J Appl Phycol (2014) 26:10191027

DOI 10.1007/s10811-013-0147-8

Nutritional and bioactive properties of three edible species

of green algae, genus Caulerpa (Caulerpaceae)
Thilahgavani Nagappan & Charles Santhanaraju Vairappan

Received: 24 May 2013 / Revised and accepted: 5 September 2013 / Published online: 15 September 2013
# Springer Science+Business Media Dordrecht 2013

Abstract The green algae genus Caulerpa is coenocytic, and

the thallus consists of only one cell with many nuclei. It is
widely distributed in the tropical seas. In the Southeast Asian
waters, there are at least ten known species. Three species,
particularly Caulerpa racemosa var. clavifera f. macrophysa
(Ktzing) Weber-van Bosse, C. racemosa var. laetevirens
(Montagne) Weber-van Bosse, and Caulerpa lentillifera J.
Agardh are widely consumed. The proximate analysis and
secondary metabolite composition of these three species were
determined to describe their lipid and nutritional values.
Glycolipids and phospholipids were the major lipid classes,
with significant levels of triacylglycerol. Polyunsaturated fatty
acids (PUFA) were the major fatty acids of all the three species.
Typical n-3 and n-6 PUFA such as 18:3n-3, 18:4n-3, 20:5n-3,
18;2n-6, and 20:4n-6 were found in significant amount in all
these three species. All three species contained a red-pigmented
secondary metabolite determined as caulerpin. All three extracts exhibited potent antimicrobial activity against human
food pathogenic bacteria and anti-inflammatory activity against
the murine macrophage cell line, RAW 264.7.
Keywords Seaweed . Caulerpa . Caulerpin . Fatty acids .
Anti-inflammatory

Introduction
Seaweeds are an important coastal produce that has been a
valuable resource for human consumption and extraction of
industrial chemicals (Kumari et al. 2013). Edible seaweeds are
T. Nagappan : C. S. Vairappan (*)
Laboratory of Natural Products Chemistry, Institute for Tropical
Biology and Conservation, Universiti Malaysia Sabah,
88 440 UMS Road, Kota Kinabalu, Sabah, Malaysia
e-mail: csv@ums.edu.my

widely consumed in Asian countries as fresh, dried, salted, or as

ingredients in prepared foods (Hong et al. 2007). In recent
years, globalization of consumer markets has brought about
an increasing demand of a once traditional food, diminishing
the boundaries of human food ingredients to certain geographical regions. In addition, there is also an urgent quest for
nonconventional food sources to enhance and supplement the
nutritional quality of human foods. Interest in supplementing
human food with antioxidants and neutraceuticals from natural
resources has been on the rise as synthetic agents have been
suspected to be a possible cause of liver damage and carcinogenesis (Farag et al. 2003; Tang et al. 2001).
To date, algal species that have been widely utilized as sea
vegetables are mainly red and brown algae. The green algae are
comparatively uncommon species utilized directly as food
aside of the genus Monostroma. Recent reports revealed that
Ulvales are used in Asia as food condiment and as a nutritional
supplement in China, Japan, USA, France, and Chile. Members
of this genus are harvested to prepare aonori, which is
included in a great variety of dishes (Pena-Rodriguez et al.
2011). However, green algae of the genus Caulerpa
(Caulerpaceae), particularly Caulerpa racemosa (Forsskl) J.
Agardh and Caulerpa lentillifera J. Agardh are widely consumed in Southeast Asian countries and Japan. Caulerpa is a
siphonaceous green alga that grows on sandy rock bottoms in
the upper sublittoral zone of tropical coral reefs (Horstmann
1983; Mao et al. 2011). In some Asian countries like the
Philippines and Malaysia (Borneo), this seaweed has almost
been eradicated in nature due to intensive harvesting. When
prepared as a salad or vegetable, it is considered a delicacy in
the Philippines, Indonesia, Malaysia (Sabah), and Japan
(Okinawa). Due to its demand, culture of these varieties in
shallow tidal ponds was initiated over 30 years ago in Mactan
Island, Cebu (Philippines) and distributed widely in the
Philippines, even exported to Japan and Taiwan. In Thailand,
C. lentillifera is often cultured in shrimp ponds as a water

