The Blue Jellyfish: A Promising

Autoimmune Stimulant
RIRDC Publication No. 09/035

RIRDC Innovation for rural Australia

 The Blue Jellyfish
a promising autoimmune stimulant

by B R Rich and P A Cheras

July 2009

RIRDC Publication No 09/035

RIRDC Project No PRJ-000835
 2009 Rural Industries Research and Development Corporation.
All rights reserved.

ISBN 1 74151 839 3

ISSN 1440-6845

The Blue Jellyfish a promising autoimmune stimulant

Publication No. 09/035
Project No. PRJ-000835

The information contained in this publication is intended for general use to assist public knowledge and discussion
and to help improve the development of sustainable regions. You must not rely on any information contained in
this publication without taking specialist advice relevant to your particular circumstances.

While reasonable care has been taken in preparing this publication to ensure that information is true and correct,
the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication.

The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the
authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability
to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or
omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the
part of the Commonwealth of Australia, RIRDC, the authors or contributors.

The Commonwealth of Australia does not necessarily endorse the views in this publication.

This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are
reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights
should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.

Researcher Contact Details

Bruce Rich Phillip Cheras
20 Tierney Terrace Russell Island 2 Falcon Avenue, Thornlands 4164
Queensland Australia Queensland Australia
Phone: 0417 700 146 Phone: 0412562250
Fax: Fax: 07 38210031
Email: brucerich@dodo.com.au Email: philcheras@yahoo.com.au

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

RIRDC Contact Details

Rural Industries Research and Development Corporation
Level 2, 15 National Circuit
BARTON ACT 2600
PO Box 4776
KINGSTON ACT 2604

Phone: 02 6271 4100

Fax: 02 6271 4199
Email: rirdc@rirdc.gov.au.
Web: http://www.rirdc.gov.au

Electronically published by RIRDC in July 2009

Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au
or phone 1300 634 313

ii
Foreword
RIRDC conducts feasibility studies of prospective animal industries to stimulate consideration of
potential commercialisation. Products derived from natural sources for both cosmetic and therapeutic
uses have experienced extraordinary popularity and rapid growth in global sales over the past several
decades. It has become clear that there are of untapped resources in our native flora and fauna, both
terrestrial and marine. The common blue jellyfish Catostylus mosaicus has been largely overlooked in
terms of scientific investigation, largely because it poses no lethal threat to people. However, limited
previous studies have indicated that the venom does have potential activity in altering haemostasis and
a number of other physiological functions. Modulators of immune function have also gained increased
importance in the fight against disease as almost all disease states manifest either inappropriately
raised or lowered immune function. Given these findings, together with the abundance and ease of
collection of Catostylus mosaicus the current studies were performed in order to identify potential
biological activities, and specifically immune function modulatory activity.

The findings of this preliminary research indicate that extracts of this jellyfish do appear to show
potential immune function modulatory activity. The next logical step would be to confirm and expand
upon this encouraging preliminary data. The result could be the commercialisation of this inexpensive
and abundant natural resource with the development of a suite of commercially valuable products.

The project was funded by RIRDC Core funds and the report is an addition to RIRDCs diverse range
of over 1800 research publications, forms part of our New Animal Products R&D program, which
aims to accelerate the development of viable new animal industries.

Most of RIRDCs publications are available for viewing, downloading or purchasing online at
www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.

Peter OBrien
Managing Director
Rural Industries Research and Development Corporation

iii
Acknowledgments
This project could not have been conducted without the assistance, cooperation and support of a
number of organisations and individuals.

In this regard acknowledgement is gratefully extended to the following:

Professor Paul Alewood - Chair of Chemical and Structural Biology, Institute for Molecular Bioscience, The
University of Queensland.

Professor Alewood oversaw the extraction and concentration of the jellyfish extract used in this project. He
was also responsible for all the testing reported here under extraction and concentration and he authored
the commentary to that section.

Associate Professor David Leach, Dr. Carrol Morris and Dr. Paul Connellan Southern Cross University,
conducted the bioassays included in this report and authored the commentary associated with the bioassay
component.

Professor Deon Venter Co-Director Pathology, and Gareth Price - Mater Health Services, were responsible
for the genomic study into the stimulatory and inhibitory effects of the extract on the human genome
utilizing peripheral blood mononucleocytes (PBMC). They also authored the commentary associated with
this component of the study.

Mr. Jim McLeod Sandgate Fishermans Co-Op Society Ltd, for facilitating collection of jellyfish
specimens.

Dr. Peter McInnes Rural Industries Research and Development Corporation for his support and input
into the project.

Abbreviations
ACN acetonitrile solution
CTE crude tentacle extract
FCS fetal calf serum
MALDI-TOF MS Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass
Spectrometry
PBMC peripheral blood mononuclear cell
PBS phosphate buffered saline
PCTE purified crude tentacle extract
TFA trifluoroacetic acid

iv
Contents
Foreword.......................................................................................................................................................................ii

Abbreviations .............................................................................................................................................................. iv

List of Figures.............................................................................................................................................................. vi

List of Tables ..............................................................................................................................................................vii

Executive Summary ..................................................................................................................................................viii

1. Introduction ......................................................................................................................................................... 1

2. Objectives ............................................................................................................................................................. 3

3. Methodology......................................................................................................................................................... 4
3.1. Specimen Collection and Handling................................................................................................. 4
3.2. Extraction, Purification and Characterisation Analysis .................................................................. 4
3.3 Bioassays.......................................................................................................................................... 6
3.4. In vitro effects of extract on genes in cells of the circulating immune system ............................... 7

4. Results................................................................................................................................................................. 10
4.1. Specimen collection ...................................................................................................................... 10
4.2. Extraction and Purification ........................................................................................................... 10
4.3. Bioassays....................................................................................................................................... 30
4.4. In Vitro Effects of Extract On Cells Of The Circulating Immune System ................................... 33

5. Discussion ........................................................................................................................................................... 34

6. Implications........................................................................................................................................................ 35

7. Recommendations.............................................................................................................................................. 36

8. References .......................................................................................................................................................... 37

