Red algae of the genus Laurencia Harvey
(Ceramiales, Rhodomelaceae) is a complex genus,
encompassing a large variety of morphologicallycomplex
algal species. In continuation of our work on
chemistry and biological activity studies of some Sri
Lankan seaweeds, we examined the chemistry of
MeOH extract of Laurencia hetroclada collected from
Tangalle coast. Above extract was subjected to column
chromatography followed by PTLC gave a new
brominated sesquiterpene 1 along with two known
compounds, algoane 2, and caulerpin 3. The 13C NMR
spectrum along with the DEPT experiments of 1
revealed only 15 carbon signals corresponding to four
methyls, five methylene, one methine and five
quaternary carbons. The 1H NMR (CD3OD, 500 MHz)
spectrum of 1 showed signals due to three methyls,
four methylenes, and two methine protons. The
singlets resonated at 1.25 (3H, s), 1.27 (3H, s), and
1.85 (3H, s) were assigned to Me-8’, Me-6’, and Me-9’,
respectively. The characteristic downfield proton at
4.83 (dd, J = 4.2,15 Hz) was assigned to H-4, geminal
to the Br atom, whereas the proton that resonated at
3.96 (d, J = 1.7 Hz) was assigned to the oxymethine H-
1. Furthermore, the olefinic singlet resonated at 5.26
was assigned to Ha-7’ and Hb-7. The down field
carbon atoms resonated at 110.2 and 165.2 were
assigned to olefinic carbon atoms. The positive CI MS
of 1 showed the molecular ion peak [M+H]+ at m/z
349 along with isotopic peak at m/z 351 (1:1),
indicating the presence of a Br atom in the molecule.
The above MS data were found to be consistent with
the molecular formula C15H25O4Br with 3 degrees
of unsaturation. A comparison of the NMR
spectroscopic data of 1 with those of 2 suggested a
similar skeleton with differences in substitution pattern
and unsaturation sites. From the above spectral data,
the structure of 1 was confirmed as a new natural
product 4-bromo-5-methyl-2-(3’-hydroxy-1’,3’-
dimethyl-2’ methylenecyclopentyl) cyclohexane-1,2,5-
triol. Caulerpin (3) showed a significant dose
suppressive effect with an IC50 5.8 ± 1.0 μg/mL on Tcell
proliferation assay

Dharmaratne, W.

Haroon, M.H.

Ranjithm, H.