1020

treatment measure. It is also a popular material for animal feed

among the coastal villages in Southern Thailand (Ratanaarporn and Chirapart 2006).
Nutritional evaluations of C. lentillifera and C. racemosa
have been reported by Kumar et al. (2011) and Ratana-arporn
and Chirapart (2006). Nutritional data for C. lentillifera were
for populations from shrimp culture ponds and might not
reflect the natural nutritional composition in wild or cultured
populations. In addition, values reported for C. racemosa by
Kumar et al. (2011) could not be attributed to any particular
variety since C. racemosa is reported to exist in many varieties. Out of these varieties, only C. racemosa var. clavifera f.
macrophysa (Sonder ex Ktzing) Weber-van Bosse and C.
racemosa var. laetevirens (Montagne) Weber-van Bosse are
utilized as edible species.
Edible seaweeds have often been associated with various
health benefits (Cengiz et al. 2011). In this regard, only the
antioxidant potential of the edible Caulerpa is reported. More
information is needed to better understand the nutritional
profiles of the exact species that are consumed and their
biological potentials. An in-depth evaluation of the edible
varieties describing their nutritional values and the presence
of secondary metabolites would be an important aspect to
better understand their importance as marine salad and functional food.
In the north Borneo Island (Sabah, Malaysia), three species
from the genus Caulerpa are widely consumed: C. racemosa
var. clavifera f. macrophysa, C. racemosa var. laetevirens,
and C. lentillifera. They are considered as delicacies and eaten
raw as salad especially by locals in Sabah and Sarawak. C.
lentillifera is one of the favored species due to its bright green
color and soft and succulent texture. One the other hand, C.
racemosa var. clavifera f. macrophysa and C. racemosa var.
laetevirens is light green, with simple radical branches bearing
transluscent spherical overlapping grape-like structure (Shafik
and Manawy 2008). However, not much of their nutritional
values, secondary metabolite composition, or their biological
potentials is documented. This paper is aimed to address those
issues.

J Appl Phycol (2014) 26:10191027

rinsed three times in double-distilled water (DDW) and airdried at 24 C away from direct sunlight.
Proximate analysis Proximate analysis of moisture and ash
was determined based on AOAC methods (1995). Moisture
content in the specimens was determined by heating the
seaweeds at 105 C until constant weight was obtained. The
ash contents were estimated by heating the seaweeds overnight in a furnace at 525 C. To evaluate total lipid content,
lipids were extracted from the samples with 100 % petroleum
ether as described by Yaniv et al. (1999) with slight modification. Total protein content was determined through nitrogen
analysis following the standard micro-Kjeldahl method system (N 6.25) (Kumari et al. 2011). To analyze the composition of fatty acids, 10 g of extracts was subjected to Si gel
column chromatography with gradient hexane (Hex)/ethyl
acetate (EtOAc) (9:1, v/v) to obtain the lipid fraction. Then,
the lipid was converted to the methyl esters (FAME) by
transmethylation using sodium methoxide as described by
Christie (1990) with minor modification. The converted fatty
acids were analyzed using a Shimadzu QP-2010 gas chromatograph (GC) equipped with a silica BPX70 capillary column (60 m, with film thickness of 0.25 m) coupled with
mass chromatography (MS) detector using helium as carrier
gas. The run method was through a temperature gradient from
150 up to 240 C and total analysis time of 60 min.
Chemical analysis Partially dried algal thallus of C. racemosa
var. clavifera f. macrophysa, C. racemosa var. laetevirens,
and C. lentillifera (20 g) was soaked in MeOH for 3 days. The
MeOH solution was concentrated in vacuo and partitioned
between EtOAc and water (H2O). The EtOAc solution was
washed with distilled water, dried over anhydrous sodium
sulfate, and evaporated to leave dark-green oil. Chemical
profiles of all the crude extracts were done by spotting crude
extracts on silica gel F254 thin layer chromatography plates
(TLC) and developed in toluene (100 %) and Hex/EtOAc
(3:1) solvent systems, visualized by UV light (254 nm) and
molybdophosphoric acid, and heated. The crude extracts of
these three seaweeds (1.0 g) were then fractionated by silica
gel column chromatography with a step gradient (Hex/EtOAc
in the ratio of 9:1, 8:2, 7:3, 5:5, and 100 % EtOAc).

