Eugenin
An immunomodulator used to protect young in the pouch of the
Tammar wallaby, Macropus eugenii
Russell V. Baudinette
1,
*, Pinmanee Boontheung
2
, Ian F. Musgrave
3
, Paul A. Wabnitz
2
,
Vita M. Maselli
2
, Jayne Skinner
1
, Paul F. Alewood
4
, Craig S. Brinkworth
2
and John H. Bowie
2
1 Department of Environmental Biology, The University of Adelaide, South Australia
2 Department of Chemistry, The University of Adelaide, South Australia
3 Department of Clinical and Experimental Pharmacology, The University of Adelaide, South Australia
4 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
Marsupials are born in an immature state and many
of the developmental processes that occur in these
mammals take place during pouch life [1]. After a
short period of intrauterine development, the young
marsupial crawls unaided to the mother’s pouch, atta-

Tel: +61 08 83035767
E-mail: [email protected]
*Author deceased
(Received 30 August 2004, revised 18
October 2004, accepted 16 November
2004)
doi:10.1111/j.1742-4658.2004.04483.x
Eugenin [pGluGlnAspTyr(SO
3
)ValPheMetHisProPhe-NH
2
] has been
isolated from the pouches of female Tammar wallabies (Macropus eugenii)
carrying young in the early lactation period. The sequence of eugenin
has been determined using a combination of positive and negative ion
electrospray mass spectrometry. This compound bears some structural
resemblance to the mammalian neuropeptide cholecystokinin 8
[AspTyr(SO
3
)MetGlyTrpMetAspPhe-NH
2
] and to the amphibian caerulein
peptides [caerulein: pGluGlnAspTyr(SO
3
)ThrGlyTrpMetAspPhe-N H
2
].
Eugenin has been synthesized by a route which causes only minor hydrolysis
of the sulfate group when the peptide is removed from the resin support. Bio-
logical activity tests with eugenin indicate that it contracts smooth muscle at a

immunoglobulins, lysozyme and other antibacterial
enzymes, in the milk of higher animals [7,10–18]. In
this context, marsupial whey proteins have been exam-
ined as a function of the time when they are present
during the lactation period [19–25]; generally the pro-
tein content varies significantly from the early to the
late lactation period.
Female wallabies produce a waxy secretion in the
pouch, and the constituency of this secretion appears
to depend upon the oestrus cycle and the time the
young has spent in the pouch [6]. There is also evi-
dence that polyprotodont opossums produce immuno-
globulins in the pouch [26]. Whether immunoglobulins
are secreted into the pouch of diprotodonts such as
the Tammar wallaby is yet to be established.
In this paper we report a study of the low molecular
mass (< 2000 Da) water-soluble components of swabs
taken from the pouch of the Tammar wallaby [27],
with a view to identifying any maternal defence com-
pounds (e.g. antimicrobial agents and ⁄ or neuropep-
tides) in the pouch. We describe a unique mammalian
cholecystokinin (CCK)-like peptide, eugenin, which
may act as an immunomodulator.
Results
The pouch swabs of female wallabies with or without
young in the pouch, contain low molecular mass
(< 2000 Da) water-soluble compounds. Figure 2 shows
a typical HPLC separation. MS and MS ⁄ MS data on
the components of all HPLC fractions indicate the
presence of a variety of lipid, sugar and phosphate

Because eugenin has an N-terminal pGlu residue,
automated Edman sequencing [28] cannot be used to
determine the amino acid sequence of this peptide.
Sequence analysis was effected using positive and neg-
ative ion electrospray mass spectrometry.
The negative ion mass spectrum of eugenin gives
peaks corresponding to (M-H)
–
and [(M-H)
–
-SO
3
]
–
at
m ⁄ z 1371 and 1291, respectively, indicating that euge-
nin has a molecular mass of 1372 Da, and that it con-
tains a sulfate group. The positive ion mass spectrum
shows a small MH
+
ion at m ⁄ z 1373, and a pro-
nounced peak corresponding to an [MH
+
-SO
3
]
+
spe-
cies at m ⁄ z 1293. The collision induced mass spectrum
(MS ⁄ MS) of the [MH

