomed. Anal. 74 (2013) 141–155. chemical profile of bufo marinus cane toad skin extracts. [18] Y. Liu, J. Feng, Y. Xiao, Z. Guo, J. Zhang, X. Xue, J. Ding, X. Zhang, X. Liang, The aqueous and 60% ethanolic (aq) skin extracts of bufo marinus Purification of active bufadienolides from toad skin by preparative reversed-phase liquid chromatography coupled with hydrophilic interaction have become our ‘extracts of focus’ in ongoing investigations of the chromatography, J. Sep. Sci. 33 (2010) 1487–1494. therapeutic potential of the Australian cane toad in the treatment [19] X. Wu, H. Zhao, H. Wang, B. Gao, J. Yang, N. Si, B. Bian, Simultaneous of neuropsychiatric disorders. determination of eight bufadienolides in cinobufacini injection by HPLC coupled with triple quadrupole mass spectrometry, J. Sep. Sci. 35 (2012) 1893–1898. [20] H. Kasai, M. Tsubuki, K. Shimada, T. Nambara, T. Honda, Analyses of Acknowledgements biologically active steroids: antitumor active OSW-1 and cardiotonic marinobufotoxin by matrix-assisted laser desorption/ionization quadrupole A.Z. is thankful to Griffith University and the Australian Govern- ion trap time-of-flight tandem mass spectrometry, Chem. Pharm. Bull. ment for GUPRS & GUIPRS scholarships as well as support from the (Tokyo) 57 (2009) 948–956. [21] X. Xia, H. Jin, S. Yan, W. Zhang, Analysis of the bioactive constituents of School of Medical Science for carrying out this research. We also ChanSu in rat plasma by high performance liquid chromatography with mass thank Professor A. Carroll for his valued comments. spectrometric detection, J. Pharm. Biomed. Anal. 53 (2010) 646–654. 272 A.H.Md. Zulfiker et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 260–272

[22] K. Shimada, Y. Sato, T. Nambara, Occurrence of marinobufotoxin and [27] H. Gao, M. Zehl, A. Leitner, X. Wu, Z. Wang, B. Kopp, Comparison of toad telocinobufotoxin homologs in the skin of Bufo bankorensis BORBOUR, Chem. venoms from different Bufo species by HPLC and LC-DAD-MS/MS, J. Pharm. Bull. (Tokyo) 35 (1987) 2300–2304. Ethnopharmacol. 131 (2010) 368–376. [23] R.A. Hayes, A.M. Piggott, K. Dalle, R.J. Capon, Microbial biotransformation as a [28] D.L. Wang, F.H. Qi, W. Tang, F.S. Wang, Chemical constituents and bioactivities source of chemical diversity in cane toad steroid toxins, Bioorg. Med. Chem. of the skin of Bufo bufo gargarizans Cantor, Chem. Biodivers. 8 (2011) Lett. 19 (2009) 1790–1792. 559–567. [24] M. Ye, D.a. Guo, Analysis of bufadienolides in the Chinese drug ChanSu by [29] P. Jiang, S. Dou, L. Liu, W. Zhang, Z. Chen, R. Xu, J. Ding, R. Liu, Identiﬁcation of high-performance liquid chromatography with atmospheric pressure multiple constituents in the TCM-formula Shexiang Baoxin pill by LC coupled chemical ionization tandem mass spectrometry, Rapid Commun. Mass with DAD-ESI–MS-MS, Chromatographia 70 (2009) 133–142. Spectrom. 19 (2005) 1881–1892. [30] Q. Zhang, Y. Li, D. Chen, Y. Yu, L. Duan, B. Shen, W. Liu, Radical-mediated [25] T. Rezanka,ˇ K. Sigler, Odd-numbered very-long-chain fatty acids from the enzymatic carbon chain fragmentation-recombination, Nat. Chem. Biol. 7 microbial, animal and plant kingdoms, Prog. Lipid Res. 48 (2009) 206–238. (2011) 154–160. [26] X. Cao, S. Chen, P. Xiao, S. Chen, Non-alkaloid constituents in Fritillaria cirrhosa and fritillaria delarayi, World Sci. Technol. Modern. Trad. Chin. Med. Materia Med. 10 (2008) 83–88. Chapter 4: Functional characterisation of

Australian cane toad skin extract

97 This chapter represents a co-authored paper, with the bibliographic details as follows:

Title: Cane toad skin extract - induced upregulation and increased interaction of serotonin 2A and D2 receptors via Gq/11 signaling pathway in CLU213 cells

Authors: Abu Hasanat Md. Zulfiker, Saeed M. Hashimi, David A. Good, I. Darren Grice, and Ming Q. Wei

Journal: Journal of Cellular Biochemistry

Article type: Research paper

Publication status: Accepted

Publication link: http://onlinelibrary.wiley.com/doi/10.1002/jcb.25627/full

I am the first author of this paper and my significant contribution to this paper involved: research design, execution of all laboratory experiments including extraction of toad skins, conduction of assays, results interpretation and manuscript writing.

14/06/2016

Signed------(Date) ------(Abu Hasanat Md. Zulfiker) 14/06/2016

Signed------(Date) ------(Associate supervisor: Dr. I. Darren Grice) 14/06/2016

Signed------(Date) ------(Corresponding author and Principal supervisor: Dr. Ming Q. Wei)

98 Abu Zulfiker

Permission to include accepted article into PhD thesis 2 messages

Abu Zulfiker Tue, Jun 14, 2016 at 7:28 PM To: [email protected] Cc: Ming Wei , Darren Grice Cosupervisor

Dear Sir

I am going to submit my PhD thesis on 15 June 2016. I want to include the accepted paper (please see the attached file) or online version entitled "Cane toas skin extract-induced upregulation of serotonin 2A and Dopamine D2 receptors via Gq/11-PLC β signalling pathway" which is going to be published in your reputed Journal (Journal of Cellular Biohemistry).

I am wondering about, may I include this paper as a chapter (as it is one of the original work of my PhD thesis).

Would you please let me know ASAP as I am in a hurry to submit my PhD thesis (on 15 June). It is highly appreciable if you kindly give me your decision on or before 15 June.

Hope to hearing from you soon.

With best regards

Abu Hasanat Md. Zulfiker PhD Candidate Professor Ming Wei group School of Medical Science & Menzies Health Institute Queensland Griffith University, Gold Coast, Australia email: [email protected] Phone: +61 756780919 (G40_9.12) Mobile: +61 469375148 (personal)

Zulfiker_et_al-2016-Journal_of_Cellular_Biochemistry (1) (1).pdf 3029K

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From: Abu Zulfiker [mailto: [email protected] ] Sent: 14 June 2016 14:29 To: Wiley Global Permissions Cc: Ming Wei; Darren Grice Cosupervisor Subject: Permission to include accepted article into PhD thesis

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John Wiley & Sons Limited is a private limited company registered in England with registered number 641132. Registered office address: The Atrium, Southern Gate, Chichester, West Sussex, United Kingdom. PO19 8SQ. Article

Cane toad skin extract - induced upregulation and increased interaction of serotonin 2A and † D2 receptors via Gq/11 signaling pathway in CLU213 cells

Abu Hasanat Md. Zulfiker,1 Saeed M. Hashimi,1,2 David A. Good1,3, I. Darren Grice,4 and Ming Q. Wei1*

1School of Medical Science and Menzies Health Institute Queensland, Gold Coast campus, Griffith University, 4222 Queensland, Australia 2Biology Department, Deanship of Preparatory Year, University of Dammam, Dammam, Kingdom of Saudi Arabia 3School of Physiotherapy, Banyo Campus, Australian Catholic University, 4014 Queensland, Australia 4Institute for Glycomics and School of Medical Science, Gold Coast campus, Grifﬁth University, 4222 Queensland, Australia

Running head: Cane toad skin extract upregulates serotonin 2A and D2 receptors

KEY WORDS: AQUEOUS EXTRACT; AUSTRALIAN CANE TOAD SKIN; CLU213 CELLS; Gq/11 SIGNALING PATHWAY

Grant sponsor: Griffith University Grant number: s2864476

*Correspondence to: Professor Ming Q. Wei, School of Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast, Australia, QLD, 4222. E-mail address: [email protected], Tel: +61 (07)56780745, Fax: +61 (0)755528908

†This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcb.25627]

Additional Supporting Information may be found in the online version of this article.

Recent evidences show that activation of serotonin 2A receptors (5-HT2AR) by agonists is significant in improving therapeutic activity of disease conditions, such as obsessive-compulsive disorder (OCD). Though the exact molecular mechanism is still not well understood, it is thought to involve agonist-driven, enhanced expression of 5-HT2AR in certain areas of brain, such as the prefrontal cortex (PFC). Several other reports have also demonstrated association of OCD with lower dopamine receptor (D2R) availability, primarily in the striatum of the brain along with dysfunction of

5-HT2AR-D2R heteromer regulation. We thus hypothesised that compound(s) interacting with this molecular mechanism could be developed as drugs for long-term beneficial effects against OCD.

In the present study, we have obtained experimental evidence in cultured neuronal cells (CLU213) that aqueous extract (AE, 50 µg/mL, p<0.05) of the Australian cane toad skin significantly increased the levels of 5-HT2AR and D2R protein and mRNA expression. AE was also found to enhance the interaction between 5-HT2AR and D2R and formation of expression of 5-HT2AR-D2R heteromer using co-immunoprecipitation and Western blot. Further investigation showed the involvement of classical signalling pathway (Gq/11-PLCβ) along with c-FOS transcription factor preferentially in 5-HT2A-mediated agonist activation. These results obtained demonstrated that AE upregulates 5-HT2AR by a mechanism that appears to involve Gq/11-PLCβ signalling pathway and c-

FOS transcription factor activation. We indicate this enhanced 5-HT2AR and D2R expression and their interaction to induce increased 5-HT2AR-D2R heteromer formation by exposure to AE might provide a molecular mechanism to develop potential novel drug candidates to ameliorate OCD symptoms. This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved The Serotonin receptor 2A (5-HT2AR) is a postsynaptic G protein-coupled receptor that is abundantly present in the central nervous system (CNS) [Klein et al., 2010]. It is involved in the pathophysiology of various mental disorders including schizophrenia [Gonzalez-Maeso and

Sealfon, 2009; Moreno et al., 2016], depression [Xu et al., 2016], anxiety [Farzaei et al., 2016] and obsessive-compulsive disorder (OCD) [Simpson et al., 2011]. For instance, there are several reports that drugs such as psilocybin, lysergic acid diethylamide (LSD), quipazine and lisuride have

5-HT2A agonist effects and may therefore play a role in the treatment of severe, treatment-resistant

OCD [Shapiro and Roth, 2001]. OCD is a chronic and debilitating condition with obsessive thoughts and compulsive behaviours which is reportedly the fourth most prevalent life time psychiatric disorder in the general population [Fontenelle and Hasler, 2008; Mehraban et al., 2014].

To date the only antidepressant agents found effective in OCD treatment are serotonin (5-HT) reuptake inhibitors (SRIs) [El Mansari and Blier, 2006], with a success rate of 50% in patients

[McDougle et al., 2000]. Several other drugs including lithium [McDougle et al., 1991], buspirone

[McDougle et al., 1993] and pindolol [Blier and Bergeron, 1996] were found to have little success in

SRI-resistant patients. Thus there is a serious need for the development of new drugs for the treatment of OCD. OCD is also known to be a consequence of abnormalities in dopamine neurotransmission, in addition to 5-HT. It is reported to have decreased D2/3 receptor availability in

OCD patients [Denys et al., 2013]. Moreover, recent reports suggest that post-synaptically located

5-HT2A and D2 receptors form into functionally interacting heteromers in the pre-frontal cortex

(PFC) of the brain [Albizu et al., 2011]. Although the molecular mechanisms that control this 5-HT2A and D2 receptor interaction are still unclear [Lukasiewicz et al., 2011], it is assumed that OCD has an association with dysfunctional 5HT2-D2 receptor regulation [Harvey et al., 2002]. Agonists working on 5-HT2AR may stimulate several signal transduction pathways, such as activation of

MAPK, PLC-β and PLA2 [Kurrasch-Orbaugh et al., 2003] leading to long-term transcriptional changes. However, the classical Gq/11-PLC-β signalling pathway is reportedly preferentially followed by most of the agonists acting on 5-HT2AR [Barnes and Sharp, 1999; Gonzalez-Maeso et al., 2007].

Hawaii in 1935, with the intention of controlling the destructive cane beetle population. But they failed to control the cane beetle and today they are regarded as feral pests in Australia. Cane toad skin has a long history in its human use, including euphoric use (where people have described it to be like ‘smoked chicken’) [Radford et al., 1986], drinking of ‘thickened juice’ of boiled cane toads

[Keup, 1995] as well as incidents of toad lickin’ [Lyttle, 1993]. While some researchers have examined the biological effects of cane toad parotid venom secretions [Bagrov et al., 1993], investigations on the potential of cane toad skin extracts in the treatment of OCD are an unexplored focus. Interestingly, cardiac glycoside-like activity has been reported using human and animal isolated organs in vitro as bioassay tissues of aqueous and chloroform extracts of

Australian cane toad skin [Leitch et al., 2000]. In Traditional Chinese Medicine (TCM) there are however reports on the use of extracts of toad skin species in the treatment of cancer, inflammation and various diseases [Wang et al., 2012]. In the present study, we used cultured cells to investigate whether cane toad skin extracts could show agonist effects on 5-HT2A and D2

receptors by upregulating their expression and enhance the formation and activity of 5-HT2A-D2 receptor heteromers. This could provide a molecular mechanism by which cane toad skin extracts might be developed for potential therapeutic treatments of OCD.

MATERIALS AND METHODS

CHEMICALS AND REAGENTS

Fetal bovine serum (FBS) was purchased from Bovogen Biologicals, Australia. Dulbecco′s

Modified Eagle′s Medium (DMEM), N-2-hydroxyethyl-pipera ine- '-2-ethane sulfonic acid

(HEPES), penicillin, streptomycin were purchased from Sigma Aldrich, Australia. Bufotenine (BT) was purchased from Toronto Research Chemicals (Canada). (±)-1-(2,5-Dimethoxy-4-iodophenyl)-

2-aminopropane hydrochloride, (±)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride [(±)-DOI],

M100907 (Selective 5-HT2A receptor antagonist), U73122 hydrate (PLC-β inhibitor) were

University of Queensland, Australia.

CELL CULTURE AND IN VITRO TREATMENT

CLU213 (rat hypothalamus cell line) cells were purchased from Cedarlane Laboratories

(Burlington, NC, USA) (neuronal cell line selected as a validated source of expression of 5-HT2A and D2 receptors [Franklin and Carrasco, 2012]). Cells were cultured in 1x DMEM with 10% fetal bovine serum (FBS), 25 mM glucose and 1% penicillin/streptomycin. Cells were maintained at

37ºC in a humidified incubator with 5% CO2 then used for experiments between passages 12 and

20. For most of the assays, CLU213 cells were treated with AE, EE or other drugs as indicated, at specific concentrations for an incubation period of 24 h. However, CLU213 cells were pre-treated with 5-HT2A selective antagonist, M100907 (10 µM, 1 h) and PLC-β blocker, U73122 (5 µM, 30 min) before addition of AE for 24 h. After pre-treatment, cells were washed with PBS two times to ensure no inhibitor was present.

EXTRACTION OF AUSTRALIAN CANE TOAD SKINS

The extraction method utilised in this study was based on the commonly used method for the preparation of Chinese medicines from toad skins [Oka et al., 1985; Roseghini et al., 1976]. Cane toad skins (10 g) were dried, chopped, then immersed into 200 mL MilliQ water in a flask with a water-jacketed condenser. The mixture was brought to gentle reflux for 8 hours, then allowed to cool overnight. The skins were removed by filtration using Whatmann filter paper (Bibby RE200,

Sterilin Ltd., UK), then the filtrate divided into two equal parts. One part was centrifuged at 1500g x

10 minutes and the supernatant freeze-dried to give the aqueous extract (AE) (24.4% w/w). The second portion of supernatant was freeze-dried, then treated with 60% ethanol (aqueous), centrifuged at 1500g x 10 minutes, supernatant removed and freeze-dried to give the 60% ethanol

(aqueous) extract (EE) (4.8% w/w).

SAMPLE PREPARATION

The freeze dried extracts (AE and EE) were stored at 2-8ºC. Separate solutions of AE, EE and BT were diluted in MilliQ water, 60% ethanol and absolute methanol, respectively followed sterilisation

This article is protected by copyright. All rights reserved through a 0.22 µm membrane filter (Merck Millipore Ltd., Darmstadt, Germany). All stock solutions of AE, EE and BT for cell culture of the desired concentration were prepared in media where the final maximum concentration of organic solvent such as ethanol in EE or methanol in BT was kept within 0.1%.

WST-8 ASSAY

Cell proliferation was evaluated using the WST-8 assay. CLU213 cells (1x104 cells/well) were seeded into 96 well plates with 100 µL of culture medium overnight, and then treated with various concentrations of AE, EE, BT, DOI, M100907 and U73122. After 24 hours, 10 µL of WST-8 reagent solution (Cell Counting kit, Sigma Aldrich, Australia) was added and incubated for 4 hours.

The optical density (OD) value was measured according to manufacturer’s instructions at 450 nM.

The percentage of ‘inhibition of proliferation’ was calculated as follows: percentage of inhibition =

(mean control OD - mean experimental OD)/mean control OD x 100) %.

CO-IMMUNOPRECIPITATION OF 5-HT2A AND D2 RECEPTOR PROTEIN IN CLU213 CELLS

Co-immunoprecipitation was carried out using the Thermo Scientific Pierce co-IP kit (Rockford, IL,

USA) following the manufacturer’s protocol. Cells were lysed in IP Lysis/Wash buffer containing

0.025 M tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol; pH 7.4, supplied with the kit.

Aliquots of both vehicle control and aqueous extracts (AE) containing equal amounts of protein (1 mg) were precipitated at 4ºC overnight with 50 µL of AminoLink Plus coupling resin pre-bound with

10 µg of either 5-HT2A or D2 antibodies (Santa Cruz Biotechnology, Inc., USA). Before performing the co-IP, the extracts were pre-cleared using the control agarose resin to avoid non-specific binding. After overnight incubation, the resins were washed four times using the IP Lysis/Wash buffer and the immuno-precipitated proteins were eluted using elution buffer. Samples were analysed by western blot using rabbit polyclonal 5-HT2A primary antibody (1:1000) (M-75; Santa

Cruz Biotechnology, Inc., USA) or rabbit polyclonal D2DR primary antibody (1:1000) (H-50; Santa

Cruz Biotechnology, Inc., USA).

PROTEIN EXTRACTION AND WESTERN BLOT ANALYSIS

This article is protected by copyright. All rights reserved CLU213 cells were harvested and washed with phosphate-buffered saline (PBS). Membrane bound proteins were extracted using RIPA Buffer (Thermo Scientific, Rockford, IL, USA) following manufacturer’s protocol. uclear-associated proteins were isolated using NE-PER® Nuclear and

Cytoplasmic Extraction Reagents (Thermo Scientific, IL, USA) following manufacturer’s protocol.