Materials and methods

The Caulerpa species are commonly found in a variety of
habitats including reef flats, reef slopes, dead coral beds,
sandy lagoons, and sea grass beds. All the three studied
species of Caulerpa were collected by scuba divers at two
sandy intertidal seaweed beds at Sepanggar Bay (6050N
116090E), Kota Kinabalu, Sabah. Specimens were cleaned
of epiphytes, sand, and organic debris, thoroughly washed in
seawater at site before transported in sterile bags at 4 C in a
chiller to the laboratory. In the laboratory, all the algae were

Bioactive potential evaluations

Minimum inhibitory concentration assay The minimum inhibitory concentration of three Caulerpa extracts and caulerpin
were determined based on Nagappan et al. (2011) with slight
modifications. Minimum inhibitory concentration (MIC) was
assayed using twofold microdilution broth method. About
0.1 mL of dilutions was dispensed into each of the sterile 96
wells of a standard microdilution tray. Each well constitute of

J Appl Phycol (2014) 26:10191027

5105 colony forming units (CFU mL1) of test bacteria,

serially diluted samples, and respective growth medium.
Trays were incubated at 37 C for 24 h and checked for the
presence/absence of whitish sedimentation that indicates dead
bacterial cells. The growth end points were determined by
comparing the amount of growth in the wells containing test
samples with that in the control wells. The acceptable growth
(2 mm button or definite turbidity) must occur in the positive
control well. The highest MIC value was determined when a
single skipped well occurred. Dimethyl sulfoxide (DMSO)
and gentamicin were used as negative and positive controls
for this assay, respectively. A total of 50 L of DMSO was
used to dissolve the chemical into the assay matrix, and it is
the lowest amount with no cytotoxic effect. The food pathogenic bacterial strains tested were Escherichia coli ,
Staphylococcus aureus, Streptococcus sp., and Salmonella
sp. Assays were carried out in triplicate for each concentration
tested.
Cytotoxicity assay Cytotoxicity of extracts and caulerpin was
evaluated based on 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma Co., USA) assay described by Vairappan et al. (2013) with minor modifications.
Murine macrophage cell line (RAW 264.7) was cultured in
Dulbecco modified Eagle's medium (DMEM) (Gibco Inc.,
USA) supplemented with 100 U mL 1 of penicillin,
100 g mL1 of streptomycin, and 10 % fetal bovine serum
(FBS) (Gibco Inc., USA). The cells were maintained and
incubated at 37 C with 5 % of CO2 supply. These cells were
seeded in 96-well plate at a concentration of 1.0 105
cells mL1. Prior to 16 h of incubation period, the cells were
treated with 10 L of extracts and caulerpin (20 g mL1) and
incubated at 37 C under a humidified atmosphere for 24 h.
Soon after incubation, 50 L (2 mg mL1) of MTT stock
solution was added to each of the wells to a total reaction
volume of 200 L. After 3 h of incubation, the plates were
centrifuged for 5 min at 800 rpm, and the supernatant was
aspirated. The formazan crystals in each well were rinsed with
150 L of DMSO, and absorbance was measured using an
enzyme-linked immunosorbent assay (ELISA) reader
(Sunrise; Tecan Co. Ltd., Australia) at 540 nm. Relative cell
viability was determined in accordance with the quantity of
MTT converted to the insoluble crystals. DMSO was used as
control. The data were expressed as mean percentages of the
viable cells versus the respective control.
Determination of nitric oxide production Detection of nitric
oxide production was carried out by plating RAW 264.7 cells
(1105 cells mL1) on 24 well plates, and after 16 h, the cells
were preincubated with the extracts and caulerpin at 37 C for
1 h. The same plate was further incubated with LPS
(1 g mL1) under the same condition. The quantity of nitrite
accumulation in the culture medium was measured after the