v
List of Figures
Figure 1: Picture of Catostylus mosaicus (taken from reference 3) .................................................................... 1
Figure 2: Schematic showing preparation of tentacle extract for genomic analyses ............................................ 8
Figure 3: RP-HPLC trace of 50uL of 1mg/mL C. mosaicus tentacle extract
run to a gradient of 40% B at 1%/min. ............................................................................................... 11
Figure 4: RP-HPLC trace of 50uL of 10mg/mL C. mosaicus tentacle extract run to a
gradient of 40% B at 1%/min.. ........................................................................................................... 11
Figure 5: RP-HPLC trace of 50uL of 10mg/mL C. mosaicus tentacle extract run to a
gradient of 70% B at 1%/min. ............................................................................................................ 12
Figure 6: RP-HPLC trace of C. mosaicus tentacle extract run at 0-40%B at 1%
ACN/min on the Promix MP column at a flow rate of 0.25mL/minute.............................................. 13
Figure 7: HPLC trace of fraction 6 (refer to figure 6) from the crude C. mosaicus
tentacle extract run at a gradient of 0-40%B at 1% ACN/min on the
Vydac C18 column at a flow rate of 0.25mL/min .............................................................................. 14
Figure 8: Biorad 4-20% Tris/HCl precast gel with the lane number marked above........................................... 16
Figure 9: Results of the RP-HPLC analysis of the CTE lyophilised in 30% ACN,
0.1% TFA, run over a gradient of 0-40% B over 36 minutes. ............................................................ 17
Figure 10: Results of the RP-HPLC analysis (including pre- and post-run blanks) of the
CTE lyophilised in 30% ACN, 0.1% TFA, run over a gradient of 0-40% B over 36 minutes. .......... 17
Figure 11: Results of the RP-HPLC analysis of the CTE lyophilised in 30% ACN, 0.1% TFA,
run over a gradient of 0-40% B over 72 minutes. ............................................................................... 19
Figure 12: Results of the RP-HPLC analysis (including pre- and post-run blanks) of the
CTE lyophilised in 30% ACN, 0.1% TFA, run over a gradient of 0-80% B over 72 minutes. .......... 19
Figure 13: RP-HPLC trace of 7.6mg/mL CTE lyophilized in 0.1% TFA, 30% CN, run to a gradient
of 0-40% B over 72 minutes (0.5% CAN/minute) with 10 minutes at 100% ................................. 21
Figure 14: RP-HPLC trace showing the peaks corresponding to fractions 14-17................................................ 22
Figure 15 : MALDI-TOF results for fractions 14 to 17 using DHB as the matrix in reflector mode.................... 23
Figure 16 : MALDI-TOF results for fractions 14 to 17 using SA as the matrix in reflector mode. ...................... 24
Figure 17 : RP-HPLC trace of PCTE using a Shimadzu SPD-M10A VP detector with
a diode array detector over a gradient of 0-40% B in 36 minutes, with
10 minutes of 100% A initially........................................................................................................... 26
Figure 18 : Absorbance of the component that eluted at 11.84 minutes in figure 17............................................ 27
Figure 19 : Absorbance of the component that eluted at 19.42 minutes in figure 17............................................ 27
Figure 20 : Spontaneous contraction of the proximal guinea pig colon in response to the
addition of 10ug/mL and 20ug/mL of PCTE. ..................................................................................... 29
Figure 21 : Cytotoxicity of Jellyfish extract against P388 cells (Mean SE)....................................................... 30
Figure 22: Cytotoxicity of Jellyfish extract against MCF7 cells (Mean SE) .................................................... 30
Figure 23: Effect of Jellyfish Extract on NK Cell Cytotoxic Activity (Mean SE)............................................ 31
Figure 24: Effect of Jellyfish Extract on Phagocytic Activity of Granulocytes (Mean SE).............................. 32
Figure 25: Effect of Jellyfish Extract on Phagocytic Activity of Monocytes (Mean SE) ................................. 32

vi
List of Tables
Table 1: Concentration and conditions of the samples loaded onto the 1D-gel. ................................................. 5
Table 2: Mass of crude tentacle extract (CTE) remaining after lyophilisation under different conditions. ...... 10
Table 3: Fractions collected from Figure 6. ...................................................................................................... 13
Table 4: Fractions collected from Figure 7. ...................................................................................................... 14
Table 5: Major peaks observed in the fractions analysed by MALDI-TOF MS............................................... 15
Table 6: Fractions collected with corresponding times from figure 9............................................................... 18
Table 7: Fractions collected with corresponding times from figure 11............................................................. 20
Table 8: Major peaks in fractions analysed by MALDI-TOF MS from figure 11............................................ 25
Table 9: Description of absorbance of components of PCTE obtained with a diode array detector................. 28

vii
Executive Summary
What the report is about

This report presents the results of novel investigations into the properties of the venom from the
jellyfish Catostylus mosaicus.

The study has involved three distinct phases:

Extraction and concentration
Bioassays of the extract
Studies of the in vitro effects of the extract on genes within cells of the circulating immune
system.

Who is the report targeted at?

This report is targeted at investors or people interested in developing therapeutic products from natural
resources. The report provides preliminary findings of an investigation to establish autoimmune
properties of the venom under study and provide a foundation for subsequent investigations.

Background

The establishment of a Queensland developmental jellyfish fishery meant for the first time that
Catostylus mosaicus (commonly known as the blue jellyfish) could be harvested commercially. This
project forms part of the establishment of a new commercial sector targeting dried food products and
value added products, such as therapeutic products.

This toxin of C.mosaicus exhibits physiological activity such as hemolysis and autoimmune
stimulation by elevation of IgM and IgG serum levels in humans.

For the commercial sector to enhance commercial viability and realise the true potential of the
resource, the value added products are viewed as a vital component.

Aims/Objectives

This study set out to determine the handling requirements of C.mosaicus to preserve venom activity
and to demonstrate that physiological activity, in particular autoimmune stimulation, was present.

It sought to establish a foundation base for further study.

viii
Methods used

The study was co-ordinated by the authors and involved collaboration between researchers from the
following tertiary institutions:

Chemical and Structural Biology, Institute for Molecular Bioscience. The University of
Queensland
Southern Cross University and
Mater Health Services.

Results/key findings

The results have shown that:

Biological activity was demonstrated by the jellyfish extract in the form of spontaneous
contraction of guinea pig colon by 10g/mL of the extract.
The extract is not directly cytotoxic. This was demonstrated by lack of observed toxicity due
to the extract on P388 and MCF7 cell lines.
The extract showed innate immune function stimulation, demonstrated by apparent stimulation
of phagocytosis of granulocytes by the extract in bioassay studies. In addition, a range of gene
activities associated with immune function were observed in studies of peripheral blood
mononuclear cells. Functions of these genes include phagocytosis, antigen binding, cell
adhesion and the cellular defense response. Other genes stimulated by the extract reflect the
processes associated with cell proliferation.

Implications for relevant stakeholders

This study has provided a comprehensive overview of the characteristics of the venom of. C. mosaicus
It has for the first time, provided solid data on the nature of the venom attributes. The investigation
has shown that activity relating to the immune system exists and the results warrant further
investigation. It has provided a sound platform for further investigations as a prelude to investment for
commercial development and a way forward for the utilisation of this under utilised natural resource.

Recommendations

We recommend that a second study be undertaken to further evaluate the effects of the tentacle extract
on peripheral blood mononuclear cell derived from multiple individuals, using multiple technical and
biological replicates, in order that the molecular changes identified can be confirmed and pursued as
potential commercial therapeutic targets.

ix
1. Introduction
The Queensland Government for the first time issued collection permits for Catostylus mosaicus
(commonly known as the blue jellyfish) in April 2005. This allowed the establishment of a new
commercial sector targeting dried food products and value added products, such as therapeutic agents.

Catostylus mosaicus belongs to the phylum Cnidaria which consists of a group of animals that exhibit
common characteristics (radially symmetrical, body multicellular few tissues, some organelles,
contains internal cavity and mouth, two different forms exist medusa and polyp), One of the most
important distinguishing characteristics is the presence of nematocysts, from which coiled thin threads
can be triggered to penetrate the skin of its prey or predator and inject a toxin. This toxin exhibits
physiological activity such as hemolysis and autoimmune stimulation by elevation of IgM and IgG
serum levels in humans.

For the sector to enhance commercial viability and realise the true potential of the resource, the value
added products are viewed as a vital component.

To date there has been much research into the venoms of those jellyfish that attract significant medical
attention, such as the box jellyfish (Chironex fleckeri) and the irukandji (Carukia barnesi), in the hope
of finding antivenom, or a molecule or peptide of medical importance. As a consequence, components
of their venoms have been isolated, and their actions extensively studied. Catostylus mosaicus is a
jellyfish that has attracted much less medical attention due to the fact that it causes a less severe
reaction upon contact [1]. Because of this, C. mosaicus and its venom components have only been
sparsely studied.

C. mosaicus is a scyphozoan, edible jellyfish that is mainly found along the eastern coast of Australia,
with some sightings along the northern Australian coast and around the Malaysian peninsula [1,2].
They are commonly blue in colour in Queensland waters (refer to figure 1) but are coloured brown
further south due to the presence of symbiotic zooxanthellae within their bodies [1]. The bell of the
jellyfish ranges in size from 25 to 35cm in diameter [1]. The nematocysts of C. mosaicus are located
on eight oral arms, which also are used for the ingestion of food [1].