Protein concentration was determined using a Bio‑Rad DC protein assay kit (Bio‑Rad, Hercules,

CA, USA). Protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition (10% β-mercaptoethanol). Proteins (25-50

µg for membrane bound proteins or 10 µg for nuclear proteins) were loaded per lane on 10% stain- free Tris-Glycine gels (Bio‑Rad, Hercules, CA, USA). All proteins were transferred to PVDF

(polyvinylidene difluoride) (Bio‑Rad, Hercules, CA, USA) membranes and then blocked for 1 h at room temperature with 5% non-fat dried milk dissolved in TBST (Tris-buffered saline-Tween) (50 mM Tris, 150 mM NaCl and 0.1% Tween-20). The membranes were then incubated with rabbit polyclonal 5-HT2A primary antibody (1:1000) (M-75; Santa Cruz Biotechnology, Inc., USA), rabbit polyclonal primary D2L antibody (1:500) (AB1792P, EDM Millipore, CA, USA), rabbit polyclonal D2S primary antibody (1:1000) (BP122S, Acris Antibodies GmbH, Germany), rabbit polyclonal GAPDH primary antibody (1:1000) (FL-335; Santa Cruz Biotechnology, Inc., USA), rabbit polyclonal actin primary antibody (1:500) (H-196; Santa Cruz Biotechnology, Inc., USA), rabbit polyclonal c-FOS primary antibody (1:1000) (sc-52; Santa Cruz Biotechnology, Inc., USA), rabbit polyclonal D2DR primary antibody (1:1000) (H-50; Santa Cruz Biotechnology, Inc., USA). The incubation with primary antibodies was followed by washing and incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, 1 h at room temperature) (Santa

Cruz Biotechnology, Inc., USA). Blots were developed using an ECL detection kit (Bio‑Rad,

Hercules, CA, USA) and CCD imager ChemiDoc system (Bio-Rad, Hercules, CA, USA). Protein loading for each lane was verified using GAPDH (membrane protein) or actin (nuclear protein) or total protein. The optical density of the bands was analysed by Iamge Lab software (version 5.2;

Bio-Rad, Hercules, CA, USA) and the western blot data was expressed as relative optical density units on each gel normalized to vehicle controls

QUANTITATIVE REAL-TIME (qRT)-PCR

CA, USA) according to the manufacturer’s instructions. First strand cD A was generated from total

RNA using iScript™ Reverse Transcription Supermix (Bio‑Rad, Hercules, CA, USA) following the manufacturer’s protocol. The mRNA levels were measured by Quantitative real time (qRT)-PCR prepared using iQ Syber Green Supermix (Bio‑Rad, Hercules, CA, USA) with forward and reverse primers at a final concentration of 500 nM. All reactions were performed in triplicate in a final reaction volume of 10 µL using a Rotor Gene Q cycler (QIAGEN, Frederick, MD, USA).

A negative control without cDNA or any known DNA template and a minus Reverse Transcription control (-RT control) was included for each primer pair during each qRT-PCR experiment. The primers used in this study were: 5HT2A (NM_017254.1; F:5´-

AACGGTCCATCCACAGAG-3´ and R:5´-AACAGGAAGAACACGATGC-3´, D2 (NM_012547.1;

F:5´-CACCACGGCCTACATAGCAA-3´ and R:5´-GGCGTGCCCATTCTTCTCT-3´), GAPDH

(NM_017008.4; F:5´-TGGAGTCTACTGGCGTCTTCAC-3´ and R: 5´-

GGCATGGACTGTGGTCATGA-3´), c-FOS (NM_022197.2; F:5´-

GGGGCAAAGTAGAGCAGCTATC-3´ and R:5´-GAGCGTATCTGTCAGCTCCC-3´). Quantitative gene expression levels were measured using a two-step cycling protocol. The reaction parameters were as follows: 95ºC for 3 min, (95ºC for 15 sec, 57ºC for 1 min) for 40 cycles. Melting curves were included to ensure that only a single product was amplified. Relative gene expression levels were calculated using the 2[-∆∆C(T)] method. For each sample, the amount of targeted mRNA was normalized to that of the reference transcript GAPDH mRNA.

IMMUNOFLUORESCENCE MICROSCOPY

Immunofluorescence microscopy was carried out on cells (0.2 x 105/ well) that were seeded in sterile 13 mm tissue culture cover slips (Sarstedt Inc, Newton, NC, USA) within a 24 well plate. The cells were fixed using 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in

PBS for 5 min, blocked for 1 hour with 5% bovine serum albumin (BSA) in PBST and incubated overnight at 4ºC with rat polyclonal c-FOS primary antibody (Santa Cruz Biotechnology, USA) at a concentration of 1:300 in 1% BSA/PBST. Following primary antibody incubation, cells were further

This article is protected by copyright. All rights reserved incubated with anti-rabbit Alexa-fluor 488-conjugated secondary antibody (Invitrogen) at a concentration of 1:1000 in 1% BSA/PBS for 1 hour at room temperature in the dark. Cells were then stained with Hoechst 33342, trihydrochloride, trihydrate (Thermo Fisher Scientific, Australia) for 5 min at room temperature in the dark. A minimum of three washes was performed between each of the above mentioned steps. A Nikon A1R+ confocal microscope was used with 60x objective lens and Images were acquired with a Nikon digital camera and NIS-Elements acquisition software (version 4.13). Images taken were processed with Adobe Photoshop CS8 to reduce file size.

STATISTICAL ANALYSIS

The results represent the mean ± standard error of mean (SEM) of the values obtained of at least three independent experiments, carried out in triplicate. Comparisons between groups were made with A OVA and Dunnette’s or Tukey’s multiple comparison test using GraphPad Prism software

(version 6.01, La Jolla, CA). A value of p< 0.05 was considered significant.

RESULTS

EFFECTS OF AE AND EE ON CELL VIABILITY

The effects of AE and EE on the viability of CLU213 cells were evaluated using the WST-8 cell- viability assay. The cell survival was not affected significantly at various concentrations of AE and

EE ranging from 0.1 to 1000 µg/mL over 24 hours. The effects of BT (0.1 to 1000 nM for 24 hours),

DOI (0.1 to 1000 nM for 24 hours), M100907 (0.1 to 20 µM for 1 hour) and U73122 (0.1 to 5 µM for

1 hour) on the viability of CLU213 cells were also evaluated and found to exert no significant toxic effects (Supplementary Fig. 1) during the specified time. However, higher concentrations and a longer duration of exposure of all the drugs on CLU213 cells resulted in an evident decrease in cell survival. Consequently, non-cytotoxic concentration of the extracts and other drugs were used in subsequent experiments.

The effects of AE and EE on 5HT2A receptor protein levels in CLU213 cells were measured using

Western blot (Fig. 1). It was observed that 5HT2A receptor protein levels were increase significantly

(p<0.05) upon treatment of CLU213 cells with AE for 24 hours in a concentration dependent manner. At the highest concentration of AE (50 µg/mL), the maximal upregulation of 5HT2A receptor protein levels (214.9%; p<0.05) was found when compared with vehicle control (100%)

(Fig. 1A). This observed increase of 5HT2A receptor protein levels by AE at 50 µg/mL was similar to both BT (Fig. 1C, 211.9%; p<0.05) and DOI (Fig. 1D, 262.9%; p<0.05) at 0.1 nM, used as positive controls. The concentration of up to 0.1 nM for both BT and DOI were used which seemed sufficient to increase the 5HT2A receptor protein levels as pure standard compounds. On the other hand, EE also showed that it induced an increase in the expression of 5HT2A receptor protein levels in a concentration dependent manner, though the upregulation was small (Fig. 1B, 134.8%; p<0.001) even at 50 µg/mL when compared with vehicle control (100%).

EFFECTS OF AE AND EE ON 5-HT2A RECEPTOR mRNA LEVELS IN CLU213 CULTURED CELLS

The effects of AE and EE on 5HT2A receptor mRNA levels in CLU213 cells were measured using qRT-PCR (Fig. 2). To examine their effects, the cells were treated with AE and EE for 24 hours. It was found that 5HT2A receptor mRNA levels were significantly (p<0.0001) increased after 24 hours incubation of CLU213 cells with AE in a concentration dependent manner. At 50 µg/mL, AE showed higher upregulation of 5HT2A receptor mRNA levels (Fig. 2A, 2.33 fold; p<0.0001) when compared with vehicle control. Interestingly, this observed increase of 5HT2A receptor mRNA levels was greater than that seen for BT (0.01 nM) (Fig. 2C, 1.89 fold; p<0.05) and DOI (0.01 nM) (Fig.

2D, 2.29 fold; p<0.05) positive controls. In contrast, EE showed very negligible effects (p>0.05) in the upregulation of 5HT2A receptor mRNA levels, even at the highest concentration (50 µg/mL), it displayed only 1.27 fold increase (Fig. 2B) when compared with vehicle control.

EFFECTS OF AE AND EE ON D2 RECEPTOR PROTEIN LEVELS IN CLU213 CULTURED CELLS

Western blot (Fig. 3). There are two different splice variants of D2 receptor in humans and rodents that are codified for the same gene. These are D2 receptor long (D2L) and D2 receptor short (D2S) isoforms containing 414 and 443 amino acids, respectively [Dal Toso et al., 1989; Fishburn et al.,

1995; Usiello et al., 2000]. A variation in the third cytoplasmic loop is present, which accounts for the difference in the length of the two splice isoforms. The D2S receptor (molecular weight 48 kDa) is reported to localize pre-synaptically while D2L (molecular weight 52 kDa) receptor mainly post- synaptically [Khan et al., 1998; Usiello et al., 2000]. Treatment of CLU213 cells with AE for 24 hours produced significant increases of D2S receptor levels (Fig. 3A) and D2L receptors (Fig. 3B) protein expression when compared with vehicle control in a concentration dependent manner. At

50 µg/mL, the level of D2S receptors and D2L receptors protein expression were increased between 160.5% (Fig. 3A, p<0.05) and 719.6% (Fig. 3B, p<0.01) when compared with vehicle control. On the other hand, after 24 hours treatment of CLU213 cells with EE (50 µg/mL), no noticeable increase in D2S receptor protein levels was observed (Fig. 3C, 108.7%; p>0.05), though a very modest increase in expression of D2L receptor protein was evident (Fig. 3D, 141.81%; p<0.05) when compared with vehicle control.

EFFECTS OF AE AND EE ON D2 RECEPTOR mRNA LEVELS IN CLU213 CULTURED CELLS

The effects of AE and EE on D2 receptor mRNA levels in CLU213 cells were measured using qRT-

PCR (Fig. 4). To determine the level of D2 receptor mRNA, CLU213 cells were incubated with AE and EE for 24 hours. It was observed that, cells treated with AE exhibited substantial increase in

D2 receptor mRNA levels when compared with controls (Fig. 4A). At 50 µg/mL, AE showed more than six-fold increase (Fig. 4A, p<0.01) in D2 receptor mRNA levels when compared with controls.

EE also induced increases of D2 receptor mRNA levels concentration dependently when compared with controls (Fig. 4B). Approximately 1.71 fold increase of D2 receptor mRNA levels (Fig. 4B, p<0.01) was observed at the highest concentration of EE (50 µg/mL) when compared with vehicle control.

CULTURED CELLS

In order to examine the physical interaction between 5HT2A and D2 receptors we conducted a co- immunoprecipitation assay (Fig. 5). Several reports suggest that 5HT2A and D2 receptors are co- localised which could form the heterodimers of 5HT2A-D2 receptor [Albizu et al., 2011; Borroto-

Escuela et al., 2010; Lukasiewicz et al., 2011]. In order to assess this phenomenon, CLU213 cell lysate treated with either vehicle or AE (50 µg/mL) were used for this purpose. We used either D2 or 5HT2A receptor antibodies as baits in two different co-immunoprecipitation experiments. In the first experiment, active columns were used to precipitate 5HT2A receptors using D2 receptor antibodies (bait) (Fig. 5A). It was found that 5HT2A receptors co-precipitate with D2 receptors when

D2 receptor antibodies were used as bait (Fig. 5A). This co-immunoprecipitation was higher in AE- treated cells when compared with vehicle control-treated cells. Similarly, increased co- immunoprecipitation of D2 receptors with 5HT2A receptors in AE treated CLU213 cells when compared with vehicle control was found when 5HT2A receptors antibodies were used as bait (Fig.

5B). The co-immunoprecipitation between 5HT2A and D2 receptors were enhanced approximately

200% in AE (50 µg/mL) treated cells, compared to vehicle control (Fig. 5B). These results suggest that treatment of CLU213 cells with AE enhances the formation of 5HT2A-D2 receptor complex.

EFFECTS OF A SELECTIVE 5-HT2A RECEPTOR ANTAGONIST (M100907) ON AQUEOUS EXTRACT-INDUCED

INCREASES IN 5-HT2A RECEPTOR mRNA AND PRTEIN LEVELS IN CLU213 CULTURED CELLS

To confirm the effects of AE mediating on 5HT2A receptors, a selective 5HT2A receptor antagonist

(M100907) was used. In this experiment, CLU213 cells were pre-treated with M100907 (10 µM) for

1 hour before incubating the cells with AE (50 µg/mL) for 24 hours (Fig. 6 A & B). It was found that

AE-treated cells significantly increased 5HT2A receptor protein levels (Fig. 6A, approximately

198.8%; p<0.05) when compared with vehicle treated control. The effects of AE were significantly blocked (128.5%; p<0.05) in cells pre-treated with M100907. However, the cells treated with only

M100907 had no significant effect (117.5%; p>0.05) on 5HT2A receptor protein level.

(50 µg/mL) was added. The expression of 5HT2A receptor mRNA level was measured in these cells after 24 hours of incubation. It was observed that AE-treated cells significantly increased 5HT2A receptor mRNA level (Fig. 6B, approximately 1.78 fold increase; p<0.001) when compared with vehicle treated control. The effect of AE was blocked significantly (1.16 fold; p<0.01) in cells pre- treated with M100907. However, the cells treated with only M100907 had no significant effect (1.03 fold; p>0.05) on 5HT2A receptor mRNA level.

EFFECTS OF PHOSPHOLIPASE C-β (PLC-β) INHIBITOR (U73122) ON AE-INDUCED INCREASES IN 5-HT2A

RECEPTOR PROTEIN and mRNA LEVELS IN CLU213 CULTURED CELLS

Fig. 6C and 6D represent the effects of PLC-β inhibitor on AE-induced upregulation of 5HT2A receptor protein levels and mRNA levels. Previous reports suggested that 5HT2A receptor agonists trigger multiple signal transduction pathways via heterotrimeric Gq/11 proteins, that activate phospholipase C-β (PLC-β) [Gonzalez-Maeso et al., 2007] resulting in the increased accumulation of inositol phosphates and intracellular calcium mobilization [Barnes and Sharp, 1999]. This ultimately regulates gene transcription and induces cellular responses [Ruf and Sealfon, 2004].

CLU213 cells were pre-treated with either vehicle or the PLC-β inhibitor U73122 (5 µM) for half an hour before incubating the cells with AE (50 µg/mL) for 24 hours. It was found that AE-treated cells significantly increased 5HT2A receptor protein levels (Fig. 6C, approximately 238.9%; p<0.05) when compared with vehicle treated control. The effects of AE were blocked significantly (123.3%; p<0.05) in cells pre-treated with U73122. However, no significant effects on 5HT2A receptor protein levels were observed in cells treated with only U73122 (114.4%; p>0.05)

CLU213 cells were treated with either vehicle or U73122 (5 µM), then half an hour later vehicle or

AE (50 µg/mL) was added. The level of expression of 5HT2A receptor mRNA was evaluated in these cells after 24 hours of incubation. It was found that AE-treated cells significantly increased

5HT2A receptor mRNA levels (Fig. 6D, approximately 3.7 fold increase; p<0.05) compared to vehicle treated control. The effect of AE was blocked significantly (1.23 fold; p<0.05) in cells pre-

(1.06 fold; p>0.05) on 5HT2A receptor mRNA levels. These results suggest that the upregulation of

5HT2A receptor follows the classical Gq/11-PLC- β signalling pathway.

EFFECTS OF PHOSPHOLIPASE C-β (PLC-β) INHIBITOR (U73122) ON AE-INDUCED INCREASES IN NUCLEAR

LEVELS OF c-FOS mRNA AND PROTEIN LEVELS IN CLU213 CULTURED CELLS

Figs. 7A, 7B & 7C represent the effect of PLC-β inhibitor pre-treatment on AE-induced increases of nuclear c-FOS transcription factor protein and mRNA levels in CLU213 cells. Recent reports suggested that the regulation of PLC-β response through the Gq/11 signalling pathway by agonists acting at 5HT2A receptors induces upregulation in expression of the c-FOS gene [Gonzalez-Maeso et al., 2007; Santini et al., 2011].

CLU213 cells were pre-treated with either vehicle or the PLC-β inhibitor, U73122 (5 µM) for half an hour before adding AE (50 µg/mL) for 24 hours. It was found that AE-treated cells significantly increased nuclear c-FOS protein levels (Fig. 7A, 162.7%; p<0.01) when compared with vehicle treated control. The effect of AE was blocked significantly (107.1%; p<0.01) in cells pre-treated with U73122. However, no significant effects were seen in cells treated with only U73122 (105.5%; p>0.05) on c-FOS protein levels.

CLU213 cells were treated with either vehicle or U73122 (5 µM), then half an hour later vehicle or

AE (50 µg/mL) was added. The expression of nuclear c-FOS mRNA level was measured in these cells after 24 hours of incubation. It was observed that AE-treated cells significantly increased the level of nuclear c-FOS mRNA (Fig. 7B, approximately 1.74 fold increase; p<0.01) when compared with vehicle treated control. The effect of AE was blocked significantly (1.01 fold; p<0.01) in cells pre-treated with U73122. However, the cells treated with only U73122 had no significant effect

(0.99 fold; p>0.05) on nuclear c-FOS mRNA level.

CLU213 cells were pre-treated with either vehicle or the PLC-β inhibitor, U73122 (5 µM) for half an hour. AE (50 µg/mL) was then added and incubated for 24 hours. It was observed that AE-treated cells significantly increased nuclear c-FOS protein levels (Fig. 7C) when compared with vehicle

This article is protected by copyright. All rights reserved treated control (untreated). The effect of AE was blocked in cells pre-treated with U73122. These results suggest that the gene response induced by exposure to AE require the Gq/11, c-FOS transcription factor.

DISCUSSION

The effects of AE and EE from the skins of the Australian cane toad Bufo marinus on the activation and interaction of 5-HT2A and D2 receptors in rat hypothalamic cells (CLU213) were characterized in the present study. It has been reported that 5-HT2A represents a major molecular target for drugs used to treat numerous diseases including schizophrenia, depression, anxiety, eating disorders and OCD [Shapiro and Roth, 2001]. On the other hand, agonist activation of 5-HT2A is also implicated with the stimulation of the dopaminergic system and it is reported that abnormalities of dopamine neurotransmission is involved in the pathophysiology of various neuropsychiatric disorders including OCD [Bortolozzi et al., 2005; Koo et al., 2010]. In this research, a neuronal cell line (CLU213) has been used to determine the impact of AE and EE on the regulation of 5HT2A and

D2 receptor protein and mRNA levels. The focus of the work is to establish the potential of AE and

EE for the development of a therapeutic treatment of OCD and/or other neuropsychiatric disorders.