(MS ⁄ MS) of the [(M-H)
–
-SO
3
]
–
ion of eugenin is
shown in Fig. 4. There are a number of backbone
cleavages in negative ion spectra which provide
sequencing information. These have been described
previously [30]. Two of these (a and b cleavages) are
fragmentations of amide moieties, and give infor-
mation analogous to that provided by B and Y+2
cleavages in the corresponding positive ion spectra.
The other backbone cleavages (d and c processes) ori-
ginate from Asp, Asn, Glu or Gln side chains and
provide specific information concerning the positions
of these four residues. The d and c fragmentations
are particularly important in identifying Gln residues,
because isobaric Gln and Lys cannot be differentiated
by low resolution positive ion mass spectrometry. The
a and b derived sequences are indicated schematically
above and below the negative ion spectrum shown in
Fig. 4, while d and c cleavages are indicated on the
spectrum. The data shown in Fig. 4 gives the
sequence of eugenin except that it does not indicate
the relative orientation of residues 6 and 7. The spec-
trum identifies pGlu as residue 1 and shows that resi-
due 2 is Gln rather than Lys. A combination of the
fragmentation data from the negative and positive ion

Phe-NH
2
Caerulein [39,40]
pGluGlnAspTyr(SO
3
)ThrGlyTrpPheAsp-
Phe-NH
2
Caerulein 1.2 [41]
pGluAsnAspTyr(SO
3
)LeuGlyTrpMetAsp-
Phe-NH
2
D
2
L
5
-Caerulein [58]
pGluGluTyr(SO
3
)ThrGlyTrpMetAspPhe-NH
2
Phyllocaerulein [59]
Fig. 4. Negative ion mass spectrum of the [(M-H)
–
-SO
3
]
–

As eugenin had similar structural elements to both
CCK and caerulein, known CCK receptor agonists, we
performed biological activity screening in tissues with
well-characterized CCK responses.
Contraction studies
Acetylcholine contracted guinea pig ileal segments in
a concentration-dependent fashion (data not shown).
The mixed CCK
1
CCK
2
receptor agonist and standard
CCK-8 produced potent increases in contraction, was
maximally effective at 10
)9
m, but produced only
about 60% of the contraction produced by the
maximally effective concentration of acetylcholine
(Fig. 5A). The CCK
2
agonist and standard cholecy-
stokinin 8 nonsulfated (CCK-8-NS) also produced
increases in contraction, but was less potent and less
effective than CCK-8 (Fig. 5A). These results are con-
sistent with previous studies [33]. Eugenin also pro-
duced an increase in contraction, and was equieffective
and equipotent with CCK-8-NS (Fig. 5A). This sug-
gested that eugenin might be acting as a CCK
2
agon-

CCK-8 produced a concentration dependent increase
in lymphocyte proliferation in both the presence
(Fig. 6A) and absence (data not shown) of the mito-
gen concanavalin A. CCK-8-NS was less effective
(Fig. 6A). This is consistent with previous studies
[35,36]. Eugenin (and to a lesser extent, desulfated
eugenin) also produced a concentration dependent
increase in lymphocyte proliferation in both the pres-
Fig. 5. (A) CCK-8 (d), CCK-8-NS (h) and eugenin ( ) concentration–
response curves in guinea pig ileum. Ileal segments were exposed
to increasing concentrations of CCK-8, CCK-8-NS and eugenin. Con-
tractions were measured on a Maclab data recorder (Maclab, Castle
Hill, New South Wales, Australia) and expressed as a percentage
of the maximal acetylcholine response (10
)6
M; 56 ± 15 mm). Data
are expressed as mean ± SD of three independent experiments.
(B) The effect of atropine on contractions produced by CCK-8 and
eugenin in guinea pig ileum. Ileal segments were exposed to either
vehicle or atropine (10
)6
M) for 15 min then CCK-8 or eugenin
applied. Contractions were measured on a Maclab data recorder
and expressed as a percentage of the maximal acetylcholine
response (10
)6
M; 86 ± 15 mm). Data are expressed as mean ± SD
of two experiments, except for eugenin vehicle, where n ¼ 1.
R. V. Baudinette et al. Protection by eugenin of Macropus eugenii young
FEBS Journal 272 (2005) 433–443 ª 2005 FEBS 437