1021

incubation as an indicator of NO production. Briefly, 100 L

of cell culture medium was mixed with 100 L of Griess
reagent (1 % sulfanilamide and 0.1 % napthylethylenediamine
dihydrochloride in 2.5 % phosphoric acid). The mixture was
then incubated at room temperature for 1 min before the
optical density at 540 nm was measured using an ELISA
microplate reader.
Lactate dehydrogenase cytotoxicity assay Lactate dehydrogenase activity was investigated by plating RAW 264.7 cells
(1.5105 cells mL1) on 96-well plates, and after 16 h, the
cells were on extracts and caulerpin at 37 C for 1 h. Then, the
cells were further incubated for another 24 h with LPS
(1 g mL1) at 37 C. Immediately after incubation, the
production of lactate dehydrogenase (LDH) level in the culture medium by using a LDH cytotoxicity detection kit
(Promega, USA) was determined. A volume of 50 L of
reaction mixture was added to each well, and the reaction
was incubated for 30 min in the dark at room temperature.
To terminate the reaction, 50 L of stop solution was added to
each well, and the absorbance was measured at 490 nm using
a microplate reader.
Determination of pro-inflammatory cytokines (TNF- and IL6) The inhibitory effect of extract on the production of proinflammatory cytokines from LPS-stimulated RAW 264.7
cells was determined according to the method described by
Cho et al. (2000) and Vairappan et al. (2013). Briefly, RAW
264.7 cells (1105 cells mL1) were pretreated with caulerpin
for 2 h and then treated with LPS (1 g mL1) to allow
production of pre-inflammatory cytokines for 24 h.
Supernatants were used for the assay by using an ELISA Kit
(R&D Systems, USA) according to the manufacturer's
instructions.

Statistical analysis
All data are expressed as mean standard deviation of three
determinations. The statistical significance of differences
(P < 0.05) was estimated by one-way analysis of variance
(ANOVA) using SPSS 10.0.

Results
Proximate composition
The proximate compositions based on the dry weight of the
specimens analyzed are shown in Table 1. Measurable differences in nutritional composition were evident between the three
edible Caulerpa species. The moisture content was significantly higher in C. racemosa var. laetevirens (93.20 %) followed

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J Appl Phycol (2014) 26:10191027

Table 1 Proximate composition of moisture, carbohydrate, ash, lipid, fiber, and protein in different species of Caulerpa
Species

Moisture (% WW)

Carbohydratea (% DW)

Ash (% DW)

Lipid (% DW)

Fiber (% DW)

Protein (% DW)

C. lentillifera
C. racemosa var. cm
C. racemosa var. lv

87.050.50
91.361.09
93.200.81

44.022.01
52.811.89
50.191.35

29.611.50
23.811.35
26.742.17

2.870.03
2.210.05
2.110.04

4.120.16
3.110.32
3.180.38

19.381.48
17.360.72
17.280.63

Calculated by difference (100 proteinlipidashfiber). All values are mean standard deviations; n =3
DW dry weight, WW wet weight
a

C. racemosa var. clavifera f. microphysa (C. racemosa var. cm) and C. racemosa var. laetevirens (C. racemosa var. lv)

by C. racemosa var. clavifera f. macrophysa , and C.

lentillifera with 91.36 and 87.05 %, respectively. Ash was the
most abundant component of dried seaweed material for all the
three Caulerpa species, and it ranged between 23.81 and
29.61 % of dried weight. The highest ash value was recorded
for C. lentillifera (29.61 %) followed by C. racemosa var.
laetevirens (26.74 %) and C. racemosa var. clavifera f.
macrophysa (23.81 %). The protein contents in all three specimens varied, but C. racemosa var. clavifera f. macrophysa
and C. racemosa var. laetevirens had a very similar value
17.36 and 17.28 %, respectively. C. lentillifera had a higher
protein value of 19.38 %. The total lipid content in all three
species was low (2 %) and not significantly different. The
highest lipid content was for C. lentillifera with a value of
2.82 %. Total fiber content has always been an important
parameter in any nutritional evaluation of food ingredient or
organic food. The fiber content in C. lentillifera was the highest
with a value of 4.12 %, with both the C. racemosa having a
similar value of approximately 3 %. Carbohydrate level was
lowest in C. lentillifera (44.02 %), followed by C. racemosa
var. laetevirens (50.19 %) and C. racemosa var. clavifera f.
macrophysa (52.81 %), respectively.
Fatty acid composition
Data on fatty acid compositions of the three species of
Caulerpa are presented in Table 2. Fatty acids ranged from
C6 to C24, which included 17 types of saturated fatty acids
(SFA), 9 types of monounsaturated fatty acids (MUFA), and 8
types of polyunsaturated fatty acids (PUFA). The best fatty
acid profile could be seen in C. lentillifera with 42.71 %
saturated fatty acids, 18.29 % monounsaturated fatty acids,
and 38.07 % polyunsaturated fatty acids. In contrast, both the
varieties of C. racemosa exhibited higher amount of SFA and
lower amount of MUFA and PUFA as shown in Table 2.