The 5-HT2AR is a postsynaptic G-protein coupled receptor that is reported to be expressed widely in the CNS [Klein et al., 2010]. 5-HT2ARs are reportedly highly expressed in pyramidal neurons in middle layers of the medial prefrontal cortex [Miner et al., 2003]. They are also present in the frontal cortex, cerebral cortex, basal forebrain, hippocampus, amygdala, dorsal thalamus, hypothalamus, superior colliculus, substantia nigra, pedunculopontine nucleus, legmental area, and myelencephalon [Leysen, 2004]. 5-HT2ARs are involved in mediation of normal and psychotic states, working memory, regulation of GABAergic and cholinergic neuronal cells, sleep, peripheral pain, and cardiovascular functions. In addition, 5-HT2AR are implicated in several psychiatric disorders, such as schizophrenia, depression, and anxiety i.e., OCD [Naughton et al., 2000;

Nichols et al., 1994]. Thus, there is a need for selective ligands to investigate the pharmacological role of 5-HT2A receptors [Leung, 2004]. The present study showed that AE-induced significant

This article is protected by copyright. All rights reserved (p<0.05) upregulation of 5-HT2A receptor protein (Fig. 1A) and mRNA (Fig. 2A) levels, when compared with vehicle treated controls. On the other hand, EE-induced upregulation of 5-HT2A receptor protein (Fig. 1B) and mRNA (Fig. 2B) levels was much lower, when compared with vehicle treated controls. It was observed that the activity of AE (50 µg/mL) to enhance 5-HT2A receptor protein and mRNA levels was similar to BT (Fig. 1C and 2C respectively) and DOI (Fig. 1D and 2D respectively), which were used as positive controls of 5-HT2A receptor agonists [Shen et al., 2010;

Yu et al., 2008]. BT, is an indolealkylamine and a 5HT2A receptor agonist. BT was the first choice to use as a positive control in this study because it has previously been reported to be present in various toad skins, including Cinobufacini (an aqueous extract of Chinese toad skins) [Md Zulfiker et al., 2015; Zhao et al., 2013]. We have previously identified seven BT-like compounds

(indolealkylamines) in several aqueous-based extracts of Australian cane toad skins in our ongoing investigations of the therapeutic potential of these extracts (Zulfiker et al., unpublished). For several years, it was reported that agonist activation of 5-HT2AR by various hallucinogenic indolealkylamines such as psilocybin, lysergic acid diethylamide (LSD) might have potential for the treatment of anxiety disorder such as OCD. The activation of 5-HT2A receptors by hallucinogens may lead to acute reduction of, and possible longer-term beneficial effects of OCD symptoms. It is likely that the biological mechanism of these effects involves agonist activation of 5-HT2A receptors, but it is still unclear whether this 5-HT2A receptor activation occurs due to the acute physiological response or the adaptive changes [Delgado and Moreno, 1998]. The involvement of neuronal circuitry in the orbitofrontal cortex (OFC), the head of the caudate nucleus and the thalamus in

OCD has been confirmed anatomically using PET (positron emission tomography) [Nordahl et al.,

1989]. It was also demonstrated that that the activation of normo-sensitive postsynaptic 5-HT2-like receptors may underlie the enhanced 5-HT release in the OFC [El Mansari and Blier, 2005] because increased release of 5-HT by the serotonergic neurons of the OFC would decrease the clinical symptoms of OCD [Husted and Shapira, 2004]. Thus the anti-OCD effects could be mediated by an increased 5-HT neurotransmission in the OFC as a result of an increased 5-HT release, because 5-HT exerts its effects mainly via 5-HT2-like receptors in the OFC. Since postsynaptic 5-HT2 receptors induce the effect of increased 5-HT release in OFC, a 5-HT2 agonist having capability to selectively activate these cortical receptors should produce a rapid anti-OCD

This article is protected by copyright. All rights reserved effect [Blier and de Montigny, 1998; El Mansari and Blier, 2006]. So the results presented here suggest that AE, but not EE, mediate the upregulation of 5-HT2AR significantly which suggests the potential of AE in the development of a therapeutic treatment for OCD. We assume that the presence of BT [Md. Zulfiker et al., 2016] and BT-like compounds in AE (we have identified some of these by HPLC-Q-TOF-MS/MS already in our ongoing studies) might be the reason for the significant upregulation of 5-HT2AR. In addition, recently we have reported the concentration of BT

(0.70 ng/mL) detected in AE using commercial BT standard by HPLC-Q-TOF-MS/MS [Md. Zulfiker et al., 2016]. AE (50 µg/mL) is estimated to contain 0.035 ng/mL or 0.17 nM BT, accordingly which is approximately the concentration of BT (0.1 nM) used as positive control in the present study.

This result indicates that 50 µg/mL concentration of AE is sufficient to upregulate 5-HT2AR and also supports our assumption about AE containing BT and BT-like compounds as well as induction of 5-

HT2AR upregulation. However, initial quantification of BT [Md. Zulfiker et al., 2016] and BT-like compounds in AE and EE was performed on fresh extracts stored at 4ºC for a maximum of one week prior to analysis, clearly showing their presence. Interestingly after sample storage (at -20˚C) in our ongoing study, BT along with some of the BT-like compounds could not be detected in EE. It is evident then that BT and some BT-like compounds are not stable in long term storage, even at -

20ºC in organic solvent. This might be the reason for little or negligible effect of EE on the upregulation of 5-HT2AR protein and mRNA level. Furthermore, it is possible that the presence of some compounds in EE having potent antagonistic properties towards 5-HT2AR might contribute for little or negligible effect of EE.

Like 5-HT2AR, the dopamine (DA) receptor is also a G-protein coupled receptor which has two splice variants in humans and rodents known as D2 short (D2S) and D2 long (D2L) that differ by 29 amino acids in the third cytoplasmic loop [Fishburn et al., 1995]. D2S is mainly expressed in cell bodies and axons of mesencephalon and hypothalamus, while D2L is more strongly expressed by neurons in the striatum and nucleus accumbens. D2S is reportedly an auto-receptor, whereas the

D2L isoform is primarily a postsynaptic receptor [Khan et al., 1998]. Recently preclinical, neuroimaging and neurochemical studies have demonstrated the association of the dopaminergic system with OCD in addition to the serotonergic system [Goodman et al., 1990; Koo et al., 2010].

(D2R) protein expression in CLU213 cells (Fig. 3). Using immunoblotting, both D2S and D2L receptor protein levels in CLU213 cells were found to increase significantly (p<0.05) in a concentration dependent manner (Fig. 3). The results presented here suggest that AE mediates the upregulation of D2 receptor protein and mRNA levels significantly (Fig. 3A and 3 B; P<0.05), whereas EE shows inconsistent effects in the upregulation of D2 receptor protein and mRNA levels

(Fig. 3C and 3D). This upregulation of D2 receptor protein by AE appears to be due to increased trafficking of D2 receptors from the cytosol to the membrane and/or through decreased degradation of D2 receptors [Franklin and Carrasco, 2012] though the mechanism of upregulation of D2 mRNA levels is still unclear in the present study . However, our results presented here support a previous report where several typical and atypical antipsychotic drugs such as chlorpromazine, clozapine, haloperidol, molindone, olanzapine, pimozide, remoxipride and risperidone were found to upregulate both D2S and D2L receptor mRNA levels in the prefrontal cortex and striatum, thereby demonstrating therapeutic effects [Lidow and Goldman-Rakic, 1997]. Recently low levels of dopaminergic D2 receptor in the striatum were identified in OCD patients. In animal models these low D2 receptor levels were reportedly improved by the use of agonists on both the 5HT2A and D2 receptor [Denys et al., 2004; Korff et al., 2008]. The presence of compounds having D2 receptor agonist-like affects along with BT-like compounds in AE might be the reason for its upregulation of

D2 receptors. From this point of view, the inconsistent effects of EE on upregulation of D2 receptors

(Figs. 3 and 4) and 5HT2A (Figs. 1 and 2) are likely due to the decomposition of BT-like compounds from storage in the presence of an organic solvent or it is possible that some compounds have antagonistic effects on D2 and 5HT2A receptors. Moreover, the difference of upregulation in protein and mRNA levels by EE (as compared to AE) might be due to differences in synthesis rates and stability of proteins and mRNA as well as their possible post-transcriptional regulation. In addition, culture to culture variations, differences in translation efficiency, diversity in degradation rates among different genes and also between mRNA and proteins may be factors [Jayapal et al., 2008].

These findings suggest that, AE but not EE, could be a potential candidate for the development of a therapeutic treatment for OCD. As the effect of EE on the upregulation of both 5-HT2A and D2 receptor protein and mRNA levels was found either negligible or little, no further experiments were

This article is protected by copyright. All rights reserved carried out on EE. These findings suggest that, AE, but not EE could be a potential candidate for the development of a therapeutic treatment for OCD.

The existence of dimers or parts of larger oligomeric complexes of different G-protein coupled receptors (GPCRs) in their respective downstream signalling pathways has now been well established [De Bartolomeis et al., 2013; Ferre et al., 2009; Lohse, 2006]. Especially in recent studies support exists for the view that 5-HT2A and D2 receptors are co-expressed in the same cells and could lead to the formation of 5HT2A-D2 receptor heterodimers [Albizu et al., 2011; Lukasiewicz et al., 2011]. These functional heterodimers between 5-HT2A and D2 receptors are reported to be found in cultured cells co-expressing these receptors, such as CLU213 cells as well as in cells in several brain areas including the prefrontal cortex, substantia nigra and striatum [Albizu et al.,

2011; Franklin and Carrasco, 2012; Lukasiewicz et al., 2010]. In the present study, AE (50 µg/mL) increased the interaction between 5-HT2A and D2 receptors in CLU213 cells compared to vehicle treated controls (Figure 5). This increased interaction between 5-HT2A and D2 receptors appears to be due to an associated increase in protein levels of membrane-associated 5-HT2A and D2 receptors, as shown in Figures 1 and 3. Moreover, the tendency of D2 receptor activation by drugs to increase agonistic activity of 5-HT2A receptors and vice versa might underlie an enhanced interaction between them [Albizu et al., 2011; Borroto-Escuela et al., 2010; Bortolozzi et al., 2005].

Our results support previously investigated drugs (in co-immunoprecipitation studies in cultured cells and in vivo) that have been shown to induce increased interaction of 5-HT2AR-D2R heteromers [Albizu et al., 2011; Lukasiewicz et al., 2010]. Though the exact mechanism and nature of the co-localization of 5-HT2A and D2 receptors in various areas of brain is still unclear, their enhanced interaction after treating with drugs in vitro and in vivo is evident. Hence it is suggested that the profile of 5-HT2AR-D2R complex formation might be considered in the mechanism of action of current and future novel drug development against various neuropsychiatric disorders including

OCD. In a recent report, the association of OCD with dysfunctional 5-HT2AR-D2R heterodimer also supports this view [Albizu et al., 2011; Harvey et al., 2002].

This article is protected by copyright. All rights reserved To ensure the effects of AE activation were mediated through 5-HT2A receptors, we used a selective 5-HT2A receptor antagonist (M100907). It was found that pre-treatment with M100907 (10

µM, 1 hour) before AE (50 µg/mL, 24 hours) blocked the effects of AE significantly (Fig. 6 A and B).

These results suggest that AE exert its effect via the 5-HT2AR. We next investigated the signalling pathway involved in AE-induced enhanced 5-HT2A receptor upregulation. Agonist activation of the

5-HT2A receptor is reported to initiate preferentially phospholipase C-β (PLC-β) through the heterotrimeric Gq/11 signaling pathway [Gonzalez-Maeso et al., 2007]. To examine these effects,

CLU213 cells were pre-treated with a PLC-β inhibitor (U73122, 5 µM, 30 min) before adding AE

(50 µg/mL, 24 hours). Inhibition of PLC-β eliminated the gene response obtained by AE (Figure 6

C and D). These results indicate that the 5-HT2A receptor produces a heterotrimeric G protein- mediated signalling response when stimulated by AE. Several hallucinogenic compounds such as

DOI, LSD, psilocybin and antiparkinsonian compounds including ergot derivatives elicit their agonistic effects in 5-HT2A by activating PLC-β via Gq/11 proteins [Cussac et al., 2008; Gonzalez-

Maeso et al., 2007].

Gq/11 -dependant PLC-β activation induces increased c-FOS expression which is common to all 5-

HT2A receptor agonists [Gonzalez-Maeso et al., 2007]. The c-Fos gene is a member of FOS protein family and encodes a 55 kDa product which is post-translationally modified to generate a product that ultimately migrates as a 62 kDa product. In response to cell surface signals, c-FOS functions rapidly by altering gene transcription [Hoffman et al., 1993] which is reportedly blocked after pre- treatment with the PLC-β inhibitor (U73122) [Gonzalez-Maeso et al., 2007]. Therefore, we tested the effects of this inhibitor on the c-FOS gene response linked to AE-induced upregulation of 5-

HT2A receptor (Fig. 7). Our results demonstrate that AE-induced (50 µg/mL, 24 hours) c-FOS activation was significantly (p<0.01) inhibited by pre-treatment of PLC-β inhibitor (U73122) (5 µM,

30 min). On the other hand, activation of D2 receptors is reported to regulate Gi/o signalling pathway (Fig. 8B) and further D2R expression enhanced the efficacy of 5-HT2AR agonists that mediate the Gq/11-PLC-β signalling pathway (Fig. 8C) [Albizu et al., 2011]. The reason for this increased efficacy of 5-HT2AR agonists to mediate preferentially the Gq/11-PLC-β signalling pathway

This article is protected by copyright. All rights reserved (Fig. 8C) appears to be the allosteric crosstalk between 5-HT2AR and D2R [Albizu et al., 2011; Fuxe et al., 2014]. Taken together, it is suggested that AE upregulates 5-HT2AR in a classical Gq/11 dependant PLC-β signalling pathway, which is involved in the activation of the c-FOS transcription factor (Fig. 8). Thus based on the experimental results obtained, we hypothesize that this aqueous extract induced -5-HT2A upregulation could provide a molecular mechanism for the development of potential novel drug candidates to improve OCD.

Activation of 5-HT2AR is also significant in inflammation along with OCD and it was found that upon stimulation of 5-HT2AR by agonists inflammation was inhibited via the NF-κβ signalling pathway [Yu et al., 2008]. From our laboratory, we have already reported the anti-inflammatory potential of AE and EE by suppressing pro-inflammatory cytokine secretion in U937 Cells via NF-κB signalling pathway [Md. Zulfiker et al., 2016]. Recently inflammation is reported as a common condition in various diseases including neuropsychiatric disorders such as depression, anxiety and OCD [Czirr and Wyss-Coray, 2012]. In the present work, we have demonstrated the potential of AE for novel drug development in the treatment of OCD, preferentially via 5-HT2AR activation. Thus to explore the anti-inflammatory potential of AE via the NF-κB signalling pathway through 5-HT2AR activation is an alternative approach.

Finally, 5-HT2AR and D2R which are high affinity G protein-coupled receptors has been implicated in several neuropsychiatric disorders such as schizophrenia, depression, and anxiety i.e., OCD due to their altered neurotransmission [Naughton et al., 2000; Nichols et al., 1994]. As stated above, OCD is strongly associated with the dysregulation of 5-HT2AR and D2R and their complex formation. Recent studies supported this view and report that OCD symptoms are alleviated after agonist activation of 5-HT2AR [Moreno et al., 2006]. The present study also provides evidence on the upregulation of 5-HT2AR and D2R and their increased heterodimers formation by AE which are indicative in the development of drugs not only in OCD but also other neuropsychiatric disorders following similar mechanism. Following on from our present work we aim to identify and isolate compounds from AE that are responsible for the early anti-anxiety therapeutic activity we are

This article is protected by copyright. All rights reserved observing. Moreover, further in vivo assays need to be conducted in a similar fashion to establish the potential of AE in OCD or other disease conditions having the same mechanism.

AUTHORS’ CONTRIBUTION

AHMZ designed and conducted the experiments, interpreted the data and drafted the manuscript.

SMH supported experiments, was consulted on experimental protocols and revised the manuscript. DG supported and supervised the extraction and preparation of extracts as well as revised the manuscript. DAG and MQW supported and supervised the overall work, assisted with conduction of experiments, proof read and revised the manuscript.

ACKNOWLEDGEMENT

Thanks go to Griffith University for GUIPRS and GUPRS scholarships to AZ to enable this study to be conducted.

DISCLOSURES

The authors declare no financial or commercial conflict of interests.

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Zhao H, Wu X, Wang H, Gao B, Yang J, Si N, Bian B. 2013. Qualitative and quantitative analysis of cinobufacini injection using rapid separation liquid chromatography coupled with quadrupole- time-of-flight mass spectrometry and HPLC-photodiode array detection, a feasible strategy for the quality control of Chinese medicine injections. J. Sep. Sci 36:492-502.

Figure 6

Australian cane toad skin extracts (Continued)

141 This chapter presents a co-authored paper, the bibliographic details of which are as follows:

Title: Aqueous and ethanol extracts of Australian cane toad skins suppress proinflammatory cytokine secretion in U937 cells

Authors: Abu Hasanat Md. Zulfiker, Saeed M. Hashimi, Ji Qi, I. Darren Grice and Ming Q. Wei

Journal: Journal of Cellular Biochemistry

Article type: Research paper

Publication status: Accepted

Publication link: http://onlinelibrary.wiley.com/doi/10.1002/jcb.25577/full

14/06/2016

Signed------(Date) ------(Abu Hasanat Md. Zulfiker) 14/06/2016

Signed------(Date) ------(Associate supervisor: Dr. I. Darren Grice) 14/06/2016

Signed------(Date) ------(Corresponding author and Principal supervisor: Dr. Ming Q. Wei)

142 Abu Zulfiker

Thank you for your RightsLink / John Wiley and Sons transaction

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Dear Abu Hasanat Md. Zulﬁker

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From: Abu Zulfiker [mailto:[email protected]] Sent: 07 June 2016 01:46 To: Wiley Global Permissions Cc: Ming Wei; Darren Grice Cosupervisor Subject: Re: Thank you for your RightsLink / John Wiley and Sons transaction

Dear Sir

I am going to submit my PhD thesis on 15 June 2016. I want to include the accepted paper (please see the attached file) or online version (if it is available on or before 15 June) entitled "Aqueous and Ethanol Extracts of Australian Cane Toad Skins Suppress Pro‐Inflammatory Cytokine Secretion in U937 Cells via NF‐κB Signalling Pathway" which is going to be published in your reputed Journal (Journal of Cellular Biohemistry). I have also ordered the permission using Wiley online copyright clearance centre (please see the email below).

I am wondering about, may I include this paper as a chapter (as it is one of the original work of my PhD thesis).

Would you please let me know ASAP as I am in a hurry to submit my PhD thesis (on 15 June). It is highly appreciable if you kindly give me your decision on or before 15 June.