Figure 1: Picture of Catostylus mosaicus (taken from reference 3)

Human contact with this form of jellyfish has resulted in mixed responses, with the most common
being a mild, non-irritating, skin reaction. Some patients have also reported acute pain associated with
a skin irritation, with the pain taking hours to subside. It has been suggested that the increasing
severity of the sting is associated with different times of the year, with it being most severe during the
breeding season. During this season reports of cyanosis, sharp pain and breathing difficulties are
common [1].

1
Studies into the effects of C. mosaicus venom in mice have shown that it possesses haemolytic,
oedema and haemorrhage inducing activities, with some of these effects possibly due to the presence
of phospholipase A [2].

As only very limited research has been performed on C. mosaicus, it is possible that this species of
jellyfish could possess novel proteins or peptides that could be used for products of medical
significance. One aim of this research was to identify the protein composition of extract from the
Catostylus mosaicus oral arms to act as a basis for future investigation.

This report also describes bioassays performed to determine potential cytotoxicity of the extracts and
also potential immune function stimulation.

Effects of the extract on the human genome were also assessed in a preliminary manner for the first
time. The tentacle extract was used to stimulate PBMCs at different time points and these were
analysed on a GeneChip genomics platform, representing probes to essentially all genes in the human
genome.

2
2. Objectives
This was an ambitious study that was undertaken to meet the following objectives:
Establish and refine handling and processing procedures for Catostylus mosaicus.
Produce a crude extract from the oral tentacles and perform compositional characterization
studies.
Determine the degree of biological activity of the extract across a range of parameters
including immune function stimulation through the use of bioassays.
Determine the potential actions of the extract on genes derived from human peripheral blood
mononuclear cells particularly in relation to immune function, thereby providing information
on the effects of the extract on the complete human genome.
Gather sufficient data to make Go/No Go decisions regarding additional studies prior to
commercial development.

3
3. Methodology
Research strategies were developed in order to meet the previously stated objectives.

3.1. Specimen Collection and Handling

Specimens were collected using a scoop net. The jellyfish was held by the bell, the tentacles where cut
free and collected in a plastic specimen bag. Bags were placed on metal prawn trays and snap frozen.

Specimens were stored at -80 C until required for use.

3.2. Extraction, Purification and Characterisation Analysis

3.2.1 Preparation of crude C. mosaicus tentacle extract (CTE)

Five kilograms (5 kg) of the frozen jellyfish was fully thawed overnight. The oral arms were removed
from the exumbrella region before approximately 600 grams of the removed oral arms were
homogenised with 300mL of cold milliQ water using an Ultra Turrax homogeniser. The homogenate
was centrifuged for 15 minutes at 5000 rpm and 4C. The supernatant (500mL) was lyophilised
overnight. The dried crude tentacle extract (CTE) was stored at -18C until use.

3.2.2 Purification of Crude C. mosaicus tentacle extract (PCTE)

0.5mg of the crude tentacle extract was mixed with a 0.1% solution of trifluoroacetic acid (TFA), and
with a 0.1% trifluoroacetic acid and 30% acetonitrile (ACN) solution, before being centrifuged with
the supernatant removed from both tubes and freezedried overnight. The resulting dried extract will be
referred to as the purified crude tentacle extract (PCTE).

3.2.3 RP-HPLC analysis of the CTE

The crude tentacle extract was mixed with buffer A (0.05% TFA) in a 1mg:1mL ratio before a RP-
HPLC was run on this sample (50uL injection) at a gradient of 0-40% buffer B (90% ACN, 0.043%
TFA) at 1%/min on a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A). This was performed
on a Shimadzu HPLC with a Shimadzu SPD-M10A VP detector, detecting at 214 and 280nm.

Based on the results obtained, a second RP-HPLC was run on the crude tentacle extract under the same
conditions as the previous run, however the concentration was increased to 10mg/mL to increase peak
intensity. A run was also performed at 0-70% B at 1%/min using the conditions previously mentioned.

The extract was then analysed using a Promix MP 5uM column, with a flow rate of 0.25mL/min, at a
gradient of 0-40% B over 36 minutes (1% ACN/min), and an injection volume of 10uL. This was
performed on a Shimadzu HPLC system (Shimadzu SPD-10A VP detector) with the UV set to detect
at 214 and 280nm. Fractions that required further separation were separated with a Vydac C18 column
(5uM pore size, 2.1 x 250mm, 300A) under the same conditions as above. The fractions were dried,
mixed with 50% ACN in a 1:1 ratio and subjected to MALDI-TOF mass spectrometry.

3.2.4 MALDI-TOF mass spectrometry analysis of the extract CTE

The mass of the fractions collected in the RP-HPLC traces (refer to section 2.3) were then determined
using MALDI-TOF MS on a Voyager-DE STR workstation in reflector mode with DHB (gentisic
acid) as the matrix solution. 0.5uL of each fraction was analysed with 0.5uL of the matrix.
3.2.5 1D-gel electrophoresis of CTE
1D-gel electrophoresis was performed in order to estimate the mass of the tentacle extract components.
This was done using a 4 to 20% pre-cast Biorad Tris/HCl gel. 20uL of the samples were added, with
the conditions and concentrations of the samples being varied. Table 1 outlines the concentrations and
conditions of the samples that were loaded. The gel was run for 10 minutes at 15mA, and 50 minutes
at 20mA. It was stained in coomassie blue, before being destained in acetic acid.

Table 1: Concentration and conditions of the samples loaded onto the 1D-gel.
Well Number Sample Identity Amount of Reduced/ Amount
sample (ug) non-reduced loaded (uL)
1 - - - -
2 PCTE 10 Non-reduced 20
3 Marker - - 5
4 MilliQ water - Reduced 20
(negative control)
5 MilliQ water - Non-reduced 20
(negative control)
6 CTE 10 Reduced 20
7 CTE 20 Reduced 20
8 CTE 30 Reduced 20
9 CTE 10 Non-reduced 20
10 CTE 20 Non-reduced 20
11 CTE 30 Non-reduced 20
12 PCTE Reduced 20
13 PCTE Reduced 20
14 PCTE Non-reduced 20
15 Another persons - - -
sample

3.2.6 RP-HPLC analysis of the PCTE

RP-HPLC analysis was performed on the CTE that was lyophilised in a 30% ACN, 0.1% TFA solution
(refer to section 1.2). The sample was prepared through the procedure mentioned above, with the
lyophilised extract being mixed with 10% ACN, in a concentration of 7.6mg/mL. Spectra were
collected at 214 and 280nm on a Shimadzu HPLC system (SPD-10A VP detector), with the analyses
performed using a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A). Initially a gradient of 0-
40% buffer B was run over 36 minutes, with a 10-minute period of 100% buffer A at the start of the
run. 50uL of the sample was injected and a flow rate of 0.25mL/minute was used. Fractions were
collected that corresponded to the peaks.

After this trace was collected, a gradient of 0-80% B was run over 72 minutes, with a 10-minute period
of 100% A prior to the start of the gradient. The same conditions as the previous run were used.
Fractions were again collected for further spectral analysis.

In order to obtain better peak separation, the sample was subjected to another RPHPLC run. This was
run at a gradient of 0.5% ACN/minute (0-40% B over 72 minutes). The flow rate and other conditions
were kept consistent with the above runs.