CCK
2
, differing in anatomical locations and actions
[43]. The sequences of the CCK receptors are known
[44] and representations of their 3D structures have
been reported [44,45]. Both NMR and other experi-
mental data have been used to determine where CCK-
8 binds on the receptors [44–47]. In the present study
we use CCK-8 and its desulfated analogue (CCK-8-
NS) as standards.
CCK-8 and caerulein activate both CCK receptors:
perhaps eugenin may act via one or both CCK receptor
subtypes. In the guinea pig ileum, CCK receptor agon-
ists act to cause contraction of smooth muscle [33].
CCK
1
receptors are present on the smooth muscle, and
contract the smooth muscle directly. In contrast, CCK
2
receptors act indirectly, by causing the release of acetyl-
choline from cholinergic nerves in the myenteric plexus,
which activates muscarinic receptors on smooth muscle
[33]. In the present study, the standard neuropeptide
CCK-8, which activates CCK
1
and CCK
2
receptors,
produced a concentration dependent increase in
contraction of guinea pig isolated ileal segments. CCK-

curves in mouse splenocytes. Splenocytes were exposed to
increasing concentrations of CCK-8, or a single concentration of
CCK-8-NS in the presence of the mitogen concanavalin A. Lympho-
cyte proliferation was measured by the increase in fluorescence
due to conversion of Alamar Blue [37]. Data shown are from a sin-
gle experiment performed in quadruplicate, representative of two
experiments carried out in quadruplicate. (B) Eugenin (
) and euge-
nin-NS (h) concentration-response curves in mouse lymphocytes.
Lymphocytes were exposed to increasing concentrations of euge-
nin in the presence of the mitogen concanavalin A. Lymphocyte
proliferation was measured by the increase in fluorescence due to
conversion of Alamar Blue. Data were expressed as a percentage
of the CCK maximum response (10
)5
M, performed in the same
time period with each run). Values are the mean ± SD of four
experiments for eugenin.
Protection by eugenin of Macropus eugenii young R. V. Baudinette et al.
438 FEBS Journal 272 (2005) 433–443 ª 2005 FEBS
ation. In these experiments, exposure of mouse spleno-
cytes to the standard neuropeptide CCK-8 resulted in
a concentration dependent increase in proliferation, as
measured by the Alamar Blue assay [34]. These results
are consistent with previous studies [35,36] (although
Medina et al. [36] found CCK-8 to be more potent
than in the current study, the cells were exposed to
CCK agonists for 72 h compared to 24 h in this
study). CCK-8-NS produced proliferation, but was less
potent than CCK-8. Eugenin also produced an

Pouch swabs
Cotton wool swabs of the pouches of three female M. euge-
nii were taken at two day intervals, from two days before
the young occupies the pouch until the pouch had been
occupied for two weeks, and then weekly for the next four
weeks. Each swab was shaken with deionized water
(50 mL), the mixture diluted with an equal volume of meth-
anol, centrifuged, filtered through a Millex HV filter unit
(0.45 lm), and lyophilized (the methanol was added to
denature and precipitate any enzymes which may effect
degradation of active pouch components). This procedure
provided, on average, 1–2 mg of solid material from each
swab. Swabs were also taken, for comparison, from
pouches of female M. eugenii that were not bearing young.
HPLC separation of pouch material
HPLC separation of pouch material was achieved using a
VYDAC C18 HPLC column (5l, 300A, 4.6 · 250 mm)
(Separations Group, Hesperia, CA, USA) equilibrated with
10% acetonitrile ⁄ aqueous 0.1% TFA. The lyophilized mix-
ture (generally  1 mg) was dissolved in deionized water
(50 lL), of which a 10 lL fraction was injected into the
column. The elution profile was generated using a linear
gradient produced by an ICI DP 800 Data Station control-
ling two LC1100 HPLC pumps, increasing from 10 to 75%
(v ⁄ v) acetonitrile over a period of 60 min at a flow rate of
1mLÆmin
)1
. The eluant was monitored by ultraviolet
absorbance at 214 nm using an ICI LC-1200 variable wave-
length detector (ICI Australia, Melbourne, Australia). An