Hope to hearing from you soon. ARTICLE Journal of Cellular Biochemistry 9999:1–12 (2016)

Aqueous and Ethanol Extracts of Australian Cane Toad Skins Suppress Pro-Inflammatory Cytokine Secretion in U937 Cells via NF-kB Signaling Pathway Abu Hasanat Md. Zulfiker,1 Saeed M. Hashimi,1,2 Ji Qi,1 I. Darren Grice,3 and Ming Q. Wei1* 1School of Medical Science and Menzies Health Institute Queensland, Gold Coast Campus, Griffith University, Queensland 4222, Australia 2Department of Biology, Deanship of Preparatory Year, University of Dammam, Dammam, Kingdom of Saudi Arabia 3Institute for Glycomics and School of Medical Science, Gold Coast Campus, Griffith University, Queensland 4222, Australia

ABSTRACT Toad skin extracts, such as aqueous extracts (AE) of Chinese toad skins, have demonstrated therapeutic benefits for a range of diseases including pain, inflammation, swelling, heart failure, and various types of cancers. In this study, we investigated the anti-inflammatory potential of an AE (0.1–10 mg/mL) and a 60% ethanol extract (EE; 0.1–10 mg/mL) from Australian cane toad (Bufo marinus) skins and the known bioactive compound, bufotenine (BT; 0.1–10 nM). The assay employed a model of the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) for the release of tumor necrosis factor (TNF)-a and interleukin (IL)-6. We demonstrated that AE, EE, and BT significantly inhibited the release and expression of TNF-a and IL-6 in a dose-dependent manner when the cells were pre-treated at non-cytotoxic concentrations. Further investigation revealed that the inhibition of TNF-a and IL-6 release and expression was associated with the suppression of nuclear factor (NF)-kappa (k)B activation. These results indicate that AE, EE, and BT are strong inflammation inhibitors, thus have the potential for further development as anti-inflammatory therapeutic agents from a natural source regarded as a feral pest in Australia. J. Cell. Biochem. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.

KEY WORDS: AQUEOUS EXTRACT; 60% ETHANOL EXTRACT; BUFOTENINE; AUSTRALIAN CANE TOAD SKIN; U937 CELLS

nflammation is considered a general phenomenon in various pro-inflammatory cytokines. Several reactive cascades occur in the I disease conditions, including cardiovascular disease, diabetes, NF-kB signaling pathway during this activation and release, cancers, and neuropsychiatric disorders [Couzin-Frankel, 2010; including phosphorylation, inactivation of IkB, and resulting Czirr and Wyss-Coray, 2012]. The monocyte/macrophage cells play a translocation of NF-kB (p50/p65) from the cytosol to nucleus, pivotal role in the progression of these inflammatory states by thus binding to the kB motif of associated target genes, leading to producing various cytokines, including TNF-a, IL-1b, IL-6, IL-10, induction of inflammatory cytokines [Sodsai et al., 2007; Habte- IL-12, etc., that are important regulators or modulators of mariam, 2013]. Many natural and synthetic compounds are reported inflammation [Østerud and Bjørklid, 2003]. Specific genes need to to suppress NF-kB activation [Surh, 2002]. In Traditional Chinese be activated to express these cytokines in macrophages. It has been Medicine (TCM), aqueous extracts (AE) of Chinese toad skins have observed that the transcriptional control and release of pro- also been demonstrated to inhibit the stimulation of NF-kB [Wang inflammatory cytokines are triggered primarily via the nuclear et al., 2012]. At present, NF-kB regulation has gained much interest factor (NF)-kappa (k)B system. It is now established that NF-kB as a new drug therapy for controlling inflammation [Sodsai et al., exists in macrophages as an inactive heterodimer comprising p50 2007]. and p65 bound with an inhibitory kappa (Ik)B subunit. In response to The Australian cane toad, Bufo marinus, is an introduced species. various inflammatory stimuli, such as bacterial LPS, stress-inducing Originally, it was found in Central and South America; however, in agents, and PMA, monocytes are activated resulting in the release of 1935 they were introduced to Queensland, Australia from Hawaii,

*Correspondence to: Prof. M. Q. Wei, School of Medical Science, Menzies Health Institute Queensland (G40), Gold Coast campus, Griffith University, QLD 4222, Australia. E-mail: m.wei@griffith.edu.au Manuscript Received: 11 January 2016; Manuscript Accepted: 29 April 2016 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2016 DOI 10.1002/jcb.25577 © 2016 Wiley Periodicals, Inc. 1 with the intention that they would control the destructive sugar cane EXTRACTION OF AUSTRALIAN CANE TOAD SKINS beetles and their larvae in Queensland. This was an infamous The extraction method utilized in this study was based on the disaster, with the cane toads being remarkably successful at commonly used method for the preparation of Chinese medicines reproducing and spreading themselves due to lack of natural from toad skins [Roseghini et al., 1976; Oka et al., 1985]. Cane toad predators, along with an abundant food supply [Letnic et al., 2008]. skins (10 g) were dried, chopped, then immersed into 200 mL MilliQ Today, they are regarded as feral pests with the government having water in a flask with a water-jacketed condenser. The mixture was resident eradication efforts in place, include asking residents to aid brought to gentle reflux for 8 h, then allowed to cool overnight. The in their collection and disposal. Although in TCM there are some skins were removed by filtration by whatmann filter paper (Bibby reports of the use of extracts of toad species in the treatment RE200, Sterilin Ltd., UK), then the filtrate was divided into two equal of inflammation and various diseases [Wang et al., 2012], no parts. One part was centrifuged at 1,500g for 10 min and the investigations have been reported to date on the potential anti- supernatant freeze-dried to give the AE (24.4% w/w). The second inflammatory activity of Australian cane toad skin extracts. In the portion of supernatant was freeze-dried, then treated with 60% present study, we have investigated the potential anti-inflammatory ethanol (aqueous), centrifuged at 1,500g 10 min, supernatant activity of AE, ethanol extract (EE) of Australian cane toad skins and removed and freeze-dried to give the 60% ethanol (aqueous) extract BT (Fig. 1), for the first time, on the release of TNF- a and IL-6 along (EE; 4.8% w/w). with their effects on the activation of the NF-kB system in the monocyte/macrophage-derived cell line U937 . SAMPLE PREPARATION The freeze-dried extracts (AE and EE) were stored at 2–8°C. Separate MATERIALS AND METHODS solutions of AE, EE, and BT were diluted in MilliQ water, 60% ethanol, and absolute methanol, respectively, followed sterilization through a 0.22 mm membrane filter (Merck Millipore Ltd., Darmstadt, CHEMICALS AND REAGENTS Germany). All stock solutions of AE, EE, and BT for cell culture of the Fetal bovine serum (FBS) was purchased from Bovogen Biologicals, desired concentration were prepared in media where the final Australia. Roswell Park Memorial Institute (RPMI)-1640 medium, 0 maximum concentration of organic solvent such as ethanol or N-2-hydroxyethyl-piperazine-N -2-ethane sulfonic acid (HEPES), methanol was kept within 0.1%. penicillin, streptomycin, LPS from Escherichia coli 055:B5, and PMA were purchased from Sigma–Aldrich, Australia. Enzyme-linked WST-8 ASSAY immunosorbent assay (ELISA) TNF-a and IL-6 kits were purchased Cell proliferation was evaluated using the WST-8 assay. U937 cells from Elisakit.com (Australia). BT was purchased from Toronto (1 104 cells/well) were seeded into 96-well plates with 100 mLof Research Chemicals (Canada). Huachansu (500 mg/mL) was kindly culture medium overnight, then treated with various concen- provided by Anhui Jingchan Pharmaceutical Co., China. Cane toad trations of AE and EE. After 24 h, 10 mL of WST-8 reagent solution skins were obtained from Dr. Jing Jing, University of Queensland, (Cell Counting kit, Sigma–Aldrich, Australia) was added and Australia. incubated for 4 h. The optical density (OD) value was measured according to manufacturer0s instructions at 450 nM. The percent- CELL CULTURE AND IN VITRO TREATMENT age of “inhibition of proliferation” was calculated as follows: Human pro-monocytic U937 cell line was obtained from American percentage of inhibition ¼ (mean control OD mean experimental Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in OD)/mean control OD 100) %. RPMI-1640 medium supplemented with 10 mM HEPES, 2 mM L-glutamine, 10% FBS, 100 U/mL of penicillin, and 100 mg/mL of ELISA streptomycin. Cells were maintained at 37°C in a humidified For evaluation of secreted TNF-a and IL-6, U937 cells incubator with 5% CO2 then used for experiments between passages (5 105 cells/mL) were treated with various concentrations of 12 and 20. AE and EE for 6 h, then treated with or without LPS (1 mg/mL) þ PMA (30 ng/mL) in a final volume of 2 mL followed by culturing for 24 h. Cell culture supernatants were then collected and stored at 80°C until analyzed. The concentration of TNF-a and IL-6 were determined using ELISA kits according to the manufacturer0s instructions. Absorbance readings were taken with Polarstar Omega 96-well microplate reader (BMG Labtech GmBH, Germany). The limit of sensitivity for detection of TNF-a and IL-6 was <5and <1 pg/mL, respectively, with both intra-assay and inter-assay variability <10%.

RT-PCR FOR THE DETECTION OF TNF-a AND IL-6 mRNA EXPRESSION Total RNA was extracted from U937 cells using an RNeasy plus mini Fig. 1. Chemical structure of bufotenine (BT)—an active constituent reported to be present in Australian cane toad skins (unpublished). kit (Qiagen GmbH, Hilden, Germany) according to the man- ufacturer0s instructions. First strand cDNA was generated using

2 AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES JOURNAL OF CELLULAR BIOCHEMISTRY RT2 First Strand Kit (Qiagen Pty Ltd., Victoria, Australia) following Tris–Glycine gels (Bio-Rad, Hercules, CA). All proteins were the manufacturer0s protocol. The mRNA level of TNF-a and IL-6 was transferred to PVDF (polyvinylidene difluoride; Bio-Rad, Hercules, measured by RT-PCR. The primers were synthesized, optimized, and CA) membranes and then blocked for 1 h at room temperature with validated by QIAGEN (Qiagen Pty Ltd., Australia), with details given 5% non-fat dried milk dissolved in TBST (Tris-buffered saline- in Table I. Quantitative determination of gene expression levels Tween; 50 mM Tris, 150 mM NaCl, and 0.1% Tween-20). The using a two-step cycling protocol was performed in a Rotor Gene Q membranes were then incubated with rabbit polyclonal NF-kb cycler (Qiagen Pty Ltd., Australia) using RT2 SYBR Green ROX qPCR primary antibody (1:1,000; Sc-372; Santa Cruz Biotechnology, Inc., Mastermix (QIAGEN, Frederick, MD). The reaction parameters were USA), rabbit polyclonal GAPDH primary antibody (1:1,000; FL-335; as follows: 95°C for 10 min, (95°C for 15 s, 60°C for 1 min) for 40 Santa Cruz Biotechnology, Inc., USA), rabbit polyclonal actin cycles. Relative gene expression levels were calculated using primary antibody (1:500; H-196; Santa Cruz Biotechnology, Inc., 2[DDC(T)] method. The test sample target levels were normalized to USA). The incubations with primary antibodies were followed by human HPRT1 expression. washing and incubations with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, 1 h at room IMMUNOFLUORESCENCE MICROSCOPY temperature; Santa Cruz Biotechnology, Inc., USA). Blots were Immunofluorescence microscopy was carried out on cells that were developed using ECL detection kit (Bio-Rad, Hercules, CA) and CCD seeded in a 35 mm sterile dish (Sarstedt, Germany). The cells were imager ChemiDoc system (Bio-Rad, Hercules, CA). Protein loading fixed using 4% paraformaldehyde for 10 min, permeabilized with for each lane was verified using GAPDH (cytoplasmic) or actin 0.1% Triton X-100 in PBS for 5 min, blocked for 1 h with 5% bovine (nuclear protein) or total protein. The optical density of the bands serum albumin (BSA) in PBST and incubated overnight at 4°C with was analyzed by Image Lab software (version 5.2; Bio-Rad, Hercules, human NF-kB p65 primary antibody (Santa Cruz Biotechnology, CA) and the western blot data was expressed as relative optical USA) at a concentration of 1:300 in 1% BSA/PBST. Following density units on each gel normalized to vehicle controls. primary antibody incubation, cells were further incubated with anti- human Alexa-fluor 488-conjugated secondary antibody (Invitro- HPLC-MS ANALYSIS gen, Australia) at a concentration of 1:1,000 in 1% BSA/PBS for 1 h (1) Preparation of standard solutions and samples at room temperature in the dark. Cells were then stained with A standard stock solution of BT was prepared by dissolving 5 mg Hoechst 33342, trihydrochloride, trihydrate (Thermo Fisher Scien- into 200 mL of absolute methanol to yield a concentration of 25 mg/ tific, Australia) for 5 min at room temperature in the dark. A mL, then further diluted with methanol to a final concentration of minimum of three washes was performed between each of the above- 5 mg/mL. The freeze-dried extracts AE and EE were stored at 2–8°C. mentioned steps. A Nikon A1Rþ confocal microscope was used with Prior to HPLC-MS analysis, the samples (AE and EE) were dissolved in 60 objective lens, then Images were acquired with a Nikon digital methanol (to 0.5 g/mL), then centrifuged (13,000g, 10 min), followed camera and NIS-Elements acquisition software (version 4.13). by filtration (0.22 mm membrane filter; Merck Millipore Ltd.). Filtrates Images taken were processed with Adobe Photoshop CS8 to reduce were then transferred to an auto-sampler vial for analysis. file size. (2) HPLC-Q-TOF-MS/MS Initial chromatographic separation was performed on an Agilent PROTEIN EXTRACTION AND WESTERN BLOT ANALYSIS 1290 HPLC, using an Agilent Eclipse plus C18 column U937 cells were harvested and washed with phosphate-buffered (2.1 100 mm, 1.8 mm particle size). In brief, an injection volume saline (PBS). Nuclear-associated and cytoplasmic proteins were of 5 mL was used with a mobile phase flow rate of 0.6 mL/min. Mobile 1 isolated using NE-PER Nuclear and Cytoplasmic Extraction phases consisted of ultrapure water, 0.1% (v/v) formic acid (A), and Reagents (Thermo Scientific, IL) following manufacturer0s protocol. 95/5 (v/v) acetonitrile/water with 0.1% (v/v) formic acid (B). The Protein concentration was determined using a Bio-Rad DC protein method consisted of an initial isocratic phase at 5% B for 5 min, assay kit (Bio-Rad, Hercules, CA). Protein extracts were subjected to followed by a gradient from 5% to 30% B from 5 to 13 min, then a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gradient from 30% to 100% B over 13–20 min, finished by column (SDS–PAGE) under reducing conditions (10% b-mercaptoethanol). washing and re-equilibration steps. Protein extracts (25 mg [for cytoplasmic proteins] or 10 mg Mass spectrometry was carried out in positive-ion mode on an [for nuclear proteins]) were loaded per lane on 10% stain-free Agilent 6530 Quadrupole Time-Of-Flight (Q-TOF) mass spectrometer equipped with a Jet Stream Electrospray Ionization (ESI) source. Source ionization conditions were as follows: drying gas temperature, 300°C; drying gas flow, 10.0 L/min; sheath gas temperature TABLE I. Primers Used for RT-PCR Analysis 300°C; capillary voltage, (þ) 3,000 V and () 1,500 V; skimmer Gene RefSeq PCR product voltage, 65 V; fragmenting (cone) voltage, 110 V and nebulizer at symbol Catalog no. accession no.a size (bp) 25 psi. The mass spectrometer was operated with a range of m/z 100– 1,700 with an acquisition rate of 1.4 spectra/s. TNF-a 330001 PPH00341F NM_000594.3 110 IL-6 330001 PPH00560C NM_000600.3 98 (3) Bufotenine quantification HPRT-1 330001 PPH01018C NM_000194.2 57 The analysis was performed using the MassHunter Qualitative aThe RefSeq accession no. refers to the sequence used to design the RT2 qPCR analysis and Quantitave analysis software platforms (Agilent Primer Assay by QIAGEN. Technologies). In brief, the chromatographic retention time for

JOURNAL OF CELLULAR BIOCHEMISTRY AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES 3 bufotenine (BT) was determined using by extracting the ion to huachansu (about 58%; P < 0.0001; Fig. 2 E, F, and H). The chromatogram for the ionized accurate mass of a standard injected concentration of TNF-a and IL-6 was also diminished significantly at 1 mg/mL. After range finding analysis of the target samples, a in the presence of bufotenine (10 nM) by 68% (P < 0.001) and 56% 6-point standard curve was prepared using the peak area of extracted (P < 0.0001), respectively (Fig. 2C and G). These results indicate that ion chromatogram of M-H of bufotenine ranging from 40 to 1 ng/mL AE, EE, and BT decrease the production of TNF-a and IL-6 in U937 (R2 ¼ >0.995). This was used to quantify bufotenine in the extracts cells stimulated with LPS þ PMA, but do not induce their secretion in by direct comparison of peak areas. All putative bufotenine peaks unstimulated cells. detecting in the samples where confirmed through using collision- induced dissociation MS/MS analysis (CID 20V) and direct EFFECTS OF THE AQUEOUS EXTRACT, 60% ETHANOL EXTRACT, AND comparison of the fragmentation spectra with that obtained from BUFOTENINE ON TNF-a AND IL-6 mRNA EXPRESSION IN LPS AND the commercial bufotenine standard. PMA-STIMULATED U937 CELLS To determine the effect of AE, EE, and BT on U937 cell gene STATISTICAL ANALYSIS expression of TNF-a and IL-6, U937 cells were incubated separately The results represent the mean standard error of mean (SEM) of the with various concentrations of AE, EE, BT, and huachansu for 6 h, values obtained of at least three independent experiments, carried then treated with LPS (1 mg/mL) þ PMA (30 ng/mL) for a further 24 h. out in triplicate. Comparisons between groups were made with Compared with untreated cells, the presence of LPS (1 mg/mL) and ANOVA and Dunnette0s multiple comparison test using GraphPad PMA (30 ng/mL) strongly induced the gene expression of TNF-a and Prism software (version 6.01, La Jolla, CA). A value of P < 0.05 was IL-6 (70- and 900-fold, respectively) after 24 h incubation considered significant. (Fig. 3). In the absence of LPS and PMA, the gene expression level of TNF-a and IL-6 was found to be negligible at the highest m m RESULTS concentration of AE (10 g/mL), EE (10 g/mL), huachansu (10 mg/mL), and BT (10 nM) over the same incubation period of 24 h (Fig. 3). It is noteworthy that the induced TNF-a and IL-6 mRNA EFFECTS OF AQUEOUS EXTRACTS, 60% ETHANOL EXTRACTS, AND expression levels were suppressed by the addition of various BUFOTENINE ON CELL VIABILITY concentrations (0.1–10 mg/mL) of AE, EE, huachansu, and BT (0.1– The effect of AE, EE, and BT on the viability of U937 cells was 10 nM; Fig. 4). evaluated using the WST-8 cell-viability assay. The cell survival was not affected significantly at various concentrations of AE, EE, and BT ranging from 0.1 to 10 mg/mL for over 12 h (data not shown). Higher EFFECTS OF THE AQUEOUS EXTRACT, 60% ETHANOL EXTRACTS, concentrations of AE, EE (100–1,000 mg/mL) and BT (100–1,000 nM) AND BUFOTENINE ON NF-kB ACTIVATION IN U937 CELLS resulted in; however, an evident decrease in cell survival. An investigation into the contribution of AE, EE, and BT to the Consequently, non-cytotoxic concentrations of the extracts and regulation of the transcription factor NF-kB for cytokine secretion in BT were used in subsequent experiments. U937 cells was performed by immunofluorescence microscopy (Fig. 5). The levels of activation of NF-kB p65 in U937 cells treated EFFECTS OF AQUEOUS EXTRACT, 60% ETHANOL EXTRACT, AND with LPS (1 mg/mL) þ PMA (30 ng/mL) and the inhibitory effects of BUFOTENINE ON TNF-a AND IL-6 SECRETION IN U937 CELLS the separate extracts (AE and EE) and BT compared to huachansu are The effect of AE, EE, and BT on the secretion of pro-inflammatory shown in Figure 5. It was observed that the addition of LPS þ PMA cytokines TNF-a and IL-6 in U937 cells was measured using ELISA enhanced the translocation of NF-kB p65 from the cytoplasm to (Fig. 2). The release of TNF-a and IL-6 was not observed in the nucleus by activating it compared to untreated cells. After pre- control (untreated cells) without LPS þ PMA. The secretion of TNF-a treatment of cells with AE, or EE or BT for 6 h and then stimulated and IL-6 was negligible at the highest concentration of AE with LPS þ PMA for a further 24 h, the activation of NF-kB p65 was (10 mg/mL), EE (10 mg/mL), huachansu (positive control-TCM; reduced compared to huachansu (positive control; Fig. 5). Further- 10 mg/mL), and BT (10 nM) in the absence of LPS þ PMA. However, more, the inhibition of LPS þ PMA-induced NF-kB p65 activation in a significant increase (P < 0.0001) in secretion of cytokines was U937 cells by AE, EE, and BT was confirmed using western blot assay found in the presence of LPS (1 mg/mL) þ PMA (30 ng/mL) (TNF-a, (Fig. 6). The Western blot analysis presented in Figure 6A showed 1613.12 69.85 pg/mL and IL-6, 3090.94 175.72 pg/mL) after that the level of NF-kB p65 in the cytoplasm of LPS þ PMA-treated 24 h incubation. When the cells were treated with various cells was markedly reduced by inducing translocation into the concentrations of AE, EE, and BT for 6 h, then stimulated with nucleus, whereas pre-treatment with AE, EE, and BT before addition LPS þ PMA for 24 h, the pro-inflammatory cytokine concentrations of LPS þ PMA blocked the translocation of NF-kB p65 level. On the were reduced significantly compared to huachansu (positive control; other hand, Figure 6B displayed increased levels of NF-kB p65 in the Fig. 2). In separate experiments, at a concentration of 10 mg/mL, both nucleus of LPS þ PMA-treated cells, while pre-treatment with AE, AE and EE significantly (P < 0.0001) decreased TNF-a secretion by EE, and BT before addition of LPS þ PMA inhibited the translocation about 58% and 85%, respectively, compared to huachansu (60%; effectively. It was observed that almost 191.68% NF-kB p65 was P < 0.01; Fig. 2 A, B, and D). At the same concentration (10 mg/mL) translocated into the nucleus, where this translocation was inhibited of AE and EE, the IL-6 release was also significantly reduced by effectively by pre-treatment of U937 cells with AE, EE, and BT before about 51% (P < 0.0001) and 80% (P < 0.01), respectively, compared addition of LPS þ PMA (Fig. 6).