3.2.7 Mass spectrometry of the collected fractions from RP-HPLC analysis of the PCTE
The fractions that were collected from the trace that was run over a gradient to 80% B (refer to section
2.6) were prepared for spectral analysis by drying using a speedyvac concentrator. 10uL of 50% ACN

5
was added to each fraction. The fractions were analysed using MALDI-TOF MS (Voyager-DE STR
workstation) with a matrix of DHB (gentisic acid) and SA (sinapic acid). 0.5uL of the fraction and the
matrix were used. The samples were analysed in reflector mode in the mass range of 500 to 8000Da
and 19000 to 23000Da.

The fractions that corresponded to the peaks of greater intensity were also analysed using electrospray
MS (API 2000 LC/MS/MS system). 10uL of fractions 1, 2, 3, 20, 21, and 24 that were collected in the
0-40% B trace (refer to section 2.6) were analysed, detecting in the range of 400 to 1800Da.

3.2.8 RP-HPLC analysis of the PCTE using a diode array detector

The PCTE sample was subjected to further RP-HPLC analysis, using a diode array detector (Shimadzu
SPD-10A VP detector). 50uL of the sample was injected for a 0-40% B gradient run (1%
ACN/minute). Initially there was a period of 100% A for 10 minutes before the gradient began. This
was performed on a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A), at a flow rate of
0.25mL/minute. The sample was in a concentration of 7.6mg/mL in 10% ACN. The spectra were
collected in a range from 190-350nm.

3.2.9 Spontaneous contraction assay of PCTE

The PCTE was tested for activity upon the spontaneous contraction of the proximal colon (isolated
from a guinea pig). Dunkin Hartley male guinea pigs were sacrificed by carbon dioxide followed by
cervical dislocation. The proximal colon was dissected 5-10cm caudal to the colon-caecal junction
before being placed in ice-cold Krebs-Hensleit buffer. 2cm segments were set up longitudinally with
1g resting tension in a 5mL organ bath containing Krebs-Hensleit buffer. The bathing medium was
constantly perfused with carbogen and maintained at 37C. The tissue was equilibrated for
approximately 1 hour before testing and the buffer was replaced every 15 minutes.

3.3 Bioassays
A lyophilised crude peptide extract (CTE) from the jellyfish Catostylus mosaicus was supplied by the
Institute for Molecular Bioscience at the University of Queensland.

Two 600mg vials of the extract were submitted to determine any immunomodulatory activity in vitro.
Initially, the extract was screened for cytotoxicity to determine appropriate concentration levels for
immune testing. Then, the extract was tested using the Phagocytosis and Natural Killer (NK) cell
activity assays. Both these assays represent important components of the innate immune system.

The jellyfish samples were stored at -20C prior to testing and identified as CPR070129.

3.3.1 Cytotoxicity Assay

Cytotoxicity was measured against two cell lines. P388D1 cells (mouse lymphoblast) are routinely
used for cytotoxicity screening as they have a reputation for a low incidence of false negatives. MCF7
cells (Human mammary gland adenocarcinoma) were assayed for additional validation of results.
Jellyfish extract solutions in phosphate buffered saline (16.3mg/mL) and DMSO (28.9mg/mL) were
prepared. A series of dilutions of chlorambucil and curcumin were included as positive controls. Serial
dilutions of each of the samples and standards were prepared and added to the cells such that DMSO
never exceeded 1% final concentration. All samples were assayed in triplicate. Following the addition
of samples, the cells were incubated for 24 hours (37 C, 5% CO2). After this time, cytotoxicity was
determined using the ATPliteTM assay, which measures the amount of ATP present in wells,
indicative of the number of live cells. The luminescence produced, proportional to the number of
viable cells, was read on a Victor2 multilabel plate reader (PerkinElmer).

6
3.3.2 Natural Killer (NK) Cell Activity
NK cell cytotoxic activity on K562 target cells was determined using ficoll prepared suspensions of
peripheral blood mononuclear cells (PBMCs) from fresh lithium heparinised blood collected from a
healthy male volunteer.

The jellyfish extract was carefully reconstituted in phosphate buffered saline (PBS) to give final
concentrations of 0.1, 1.0 & 10.0 g/ml. The sample/PBMC mixtures were preincubated for two hours
at 37C (5% CO2).

Aliquots of each mixture were then incubated with K562 target cells at a PBMC:K562 ratio of 25:1 for
two hours at 37C (5% CO2). Target cell controls were also run to monitor spontaneous K562 cell
death. The cell suspensions were then stained with a fluorescent viability dye. The NK cell cytotoxic
activity for each mixture was obtained by determining the percentage of dead target cells minus the
target control value. Testing was performed in triplicate. Sample effects were determined with respect
to PBS controls.

The samples were analysed by flow cytometry using a BD FACSCalibur instrument.

3.3.3 Phagocytosis Assay

Phagocytosis of E.coli in human peripheral blood was measured using the Phagotest Kit (Orpegen
Pharma) according to the manufacturers protocol. Fresh lithium heparinised blood was collected from
the same healthy male volunteer.

The jellyfish extract was carefully reconstituted in PBS to give final concentrations of 0.1, 1.0 & 10.0
g/ml.

The sample/blood mixtures were preincubated for two hours at 37C (5%
CO2). E.coli (FITC labelled) was added to an aliquot of each mixture and then incubated for ten
minutes at 37C (5% CO2). The percentage of phagocytes (granulocytes & monocytes) which had
ingested the fluorochrome labelled bacteria (E.coli) was then determined for each mixture by flow
cytometry using a BD FACSCalibur instrument. Testing was performed in triplicate and 0C controls
(no phagocytic activity) were run to determine any background fluorescence. Test results were
corrected for any background fluorescence levels. PBS controls were also run and were the reference
point for calculation of the sample effects.

3.4. In vitro effects of extract on genes in cells of the circulating immune

system

3.4.1 Preparation of tentacle extract

Tentacle extracts for genomic analyses were prepared as shown in Figure 2.

7
 Jellyfish
C.Mosaicus
Crude Tentacle extract, 0.61g + 0.54g

PBMCs
Time Points:

Grind up 0.5g of tissue (dry ice or liq N2) 0hr

Resuspend in PBS 4hr
Spin at 4oC for 15min, max g 24hr
0.22uM filter 48hr
Use at 2 concentrations, relative to TC plate or flask used

Figure 2: Schematic showing preparation of tentacle extract for genomic analyses

8
3.4.2 Genomics Analysis

Total RNA extraction

PBMCs were pelleted at 500g for 5min and resuspended in 1mL cold Trizol. Trizol samples were
stored over night at 4C for total RNA isolation within 24hrs,as per manufacturer directions. RNA was
resuspended in RNase free water and stored at -20oC.

Affymetrix Gene Chip Array

RNA was reverse transcribed using the One Cycle Labeling Kit from Affymetrix after pre-
amplification using an Ambion RNA amplification kit. In brief, 500ng of RNA was reverse transcribed
using a T7-oligodT primer I the presence of controls to track the reverse transcription and labeling
protocol. An in vitro transcription reaction is then performed to produce a pool of biotin-dUTP labeled
cRNA. The cRNA was fragmented by metal-induced hydrolysis for 35 minutes and stored at -20C
until hybridization.

Gene Chip Hybridization and Scanning

Each sample was hybridized to a U133 Plus 2.0 Array Human Genome Gene Chip at 45C, 60rpm for
16 hours. Eukaryotic hybridization controls (bio B, bio C, Bio D and cre) were included in this step.
The GeneChip was washed and stained on a FS450 fluidics station prior to image acquisition on an
Affymetrix G7 GeneChip scanner.

Gene Chip Analysis and Interpretation:

GCOS 1.4 software was utilized for image acquisition, data analysis, extraction and normalization.