English

IR South Eastern University of Sri Lanka

[ 5 9 ]
Abstract: Red algae of the genus Laurencia Harvey
(Ceramiales, Rhodomelaceae) is a complex genus,
encompassing a large variety of morphologically-
complex algal species. In continuation of our work on
chemistry and biological activity studies of some Sri
Lankan seaweeds, we examined the chemistry of
MeOH extract of Laurencia hetroclada collected from
Tangalle coast. Above extract was subjected to column
chromatography followed by PTLC gave a new
brominated sesquiterpene 1 along with two known
compounds, algoane 2, and caulerpin 3. The 13C NMR
spectrum along with the DEPT experiments of 1
revealed only 15 carbon signals corresponding to four
methyls, five methylene, one methine and five
quaternary carbons. The 1H NMR (CD3OD, 500 MHz)
spectrum of 1 showed signals due to three methyls,
four methylenes, and two methine protons. The
singlets resonated at  1.25 (3H, s), 1.27 (3H, s), and
1.85 (3H, s) were assigned to Me-8’, Me-6’, and Me-9’,
respectively. The characteristic downfield proton at
4.83 (dd, J = 4.2,15 Hz) was assigned to H-4, geminal
to the Br atom, whereas the proton that resonated at
3.96 (d, J = 1.7 Hz) was assigned to the oxymethine H-
1. Furthermore, the olefinic singlet resonated at  5.26
was assigned to Ha-7’ and Hb-7. The down field
carbon atoms resonated at  110.2 and 165.2 were
assigned to olefinic carbon atoms. The positive CI MS
of 1 showed the molecular ion peak [M+H]+ at m/z
349 along with isotopic peak at m/z 351 (1:1),
indicating the presence of a Br atom in the molecule.
The above MS data were found to be consistent with
the molecular formula C15H25O4Br  with 3 degrees
of unsaturation. A comparison of the NMR
spectroscopic data of 1 with those of 2 suggested a
similar skeleton with differences in substitution pattern
and unsaturation sites. From the above spectral data,
the structure of 1 was confirmed as a new natural
product 4-bromo-5-methyl-2-(3’-hydroxy-1’,3’-
dimethyl-2’ methylenecyclopentyl) cyclohexane-1,2,5-
triol. Caulerpin (3) showed a significant dose
suppressive effect with an IC50 5.8 ± 1.0 μg/mL on T-
cell proliferation assay.
Keyword: Seaweeds, Laurencia hetroclada, T-cell
proliferation assay
Introduction:
Red algae of the genus Laurencia Harvey
(Ceramiales, Rhodomelaceae) is a complex genus,
encompassing a large variety of morphologically-
complex algal species with worldwide distribution and
ambiguous taxonomy (Lyakhova EG, Kalinovsky A.I,
Dmitrenok A.S, Kolesnikova S.A, Fedorov S.N,
Vaskovsky V.E, 2006). 
In continuation of our work on the chemistry
and biological activity studies of Sri Lankan
seaweeds[2], we investigated Laurencia hetroclada
which was collected in Tangalle of  the southern coast
of Sri Lanka, and isolated a new brominated
sesquiterpenoids, 4-bromo-5-methyl-2-(3’-hydroxy-
M. H. Haroon(1, 2) and  H. Ranjith W. Dharmaratne(1, 3)
New brominated sesquiterpenes from the red
alga Laurencia hetroclada Harvey,
and their immunosuppressive activity studies 
(1) Natural Products Programme, Institute of Fundamental Studies, Kandy, Sri Lanka.
(2) Faculty of Applied Sciences, South Eastern University of Sri Lanka, Sammanthuri, Sri Lanka.
(email: haroonmh@fas.seu.ac.lk)
(3) Present Address: National Center for Natural Products Research, School of Pharmacy,
University of Mississippi, University, MS 38677, USA 
Proceedings of the Third International Symposium,
SEUSL: 6-7 July 2013, Oluvil, Sri Lanka
[ 6 0 ]
1’,3’-dimethyl-2’methylenecyclopentyl) cyclohexane-
1,2,5-triol (1), along with two known compounds,
algoane (2), caulerpin (3).
Phagocytes like neutrophils and macrophages
scavenge microbes and other harmful agents entering
the body. They start their action by the release of
reactive oxygen species (ROS) to destroy invaders in a
process collectively known as oxidative burst.  T-cells
along with other white blood cells (WBC) subset play
a major role on fight against infections (Nielsen M,
Gerwien J, Geisler C, Ropke C,  Svejgaard A,  Odum
N, 1998). These cells can cause disorders in the
immune system due to changes of stimuli.  
In this juncture  immunosuppressants are
required in the case of a hyperactive immune system,
probably caused due to inflammation or a transplant
rejection. On the other hand immunostimulating
drugs are needed when the immune system stops
fighting against infections. As a considerable number
of naturally occurring diterpenes has shown
immunosuppressant activity, diterpenes 1 and 2 along
with bisindole alkaloid caulerpin (3) were tested for
their immunosuppressant activity. The effect of the