4 AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES JOURNAL OF CELLULAR BIOCHEMISTRY Fig. 2. The effect of aqueous (AE), 60% ethanol extracts (EE), and bufotenine (BT) on the secretion of TNF-a and IL-6 in LPS (1 mg/mL) þ PMA (30 ng/mL)-stimulated human monocyte cells (U937) using huachansu as a positive control by ELISA. Cells were treated with AE, EE, and BT for 6 h, prior to incubation with LPS þ PMA for 24 h. TNF-a secretion (A: AE, B: EE, C: BT, D: huachansu) and IL-6 secretion (E: AE, F: EE, G: BT, H: huachansu). Data represent the mean SEM value of three independent experiments. P < 0.05, P < 0.01, P < 0.001, P < 0.0001 were calculated by comparison with LPS þ PMA stimulation value. Statistical analysis was performed by one-way ANOVA and Dunnette0s multiple comparison test using GraphPad prism software (version 6.01, 2012).

JOURNAL OF CELLULAR BIOCHEMISTRY AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES 5 dominant fragmentation ions detected in the peak of interest from the samples were consistent with the fragmentation pattern from the bufotenine standard, providing unambiguous identiﬁcation of the BT.

DISCUSSION

The present study investigated the anti-inflammatory potential of AE and EE of Australian cane toad skins along with the known active constituent BT. These activities were also directly compared with huachansu which is best studies recipe as a positive control. All three samples suppressed the secretion of proinflammatory cytokines, TNF-a and IL-6 effectively in LPS þ PMA-stimulated U937 cells (human pro-monocytic cells). These inhibitory effects are believed to be initiated via the activation, then translocation of cytoplasmic NF- kB p65 into the nucleus. Interestingly, in TCM toad skin extracts are reported to have anti-inflammatory activity due to inhibition of proinflammatory cytokines [Wang et al., 2012]. Inflammatory process has long been considered a general phenomena in many acute and chronic human diseases, such as cardiovascular disease, diabetes, cancer, neuropsychiatric disorders [Couzin-Frankel, 2010; Czirr and Wyss-Coray, 2012]. Activation of these inflammatory processes results in the release of inflammatory cytokines into the peripheral circulation and depending on the degree or the extent of the inflammatory stimulus, they can even access the brain and alter behaviour [Miller et al., 2013]. The excessive production of inflammatory cytokines may give rise to edema, cellular metabolic stress, and tissue necrosis, although possessing highly beneficial effects during infection [Ren et al., 2010]. Therefore, agents that could better regulate the release of these inflammatory cytokines may Fig. 3. Release of TNF-a (A) and IL-6 (B) mRNA stimulated by LPS þ PMA have therapeutic effects. The most extensively studied cytokines (1 mg/mL and 30 ng/mL, respectively) in qPCR. AE (10 mg/mL), EE (10 mg/mL), in disease are TNF-a and IL-6 due to their effects on both the a and BT (10 nM). After incubation for 24 h, both TNF- ( 70-fold increase, peripheral and central nervous system [Sodsai et al., 2007; compared to untreated cells) and IL-6 (900-fold increase, compared to untreated cells) showed significantly increased release levels following Ortiz-Lazareno et al., 2008]. The present study showed that U937 stimulation by LPS (1 mg/mL) þ PMA (30 ng/mL). TNF-a and IL-6 mRNA cells were effectively induced to release TNF-a and IL-6, in showed no release in AE, EE, and BT experiments without the presence of response to LPS þ PMA stimulation, consistent with previous LPS þ PMA. Data represent the mean SEM value of three independent studies [Ortiz-Lazareno et al., 2008]. We demonstrated that AE, experiments. P < 0.0001 were calculated by comparison with untreated 0 EE, and BT (known active constituent from toads) suppressed the value. Statistical analysis was performed by one-way ANOVA and Dunnette s a þ multiple comparison test using GraphPad prism software (version 6.01, 2012). secretion of both TNF- and IL-6 in LPS PMA-stimulated U937 cells in a dose-dependent manner. It was observed in ELISA (Fig. 2) that EE was more effective (with 80% inhibition for both TNF-a and IL-6; P < 0.01) compared to AE (58% for TNF-a; IDENTIFICATION OF BUFOTENINE IN AQUEOUS (AE) AND 60% P < 0.0001 and 51% for IL-6; P < 0.0001), BT (68% for TNF-a; ETHANOL EXTRACTS OF AUSTRALIAN CANE TOAD SKINS P < 0.001 and 56% for IL-6; P < 0.0001) and even the positive BT has been reported previously in various species of toad skins, control, huachansu (60% TNF-a; P < 0.01 and 58% for IL-6; including the cane toad (Bufo marinus) [Md Zulfiker et al., 2015], P < 0.0001) at the highest concentration (10 mg/mL) in suppress- although from outside of Australia. Therefore we wanted to ing the secretion of both TNF-a and IL-6. The concentration of AE determine if BT was present in our extracts (AE and EE) of and EE (0.1–10 mg/mL) required to inhibit the release of both Australian cane toad skin (Bufo marinus), then quantify these levels TNF-a and IL-6 were similar to that of huachansu (0.1–10 mg/mL) via HPLC-MS-Q-TOF using commercial bufotenine (C12H16N2O, m/z though concentrations for BT of 0.1–10 nM were effective as a 205.1335) as a standard. BT was found to be present at 0.70 and pure standard compound. However, in the qPCR method (Fig. 4), 2.24 ng/mL in AE and EE, respectively (Table II). To confirm the the inhibition of both TNF-a andIL-6mRNAexpressionbyEE identification of BT, collision-induced disassociation MS/MS (10 mg/mL) was 59% (P < 0.01) and 75% (P < 0.0001), respec- analysis was performed. The 205.1335, 160.0765, 58.0654 m/z tively, compared to huachansu (10 mg/mL; 51% inhibition for

6 AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES JOURNAL OF CELLULAR BIOCHEMISTRY Fig. 4. The effect of AE and EE on the secretion of TNF-a and IL-6 in LPS þ PMA-stimulated human monocyte cells (U937) using huachansu as positive control by qPCR. TNF-a (A: AE, B: EE, C: BT, D: Huachansu) and IL-6 (E: AE, F: EE, G: BT, H: Huachansu). Data represent the mean SEM value of three independent experiments. P < 0.05, P < 0.01, P < 0.001, P < 0.0001 were calculated by comparison with LPS (1 mg/mL) þ PMA (30 ng/mL) stimulation value. Statistical analysis was performed by one-way ANOVA and Dunnette0s multiple comparison test using GraphPad prism software (version 6.01, 2012).

TNF-a; P < 0.01 and 54% inhibition for IL-6; P < 0.001) which is for IL-6; P > 0.05). This difference of suppression in secreted greater than both AE (10 mg/mL; 31% inhibition for TNF-a; protein and mRNA levels by the extracts and BT might be due to P < 0.05 and 43% inhibition for IL-6; P < 0.05) and BT differences in synthesis rates and stability of proteins and (10 nM; 25% inhibition for TNF-a; P > 0.05 and 29% inhibition mRNA as well as their possible post-transcriptional regulation.

JOURNAL OF CELLULAR BIOCHEMISTRY AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES 7 Fig. 5. The effect of AE, EE, and BT on nuclear factor-kb (NF-kb) translocation in LPS þ PMA-stimulated human monocyte cells (U937) using huachansu as positive control. Localization of NF-kb p65 was visualized under a fluorescence microscope after immunofluorescence staining with NF-kb p65 antibody (red). In addition, the cells were stained with Hoechst 33342 to visualize nuclei (blue). It was observed that LPS (1 mg/mL) þ PMA (30 ng/mL) enhanced the translocation of NF-kb p65 from cytoplasm to nucleus by activating it compared to untreated cells. After pretreatment with AE, EE, and BT for 6 h and later stimulation with LPS þ PMA for 24 h, the activation of NF-kb p65 was significantly reduced compared to huachansu used as positive control.

8 AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES JOURNAL OF CELLULAR BIOCHEMISTRY TABLE II. Concentration of Bufotenine (BT) in Standards and Calculated BT Concentrations in Crude AE and EE Experimental Calculated concentration of concentration of Retention Sample bufotenine (ng/mL) bufotenine (ng/mL) time (min) Response Height

Bufotenine 1 0.81 2.1 57,233 4,868 Bufotenine 2.5 1.91 2.1 146,913 12,519 Bufotenine 5 4.22 2.1 329,160 27,104 Bufotenine 10 9.68 2.1 749,615 59,925 Bufotenine 20 20.65 2.1 1,693,969 132,941 Bufotenine 40 39.89 2.1 3,576,084 287,343 Aqueous extract (AE) – 0.70 2.0 30,854 9,134 60% ethanol extract (EE) – 2.24 2.0 183,887 23,495

In addition, culture to culture variations, differences in translation dependent upon the status of the cell redox condition [Israel et al., efficiency, diversity in degradation rates among different genes, 1992; Ogata et al., 2000] LPS [Shen et al., 2015] or PMA [Li and andalsobetweenmRNAandproteinsmightbethefactors Karin, 1999; Singh and Bhat, 2004] are both capable of producing [Jayapal et al., 2008]. reactive oxygen species, but it is unclear as to the exact mechanism To shed light on the molecular mechanism of the inhibitory effects associated with the regulation of the redox state in U937 cells and of AE, EE, and BT on inflammatory cytokines, we also investigated inhibition of NF-kB. This still remains unclear after our studies here their effects on the NF-kB signaling pathway in LPS þ PMA- with AE, EE, and BT. Further studies are required to elucidate the stimulated U937 cells. NF-kB is a family of transcription factors precise mechanism of the regulation of the inflammatory process by which are expressed universally in a number of cells and modulate AA, EE, and BT. the transcription of various key inflammatory cytokines including Identification of active constituents from animal sources still TNF-a, IL-6, IL-1b, IL-10 [DiDonato et al., 2012]. LPS alone remains an important goal for pharmacology research. Toad skins [DiDonato et al., 2012; Yang et al., 2014] or in combination with world-wide are a rich source of bioactive substances including PMA [Ortiz-Lazareno et al., 2008] is reported to induce initiation of indolealkylamines, bufodienolides, alkaloids, steroids, peptides, the NF-kB signaling pathway in macrophages, phosphorylating and proteins [Giri et al., 2006]. Huachansu and in particular the IkBa leading to its ubiquitination and ultimate degradation by the bufadienolides (previously identified in huachansu) have already 26S proteasome [Iwai, 2012]. Therefore, the NF-kB signaling been reported to possess anti-inflammatory activity, targeting the pathway plays a vital role in regulating inflammation. Under NF-kB pathway [Qi et al., 2014]. Interestingly, BT, an indolealkyl- normal conditions, NF-kB binds with IkBs that impound NF-kBin amine, which has been found in the skin of various toad species, the cytoplasm. When IkBa is degraded then NF-kB (p50/p65) is including Bufo marinus (outside of Australia) [Md Zulfiker et al., prompted to localize from the cytoplasm to nucleus. As a result 2015] is also reported to induce several pharmacological activities, transcription of target genes occurs [Dyson and Komives, 2012]. In such as psychotropic effects [Gessner et al., 1960], a marker of the present study, we demonstrated that in the control group, the various psychiatric disorders including schizophrenia, autism, translocation of NF-kB p65 was high following administration of [Emanuele et al., 2010] pressor responses, and LSD-like effects LPS þ PMA compared to untreated cells (Figs. 5 and 6). However, (monkeys) [Gessner et al., 1960]. Of particular note though is that pre-treatment of cells with AE, EE, or BT for 6 h (in separate its anti-inflammatory activity has not been investigated previ- experiments) followed by treatment with LPS þ PMA for 24 h ously, to the best of our knowledge. In the present study, BT showed marked decreases in the translocation of NF-kB p65 into showed significant (P < 0.001) anti-inflammatory activity in nucleus, compared to the positive control huachansu. These data ELISA (Fig. 2C and G), though insignificant (P > 0.05) in qPCR indicate that the extracts and BT block the NF-kB signaling pathway (Fig. 4C and G), being identified in both AE and EE using HPLC-MS by preventing its translocation to the nucleus, leading to the at the concentration of 0.697 and 2.244 ng/mL, respectively (Table inhibition of release of various inflammatory factors. These results II). In our previous studies (to be published), we identified and are consistent with previous reports using RAW 264.7 macrophage reported BT along with other bioactive indolealkylamines, and cells, where results also indicated that NF-kB activation induces the bufadienoldies in both AE and EE by HPLC-Q-TOF-MS/MS. Based expression of TNF-a, IL-6, IL-1b, IL-10 genes [Barnes and Karin, on this, we propose that indolelakylamines and bufadienolides are 1997; Zhu et al., 2013; Oh et al., 2014]. As numerous anti- the major components in both AE and EE that are responsible for inflammatory drugs suppress the production of inflammatory the anti-inflammatory activity targeting the NF-kB pathway cytokines via inhibition of NF-kB activity, AE, EE, and BT show, observed in this work. Support for this comes from the elevated therefore, some early potential to be developed as anti-inflammatory TNF-a and IL-6 inhibitory-release activity evident in EE compared agents. to AE (Figs. 2 and 4), where BT concentration in EE was threefold Most normal cells are reported to have low levels of NF-kB, than that found in AE—suggesting higher levels of “like” whereas cancer cells are reported to contain elevated levels of NF-kB compounds in EE. due to dysregulation of the NF-kB pathway [Lin and Karin, 2003]. In conclusion, AE, EE, and BT induced potent inhibitory effects Interestingly, NF-kB is considered a redox-sensitive transcription on the production and expression of inflammatory cytokines factor and its activation in monocytes such as U937 cells is in LPS þ PMA-stimulated U937 cells. These effects were found to

JOURNAL OF CELLULAR BIOCHEMISTRY AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES 9 Fig. 6. The effect of AE, EE, and BT on nuclear factor-kb (NF-kb) translocation in LPS þ PMA-stimulated human monocyte cells (U937) using huachansu as positive control. (A and B) Cells were treated with AE, EE, BT, and huachansu for 6 h prior to treatment with LPS (1 mg/mL) þ PMA (30 ng/mL) for 24 h. (A) cytosolic and (B) nuclear proteins were subjected to 10% SDS–polyacrylamide stain free gels followed by Western blot analysis using rabbit anti-NF-kb p65 antibody. GAPDH and actin were used as the loading controls for cytosolic and nuclear fractions, respectively. Additionally, total protein was also used in both cytosolic and nuclear fractions to ensure the proper loading. It was found that LPS (1 mg/mL) þ PMA (30 ng/mL) increased the translocation (C) of NF-kb p65 from cytoplasm (A) to nucleus (B) by activating it compared to untreated cells. After pretreatment with AE, EE, and BT for 6 h and later stimulation with LPS þ PMA for 24 h, the activation of NF-kb p65 was signiﬁcantly reduced compared to huachansu used as positive control. Data represent the mean SEM value of three independent experiments. P < 0.05, P < 0.01, P < 0.001, P < 0.0001 were calculated by comparison with untreated and #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 were calculated by comparison with LPS (1 mg/mL) þ PMA (30 ng/mL) stimulation value. Statistical analysis was performed by one-way ANOVA and Dunnette0s multiple comparison test using GraphPad prism software (version 6.01, 2012).