9
4. Results
4.1. Specimen collection

Ten kilograms (10 kg) of tentacles were collected in open water east of Moreton Bay. The tentacles
were immediately frozen and stored at 20 C, prior to transfer to 80 C storage. Collection
procedures for future studies would ideally also include electrostimulation to induce firing of
nematocysts into collecting fluid in order to increase the concentration and purity of venom in the
resultant extracts.

4.2. Extraction and Purification

4.2.1 Preparation of crude C. mosaicus tentacle extract (CTE)

This procedure yielded 13.439 grams of lyophilised C. mosaicus tentacle, which was grey/blue in
colour and fluffy in texture.

4.2.2 Purification of crude C. mosaicus tentacle extract (PCTE)

The extract was soluble in the ACN and TFA solutions, with it forming a white, sticky solid after
lyophilisation. 73% and 76% of the tentacle extract remained after being mixed with 0.1% TFA and
0.1% TFA, 30% ACN respectively (refer to table 2).

Table 2: Mass of crude tentacle extract (CTE) remaining after lyophilisation under different
conditions.
Solvents used Mass of CTE Mass of CTE Percentage of original Observations
used (mg) obtained (mg) mass remaining
0.1% TFA 0.6 0.44 73.33% White
Sticky
Fluffy
0.1% 0.55 0.42 76.36% As above
TFA, 30%
ACN

There was no crude tentacle extract visible after mixture with chloroform.

4.2.3 RP-HPLC analysis of the CTE

The initial RP-HPLC trace with the tentacle extract in a 1mg/mL concentration showed peaks of very
small intensity (refer to figure 3). Due to the small intensity of the peaks, the concentration of the
extract was increased to 10mg/mL before being run through the HPLC. This run produced peaks of a
greater intensity, however most of the extract was eluted at the end when the gradient was rapidly
increased to 90% (refer to figure 4). This suggests that the gradient would need to be increased to 70%
of buffer B in order to determine if any peptides exist in the extract that are eluted after 40% of buffer
B.

10
Figure 3: RP-HPLC trace of 50uL of 1mg/mL C. mosaicus tentacle extract run to a gradient of
40% B at 1%/min. This was run on a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A).

Figure 4: RP-HPLC trace of 50uL of 10mg/mL C. mosaicus tentacle extract run to a gradient of
40% B at 1%/min. This was run on a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A) using a
Shimadzu HPLC with a SPD-M10A VP detector.

11
When the gradient was increased to 70% B, no additional peaks that eluted after 40% B were observed
in the trace (refer to figure 5).

Figure 5: RP-HPLC trace of 50uL of 10mg/mL C. mosaicus tentacle extract run to a gradient of
70% B at 1%/min. This was run on a vydac C18 column (5uM pore size, 2.1 x 250mm, 300A) using a
Shimadzu SPD-M10A VP detector.

The following trace run on the Promix MP column was run at a slower flow rate in the hope of
obtaining better peak resolution and to aid in fraction collection (refer to figure 6). The peaks were
more separated than the higher flow rate, however they appeared to be of a lower intensity. This could
be due to the difference in the column used or the flow rate. Select fractions corresponding to peaks in
the trace were collected for further analysis (refer to table 3). A large, broad peak corresponding to
fraction 6 was collected, dried and subjected to another HPLC run on the Vydac C18 column at a flow
rate of 0.25mL/min, as this column appeared to give better separation of the peaks of this extract.
Slightly improved separation was obtained, with two fractions being collected (refer to figure 7 and
table 4).

12
Figure 6: RP-HPLC trace of C. mosaicus tentacle extract run at 0-40%B at 1% ACN/min on the
Promix MP column at a flow rate of 0.25mL/minute. The numbered peaks were analysed using
MALDI-TOF MS. The sample was run using a Shimadzu HPLC system (SPD-10A VP detector).

Table 3: Fractions collected from Figure 6.

Fraction Time started fraction
Number collection (minutes)
1 3.91
2 4.22
3 10.21
4* 10.70
5* 11.30
6* 12.48
7* 14.35
8 15.90
9* 17.22
10* 19.58
11* 20.60
12* 21.70
13* 21.94
14* 22.57
15* 25.11
16* 30.30
17 35.01

*= Fractions were subjected to MALDI-TOF analysis

13
Figure 7: HPLC trace of fraction 6 (refer to figure 6) from the crude C. mosaicus tentacle extract
run at a gradient of 0-40%B at 1% ACN/min on the Vydac C18 column at a flow rate of
0.25mL/min. The peaks were analysed using MALDI-TOF MS. The trace was obtained using a
Shimadzu SPD-10A VP detector.

Table 4: Fractions collected from Figure 7.

Fraction Number Time started fraction
collection (minutes)
1a* 2.99
2a* 3.27

*= Fractions were subjected to MALDI-TOF analysis

4.2.4 MALDI-TOF mass spectrometry analysis of the CTE

The fractions that were subjected to MALDI-TOF MS analysis showed mixed results (all masses
reported are of the molecular ion [M+H]+). All of the masses observed were low, ranging from 500-
2000Da. There was no clear mass corresponding to the peaks, with several recurring masses such as
506.9Da and 1405.1Da (refer to table 5).

14
Table 5: Major peaks observed in the fractions analysed by MALDI-TOF MS.

Fraction Number Major peaks observed (Da)

4 581.8, 603.8, 789.6, 955.6, 1162.5,
1405.1
5 506.8, 573.9, 642.8, 780.8, 917.0,
1082.8, 1245.3, 1405.0, 1664.0
6 506.9, 628.9, 637.2, 795.0, 1029.0,
1234.3, 1405.1, 1589.0, 1585.0, 1650.8,
1946.2, 2224.4
7 506.9, 628.9, 778.9, 917.1, 1128.3,
1405.1
9 506.9, 547.9, 629.0, 762.9, 952.3,
1101.5, 1405.2, 1664.2, 1825.8, 1979.3
10 506.9, 629.0, 784.2, 952.3, 1128.3,
1405.2
11 544.9, 597.8, 952.4, 1284.3, 1383.3,
1665.2, 1966.5, 2580.8
12 629.0, 637.2, 913.2, 909.2,
1043.2,1241.3, 1405.2, 1443.1
13 506.9, 570.8, 599.0, 744.2, 970.2, 966.3,
1128.3, 1241.3, 1405.2
14 506.9, 881.1, 970.3, 1027.3, 1038.3,
1049.3, 1241.4, 1405.3, 1617.3, 2181.5
15 506.9, 574.0, 780.9, 939.9, 1097.4,
1282.3, 1405.2, 1467.4, 1660.4, 1788.4,
1842.4, 2047.4
16 572.0, 629.0, 869.0, 1049.3, 1405.2
1a 556.8, 628.9, 690.8, 882.6, 952.2,
1123.3, 1284.1, 1405.0, 1466.9
2a 506.9, 628.9, 637.1, 666.9, 935.3, 952.3,
1284.2, 1405.1

4.2.5 1D-gel electrophoresis of CTE

An estimation of the size of the components was obtained despite the quality of the gel being poor.
Some cross contamination was also experienced, indicated by the bands appearing in the negative
control lanes.

The results of the gel indicate that there are large molecular weight compounds present in the CTE and
the CTE lyophilised in 0.1% TFA, 30% ACN (refer to figure 8). The CTE had more minor bands
present than in the lyophilised CTE, however the major bands were present in both samples. The
reduced and non-reduced samples had similar bands present. Previously a minor protein band present
at around 20kDa has been shown to be strongly immunoreactive with the box jellyfish antivenom [4].

This protein band appeared to be present on this gel also.