above compounds on ROS production, by whole blood
phagocytes and in vitro activation and proliferation of
T-cells were investigated. Chemiluminescence (CL) of
these tests revealed that there is no effect of all the
tested compounds  1 and 2.  However, in the case of T-
cell proliferation, caulerpin (3) showed a significant
suppressive effect with IC50 5.8 ± 1.0 that is
comparable with the widely used immunosuppressive
drug prednisolone.
Methodology
Extraction and isolation procedure
The red alga, Laurencia hetroclada, was collected
at Tangalle. Processed alga was dried, finely powdered
and extracted with methanol using an ultrasonicator.
The MeOH extract was subjected to column
chromatography (CC) on flash Silica gel (230 -400
mess size, E-Merck) using n-hexane, ethyl acetate,
methanol and water as eluants. Further purification of
some column fractions using PTLC and gravity
column yielded three pure compounds, and their
structures were proposed using NMR and MS data. 
T-Cell proliferation assay: 
The T-cell proliferation assay was performed on
isolates as described by Nielson et al (1996). In short,
lymphocytes were isolated by gradient centrifugation
of blood in presence of lymphocyte sedimentation
medium. Cell suspension (50 μL) (106/mL) with or
without compounds (3.1-50 μg/mL) was added to each
well of a 96 well round bottom tissue culture plate. The
T-cell activator PHA (50 μL) was added in each well to
a final concentration of 5 μg/mL. The plates were
incubated at 37 oC for 72 hours (95 % humidity, 5 %
CO2). Methyl-3H thymidine 0.5 μCi was added in each
well and the plate was incubated for further 18 hours.
Cells were harvested on Type G-7 filter paper using cell
harvester. The radioactivity was measured using a -
scintillation counter. The concentration of compound
giving 50% of inhibition was calculated, comparing the
activity of compound treated cells measure as the level
of radioactivity incorporated in the proliferating cells
(CPM) with that of the non-treated cells (control cells). 
Results and Discussion
Three compounds have been isolated and the
structures of three of them were established using
spectral data.
4β-bromo-5β-methyl-2-(3’α-hydroxy-1’β,3’β-
dimethyl-2’-methylenecyclopentyl)cyclohexane-1β,2
α,5 α -triol (1)
The 1H NMR (CD3OD, 500 MHz) spectrum of 1
showed signals due to three methyls, four methylenes,
and two methine protons. The singlets resonated at
1.25 (3H, s), 1.27 (3H, s), and 1.85 (3H, s) were
assigned to Me-8’, Me-6’, and Me-9’, respectively. The
characteristic downfield proton at  4.83 (dd, J = 4.2,15
Hz) was assigned to H-4, geminal to the Br atom,
whereas the proton that resonated at  3.96 (d, J = 1.7
Hz) was assigned to the oxymethine H-1.
Furthermore, the olefinic singlet resonated at  5.26 was
assigned to Ha-7’ and Hb-7.’
M. H. Haroon and  H. Ranjith W. Dharmaratne
New brominated sesquiterpenes from the red alga Laurencia
hetroclada Harvey, and their immunosuppressive activity studies
The bromine-bearing carbon resonated at  63.0.
Four methylene carbons resonated at  34.7 (C-5’), 40.1
(C-3), 40 (C-4’), and 47.9 (C-6). These assignments of
methylene protons were supported by the COSY
spectrum of 2. The COSY spectrum showed
correlations between Ha - 3 and Hb - 3, between H -
4 and Ha - 3, between H - 4 and Hb - 3, between Hb
- 6 and Ha - 6, between Hb - 6 and H - 1, between Ha
- 6 and H – 1, and between Hb - 3 and H – 1.
Similarly, correlations were observed between Hb - 5’
and Ha - 5’, between Hb - 5’ and Ha - 4’, and between
Ha - 5’ and Hb - 4’. HMBC correlation of C-8’ ( 1.25)
with C-2 (78.9), C-1’ (53.2), C-5’ (34.7), and C-2’
(165.5) clearly indicated the connectivity of the two
structural units in the molecule (Figure 2). The relative
stereochemistry of 1was deduced by analysis of the
coupling constants and NOESY experiments. The large
coupling constants between H-4 and H-3a (15 Hz)
suggested that H-4 have an axial orientation (-
configuration). As no cross-peak could be detected
between Me (9’) and H-4, and H-1, in the NOESY
experiments, it is concluded that the Me group at the
C-5 position should be in the -configuration. Further,
the orientation of the exomethylene group in the
cyclopentane ring was predicted from the strong
NOESY correlations between H-1 and exocyclic
olefinic protons (Ha/Hb-7’). The CH3 group at C-3’
was due to have a -configuration whereas the CH3
group at C-1’ has a -configuration, as there were no
cross-peaks detected between those groups and
selected atoms in the NOESY experiments. From the
above spectral data, structure of 1 was deduced as 4-
bromo-5-methyl-2-(3’-hydroxy-1’,3’-dimethyl-2’