10 AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES JOURNAL OF CELLULAR BIOCHEMISTRY be through the inhibition of NF-kB activation. The results obtained Gessner P, Khairallah P, McIsaac W, Page I. 1960. The relationship between in this study suggest that AE and EE from Australian cane toad the metabolic fate and pharmacological actions of serotonin, bufotenine and – skins and BT could be investigated/developed further as potential psilocybin. J Pharmacol Exp Ther 130:126 133. anti-inflammatory agents. Additionally, other active constituents Giri B, Gomes A, Debnath A, Saha A, Biswas AK, Dasgupta SC, Gomes A. 2006. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad present in AE and EE could also be identified and developed as (Bufo melanostictus, Schneider) skin extract on U937 and K562 cells. Toxicon fl potential anti-in ammatory agents. 48:388–400. The results of this study are important because the deregulated Habtemariam S. 2013. Targeting the production of monocytes/macrophages- expression and biosynthesis of proinflammatory cytokines in mono- derived cytokines by anti-inflammatory herbal drugs. Phytopharmacology cytic cells impacts on numerous cellular functions that are 4:131–148. associated with several inflammatory and autoimmune diseases, Israel N, Gougerot-Pocidalo MA, Aillet F, Virelizier JL. 1992. Redox status of including rheumatoid arthritis, intestinal inflammatory diseases, cells influences constitutive or induced NF-kappa B translocation and HIV multiple sclerosis, and asthma [Garton et al., 2006; Liu and Malik, long terminal repeat activity in human T and monocytic cell lines. J Immunol 149:3386–3393. 2006]. Iwai K. 2012. Diverse ubiquitin signaling in NF-kappaB activation. Trends Cell Biol 22:355–364. AUTHORS’ CONTRIBUTIONS Jayapal KP, Philp RJ, Kok YJ, Yap MG, Sherman DH, Griffin TJ, Hu WS. 2008. Uncovering genes with divergent mRNA-protein dynamics in Streptomyces AHMZ designed and performed the experiments, analyzed the data, coelicolor. PLoS ONE 3:e2097. and wrote the manuscript. SMH supported experiments, was Letnic M, Webb JK, Shine R. 2008. Invasive cane toads (Bufo marinus) cause consulted on experimental protocols, and revised the manuscript. mass mortality of freshwater crocodiles (Crocodylus johnstoni) in tropical Australia. Biol Conserv 141:1773–1782. JQ was consulted on experimental protocols. DG supported and k supervised the extraction and preparation of extracts as well as the Li N, Karin M. 1999. Is NF- B the sensor of oxidative stress? FASEB J 13:1137–1143. analytical experiments and revised the manuscript. MW supported Lin A, Karin M. 2003. NF-kB in cancer: A marked target. Semin Cancer Biol and supervised the overall work, assisted with conduction of 13:107–114. experiments, and revised the manuscript. Liu SF, Malik AB. 2006. NF-kappa B activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol 290: ACKNOWLEDGMENTS L622-145. fi fi Md Zul ker AH, Mariottini GL, Qi J, Grice ID, Wei MQ. 2015. Indolealkyl- Thanks goes to Grif th University for GUIPRS and GUPRS amines from toad vertebrates and sea invertebrates? their identification and scholarships to AZ to enable this study to be conducted. Besides potential activities on the central nervous system. Cent Nerv Syst Agents Med authors are grateful to Ben Matthews, Lab Manager, Smart Water Chem 15:1–11. Research Centre, Griffith University, Australia for helping in Mass Miller AH, Haroon E, Raison CL, Felger JC. 2013. Cytokine targets in the spectrometry analysis and reviewing the manuscript. brain: Impact on neurotransmitters and neurocircuits. Depress Anxiety 30:297–306. Ogata N, Yamamoto H, Kugiyama K, Yasue H, Miyamoto E. 2000. 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JOURNAL OF CELLULAR BIOCHEMISTRY AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES 11 Roseghini M, Erspamer V, Endean R. 1976. Indole-, imidazole- and phenyl- Surh Y-J. 2002. Anti-tumor promoting potential of selected spice ingredients alkylamines in the skin of one hundred amphibian species from Australia and with antioxidative and anti-inflammatory activities: A short review. Food Papua New Guinea. Comp Biochem Physiol C 54:31–43. Chem Toxicol 40:1091–1097. Shen Y, Yang S, Shi Z, Lin T, Zhu H, Bi F, Liu A, Ying X, Liu H, Yu K. 2015. Wang J-Y, Chen L, Zheng Z, Wang Q, Guo J, Xu L. 2012. Cinobufocini inhibits SeMet mediates anti-inflammation in LPS-Induced U937 cells targeting NF-kB and COX-2 activation induced by TNF-a in lung adenocarcinoma NF-kB signaling pathway. Inflammation 38:736–744. cells. Oncol Rep 27:1619–1624. Singh S, Bhat MK. 2004. Carboplatin induces apoptotic cell death through YangHL,HuangPJ,LiuYR,KumarKJ,HsuLS,LuTL,ChiaYC,TakajoT,Kazunori downregulation of constitutively active nuclear factor-kappaB in human A, Hseu YC. 2014. Toona sinensis inhibits LPS-induced inflammation and HPV-18 E6-positive HEp-2 cells. Biochem Biophys Res Commun migration in vascular smooth muscle cells via suppression of reactive oxygen 318:346–353. species and NF-kappaB signaling pathway. Oxid Med Cell Longev 2014:901315. Sodsai A, Piyachaturawat P, Sophasan S, Suksamrarn A, Vongsakul M. 2007. Suppression by Curcuma comosa Roxb. of pro-inflammatory cytokine Zhu J, Luo C, Wang P, He Q, Zhou J, Peng H. 2013. Saikosaponin A mediates fl secretion in phorbol-12-myristate-13-acetate stimulated human mononu- the in ammatory response by inhibiting the MAPK and NF-kappaB – clear cells. Int Immunopharmacol 7:524–531. pathways in LPS-stimulated RAW 264.7 cells. Exp Ther Med 5:1345 1350.

12 AQUEOUS AND ETHANOL EXTRACTS SUPPRESS CYTOKINES JOURNAL OF CELLULAR BIOCHEMISTRY Chapter 6: Overall discussion

157 Since long ago toad skin extracts have demonstrated therapeutic benefits. For example, in TCM, aqueous extracts of Chinese toad skins have been reported to treat pain, swelling, heart failure as well as several cancers with minimal to no side effects, generating a ten billion USD market in China (1). Toad skins are also a good source of various bioactive substances including IAAs, bufadienolides, peptides, proteins, fatty acids and hydroxyphenylalkylamines (Table 1.1). These compounds are reported to have potential therapeutic activity in various disease conditions either as a single entity or as a group (320-323). So the present study is aimed at identification of chemical constituents in Australian cane toad skin extracts and to investigate their therapeutic relevance by performing functional characterisation studies on these extracts in cultured cells.

For compound extraction/identification, initially several aqueous-based extraction systems were designed and optimized to isolate known ‘neuro-active’ markers including mainly IAAs and bufadienolides, previously identified in toad skins. Thus aqueous, 60% ethanol and acetic acid (in two different pH: 1.78 & 2.17) were used to extract the cane toad skins based on the commonly used method for the preparation of Chinese medicines from toad skins (326). In manuscript 1 (chapter 3), these extracts of Australian cane toad skins were investigated to identify their chemical constituents using HPLC-ESI-Q-TOF-MS/MS. Forty-two constituents were identified including alkaloids, amino acids, bufadienolides, fatty acids, nucleobases, nucleosides and vitamins predominantly from the AE and EE. Of the 42 constituents identified, 29 were found in AE, 35 were found in the EE, 10 in the pH 1.78 acetic acid extract and 11 in the pH 2.17 acetic acid extract. Therefore, AE and EE present the greatest potential for ongoing development in further assays. Forty one out of the 42 constituents identified were found in either AE or EE, with 23 constituents found in both of them and 158 only 1 constituent found in both the acetic acid extracts. There were 12 constituents that were present in the combined acetic acid extracts, but overall it was clearly apparent that these acidic extracts contained a notably reduced number of ‘recognised actives’ and would therefore not be the ongoing focus of interests. These findings in the present study are in agreement with a previous investigation where 19 hydrophilic compounds were identified in TCM cinobufacini/huachansu (a boiling water extract prepared from the skin of Bufo bufo gargarizans Cantor) using HPLC-QTOF-MS (327).

Only 8 constituents identified in our study (two alkaloids and 6 nucleic bases or nucleosides) were reported in the Zhao et al (327) investigation. In another report, a total of 58 various bio-compounds including peptides, steroids (bufadienolides, cholesterols), indole alkaloids, bufogargarizanines and organic acids were identified in cinobufacini (44). Out of these 58 constituents, only 3 were identified in common with this study, namely dehydrobufotenine, bufalin and marinobufagin. Therefore, it is obvious from the previous literature that only 12 of our identified 42 compounds have been identified previously in toad skins. Interestingly, there are numerous compounds or groups of compounds such as pyrrolizidine alkaloids, fatty acids, proteins, peptides that have been identified before in the skin of various toad species (Table 1.1) that have not been detected in this study, but are reported to have potential pharmacological activities (39). Of note, is that different geographic locations sometimes play a role in the variation of the content and relative concentration of constituents even in the skin of the same toad species. Multiple reasons such as the harvest and post-harvest processing and adaption to environmental factors in different geographic locations might contribute to this variation (328). The constituents present in glands or venom of toads has been studied more extensively than toad skins with many additional constituents having been identified in various toad species (329).

Recently an investigation is reported on the potential of toad for the drug development

159 in the treatment of neuropsychiatric disorders including anxiety or depression (330).

Therefore the AE and EE were selected as the ‘extracts of focus’ to investigate their therapeutic potential for the treatment of neuropsychiatric disorders, especially OCD.

To functionally characterization, AE and EE biological assays were performed, which are described in detail in manuscript 2 (chapter 4) and manuscript 3 (chapter 5). In manuscript 2, a molecular mechanism is suggested in cultured cells (CLU213) that could be utilized therapeutically to assess AE and EE as potential targets for the development of drugs against a disease condition such as OCD. The assay targeted this molecular mechanism, as there is reported evidence that describes drugs having agonist-like activities via 5HT2A receptor (5-HT2AR) in certain areas of brain, such as the pre-frontal cortex and might therefore significantly improve OCD symptoms (331).

This was accompanied by several other findings which showed an association of OCD with lower dopamine receptor (D2R) availability, primarily in the striatum of the brain along with dysfunctional 5-HT2AR-D2R heteromer regulation (148, 279). A rat hypothalamic cell line (CLU213) was used for one of the assays as it reportedly expressed 5-HT2A and D2 receptors enabling an investigation of whether or not AE and/or EE could upregulate these receptors and therefore increase the interaction of

5-HT2AR-D2R heteromer upon treatment (284). It was found that AE (50 µg/mL), but not EE, significantly (p<0.05) increased 5-HT2AR and D2R protein and mRNA levels by western blot and qPCR, respectively (Figures 1 to 4 in manuscript 2-chapter 4). BT and

DOI were used as positive controls, respectively for this investigation. BT was reported to be present in various species of toad skins along with cane toad skins (332) and was identified in Bufo marinus skins (this research) along with several other BT-like compounds identified in both AE and EE (Table 1, manuscript 1-Chapter 3). So it is likely that the presence of BT and BT-like compounds in AE might be responsible for

160 the significant upregulation of 5-HT2AR seen in the assays. Furthermore, qualitative and quantitative determination of BT was carried out via HPLC-ESI-Q-TOF-MS/MS in both AE and EE using a BT standard. Interestingly BT was not identified in EE after sample storage (-20ºC) in our ongoing study (data not shown) though initially the presence of BT was confirmed (Table 1, manuscript 1-chapter 3) and its concentration was also found to be higher using the standard in EE (2.24 ng/mL) compared to AE

(0.70 ng/mL) (manuscript 3-chapter 5). Thus it is assumed that BT and BT-like compounds are not stable in long term storage even at -20ºC in organic solvent. This might be the reason for little or negligible effect of EE on the upregulation of 5-HT2AR protein and mRNA level. Furthermore, it is possible that the presence of some compounds in EE that were not identified in the present study have potent antagonistic properties towards 5-HT2AR and might contribute for little or negligible effect of EE.

These findings suggest that, AE, but not EE could be a potential candidate for the development of a therapeutic treatment for OCD. As the effect of EE on the upregulation of both 5-HT2A and D2 receptor protein and mRNA levels was found to be insignificant, no further experiments were carried out on EE. Further studies to support this molecular mechanism were therefore carried out on AE only. For this purpose, the assay was conducted on co-immunoprecipitation with AE (50 µg/mL) to see whether the interaction of 5-HT2AR-D2R heteromer is increased upon treatment with AE. Indeed, it was found increased interaction between 5-HT2A and D2 receptors in CLU213 cells compared to vehicle treated controls (Figure 5; manuscript 2-chapter 4). Results obtained here are in agreement with previously investigated 5-HT2AR agonists such as

DOI (in co-immunoprecipitation studies in cultured cells and in vivo) that showed enhanced interaction of 5-HT2AR-D2R heteromers (277, 333). Moreover, the classical signalling pathway (Gq/11-PLCβ) was studied which is involved in expressing the 5-HT2A mediating agonistic effects along with the transcription factor (c-FOS) in CLU213 cells

161 (Figures 6-8; manuscript 2-chapter 4). A PLCβ inhibitor (U73122, 5 µM, 30 min) was used for this investigation and found that PLCβ significantly (p<0.05) blocked the gene response and the expression of c-FOS transcription factor after treatment with AE (50

µg/mL). Taken together, it is suggested that the observed increase in 5-HT2AR and D2R protein and mRNA expression levels and their interaction to induce enhanced 5-HT2AR-

D2R heteromer formation by exposure to AE (50 µg/mL) in cultured cells might provide a molecular mechanism to develop potential novel drug candidates to improve OCD symptoms.

An anti-inflammatory study was also carried out as part of the functional characterization of AE and EE (manuscript 3-chapter 5). The basis of this investigation comes from reports that inflammation is a common mechanism in various neuropsychiatric disorders including anxiety, i.e., OCD (104). In this study, it was investigated the anti-inflammatory potential of AE (0.1-10 µg/mL) and EE (0.1-10

µg/mL) along with the known bioactive compound, BT (0.1-10 nM) using huachansu/cinobufacini (Chinese toad skin extracts) (0.1-10 µg/mL) as a positive control. The assay employed a model of the human monocyte cell line U937, whereby cells were stimulated with LPS and PMA for the release of cytokines such as TNF-α and IL-6. It was found that AE and EE both significantly (p<0.05) inhibited the release and expression of TNF-α and IL-6 in a dose dependant manner using huachansu as positive control. Cells were pre-treated at non-cytotoxic concentrations before adding

LPS + PMA (Figures 2-4; manuscript 3-chapter 5), then cytokine levels were assayed using ELISA and qPCR respectively. Interestingly, BT showed negligible release inhibition effects of TNF-α and IL-6 (p>0.05) (Figure 4; manuscript 3-chapter 5) using qPCR. However, ELISA results were different and showed significant suppression of release of TNF-α and IL-6 (p<0.001) (Figure 2; manuscript 3-chapter 5). It is assumed 162 that this difference of suppression in secreted protein and mRNA levels by the extracts and BT might be due to differences in synthesis rates and stability of proteins and mRNA as well as their possible post-transcriptional regulation. In addition, culture to culture variations, differences in translation efficiency, diversity in degradation rates among different genes and also between mRNA and proteins might be the factors

(334). The signalling pathway involved for this activity was also studied and found that the inhibition of TNF-α and IL-6 release and expression was related to the suppression of nuclear factor (NF)-κB activation. It was observed that the translocation of NF-κB p65 was high following administration of LPS+PMA compared to untreated cells

(Figures 5 and 6; manuscript 3-chapter 5). However, pre-treatment of cells with AE, EE or BT for 6 hours (in separate experiments) followed by treatment with LPS+PMA for

24 hours showed marked decreases in the translocation of NF-κB p65 into the nucleus, compared to the positive control huachansu. These data indicate that the extracts (AE and EE) and BT block the NF-κB signalling pathway by preventing its translocation to the nucleus, leading to the inhibition of release of various inflammatory factors. These results are consistent with previous reports using RAW 264.7 macrophage cells, where results also indicated that NF-κB activation induces the expression of TNF-α, IL-6, IL-

1β, IL-10 genes (335-337). As numerous anti-inflammatory drugs suppress the production of inflammatory cytokines via inhibition of NF-κB activity, AE, EE and BT show therefore some early potential to be developed as anti-inflammatory agents.

As mentioned above, the presence of BT was also identified in both AE and EE using a BT standard and further confirmed by HPLC-ESI-Q-TOF-MS/MS in AE, but showed no trace of BT in EE after sample storage (lyophilized or as a solution in 60% ethanol,

≥ 1 month) in our ongoing study. Yet we found more intense activity in EE than AE in both ELISA and qPCR assays (Figures 2 and 4; manuscript 3-chapter 5). It is likely that 163 some BT was still present, but just at levels under the instrument detection level. BT- like compounds were also identified and other additional BT-like constituents and bufadienolides are likely responsible for the evident activity of EE.

164 Chapter 7: Conclusion and future directions

165 Amphibians especially toad skins contain numerous bioactive compounds including proteins, peptides, steroids, alkaloids, opioids, etc., (Table 1.1). These compounds are reported to have wide therapeutic applications such as antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic and sleep inducing properties

(323). In TCM, a number of bioactive chemical compounds have already been identified in aqueous extracts of toad skin named cinobufacini/huachansu. Cinobufacini has a long history of use in various ailments including fever, edema, pain relief, infections, tonsillitis, sores, toothache, inflammation and various forms of cancers with minimal to no side effects, generating a ten billion USD market in China (44). Recently the use of toad for the potential drug development in neuropsychiatric disorders such as anxiety or depression has been reported in vivo (330). Cane toad skin extracts (aqueous and chloroform) have already been investigated for potential drug development of cardiac- glycoside like activity (1). While cane toad venom has been screened for various compounds (338), investigations into bioactive constituents and further functional characterisation of the cane toad skin extracts are still insufficient.

Research presented in chapter 3 (manuscript 1) was conducted to initially optimise the extraction of Australian cane toad skins in aqueous (AE), 60% ethanol (EE) and acetic acid (at two different pH: 1.78 and 2.17) based on various commonly used method for the preparation of Chinese medicines from toad skins. Following this the work focused on the establishment of a simple and reliable HPLC-Q-TOF-MS/MS method for the separation and analysis of these extracts. Analysis of the MS data relating to the four different extracts resulted in 42 known compounds being identified in the toad skins.

Twenty nine of the 42 constituents identified were found in the AE, 35 of the 42 were identified in EE. Only 1 compound was found exclusively in both the acetic acid extracts and the rest of the identified compounds in them were also commonly found in both AE 166 and EE. All of the identified compounds in the 4 extracts are known, but none have been reported before in cane toad skins or extracts of cane toad skins. Results obtained demonstrate that HPLC-ESI-Q-TOF-MS/MS is an effective method to identify constituents in cane toad skin extracts. Since acetic acid extracts presented a greatly reduced number of constituents at the levels of detection in our study, we focused on the AE and EE to carry out functional characterisation assays using in vitro cellular experiments.

For functional characterisation, an assay that targeted a molecular mechanism that is known to be closely related to neuropsychiatric disorder was utilised. As described in chapter 4 (manuscript 2), cane toad skins have a long history of recreational use for inducing euphoric effects. Therefore, it is evident that there are some constituents in cane toad skins which cause the reported euphoric sensations. From this point of view and the basis of recent evidences (manuscript 2-chapter 4), it was observed that AE

(50 µg/mL), but not EE showed significant (p<0.05) upregulation of 5-HT2AR, D2R protein and mRNA levels respectively in cultured cells (CLU213) which indicates a molecular mechanism and provides further basis to develop cane toad skin extracts as a source of drugs for improving therapeutic activity in OCD. Treatment with AE also resulted in an increase in 5-HT2AR-D2R heteromer formation, which is reportedly associated with dysfunctional and decreased in OCD. From these results it is suggested that this enhanced 5-HT2AR, D2R protein and mRNA levels and their interaction to induce increased 5-HT2AR-D2R heteromer formation following exposure to AE (50 µg/mL) in cultured cells provides a molecular mechanism that can be targeted to develop potential novel drug candidates to ameliorate OCD symptoms.