15
 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

250
150
100
75
50
37
25
20
15
10

Figure 8: Biorad 4-20% Tris/HCl precast gel with the lane number marked above. Lane 1: no
sample; 2: CTE lyophilised in TFA and ACN non-reduced (10ug); 3: marker; 4: milliQ water reduced; 5:
milliQ water non-reduced; 6: CTE reduced (10ug); 7: CTE reduced (20ug); 8: CTE reduced (30ug); 9:
CTE non-reduced (10ug); 10: CTE non-reduced (20ug); 11: CTE non-reduced (30ug); 12: CTE
lyophilised in TFA and ACN reduced (10ug); 13: CTE lyophilised in TFA and ACN reduced (20ug); 14:
CTE lyophilised in TFA and ACN non-reduced (20ug); 15: Another sample.
Marker bands: 250kDa, 150kDa, 100kDa, 75kDa, 50kDa, 37kDa, 25kDa, 20kDa, 15kDa, 10kDa.

4.2.6 RP-HPLC analysis of the purified crude tentacle extract (PCTE)

The RP-HPLC analysis of the crude tentacle extract that was lyophilised in 30% CAN and 0.1% TFA
showed several peaks of moderate intensity (refer to figure 9). This trace showed a greater number of
peaks in a more hydrophobic range than the previous runs, with the major peaks eluting after
approximately 40% of buffer B, at 37% of ACN. These peaks do not contain aromatic groups,
indicated by the 280nm trace. Conversely, one peak that eluted after approximately 12 minutes (2%
ACN) is highly aromatic.

A gradient from 0-80% B was then run due to a peak that eluted when the gradient quickly increased
to 90% in the previous spectrum (refer to figure 11). From the longer run, it was found that the highly
hydrophobic peak eluted at 70 minutes (60% ACN), but was of much lower intensity than the other
peaks. Fractions corresponding to the major peaks were collected from both traces for further analysis
(refer to tables 6 and 7). The pre- and post-run blanks indicate that the peaks observed are not a result
of the buffers (refer to figures 10 and 12).

16
Figure 9: Results of the RP-HPLC analysis of the CTE lyophilised in 30% ACN, 0.1% TFA, run
over a gradient of 0-40% B over 36 minutes. 50uL of the sample was injected in a concentration of
7.6mg/mL, using a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A). This was performed on a
Shimadzu SPD-10A VP detector.

Figure 10: Results of the RP-HPLC analysis (including pre- and post-run blanks) of the CTE
lyophilised in 30% ACN, 0.1% TFA, run over a gradient of 0-40% B over 36 minutes. 50uL of the
sample was injected in a concentration of 7.6mg/mL, using a Vydac C18 column (5uM pore size, 2.1 x
250mm, 300A). This was performed on a Shimadzu SPD-10A VP detector.

17
Table 6: Fractions collected with corresponding times from figure 9.

Fraction Number Time started fraction

collection (minutes)
1 11.30
2 11.98
3 12.43
4 13.67
5 14.56
6 15.12
7 15.77
8 17.18
9 31.24
10 31.58
11 32.87
12 34.49
13 34.67
14 37.41
15 39.17
16 39.97
17 40.90
18 42.00
19 43.20
20 46.56
21 48.27
22 49.70
23 50.36
24 50.48
25 53.18

18
Figure 10: Results of the RP-HPLC analysis of the CTE lyophilised in 30% ACN, 0.1% TFA, run
over a gradient of 0-40% B over 72 minutes. 50uL of the sample was injected in a concentration of
7.6mg/mL, using a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A). This was performed on a
Shimadzu SPD-10A VP detector.

Figure 11: Results of the RP-HPLC analysis (including pre- and post-run blanks) of the CTE
lyophilised in 30% ACN, 0.1% TFA, run over a gradient of 0-80% B over 72 minutes. 50uL of the
sample was injected in a concentration of 7.6mg/mL, using a Vydac C18 column (5uM pore size, 2.1 x
250mm, 300A). This was performed on a Shimadzu SPD-10A VP detector.
Note: The trace pictured only extends from 10 to 75 minutes. The pre-run blank was only run for 64
minutes, at a gradient of 0-40% B.

19
Table 7: Fractions collected with corresponding times from figure 11.

Fraction Time started fraction

Number collection (minutes)
1 11.35
2 11.93
3 12.27
4 13.23
5 13.52
6 14.42
7 15.27
8 16.62
9 30.94
10 32.80
11 33.80
12 34.68
13 37.38
14 39.10
15 39.90
16 40.70
17 42.27
18 43.06
19 43.20
20 46.41
21 47.42
22 48.38
23 49.60
24 50.43
25 50.80
26 51.92
27 52.60
28 53.15
29 67.90
30 68.70

The sample that was run at a gradient of 0.5% ACN/minute (refer to figure 13) did not appear to make
a difference with regards to separation of the peaks of interest (fractions 14-18 refer below). There
also were several peaks that were not in the previous traces, suggesting possible contamination.

20
Figure 13: RP-HPLC trace of 7.6mg/mL CTE lyophilized in 0.1% TFA, 30% CN, run to a gradient
of 0-40% B over 72 minutes (0.5% CAN/minute) with 10 minutes at 100% A initially. This was run
at a flow rate of 0.25mL/minute using a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A) on a
Shimadzu HPLC system (SPD-10A VP detector).

4.2.7 Mass spectrometry of the collected fractions from RP-HPLC analysis of the PCTE
The comparison between the DHB and SA matrices was useful in distinguishing between the fraction
peaks and the peaks of the solvent or matrix. Although expected, a mass was not obtained for the three
larger peaks that eluted after approximately forty minutes (fractions 21, 22, and 25). The matrix did
not crystallise when on the plate for these fractions (data not shown).

There were several recurring masses between the samples, however these were not consistent between
the two matrices. The fractions that did have recurring masses between the two matrices were 14, 15,
16, and 17, with masses of 2049, 2647, 2648, and 2082 Da respectively (refer to figures 14, 15 and
16). There was no evidence that the mass of fraction 14, 16, and 17 were the 2+ ions, however a peak
appeared at 5294Da in the spectra of fraction 15 with DHB as the matrix. This is exactly double the
mass of the main peak (2647Da). This peak was very small and did not appear in the spectra with SA
as the matrix, suggesting that it could be due to the formation of a dimer.

There also were several smaller peaks present in close proximity to these masses indicating that the
peak separation was not optimal. It is likely that there are approximately 10 different peptides present
in this broad peak (refer to figures 14, 15, and 16).

21
 Fractions 14 -17

Figure 12: RP-HPLC trace showing the peaks corresponding to fractions 14-17.

22
 Fraction 14

Fraction 15

Fraction 16

Fraction 17

Figure 13 : MALDI-TOF results for fractions 14 to 17 using DHB as the matrix in reflector mode.

23
 Fraction 14

Fraction 15

Fraction 16

Fraction 17

Figure 14 : MALDI-TOF results for fractions 14 to 17 using SA as the matrix in reflector mode.
The fractions corresponding to the peaks of the greatest intensity (fractions 1, 2, 3, 20, 21, and 24 from
figure 9 and table 6) were analysed using electrospray mass spectrometry. Although the peaks had a
high intensity on the trace, the electrospray did not reveal any significant masses.