methylenecyclopentyl) cyclohexane-1,2,5-triol.
According to the literature, compound 1 is a new
member of the non aromatic cuparane class of
secondary metabolites. The structures of known
compounds were identified by comparison of their
spectroscopic data with the reported data (McPhail KL,
Davies-Coleman MT, Copley RCB, Eggleston
D.S,1999, Anjaneyulu A.S.R, Prakash C.V.S,
Mallavadhani U.V, 1991;, Govenkar, M.B, Wahidulla,
S, 2000, Ali MS, Ahmad VU, Mazhar F, Azhar,I,
Usmanghani, K, 1999).
Figure 1: Structures of Compounds 1 ‐ 3.
Figure 2: Key HMBC Correlaons of 1
T-Cell proliferation assay:
In order to study the immunomodulatory
potential of 1, 2 and 3 isolates, their effect on ROS
production was analyzed on whole blood phagocytes.
In our investigation, no effect on ROS production was
noted with compound 1 and 2, whereas caulerpin (3)
showed a weak activity with IC50 75.6 ± 3.1 μg/mL. In
addition, they were tested for their effect on T-cell
proliferation, and only compound 3 showed a
significant dose suppressive effect with an IC50 5.8 ±
1.0 μg/mL that is comparable with the positive control
prednisolone which is a widely used
immunosuppressive drug. Compounds 1 and 2
showed no activity on T-cell proliferation and had the
IC50 value greater than 50 μg/mL. However,
Compound 2 showed a mild suppressive effect at lower
concentrations (3.12 and 12.5 μg/mL) when compared
with higher concentration 50 μg/mL . This reverse
order effect could be attributed to the poor solubility
[ 6 1 ]
Proceedings of the Third International Symposium,
SEUSL: 6-7 July 2013, Oluvil, Sri Lanka
[ 6 2 ]
of 2. Above observation of the immunosuppressive
activity of 3 is in good agreement with the recent
report on the anti-inflammatory activity of this
bisindole alkaloid  3.
Algoane (2):
The compound 2 was isolated as a white
crystalline solid with a melting point of 190-192 °(C.
Its specific rotation was found to be +70°(c = 0.5,
MeOH) which indicated the presence of chirality in the
molecule. The negative FAB MS of LH-1 showed its
molecular ion peaks at m/z 488 corresponding to the
[M-H]+ together with isotopic peaks at m/z 490, and
492 confirmed the presence of halogen atoms in the
molecule and the molecular formula was deduced to
be C17H27Br2ClO4 with three degree of unsaturation.
The 1H NMR spectrum of 2 showed signals due to five
methyl, three methylene and four methine protons.
The 1H NMR spectrum exhibited five methyl signals at
0.88 (3H, s), 1.0 (3H, s), 1.4 (3H, s), 1.74 (3H, s), and
2.06 (3H, s). Literature review (McPhail etc.,1999)
revealed this structure is a non aromatic, cuprane–type
sesquiterpene algoane-1, which has been isolated from
South African specimen of the sea hare, Aplysia
dactylomela. 
Caulerpin (3):
The compound 3 was isolated as orange red
prisms with a melting point of 313-317 °(C. The EI MS
of 3 showed a strong molecular ion peak at m/z 398
and the molecular formula was deduced to be
C24H18N2O4. The 1H NMR signals between  7.07 and
7.40 indicated the presence of ortho substituted phenyl
rings. The protons resonated at  7.28 (1H, d, J 6, 7 = 8
Hz), 7.16 (1H, t, J 6, 7 =, J 6, 5 = 7.5 Hz), 7.07 (1H, t,
J 5, 6 = J 5, 4 7.5 Hz), 7.40 (1H, d, J 4, 5 = 7.9 Hz) were
assigned to H-7/17, H-6/18, H-5/19 and H-4/20
respectively. The methoxy groups resonated at  3.88
were assigned to Me-1' and Me-2' while the NH
protons resonated at  9.17. The protons resonated at
8.03 were assigned to H-9/H-13. Based on spectral
studies (1H, 13C NMR and MS) and comparison with
the literature values the compound was identified as
caulerpin which was previously reported from green
alga Caulerpa racemosa (Govenkar etc., 2000)
Acknowledgment
M.H. Haroon thanks the Third World Academy
of Science for Developing World (TWAS), Trieste, Italy,
for providing financial support to conduct a part of his
Ph.D. research at the H. E.J. Research Institute of
Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Pakistan.
References
Ali MS, Ahmad V.U, Mazhar F, Azhar,I, Usmanghani K.
Some chemical constituents from marine algae of
Karachi coast (Arabian Sea). Turkish Journal of
Chemistry 1999; 23(2): 181-183
Anjaneyulu ASR, Prakash CVS, Mallavadhani UV. Two
Caulerpin Analogs and a Sesquiterpene from
Caulerpa racemosa. Phytochemistry 1991; 30(9):
3041-3042
Govenkar MB, Wahidulla S. Constituents of Chondrin
armata. Phytochemistry 2000: 54(8): 979-981
Haroon MH, Premaratne SR, Dharmaratne HRW .
Chemistry and Biological activity Studies of Ulva
lactuca Linnaeus and  Amphiroa anceps. Chemtech,
Colombo, Sri Lanka, 2007, July 20-23, Poster 30, pp
79
Lyakhova EG, Kalinovsky AI, Dmitrenok AS, Kolesnikova