167 In chapter 5 (manuscript 3), investigations were extended to study anti-inflammatory activity of AE and EE. The basis of this (as discussed in chapter 6), as with most disease conditions, is that patients with neuropsychiatric disorders such as OCD also experience inflammation. For inflammatory studies, TNF-α and IL-6 cytokines, were assayed using the U937 cell line as a model, whereby release was stimulated with LPS and PMA after pre-treatment with AE and EE. It was found that both AE and EE showed potent inhibitory effects on the production and expression of the inflammatory cytokines in LPS+PMA stimulated U937 cells. Commercial BT which had been identified in earlier work as present in both AE and EE (Table 1; manuscript 1-chapter 3) was also assessed for its ability to inhibit release of the cytokines, with significant activity being observed. Among, AE, EE and BT, EE showed the strongest potency in inhibitory effects on the production and expression of both the inflammatory cytokines followed by AE and BT (Figures 2 and 4; manuscript 3-chapter 5). These effects of AE, EE and

BT were found to act via the NF-κB signalling pathway. The results suggest that AE,

EE and BT from Australian cane toad skins could be investigated/developed further as potential anti-inflammatory agents.

In summary, this PhD project has advanced the knowledge base on and developed a new strategy for the qualitative characterisation of compounds present in the AE and

EE of cane toad skins using HPLC-MS. Compound identification described in manuscript 1 (chapter 3) by a HPLC-MS method is simple, easy and reliable, and could be used for routine analysis of cane toad skin extracts. Following this technique, most of the bioactive compounds could be clearly elucidated on both qualitative and quantitative basis. This type of investigation for compound identification could provide an informative motif which could lay the foundation for the discovery of essential compounds of potential therapeutic benefit. Additionally, AE and EE have been 168 investigated in functional studies to find therapeutic linkage by cellular experiments

(chapters 4 and 5). From functional studies it has been found that AE, but not EE, upregulates 5-HT2AR and D2R via the Gq/11-PLCβ signalling pathway along with the activation of c-FOS transcription factor in CLU213 cells (manuscript 2-chapter 4).

The molecular mechanisms investigated by in vitro experiments in the present study could be assessed by in vivo studies as future works. The animal studies could include either behavioural or tissue isolation. For behavioural studies, two most commonly used animal models for OCD known as “quinopirole-induced compulsive checking” and

“schedule-induced polydipsia” could be conducted. Also the specific area of brain such as orbitofrontal cortex where the lower 5-HT level was reported to cause OCD could be dissected and the lysate could be used for further in vitro like experiments which was performed in the present study. Furthermore, in manuscript 3 (chapter 5), it has been investigated that AE, EE and BT suppress pro-inflammatory cytokines including

TNF-α and IL-6 via NF-κβ signalling pathway in U937 cells. These in vitro cytokine studies could also be evaluated by in vivo cytokine assays or similar other in vivo experiments for inflammatory studies. In addition, similar in vitro or suggested in vivo work herein could be done after isolation of single or group of compounds such as BT or BT-like compounds from both AE and EE which was assumed in manuscript 2 and

3 (chapter 4 and 5 respectively) for the present activities. Moreover, the active component (s) in single or group could be identified and separated from EE as it was not seen active in the suggested molecular model of manuscript 2 (chapter 4). So further investigation with isolated active component (s) of AE or EE in this molecular model (manuscript 2-chapter 4) in vitro as well as above mentioned in vivo experiments could be much interesting.

169 Appendix

170 This appendix contains a co-authored paper, the bibliographic details of which are as follows:

Title: Indolealkylamines from toad vertebrates and sea invertebrates – their identification and potential activities on the central nervous system

Authors: Abu Hasanat Md. Zulfiker, Gian Luigi Mariottini, Ji Qi, I. Darren Grice and Ming Q. Wei

Journal: Central Nervous System Agents in Medicinal Chemistry

Article type: Review paper

Publication status: Accepted

Publication link: http://benthamscience.com/journals/central-nervous-system-agents- in-medicinal-chemistry/article/133441/

I am the first author of this paper and my significant contribution to this paper involved: platform design, literature review and manuscript writing.

14/06/2016

Signed------(Date) ------(Abu Hasanat Md. Zulfiker) 14/06/2016

Signed------(Date) ------(Associate supervisor: Dr. I. Darren Grice) 14/06/2016

Signed------(Date) ------(Corresponding author and Principal supervisor: Dr. Ming Q. Wei)

171 5/30/2016 Griffith University Student Mail publication of the part 2 of the special issue "The Activity of Products From Cnidaria: A Therapeutic Tool in Neurologica...

Abu Zulfiker

publication of the part 2 of the special issue "The Activity of Products From Cnidaria: A Therapeutic Tool in Neurological Diseases?" (CNSAMC)

Ambreen Irshad Mon, May 30, 2016 at 2:02 AM To: [email protected] Cc: Muhammad Ahmed

Grant of Permission

Dear Dr. Zulfiker:

Thank you for your interest in our copyrighted material, and for requesting permission for its use.

Permission is granted for the following subject to the conditions outlined below:

Indolealkylamines from Toad Vertebrates and Sea Invertebrates – Their Identification and Potential Activities on the Central Nervous System" which is going to be published in Journal ( Central Nervous system agents in Medicinal Chemistry) in December

To be used in the following manner:

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From: Abu Zulfiker [mailto:[email protected]] Sent: Thursday, May 26, 2016 2:18 AM To: Gian Luigi Mariottini Cc: Ming Wei; Darren Grice Cosupervisor; CNSAMC Subject: Re: publication of the part 2 of the special issue "The Activity of Products From Cnidaria: A Therapeutic Tool in Neurological Diseases?" (CNSAMC)

Dear Sir

I am going to submit my PhD thesis on or before 15 June 2016. I want to include the paper (please see the attached file) entitled " Indolealkylamines from Toad Vertebrates and Sea Invertebrates – Their Identification and Potential Activities on the Central Nervous System" which is going to be published in your reputed Journal ( Central Nervous system agents in Medicinal Chemistry) in December (As per the mail below).

I am wondering about, may I include the "Third proof" as in the attached file to my thesis or need further permission from the publisher. Please note I want to include this paper in my PhD thesis as an "Appendix".

Hope to hearing from you soon.

With best regards

Abu Hasanat Md. Zulfiker PhD Candidate

Professor Ming Wei group

School of Medical Science & Menzies Health Institute Queensland Griffith University, Gold Coast, Australia email: [email protected] Phone: +61756780919 (G40_9.12) Mobile: +61469375148 (personal)

On Tue, May 10, 2016 at 12:56 AM, Gian Luigi Mariottini wrote: https://mail.google.com/mail/u/0/?ui=2&ik=042a066692&view=pt&search=inbox&msg=154fd4104d7f3575&dsqt=1&siml=154fd4104d7f3575 2/3 Send Orders for Reprints to [email protected] Central Nervous System Agents in Medicinal Chemistry, 2016, 16, 000-000 1

Indolealkylamines from Toad Vertebrates and Sea Invertebrates - Their Identification and Potential Activities on the Central Nervous System

Abu Hasanat Md. Zulfiker1, Gian Luigi Mariottini2, Ji Qi1, I. Darren Grice3 and Ming Q. Wei1*

1School of Medical Science & Menzies Health Institute Queensland, Gold Coast campus, Griffith Uni- versity, Southport, Qld, 4222 Australia; 2Department of Earth, Environment and Life Sciences, Univer- sity of Genova, Viale Benedetto XV 5, I-16132, Genova, Italy; 3Institute for Glycomics and School of Medical Science, Gold Coast campus, Griffith University, Southport, Qld, 4222 Australia

Abstract: Indolealkylamines (IAAs) are biogenic amines and derivatives of 5-hydroxytryptamine, acting primarily on serotonin receptors. IAAs are often considered the most thoroughly investigated group of aromatic amines in the amphibian skin. On the contrary, at present the detailed knowledge of these compounds in lower organisms is still limited and the biogenic amine receptors, mediating hor- A.H. Md. Zulfiker monal and modulatory functions, are largely unknown in primitive invertebrates. However, some active research is currently underway investigating this class of biogenic amines. Notably, during the last three decades several investigations have demonstrated the biological activity of endogenous biogenic amines in cnidarians, which are known to be the lowest beings equipped with an effective, even though rudimentary, nervous system. Toads, especially those from the Bufonidae family, constitute a significant part of the amphibian family and are an identified source of IAAs. To date fourteen IAAs have been identified in the skins of toad species. All are 5-substituted IAA derivatives acting mainly on the central nervous system (CNS), with most exhibiting some degrees of 5-HT2A receptor selectivity. This selective ability presents potential for their use in the development of treatments for various disorders such as schizophrenia, depression, anxiety, obsessive-compulsive disorders and chronic pain conditions. There are indications that some IAAs may also show subclass selectivity through binding to multiple 5-HT receptor subtypes. Thus, there exists an additional promising platform for the development of therapeutics targeting multiple 5-HT receptors. In this review, IAAs occurring naturally in various species of toad skins, which have been identified and isolated since 1944 are summarized and comparisons are made with similar biogenic amines recognized in cnidarians to date. Such comparisons highlight the potential to utilize existing knowledge gathered from vertebrates, such as toads in order to improve the understanding of the activities of such compounds in lower invertebrates.

Keywords: Indolealkylamines, cnidarians, toad, marine, vertebrate, invertebrate, 5-HT, 5-HT2A

INTRODUCTION groups of aromatic amines found in the amphibian skin [3] due to their variety, abundance and widespread distribution Indolealkylamines (IAAs) are 5-hydroxytryptamine (5-HT [4]. Toads which constitute a significant part of the amphib- or serotonin) derivatives which are therefore analogues of ian family are as expected a rich source of these biogenic monoamine neurotransmitters that regulate human mood and amines. It has been reported that the IAA content in toad behaviour (Fig. 1). These types of compounds have an indole skins (per gram of dry tissue) ranges between a few µg to as moiety and a basic nitrogen atom in their structures which much as 100-150 mg [3], being typically conjugated and are generally connected by an alkyl chain of two carbons in cyclized IAA structures [5]. length [1]. IAAs are widely distributed in microorganisms, plants and animals. Interestingly most IAAs of animal origin Taxonomically, toads are found in the families of are hallucinogenic in nature, except serotonin which is pre- Bufonidae, Bombinatoridae, Discoglossidae, Pelobatidae, sent in the CNS of humans and animals [2]. Rhinophrynidae, Scaphiopodidae, and Microhylidae. So far IAAs have been discovered in toads of Bufonidae, Bombina- IAAs are biogenic amines, being the most thoroughly toridae as well as Scaphiopodidae families, but primarily in investigated group of aromatic amines of the amphibian skin, the Bufonidae family. The Bufonidae family consists of often regarded as an enormous storehouse of biogenic amines. more than 35 genera, with Bufo being the most notable genus They are one of the three most important and characteristic where IAAs have been identified. It has been reported that the skin of Bufo provides a spectacular representation of *Address correspondence to this author at the School of Medical Science & IAAs [6]. IAAs which are mainly arylalkylamines can be Menzies Health Institute Queensland, Gold Coast campus, Griffith Univer- divided into several categories such as N-substituted tryp- sity, Southport, Qld, 4222 Australia; Tel: +61(0)756780745; Fax: +61(0) tamines, α-alkyltryptamines, ergolines, lysergamides and β- 755528908; E-mail: [email protected] carbolines (Fig. 2).

R NH2 4 NR1R2 R5 4 5 3 2 1 N N N H H H

Indole Tryptamine Indolealkylamine

Fig. (1). Indole, tryptamine and IAA structures.

R R NH

NRR1 NH2 R2 R1 R1

N N N H H H N-substituted tryptamines a-Alkyltryptamines Ergolines

NR2R3

R1 O N R2 H

N N R1 H R b-Carbolines N Lysergamides R

Fig. (2). Structures of various IAAs. Among them N-substituted tryptamines which have been identified in toad skins.

Most IAAs have affinity for 5-HT2A receptors [7] and mental, nervous system. They have a simple anatomical therefore can also be categorized as hallucinogenic or psy- structure being composed of only two germ layers, epiderm chotomimetic agents [8]. Although some IAAs exhibit less and gastroderm, therefore they are diblastic. The germ layers subclass selectivity and bind to multiple 5-HT receptor sub- are separated by mesoglea, which is a connective-like tissue types [9]. It has been found that 4-hydroxylated derivatives found only in these organisms. The main feature of cnidari- of IAAs show more specificity (25-380 fold) to the 5-HT2A ans is the presence of specialized cells, namely nematocytes, receptor over the 5-HT1A, while 5-substituted derivatives which contain intracellular capsules or nematocysts, filled exhibit almost equal affinity to both 5-HT1A and 5-HT2A re- with a cocktail of venomous compounds. The nematocysts ceptor [7]. can be rapidly discharged in response to external mechanical or chemical stimuli. 5-HT is known to occur in some cnidar- The majority of IAAs identified to date in toad skins are ian venoms [12], for instance in Metridium senile where its N-substituted tryptamines, being one of the twenty classes of presence was confirmed in a Thin Layer Chromatography indole alkaloids, which reportedly includes almost 600 com- (TLC) study [13]. Furthermore, it has been reported on sev- pounds [10]. Tryptamines from toads are naturally occurring eral occasions that Cnidarians also contain non- alkaloids that are reported in many instances to possess psy- proteinaceous biogenic amines [14, 15]. choactive hallucinogenic activity. However, most N- In the early 1960s Wood and Lentz [16] showed the pres- substituted or α-alkyltryptamines can be readily produced ence of 5-HT in anthozoan nerve cells. Subsequently, per- synthetically through substitutions at selected positions of oxidase-antiperoxidase-immunohistochemistry evidenced 5- the tryptamine substrate. These derivatives retain the tryp- HT neurons in the pennatulid anthozoan Renilla koellikeri. tamine template and therefore continue to show a high affin- Notably, strong 5-HT-immunoreactivity was observed in ity for serotonin receptors. Many of the tryptamine analogs cells and in neuron-like elements, mainly in the outer ecto- (naturally occurring or synthetic) continue to induce psyche- derm or in the mesoglea and a sensory and modulatory func- delic effects including sensory deprivation, altered states of tion of 5-HT in Renilla koellikeri was suggested [17]. Addi- awareness, illusions, changes in perception [11]. tionally, 5-HT immunoreactive neurons have been repeatedly Cnidarians (phylum Cnidaria) are among the most primi- observed in the ectodermal sensory cells and in mesogleal tive animals in the phylogenetic tree and are known to be the neurons of sea pansy Renilla koellikeri [18-19]. Westfall et lowest beings provided with an effective, even though rudi- al. [20] using High Performance Liquid Chromatography Indolealkylamines from Toad Vertebrates and Sea Invertebrates Central Nervous System Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 3

(HPLC) methods studied biogenic amines in sea anemones sus and Bufo crucifer [22]. Selected IAAs found in various establishing the presence of dopamine and 5-HT in tentacles toad species (Table 1) have interestingly been quantified, and in whole body homogenates of Aiptasia pallida. They with the amounts detected varying depending on the location detected positive immunoreactivity to dopamine and 5-HT in of collection [23] (Table 2). tentacle neurons, observing the occurrence of reactive prod- Almost all of the IAAs identified to date have been from ucts in synaptic (interneuronal, neuromuscular, and neuro- the bufonidae family. IAAs have also been identified in two spirocyte) vesicles with the anti-5-HT antibody. The authors other families, namely Bombinatoridae and Scaphiopodidae. concluded that catecholamine and indolamine neurotransmit- Surprisingly, no other IAAs have been identified, with the ters occur in sea anemones, indicating the presence of multi- exception of 5-HT in toad skins outside of these two fami- ple types of transmitter substances in an early nervous sys- lies. The only exception is Scaphiopus hammondi of the tem. Furthermore it was suggested that a serotonergic path- Scaphiopodidae family where bufotenidine was found along way is involved in the control of rhythmic movements linked with 5-HT. It can be seen from Table 1 that 5-HT is a com- to their feeding behaviour. mon constituent of the skin in a number of toads, followed 5-HT immunoreactivity was observed in the cytosol of by bufotenine, dehydrobufotenine and bufotenidine. Moreo- Aiptasia pallida nematocytes and spirocytes, which was ex- ver, 5-methoxytryptamine and indole-3-acetic acid have only plained by taking into account the common origin of cnido- been found to date in the skin of Bufo alvarius. Bufoviridine cytes and neurons from interstitial cells, while nematocysts a –SO3H moiety containing compound was also reported to and spirocysts were seen to be devoid of these reaction prod- be present in several species of toad skins (Table 1). The - ucts. In the same research the authors reported the occur- SO3H group is attached to the nitrogen of the indole nucleus rence of gold particles in nematocysts and spirocysts, using (i.e. in position 1), which is unique among these indole de- an immunogold detection method. Observing 5-HT in synap- rivatives and indeed natural products [26, 32]. The other tic vesicles (from immunogold labeling experiments) of sea identified IAA constituents are fairly evenly distributed anemones suggests the authors believe the implication of amongst the various species. biogenic amines and neuropeptides in these somewhat Most of the IAAs have been separated previously using analogous nervous systems [20]. classical extraction methods with various organic-based sol- Dergham and Anctil [19] stated that in sea pansy Renilla vent systems, such as 70-80% ethanol (aq) [30], 80-99% koellikeri 5-HT is released as a neurohormone acting as a acetone (aq) [26, 38], cold methanol [39] and chloroform neuromodulator of peristaltic behaviour. Their radiolabeling [40]. Recently water has also been used as a solvent of and autoradiography studies showed higher distribution of choice to extract the aqueous soluble components from Chi- [3H]-5-HT in the ectoderm and endoderm, while mesoglea nese Bufo bufo gargarizans cantor toad skins [41]. More showed lower density. Autoradiograms of myoepithelial recently the isolation and analysis of IAAs has been achieved cells did not show clear evidence of labeling. On the con- using more efficient purification techniques, including paper trary, dense aggregates of labelled-5-HT were observed chromatography [26, 31, 42], column chromatography [29], around small cells resembling the labeled mesogleal neurons. thin layer chromatography [26, 31, 43], followed by HPLC On the basis of their results the authors stated that in sea [44, 45], or Ultra Performance Liquid Chromatography pansy 5-HT receptors are able to mediate indirectly the (UPLC) coupled with mass spectrometry [34, 46-47], along modulatory activity of 5-HT on peristalsis, while non- with gas chromatography [2, 48]. serotonergic neurons innervate the myoepithelial cells [19]. Nearly all of the identified IAAs from animal sources In additional research work it was suggested that serotonin reported to date have been found from amphibian skins [25]. binding sites in Renilla koellikeri are simplified 5-HT recep- Among them toad skins constitute the major part. The iso- tors that can be seen as diversified multiple subtype- lated quantities of nine IAAs out of the fourteen identified to receptors in higher invertebrates and in vertebrates [21]. date in the various species of toad skins have to date been The main topics addressed in this review paper are the reported (see Table 2). The levels of the constituents in the occurrence and the structural presentation of IAAs found in skins of the various toad species is evidently variable. The the skin of various toad species. These chemicals as a class reasons for this variability may well in part be due to the are known to have hallucinogenic, central stimulant activity presence of various enzyme systems encountering the IAAs and likely potential as therapeutic agents in various CNS and thereby affecting their content in the toad skins [26]. disorders. The present review summarizes the distribution of Several of the known enzyme systems that have been found fourteen IAAs identified to date in toad skins world-wide in different species of toad skins are 5-hydroxytryptophan and compares these with similar compounds recognized in decarboxylase (capable of decarboxylating 5-hydroxytryptophan lower invertebrates, such as cnidarians. This review will we to 5-HT), 5-hydroxyindole-O-methyltransferase (involved in believe help to illuminate pathways and mechanisms that the biosynthesis of dehydrobufotenine), one or two sul- may potentially provide insight into potential therapeutic phoconjugases (responsible for linking sulphuric acid to the uses for both these organisms. phenolic hydroxy group) [23] and N-methyltransferase (capable of converting 5-HT to N-methyl-5-HT leading to bufotenine IAA OCCURRENCE IN TOAD SPECIES and bufotenidine) [3]. The amount of 5-HT reported as the To date fourteen IAAs have been identified in various common and major constituent in almost every species toad species (Table 1 and Figure 3). Several other “unknown ranged from 0 to 1600 µg/g of dry toad skin tissue. Surprisingly, compounds” identified non-specifically as IAAs have also some species such as Bufo boreas halophilus, Bufo punc- been reported to be present in Bufo marinus, Bufo granulo- tatus, Bufo spinulosus chilensis, Bufo spinulosus spinulosus, 4 Central Nervous System Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 Zulfiker et al.