24
Table 8: Major peaks in fractions analysed by MALDI-TOF MS from figure 11

Fraction Major peaks with DHB matrix Major peaks with SA matrix
(Da) (Da)
574.6, 699.6, 1166.7 616.7, 655.7, 1142.3, 1700.4
1
2 551.1, 1220.6, 2053.6, 2905.5 515.5, 695.8, 1254.4
3 574.5, 596.5, 1449.9, 2016.7 515.5, 677.8, 1210.3
4 507.5, 599.9, 714.0, 883.3, 1166.7, 613.8, 595.7, 1144.4
1450.1
5 507.4, 557.4, 1166.7, 1733.5, 2301.4 613.8, 595.7, 1126.3
6 507.5, 557.5, 1036.5 537.7, 613.8, 1000.3, 1564.6
7 507.5, 599.9, 1166.8, 1336.1 613.8, 595.8, 1060.5
8 507.4, 883.3, 1166.7, 1450.1, 613.8, 595.7, 1060.3, 1733.8
1734.6
9 507.5, 643.6, 1166.8, 1734.6 563.8, 616.9, 1019.5, 1735
10 507.5, 643.6, 1450.3, 2018.1 595.8, 613.8, 1075.4
11 507.5, 517.6, 1166.8, 1733.6, 2302.4 595.8, 613.8, 1086.6, 1734.0
507.4, 523.0, 1166.6, 1734.3 595.7, 613.7, 1072.3
12
13 507.4, 1166.7 537.7, 613.7, 1282.7
14 1920.3, 2049.4, 2163.6, 2178.5, 1920.7, 2049.8, 2179.0, 2293.1, 2536.5
2292.7, 2535.8
15 1166.7, 1324.1, 2049.5, 2178.6, 616.9, 1324.9, 2049.9, 2179.1,
2292.7, 2518.0, 2647.9, 5294.1 2293.2, 2518.5, 2647.8
16 701.9, 2082.7, 2519.0, 2648.1, 595.7, 613.8, 1142.4, 2646.8,
5679.6 2648.6, 2650.8
17 507.5, 1166.7, 1564.3, 1733.6, 563.8, 616.9, 1043.5, 1564.6, 1736.1,
2066.4, 2082.8 1920.0, 2067.7, 2083.3, 2085.3
18 507.5, 1036.5, 1280.9, 1848.7 595.7, 613.7, 1450.2
19 507.5, 525.8, 1036.6 595.7, 613.8, 1097.7
20 507.5, 545.5, 1165.7 613.8, 756.0, 1226.7
21 Not significant 595.8, 613.8, 1121.5
22 Not significant 595.8, 613.8, 1142.4
23 545.5, 627.9, 1074.4, 1096.4, 1668.3, 537.7, 613.8, 1005.3
2670.7
24 523.8, 703.9, 1166.7, 1733.4 616.8, 695.8, 1164.4, 1799.7
25 Not significant 525.5, 613.6
26 623.4, 650.8, 1308.8 525.5, 595.7, 1143.3, 3492.1
27 507.4, 761.6, 1280.8 613.7, 677.8, 1142.3
28 719.0, 946.3, 1553.1 525.5, 693.8, 1158.4
29 507.5, 629.6, 1450.2, 2018.0 525.5, 547.5, 1002.2
30 507.5, 714.0, 1036.5, 1280.9, 613.8, 695.8
1848.7

25
4.2.8 RP-HPLC analysis of the PCTE using a diode array detector
The CTE was further analysed by running a 0-40% B RP-HPLC gradient using a diode array detector.
There were several peaks that appeared to be peptidic with some appearing to contain amino acids
with aromatic groups (refer to figures 17, 18 and 19 and table 9).

Figure 15 : RP-HPLC trace of PCTE using a Shimadzu SPD-M10A VP detector with a diode array
detector over a gradient of 0-40% B in 36 minutes, with 10 minutes of 100% A initially. The
column used was a Vydac C18 column (5uM pore size, 2.1 x 250mm, 300A) with a 50uL injection of a
7.6mg/mL sample of PCTE and a flow rate of 0.25mL/min.

26
Figure 16 : Absorbance of the component that eluted at 11.84 minutes in figure 17.

Figure 17 : Absorbance of the component that eluted at 19.42 minutes in figure 17.

27
Table 9: Description of absorbance of components of PCTE obtained with a diode array
detector.

Time of elution (minutes) Description of Spectra Conclusions

11.84 - 1 peak at 205nm Peptide with aromatic
- 1 peak at 270nm amino acids
13.08 - 1 peak at 214nm Peptide with aromatic
- 1 peak at 260nm amino acids
13.54 - 1 peak at 205nm
- 1 peak at 255nm
19.42 - 1 peak at 205nm
- 1 peak at 225nm
- 1 peak at 260nm
34.02 - 1 peak at 210nm
- 1 small peak at 250nm
36.28 - 1 peak at 210nm
39.97 - 1 peak at 204nm
40.58 - 1 peak at 206nm
43.24 - 1 peak at 205nm
- 1 peak at 214nm
- 1 low broad peak at
270nm
47.25 - 1 peak at 210nm
48.04 - 1 peak at 214nm Peptide
50.53 - 1 peak at 214nm Peptide
52.56 - 1 peak at 203nm

28
4.2.9 Spontaneous contraction assay with PCTE
When the extract was tested for activity upon the spontaneous contraction of the proximal guinea pig
colon, 10ug/mL produced inhibition of the amplitude of spontaneous contractions (refer to figure 20).
When 20ug/mL was used no further inhibitory effect was observed. The inhibitory activity appeared to
be transient.

Figure 18 : Spontaneous contraction of the proximal guinea pig colon in response to the
addition of 10ug/mL and 20ug/mL of PCTE.

29
4.3. Bioassays

4.3.1 Cytotoxicity assay

The jellyfish extract showed no cytotoxicity against either of the cell lines
tested (figures 21 and 22).

Figure 19 : Cytotoxicity of Jellyfish extract against P388 cells (Mean SE)

Figure 20: Cytotoxicity of Jellyfish extract against MCF7 cells (Mean SE)

30
4.3.2 Natural killer (NK) cell activity

In terms of human innate immune function, the jellyfish extract exhibited a general stimulatory effect
in vitro (figures 23, 24 and 25).

The greatest effect was noted for NK cell cytotoxic activity (figure 23), with significant increases in
mean activity of 17.1% and 19.5% at extract concentrations of 0.1 and 1.0 g/ml respectively.
Phagocytosis showed only moderate increases in mean activity (figures 24 and 25) ranging from 3.5%
to 8.2%.

Figure 21: Effect of Jellyfish Extract on NK Cell Cytotoxic Activity (Mean SE)

Jellyfish Extract (ug/ml)

31
4.3.3 Phagocytosis assay

Jellyfish Extract (ug/ml)

Figure 22: Effect of Jellyfish Extract on Phagocytic Activity of Granulocytes (Mean SE)

Jellyfish Extract (ug/ml)

Figure 23: Effect of Jellyfish Extract on Phagocytic Activity of Monocytes (Mean SE)
The stimulatory in vitro responses did not demonstrate a defined dose response pattern for the jellyfish
extract. As such, the significance of these findings is unclear.

32
4.4. In Vitro Effects of Extract On Cells Of The Circulating Immune System

4.4.1 Genomic analyses

The Genomic analysis performed on Affymetrix U133 Plus 2.0 Human GeneChip microarrays
indicated differential expression in the PBMCs of a range of genes between the different time points
following stimulation with the high-concentration tentacle extract (approx 250mg / 10ml flask).

Genes that showed a greater than 4-fold increase or decrease expression between different time points
are noted and are held in camera

Gene Ontology indicate that many of the differentially-expressed genes are involved in the immune
response, including functions such as antigen binding, phagocytosis, cell adhesion, and cellular
defense response. Many of the other genes reflect the processes associated with cell proliferation.
Whilst caution should be expressed in interpreting this preliminary data, it is none the less encouraging
and is consistent with immune response activity of the extract.

The findings derived from this technical feasibility study indicate that the use of the PBMC system
followed by genomic analysis is a valid method for evaluating the changes occurring as a result of
stimulation by natural products, such as an extract derived from the tentacles of the jellyfish Catostylus
mosaicus. This system can therefore be employed to identify molecular alterations that may be
commercially useful. Additional studies will be required to confirm and expand upon these
encouraging preliminary findings. Future studies will require evaluation of the effects of the tentacle
extract on PBMCs derived from multiple individuals, using multiple technical and biological
replicates, in order for the molecular changes to be confirmed as potential commercial therapeutic
targets.