Preparation of synthetic eugenin [pGluGlnAsp-
Tyr(SO
3
)ValPheMetHisProPhe-NH
2
]
Materials
Manual syntheses were performed with Fmoc-amino acids
purchased from Bachem, Novabiochem and Aspen (Aspen,
CO, USA). The Ramage Amide tricyclic linker was pur-
chased from Bachem. Diisopropylcarbodiimide was from
Aldrich (Castle Hill, New South Wales, Australia) and
2-(1H-benzotriazol-1-yl)-1 ,1,3,3-tetramethyl-uroniumhexafluoro-
R. V. Baudinette et al. Protection by eugenin of Macropus eugenii young
FEBS Journal 272 (2005) 433–443 ª 2005 FEBS 439
phosphate was obtained from Richelieu Biotechno-
logies (Quebec City, Quebec, Canada). N,N-diisopropyl-
ethylamine, N,N-dimethylformamide, dichloromethane,
piperidine, TFA and Fmoc-sulfotyrosine (all peptide syn-
thesis grade) were purchased from Auspep (Melbourne,
Australia). Acetone (HPLC grade) was obtained from
Water Millipore (Milford, MA, USA). High purity water
was generated by a Milli-Q
TM
purification system (Milli-
pore, Bedford, MA, USA). Screw-cap glass peptide synthe-
sis reaction vessels (20 mL) with a #2 sintered glass filter
frit and a shaker for manual solid-phase synthesis were
obtained from Embel Scientific Glassware (Brisbane,
Queensland, Australia).

2
).
Bioactivity assays
Antimicrobial testing on HPLC fractions and synthetic euge-
nin was carried out by the Microbiology Department of the
Institute of Medical and Veterinary Science (Adelaide,
Australia) using a standard procedure [56]. The microorgan-
isms used were: Bacillus cereus, Escherichia coli, Leuconostoc
lactis, Listeria innocua, Micrococcus luteus, Pasteurella multo-
cida, Staphylococcus aureus, Staphylococcus epidermidis and
Streptococcus uberis. Neither the HPLC fractions nor euge-
nin showed activity at an MIC value of 100 lg Æ mL
)1
against
any of these organisms, and is thus deemed inactive.
Contraction studies
Drugs and materials
This work was approved by The University of Adelaide
Animal Ethics Committee.
Acetylcholine, atropine, concanavalin A, CCK-8 and
CCK-8-NS were obtained from Sigma-Aldrich. Alamar blue
was obtained from Astral Scientific (Caringbar, New South
Wales, Australia).
Guinea pigs weighing approximately 300 g were used.
Immediately before the experiment, the guinea pigs were
killed by stunning and subsequent decapitation. The ileum
was dissected free and was cleansed by rinsing with physiolo-
gical salt solution (composition in mm): KCl 2.7, CaCl
2
1.0,

(1 lm) was used again to check that the response was sta-
ble. After 5 min washout and achievement of a stable base-
line, a cumulative response curve to CCK-8 (10
-10
)10
-8
m)
was performed. After another 5 min washout and achieve-
ment of a stable baseline, a cumulative concentration
response curve to either eugenin (10
-9
)10
-7
m) or CCK-8-
NS (10
-9
)10
-7
m) was performed. In some experiments, fol-
lowing washout, tissues were either pretreated with atropine
or vehicle and CCK-8 or eugenin reapplied.
Splenocyte proliferation studies
Male Balb ⁄ C mice aged 6–8 weeks were used. Lymphocytes
were prepared as described previously [57] with minor
modifications. Aseptic techniques were used during
Protection by eugenin of Macropus eugenii young R. V. Baudinette et al.
440 FEBS Journal 272 (2005) 433–443 ª 2005 FEBS
preparation of the lymphocytes. Mice were killed by cervi-
cal dislocation followed by prompt removal of the spleen.
The spleen was prepared as a single-cell suspension by mas-