Table 1. IAAs detected in the skin of various toad species.

Toad species Common name IAAs detected Family References

Bufo alvarius Colorado River Toad 5-HT, 5-Methoxytryptamine, N-Methyl-5-HT, N-Methyl- Bufonidae [3, 24-26] 5-methoxytryptamine, Bufotenine, O-Methylbufotenine, Dehydrobufotenine, Bufothionine, bufoviridine, O-Methylbufotenine 1-Sulfonic acid or O-Methylbufoviridine, Indole-3-acetic acid

Bufo americanus American Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [25, 27] Dehydrobufotenine, Bufothionine

Bufo arenarum Argentine Common Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [25, 27-30] Dehydrobufotenine, Bufothionine

Bufo bergei Striped Toad 5-HT Bufonidae [23, 25, 27]

Bufo bocourti Bocourt's Toad Bufoviridine, bufotenine-O-sulphate Bufonidae [31]

Bufo boreas boreas Boreal Toad, Western N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufothionine Bufonidae [25] Toad

Bufo boreas halophilus California Toad Bufotenine, Bufotenidine, Bufothionine Bufonidae [23]

Bufo bufo Common Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [23, 25, 27, Dehydrobufotenine, Bufoviridine, Bufothionine 32]

Bufo bufo spinosus The Spiny Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [32] Dehydrobufotenine, Bufoviridine

Bufo calamita Natterjack Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [3, 23, 25, Dehydrobufotenine, Bufoviridine, Bufotenine-O-sulphate 27,32]

Bufo canaliferus Dwarf Toad 5-HT, N-Methyl-5-HT, Bufotenidine, Bufothionine Bufonidae [25]

Bufo chilensis Concepcion Toad Bufotenine, Dehydrobufotenine, Bufothionine Bufonidae [28]

Bufo coccifer Southern Round-Gland 5-HT, Bufotenidine Bufonidae [23] Toad

Bufo cognatus Great Plains Toad 5-HT, N-Methyl-5-HT, Dehydrobufotenine Bufonidae [25]

Bufo crucifer Striped Toad 5-HT, Bufotenine, Dehydrobufotenine, Bufothionine Bufonidae [22, 28]

Bufo debilis American Green Toad, 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [3, 23, 25] Western Green Toad, and Dehydrobufotenine, Bufothionine, Bufoviridine Western Desert Toad

Bufo fernandezae Bella Vista Toad 5-HT, N-Methyl-5-HT, Bufotenine, Dehydrobufotenine, Bufonidae [23] Bufothionine

Bufo formosus Japanese Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [23, 25, 29] Dehydrobufotenine, Bufothionine

Bufo fowleri Fowler's Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [25, 27] Dehydrobufotenine, Bufothionine

Bufo funereus Angola Toad, Somber 5-HT Bufonidae [23, 25] Toad

Bufo gargarizans Chusan Island Toad, 5-HT, N-Methyl-5-HT, Bufotenidine Bufonidae [25, 27] Asiatic Toad

Bufo bufo gargarizans Chinese Giant Toad Bufotenine, Bufotenidine, Dehydrobufotenine, Bufonidae [33-34] cantor Bufothionine

Bufo granulosus Granulated Toad 5-HT, N-Methyl-5-HT, Bufotenine, Dehydrobufotenine, Bufonidae [22, 25] Bufothionine Indolealkylamines from Toad Vertebrates and Sea Invertebrates Central Nervous System Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 5

(Table 1) Contd….

Toad species Common name IAAs detected Family References

Bufo haematiticus Truando Toad 5-HT, Dehydrobufotenine Bufonidae [25]

Bufo hemiophyrs Canadian Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [23] Dehydrobufotenine

Bufo horribilis Marine Toad, Giant Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [25] Dehydrobufotenine, Bufothionine

Bufo ictericus Yellow Cururu toad 5-HT, N-Methyl-5-HT, Bufotenine, Dehydrobufotenine, Bufonidae [25] Bufothionine

Bufo kisoloensis Kisolo Toad, Montane 5-HT Bufonidae [23, 25, 27] Golden Toad

Bufo koynayensis Humbali Village Toad, Bufoviridine Bufonidae [3] Koyna Toad, Chrome-Yellow toad

Bufo luetkeni Yellow Toad 5-HT, N-Methyl-5-HT, Bufotenine Bufonidae [23]

Bufo major --- 5-HT, N-Methyl-5-HT, Bufotenine, Dehydrobufotenine, Bufonidae [23] Bufothionine

Bufo marinus Marine Toad, Giant Toad, 5-HT, N-Methyl-5-HT, Bufotenine, Dehydrobufotenine, Bufonidae [22, 25, 27, Cane Toad Bufothionine 35]

Bufo marinus poeppigi Gray Toad 5-HT, N-Methyl-5-HT, Dehydrobufotenine, Bufothionine Bufonidae [25]

Bufo marmoreus Marbled Toad 5-HT, Bufotenine, Bufoviridine, Bufotenine-O-sulphate Bufonidae [23, 31]

Bufo melanostictus Common Indian Toad 5-HT, Bufotenine, Bufotenidine, Dehydrobufotenine Bufonidae [23, 25]

Bufo mauretanicus Berber Toad, Pantherine 5-HT Bufonidae [23, 25, 27] Toad, Moroccan Toad

Bufo microscaphus Arizona Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine Bufonidae [23]

Bufo paracnemis Rococo Toad, Cururu 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [25, 27, 28] Toad Dehydrobufotenine

Bufo perplexus Confusing Toad 5-HT, Bufotenine, Bufoviridine, Bufotenine-O-sulphate Bufonidae [23, 31]

Bufo punctatus Red Spotted Toad Bufotenine, Dehydrobufotenine, Bufothionine Bufonidae [23, 25]

Bufo pygmaeus --- 5-HT, N-Methyl-5-HT, Bufotenine, Dehydrobufotenine, Bufonidae [23] Bufothionine

Bufo regularis Square-Marked Toad, 5-HT, 5-HT-O-sulphate Bufonidae [3, 23, 27] African Toad, Egyptian Toad, and Reuss's Toad

Bufo speciosus Texas Toad 5-HT, N-Methyl-5-HT, Dehydrobufotenine Bufonidae [25]

Bufo spinulosus chilensis Concepcion Toad Bufotenine-O-sulphate Bufonidae [31]

Bufo spinulosus limensis Peru Coast Toad Bufotenine, Bufotenidine, Dehydrobufotenine, Bufonidae [23] Bufothionine

Bufo spinulosus spinulosus Warty Toad 5-HT, Bufotenine, Bufotenidine, Dehydrobufotenine, Bufonidae [25, 28] Bufothionine

Bufo terrestris Southern Toad Bufotenidine Bufonidae [25]

Bufo trifolium The Peru Toad Bufotenine, Bufotenidine Bufonidae [23]

Bufo typhonius Common Forest Toad, 5-HT, N-Methyl-5-HT, Dehydrobufotenine, Bufothionine Bufonidae [25] Leaf Litter toad 6 Central Nervous System Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 Zulfiker et al.

(Table 1) Contd….

Toad species Common name IAAs detected Family References

Bufo valliceps Southern Gulf Coast Toad 5-HT, N-Methyl-5-HT Bufonidae [25]

Bufo variegatus Eden Harbour Toad Bufotenine, Bufotenidine Bufonidae [25]

Bufo viridis European Green Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [3. 23, 25, 27, Dehydrobufotenine, Bufoviridine 29, 32]

Bufo vulgaris formosus Common Toad 5-HT, N-Methyl-5-HT, Bufotenine, Bufotenidine, Bufonidae [36-37] Dehydrobufotenine, Bufothionine

Bufo woodhousii australis Southwestern Wood- 5-HT, N-Methyl-5-HT, Bufotenidine Bufonidae [23] house's Toad

Bufo woodhousei Rocky Mountain Toad 5-HT, N-methyl-5-HT, Bufotenidine, Dehydrobufotenine Bufonidae [23, 25] woodhousei

Bombina bombina European Fire-Bellied 5-HT Bombinatoridae [32] Toad

Bombinator pachypus Apennine Yellow-Bellied 5-HT Bombinatoridae [27] Toad

Bombinator igneus Firebelly Toad 5-HT Bombinatoridae [27]

Bombina variegata Yellow-Bellied Toad 5-HT Bombinatoridae [32] pachypus

Melanophryniscus Maldonado Redbelly 5-HT, Bufotenine, Bufotenidine Bufonidae [25] moreirae Toad

Scaphiopus hammondi Western Spadefoot Toad 5-HT, Bufotenidine Scaphiopodidae [25]

Scaphiopus couchi Couch's Spadefoot Toad 5-HT Scaphiopodidae [25]

Bufo trifolium and Bufo variegatus have not to date been to a number of mental disorders including schizophrenia found to contain 5-HT in their skins. The hallucinogenic [51], depression [52], anxiety [53], and obsessive- compound, O-methylbufotenine was reported to be present compulsive disorder [54]. These disorders have been associ- in a very high amount (15000-30000 µg/g of dry toad skin ated with either agonistic or antagonistic activation of tissue) in Bufo alvarius skin. The other hallucinogenic com- 5-HT1A and 5-HT2A receptor [55]. pound, bufotenine, was found in almost every species in Most of the IAAs acting on the CNS and demonstrating variable quantities (4-8600 µg/g), as was also the case for psychoactivity have alkylamine chains substituted at C3. bufotenidine (30-5200 µg/g), dehydrobufotenine (7-9000 These have free movement in contrast to substitutions on µg/g) and bufothionine (35-1680 µg/g) which were found to fused heterocyclic ring systems, such as in dehydro- be present in nearly all species. The amount of Bufoviridine bufotenine. IAAs such as bufotenine [56] and O- reported in two species namely Bufo calamita and Bufo vir- methylbufotenine [57] also have hallucinogenic activity. The idis was 450-520 and 480 µg/g, respectively. The sulfocon- extent of the hallucinogenicity of IAAs reportedly depends jugate Bufotenine-O-sulphate was found only in Bufo spinu- upon the number of single-bonded carbon atoms connecting losus chilensis at a moderately low level (270 µg/g). the amino nitrogen atom with the indole ring (ie, the distance All of the IAAs identified were 5-substituted derivatives. of the nitrogen from the indole ring). 5-Methoxytryptamine, Though there has been no extensive investigations reported identified in the skin of Bufo alvarius (Table 1), does not on the receptor selectivity of these compounds, it has been show hallucinogenic activity due to the absence of methyls observed that 5-substitution increases the activity of IAAs on the amino nitrogen [57] implying that methylation of the compared with unsubstituted derivatives (at the 5-position) alkyl amine is also required for hallucinogenic activity. Moreover, the psychotomimetic activity of IAAs is report- and that these showed equal potency at both the 5-HT1A and edly decreased when there is alkylation of the amino nitro- 5-HT2A receptors [7]. 5-HT is an important neurotransmitter that controls numerous physiological processes such as mi- gen with groups larger than a propyl group. However, higher togenesis, platelet aggregation, vascular, uterine and gastro- psychotomimetic activity is achieved in the case of dimethy- intestinal smooth muscle contraction [49-50]. It is also essen- lation rather than monomethylation, while the absence of tial in regulating various mental processes including emo- methyls on the amino of the side chain causes low potency. tion, cognition, perception and consciousness [50]. Abnor- The psychotomimetic activity of IAAs also varies when sub- malities in the serotonergic nervous system have been related stitution occurs at positions 4 or 5 with a hydroxyl or Indolealkylamines from Toad Vertebrates and Sea Invertebrates Central Nervous System Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 7

Table 2. Quantities of IAAs detected in various toad skins (µg/g of dry toad skin tissue) [23, 25].

N-Methyl- Bufo- Dehydro- Bufo- Bufotenine- O-Methyl- Toad species 5-HT Bufotenine Bufothionine 5-HT tenidine bufotenine viridine O-sulphate bufotenine

Bufo alvarius 4-6 30-40 250-1500 15000-30000

Bufo arenarum 40-250 40-200 800-2600 40-130 250-700 150-450

Bufo bergei 150

Bufo boreas boreas 24 520 31 7 68

Bufo boreas halophilus 275 50 275

Bufo bufo 0-13 10 169 54 98 320

Bufo calamita 6-8 13-15 105 35 450-520

Bufo canaliferus 3000 120 80 250

Bufo coccifer 120 10 1450

Bufo cognatus 600 20 91

Bufo debilis 24-30 50-75 15-64 1745 802

Bufo fernandezae 194 609 238 2199 1680

Bufo formosus 65 60 150 140 25 375

Bufo funereus 180

Bufo haematiticus 2100 9000

Bufo hemiophyrs 317 96 68 676 1993

Bufo ictericus 5-70 50 15 1800-4500 35-375

Bufo kisoloensis 600

Bufo luetkeni 405 20 42

Bufo major 250 130 200 550 980

Bufo marinus 30-140 40-130 4 2200-6000 30-375

Bufo marinus poeppigi 160 100-140 270-1100 210-240

Bufo marmoreus 150 770

Bufo mauretanicus 310

Bufo melanostictus 125 15 1250 8-9

Bufo microscaphus 600 160 60 5200

Bufo pygmaeus 52 99 92 2339 350

Bufo paracnemis 100 200 2500 250 1500 450

Bufo perplexus 140 1400

Bufo punctatus 10-20 543 465

Bufo regularis 540

Bufo speciosus 650 90 98

Bufo spinulosus 250 45 1000 1000 270 chilensis

Bufo spinulosus 111 350 463 720 limensis

Bufo spinulosus 230 30 180 940 spinulosus 8 Central Nervous System Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 Zulfiker et al.

(Table 2) Contd….

N-Methyl- Bufo- Dehydro- Bufo- Bufotenine- O-Methyl- Toad species 5-HT Bufotenine Bufothionine 5-HT tenidine bufotenine viridine O-sulphate bufotenine

Bufo terrestris 1600 68-80 20 1300 20-30

Bufo trifolium 550 100

Bufo typhonius 10 5-10 450 790

Bufo valliceps 1600

Bufo variegatus 8600 750

Bufo viridis 5-10 20-25 650 30 480

Bufo woodhousii 1081 154 1469 australis

Bufo woodhousei 500-800 40 950-1200 150-200 woodhousei

Melanophryniscus 25 22 3700 moreirae

COOH NHCH3 NH2 HO HO

N N N H H H Indole-3-acetic acid 5-HT N-Methyl-5-HT

NH2 NHCH3 N(CH3)2 HO H3CO H3CO

N N N H H H Bufotenine 5-Methoxytryptamine N-Methyl-5-methoxytryptamine

N(CH3)2 NH2 N(CH3)3 H3CO O3SO O

N N N H H H O-Methylbufotenine 5-HT-O-sulphate Bufotenidine

H3C H3C H C NH(CH3)2 H3C N 3 N O3SO HO O

N N H N HSO3 H Bufotenine-O-sulphate Bufoviridine Dehydrobufotenine

H3C H3C H C H3C N 3 N

O3SO H3CO

N N H HSO3 Bufothionine O-Methylbufoviridine

Fig. (3). 5-Substituted IAA derivatives identified to date in toad skins. Most have reported 5-HT2A receptor selectivity, although several also exhibit subclass selectivity to other 5-HT receptor subtypes. Indolealkylamines from Toad Vertebrates and Sea Invertebrates Central Nervous System Agents in Medicinal Chemistry, 2016, Vol. 16, No. 1 9 methoxy group. It has been found that the CNS effects are CONCLUSIONS AND FUTURE DIRECTIONS increased with methoxy group substitution, although hy- Toad skins representing a storehouse of IAAs possess doxytryptamine analogues in an ionized state can not pass certain features that make them good candidates for drug the blood brain barrier [57]. Some of the IAAs such as development. Some IAAs have shown great potential for use bufotenine [58], O-methylbufotenine [57] and indole-3- in psycho-pharmacotherapy, being utilised in the treatment acetic acid [35] have also been reported as recreational of depression, anxiety, schizophrenia, obsessive-compulsive drugs. disorders [1, 55] while others have been developed as anti- Some pharmacological activities of IAAs have been re- cancer [68, 73] and anti-migraine therapeutics [1]. Several ported, with most showing psychotomimetic and hallucino- have also indicated potential in the treatment of obesity as genic activity. 5-HT is reported to have an antagonistic activ- well as eating disorders [55]. Some have been reported as a ity at the peripheral serotonin receptors in animal experi- major class of abused hallucinogens and recreational drugs. ments [57]. Bufotenine has similar physiological and struc- Therefore, it is reasonable to think that what is known about tural properties to lysergic acid diethylamide (LSD) and acts IAAs in toads could provide the basis for gaining associated knowledge of such compounds present in ‘lower’ inverte- in a similar manner at the 5HT2 receptor [59, 60]. Bufotenine has also reportedly been used as snuff [61]. Bufotenidine has brates such as cnidarians, in order to allow their utilization reportedly exhibited hypertensive effects in the anaesthetized and the development of new drugs. Increased knowledge and dog with sodium pentobarbitone (30 mg/kg iv) [62] and also a better understanding regarding the availability and prospec- reportedly displayed acetylcholine-like activity (dose 20 tive activity of more IAAS in various CNS disorders is necessary. µg/mL) [63]. Dehydrobufotenine which has a tricyclic structure has been compared with the 1,3,4,5- CONFLICT OF INTEREST tetrahydropyrrolo[4,3,2-de]quinoline ring system [64] and showed potent in vitro cytotoxicity against human tumour The author(s) confirm that this article content has no con- cell lines [65, 66]. Dehydrobufotenine as a dry powder in- flict of interest. haler (DPI) has also been indicated in the treatment of lung tumours [67]. Bufothionine (isolated from toad skin) has ACKNOWLEDGEMENTS been presented as a cancer therapy [68], while as a powder The authors acknowledge GUIPRS and GUPRS scholar- for inhalation it is reported for use in the treatment of pul- ship for Abu Hasanat Md. Zulfiker. monary neoplasm [69]. 5-Methoxytryptamine has been reported to display antioxidant and radio-protective effects in REFERENCES various biological systems, but there exists no information on its psychoactivity [70]. [1] Yu, A.-M. 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