33
5. Discussion
An initial review of the literature, from the databases - Science Direct, Proquest, PubMed and Google;
has revealed that the majority of investigations into C. mosaicus have concentrated on either biological
aspects (growth, life cycle), environmental aspects or processing considerations as C. mosaicus is an
edible jellyfish.

A restricted number of studies have been performed on venom from a range of jellyfish. Most
Australian studies have concentrated on the Irukandji (Carukia barnesi), the Box Jellyfish (Chironex
fleckeri Southcott) and the Blubber (Catostylus mosaicus), with the Irukandji and Box Jellyfish
receiving most attention as both these species venom can cause death.

Relatively few studies have been performed on C. mosaicus venom. These have established that
Hemolysin was present and that haemolytic activity was significant[5]. Burnett reviewed the role of
immunological reactions in the pathogenesis of jellyfish envenomations and noted that stings normally
elevated IgM then IgG in human victims serum[6]. Jellyfish collagen has also been shown to
suppress the effects of antigen-induced arthritis in laboratory rats[7].

This study into the venom from the tentacles of C. mosaicus provides encouraging preliminary data
supporting potential immune stimulant activity by extracts from the tentacles.

This study has shown that:

Biological activity was demonstrated by the jellyfish extract in the form of spontaneous
contraction of guinea pig colon by 10g/mL of the extract.
The extract is not directly cytotoxic. as demonstrated by lack of observed toxicity due to the
extract on P388 and MCF7 cell lines.
The extract showed innate immune function stimulation, demonstrated by apparent stimulation
of phagocytosis of granulocytes by the extract in bioassay studies. In addition, a range of gene
activities associated with immune function were observed in studies of peripheral blood
mononuclear cells. Functions of these genes include phagocytosis, antigen binding, cell
adhesion and the cellular defense response. Other genes stimulated by the extract reflect the
processes associated with cell proliferation.

This study has provided a comprehensive overview of the characteristics of the venom of. Catostylus
mosaicus It has for the first time, provided solid data on the nature of the venom attributes. The
investigation has shown that activity relating to the immune system exists and the results warrant
further investigation. It has provided a sound platform for further investigations as a prelude to
investment for commercial development and a way forward for the utilisation of this under utilised
natural resource.

34
6. Implications
Global interest in products that promote health and well-being is ever expanding, with the global
industry approaching $50 billion in sales[1]. Functional food trends, a report published in Food
Technology (2002)[8] clearly sees this area as the next big growth sector

[n]eutraceutical marketsnew growth opportunities will emanate

from up-and coming new markets, mega market sub-segments, and
novel niches. (p.32)

In this informed, information-rich era people are taking a pro-active responsibility for their own health
and well being. This has lead to predictions that the health maintenance and prevention market
segments will continue to grow dramatically[9]. These predictions are based on two major attitudinal
trends of
i) diminishing confidence that our diet satisfies all of our nutritional needs. This belief
in diet meeting nutritional needs has decreased in women from 70% in 1994 to 46% in
2000[10].
ii) the practice of positive eating continues to grow, where 86% of consumers buy foods
that contain desirable nutritional ingredients, 80% buy foods that dont contain
undesirable ingredients and 76% purchase fortified products with specific nutritional
substances[11].

Along with the globally ageing population there is an increase in the prevalence of disease and an
increasing demand for quality of life. Diseases such as cancer, cardiovascular disease and diabetes are
prevalent and impact considerably on quality of life. Hence, there is considerable interest being shown
in products which can prevent or alleviate the prevalence of such diseases.

The ability of the immune system to function optimally plays a key role in a persons ability to deal
with almost all diseases and it ultimately has an enormous impact on quality of life. Any product able
to boost the immune system will have a large potential market.

Since C. masaicus is an underutilized and abundant natural resource,, any medicinal and other
products developed from it would have adequate sustainable and inexpensive stocks to draw from.

35
7. Recommendations
In light of the promising findings from the present study we recommend that a second study be
undertaken to further evaluate the effects of the tentacle extract on PBMCs derived from multiple
individuals, using multiple technical and biological replicates, in order that the molecular changes
identified can be confirmed and pursued as potential commercial therapeutic targets.

36
8. References
1. Williamson, J.A., Fenner, P.J., Burnett, J.W. & Rifkin, J.F. (1996) Venomous and poisonous
marine animals: a medical and biological handbook, University of New South Wales Press,
Sydney, Australia.

2. Azila, N., Siao, F.K. & Othman, I. (1991) Haemolytic, oedema and haemorrhage inducing
activities of tenticular extract of the blubber jellyfish (Catostylus mosaicus). Comp. Biochem.
Physiol.; 99(1-2): 153-6.

3. Monteray Bay Aquarium Online Field Guide, Blue Jelly: Catostylus mosaicus,
http://www.mbayaq.org/efc/living_species/default.asp?hOri=1&inhab=447
Accessed: 18/12/06 Last Updated: 2006

4. Wiltshire, C.J., Sutherland, S.K., Fenner, P.J. & Young, A.R. (2000) Optimization and preliminary
characterization of venom isolated form 3 medically important jellyfish: the box (Chironex
fleckeri), Irukandji (Carukia barnesi), and blubber (Catostylus mosaicus) jellyfish. Wilderness
Environ Med.; 11: 241-50.

5. Azila, K. 1990 Effective Catostylus mosaicus venom on erythrocytes. Toxicon 28:2

6. Burnett,J. 1991 International Society of Toxinology Meeting, Singapor, November 3

7. Hsieh,P., Leong, F., Rudloe, J. 2001 Jellyfish as Food. Hydrobiologia 451(1-3):11-17

8. Sloan Trends & Solutions (2002) The Top 10 Functional Food Trends: The Next Generation.
Food Technology 56, 4, 32-56

9. Roper/CHA (2001) Self care in the new millennium: American attitudes toward maintaining
health and treatment. Consumer Healthcare Products Assn. Washington, D.C.

10. mSI (2001) The 2001 Gallup study of nutrient knowledge and consumption. Multisponsor
Surveys, Princeton, N.J.

11. HealthFocus (2001) HealthFocusTM Trendreport, 2001. Atlanta, Ga.

37
 The Blue Jellyfish: A Promising
Autoimmune Stimulant
by B.R. Rich and P.A. Cheras
RIRDC Publication No. 09/035

This report presents the results of novel investigations into the
properties of the venom from the jellyfish Catostylus mosaicus
(commonly known as the blue jellyfish). The study involved
extraction and concentration, bioassays of the extract and
studies of the in vitro effects of the extract on genes within
cells of the circulating immune system.
The establishment of a Queensland developmental jellyfish
fishery meant that for the first time Catostylus mosaicus could
be harvested commercially. This project forms part of the
establishment of a new commercial sector targeting dried
food products and value added products, such as therapeutic
products.
For the commercial sector to enhance commercial viability
and realise the true potential of the resource, the value added
products are viewed as a vital component.
The Rural Industries Research and Development Corporation
(RIRDC) manages and funds priority research and translates
results into practical outcomes for industry.
Our business is about developing a more profitable, dynamic
and sustainable rural sector. Most of the information we
produce can be downloaded for free from our website:
www.rirdc.gov.au.
IRDC books can be purchased by phoning 1300 634 313 or
R
online at: www.rirdc.gov.au.

Contact RIRDC:
This publication can be viewed at our website Level 2
www.rirdc.gov.au. All RIRDC books can be 15 National Circuit
purchased from:. Barton ACT 2600

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