give a final volume of 200 lL, and final cell count of
50 000 cells per well.
Either vehicle or the mitogen concanavalin 1 (2.5 lgÆmL
)1
final concentration) was added to the wells, and then 10 lL
of RPMI 1640 medium containing either CCK-8, CCK-8-NS
or eugenin (to produce final concentrations of 10
-7
)10
-5
m)
was added to the plate. Plates were incubated at 37 °C, using
5% CO
2
in a humidified incubator (Thermoline, Sydney,
New South Wales, Australia) for 24 h. Twenty-five microlit-
ers of the mitochondrial activity indicator dye Alamar Blue
[34] was then added to give a final concentration of
2.5 lgÆmL
)1
, and the plates incubated as above for a further
4 h. After this, 175 lL aliquots were pipetted from each well
into a white 96 well plate, and fluorescence measured in a
Polestar Galaxy (BMG Labtechnologies, Durham, NC,
USA) fluorescent plate reader (excitation 544 nm, emission
590 nm).
Acknowledgements
We thank the Australian Research Council for provi-
ding maintenance funding for this project. The ARC
also provided the following stipends: C.S.B. (research

eugenii. Comp Immunol Microbiol Infect Dis 21, 237–
245.
7 Old JM & Deane EM (2000) Development of the
immune system and immunological protection in marsu-
pial pouch young. Dev Comp Immunol 24, 445–454.
8 Fan ZW, Eng J, Shaw G & Yalow RS (1988) Cholecys-
tokinin octapeptide purified from brains of Australian
marsupials. Peptides 9, 429–431.
9 Bobek G & Deane EM (2002) Possible antimicrobial
compounds from the pouch of the koala, Phascolartos
cinereus. Lett Peptide Sci 8, 133–137.
10 McKenzie HA & Shaw DC (1985) The amino acid
sequences of equine milk lysozyme. Biochem Int 10, 23–31.
11 Carlsson A, Bjorck L & Persson K (1989) Lactoferrin
and lysozyme in milk during acute mastitis and their inhi-
bitory effect in Delvotest P. J Dairy Sci 72, 3166–3175.
12 Koldovsky O (1989) Search for role of milk-borne bio-
logically active peptides for the suckling. J Nutr 119,
1542–1551.
13 Buts JP (1999) Bioactive factors in milk. Arch Pediatr 5,
298–306.
14 Kunz C, Rodriquez-Palmero M, Koletzko B & Jensen
R (1999) Nutritional and biochemical properties of
human milk: general aspects, proteins and carbohy-
drates. Clin Perinatol 26, 307–3033.
15 Groenink J, Walgreen-Weterings E, van’t Hof W, Veer-
man ECI & Amerongen AVN (1999) Cationic amphi-
pathic peptides, derived from bovine and human
lactoferrins, with antimicrobial activity against oral
pathogens. FEMS Microbiol Lett 179, 217–222.

during synapsid evolution. J Mammary Gland Biol Neo-
plasia 7, 225–252.
23 Simpson KJ & Nicholas KR (2002) The comparative
biology of whey proteins. J Mammary Gland Biol Neo-
plasia 7, 313–326.
24 Goldman AS (2002) Evolution of the mammary gland
defense system and the ontology of the immune system.
J Mammary Gland Biol Neoplasia 7, 277–289.
25 Trott JF, Simpson KJ, Moyle RLC, Hearn CM, Shaw
G, Nicholas KR & Renfree MB (2003) Maternal regula-
tion of milk composition, milk production, and pouch
young development during lactation in the Tammar
wallaby. Biol Reprod 68, 929–936.
26 Hindes RD & Mizell M (1976) The origin of immuno-
globulins in oppossum ‘embryos’. Dev Biol 53, 49–61.
27 The Australian Museum Trust (1995) The Australian
Museum Complete Book of Australian Mammals. Reed
Books, Chatswood, NSW, Australia.
28 Hunkapiller MW, Hewick RM, Drewer WJ & Hood LE
(1983) High sensitivity sequencing with a gas-phase
sequencer. Methods Enzymol 91, 399–406.
29 Biemann K & Martin S (1987) Mass spectrometric
determination of the amino acid sequence of peptides
and proteins. Mass Spectrom Rev 6, 1–77.
30 Bowie JH, Brinkworth CS & Dua S (2002) Collision
induced fragmentations of (M-H)
–
parent anions of
peptides. An aid to structure determination and some
unusual anion chemistry. Mass Spectrom Rev 21, 87–