SA, Fedorov SN, Vaskovsky VE. Structures and
absolute stereochemistry of nipponallene and
neonipponallene, new brominated allenes from the
red alga Laurencia nipponica. Tetrahedron Letters
2006;  47(37): 6549-6552
McPhail KL, Davies-Coleman MT, Copley RCB, Eggleston
DS. New halogenated sesquiterpenes from South
African specimens of the circumtropical sea hare
Aplysia dactylomela. Journal of Natural Products 1999;
62(12): 1618-1623
Nielsen M, Gerwien J, Geisler C, Ropke C,  Svejgaard A,
Odum N. MHC Class II Ligation Induces CD58
(LFA-3)-Mediated Adhesion in Human T Cells, Exp.
Clin. Immunogenet 1998; 15: 61-68
[ 6 3 ]
Abstract: Polypyrrole (PPY) is a prime candidate for
a polymer actuator; but has the disadvantage that the
electronic conductivity decreases by two or three
orders of magnitude as the polymer is reduced. This
reduces the actuator performance. A Standard way of
overcoming this problem is to add Au or Pt layers as
thin patterns or as helices on the polymer. The present
work is an attempt to use a second more highly
conducting polymer, poly(3,4-
ethylenedioxythiophene), (PEDOT), to enhance the
electronic conductivity. 
Bilayer and multilayers with PEDOT and PPY,
each containing dodecyl benzenesulfonate (DBS) as
immobile dopant species were synthesized and their
electrochemical behaviour was investigated using
cyclic voltammetry, optical absorption spectroscopy
and electrochemical quartz crystal microbalance
(EQCM) techniques. Bilayer results show combined
characteristics of each individual polymer. In trilayer
films, the reduction of inner and outer PPY layers takes
place at two different potentials. The oxidation occurs
at one potential only. The pentalayer film confirmed
this: three reduction peaks were observed. The
separation between the two reduction peaks depends
on the thickness of PEDOT layer, the scan rate and the
concentration of cycling electrolyte. This separation
becomes smaller with the number of cycling,
indicating the enhancement of ion diffusion through
the PEDOT layer. The optical spectra of the trilayers
show clearly distinguishable peaks belonging to
PEDOT and PPY polymers, confirming the
independent behaviour of the PEDOT in the system.
Two important conclusions of relevance for actuator
performance were reached: it is possible to make
bilayer and multilayer films that do not delaminate –
the two polymers are compatible; and both polymers
are active in the redox process. Ionic diffusion in the
trilayer which is responsible for the actuation can be
controlled by choosing appropriate thicknesses for the
three layers.
Keywords: Polypyrrole, PEDOT, actuators, EQCM,
cyclicvoltammetry
Introduction
In recent years, poly(3,4-ethylene -
dioxythiophene) (PEDOT) has emerged as one of the
most important materials in the field of conducting
polymers. PEDOT is highly insoluble in almost every
solvent, exhibits quite a high conductivity, changes in
colour depending on the applied potential, is
practically transparent in the form of thin oxidized
film, is very stable in the doped state and exhibits a low
reduced band gap (ca. 1.6-1.7 eV) [1].  All these
properties make this conducting polymer useful for
many applications. On the other hand, polypyrrole
(PPy) has been used as a prime candidate in the field
of polymer actuators since it shows great volume
changes during the redox cycling. But PPy has the
disadvantage that the electronic conductivity decreases
by two or three orders of magnitude as the polymer is
reduced. This causes a decrease in actuator
performance; since only a small part of the polypyrrole
film will then be actively contributing to the actuation
[2]. A standard solution to this problem involves the
addition of an extra electron conductor on the surface
of the polypyrrole film, e.g. Au or Pt as helices 
or as thin patterns [3,4]. The use of a corrugated gold
layer on PPy improved the performance of the actuator
[5]. 
U.L. Zainudeen(1) and M.A. Careem(2)
A Cyclic Voltammetry Study on Bi Layer and
Multi Layer Conducting Polymer Films 
(1) Department of Physical Sciences, South Eastern University of  Sri Lanka,
Sammanthurai, Sri Lanka. ((email: ulzdeen@seu.ac.lk)
(2) Centre of Ionics, University of Malaya, 50603 Kuala Lumbur, Malaysia.


New brominated sesquiterpenes from the red alga Laurencia hetroclada Harvey, and their immunosuppressive activity studies

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New brominated sesquiterpenes from the red alga Laurencia hetroclada Harvey, and their immunosuppressive activity studies

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