Methods 170, 211–224.
35 Iwata N, Murayama T, Matsumori Y, Ito M, Nagata
A, Taniguchi T, Chikara K, Matsuo Y, Minowada J &
Matsui T (1996) Autocrine loop through cholecystoki-
nin-B ⁄ gastrin receptors involved in growth of human
leukemia cells. Blood 88 , 2683–2689.
36 Medina S, Rio MD, Cuadra BD, Guayerbas N &
Fuente MD (1999) Age related changes in the modula-
tory actrin of gastrin-releasing peptide, neuropeptide Y
and sulfated cholecystokinin octapeptide in the prolifera-
tion of murine lymphocytes. Neuropeptides 33, 173–179.
37 Dockray GJ (1976) Immunological evidence of cholecy-
stokinin-like peptides in brain. Nature 264, 568–570.
38 Matsumoto M, Park J, Sugano K & Yamada T (1987)
Biological activity of progastrin post-translational pro-
cessing intermediates. Am J Physiol 252, G315–G319.
39 Anastasi A, Erspamer V & Endean R (1968) Isolation
and amino acid sequence of caerulein, the active peptide
in the skin of Hyla caerulea. Arch Biochem Biophys 125,
57–68.
40 Erspamer V (1994) Bioactive secretions of the amphib-
ian integument. In Amphibian Biology (Heatwold H &
Barthalmus GT, eds), Vol. 1, pp. 214–231. Surrey
Beatty & Sons, Chipping Norton, NSW, Australia.
41 Wabnitz PA, Bowie JH & Tyler MJ (1999) Caerulein-
like peptides from the skin glands of the Australian Blue
Mountains Tree Frog Litoria citropa. Part 1. Sequence
determination using electrospray mass spectrometry.
Rapid Commun Mass Spectrom 13, 2498–2502.
42 Varga G, Balint A, Burghardt B & D’Amato M (2004)

bulin A function. Surgery 122, 386–392.
50 Carrasco M, Hernanz A & De La Fuente M (1997)
Effect of cholecystokinin and gastrin on human
peripheral blood lymphocyte functions, implication
of cyclic AMP and interleukin 2. Regul Pept 70,
135–142.
51 Schnolzer M, Alewood PF, Jones A, Alewood D &
Kent SBH (1992) ‘In situ’ neutralisation protocols in
Boc-chemistry solid phase peptide synthesis: rapid, high
yield assembly of difficult sequences. Int J Pept Protein
Res 40, 180–193.
52 Alewood PF, Alewood D, Miranda L, Love S, Meuter-
mans WDF & Wilson D (1997) Rapid ‘in situ’ neutrali-
sation protocols for Boc and Fmoc solid-phase
chemistries. Methods Enzymol 289, 14–29.
53 Sarin VK, Kent SBH, Tam JP & Merrifield RB (1981)
Quantitative monitoring of solid-phase peptide synthesis
by the ninhydrin reaction. Anal Biochem 117, 147–157.
54 Kaiser E, Bossinger CD, Colescott RL & Olser DD
(1980) Color test for terminyl prolyl residues in the
solid-state synthesis of peptides. Anal Chem Acta 118,
149–151.
55 Pritchard CI & Auffret AD (1986) Quantitative analysis
in peptide synthesis: an improved method for proline
residues. Biochem Soc Trans 14, 1286–1287.
56 Jorgensen JH, Cleeland R, Craig WA, Doern G, Ferr-
aro MJ, Finegold SM, Hansen SL, Jenkins SG, Novick
WL, Pfaller MA, Preston DA, Reller LB & Swenson
JM (1993) Methods for antimicrobial tests for bacteria
that grow aerobically. In National Committee for Clini-