In this investigation, the prominent secondary metabolite was

studied and identified based on spectroscopic techniques.
Partially air-dried seaweeds (500 g) were exhaustively
extracted in methanol, and the resulting methanol solution
was concentrated in vacuo to yield a dark-green extract.
Crude methanol extracts were further partitioned between
diethyl ether (Et2O) and distilled water. The organic fractions
were then dehydrated in anhydrous sodium sulfate and concentrated to yield 0.8, 1.0, and 1.8 % crude extracts for C.
racemosa var. clavifera f. macrophysa, C. racemosa var.
laetevirens, and C. lentillifera, respectively.
A total of 500 mg of C. racemosa var. clavifera f.
macrophysa, C. racemosa var. laetevirens, and C. lentillifera
crude extract was subjected to silica gel normal-phase column
chromatography in a Hex/EtOAc organic solvent gradient
system. Separation of the secondary metabolite was carried
out based on polarity exclusion technique and utilization of
normal-phase column chromatography with gradient solvent
system-facilitated purification process. A total of six fractions
were collected, concentrated, and subjected to preparative thin
layer chromatography (PTLC) to isolate the active ingredient
present in all three species investigated. These were further
subjected to 1D measurements (1H-NMR, 13C-NMR) and 2D
measurements (1H-1H-COSY, HSQC, HMBC, and NOESY)
as well as high-resolution mass spectroscopy (IT-TOFMS)
spectroscopy experiments. Based on independent structural
elucidation and comparison of the spectroscopic data with
published data (Anjaneyulu et al. 1991; Mao et al. 2011), the
metabolite was identified as caulerpin (orange-red in color).
The chemical structure of caulerpin is shown in Fig. 1. The
yields of caulerpin in the investigated Caulerpa species are as
follows: 2.6 % in C. racemosa var. clavifera f. macrophysa,
1.8 % in C. racemosa var. laetevirens, and 3.5 % mg in C.
lentillifera.

Antimicrobial activity
Isolation and identification of secondary metabolites
Secondary metabolites are often attributed to interesting biological activities that could be an important component of
natural products used as human food (Radhika et al. 2012).

Antimicrobial potential of three different Caulerpa extracts

and caulerpin was evaluated against four food pathogensE.
coli, S. aureus, Streptococcus sp., and Salmonella sp. The
crude extract of C. lentillifera was found to inhibit the tested

J Appl Phycol (2014) 26:10191027

Table 2 Fatty acid compositions
in different species of Caulerpa

1023

Fatty acid

Relative retention time

Percentage of total fatty acids

C. l

C. cr

C. rl

Saturated

Composition of fatty acid is reported in means SD (% of total

FAME); n=3
C. l, C. lentillifera; C. cr,
C. racemosa var. clavifera
f. microphysa; C. rl, C. racemosa
var. laetevirens ; SFA saturated
fatty acids; MUFA monosaturated
fatty acids; PUFA polysaturated
fatty acids; nd not detected; UI
unsaturation index

C6:0
C8:0
C10:0
C11:0
C4:0
C12:0
C13:0
C14:0
C15:0
C16:0
C17:0
C18:0
C20:0
C21:0
C22:0
C23:0
C24:0
SFA
Monosaturated

10.234
11.264
12.878
14.054
15.171
16.502
17.976
20.060
22.000
24.480
25.948
27.698
32.696
35.962
36.104
38.768
41.017

0.300.06
1.100.12
6.400.81
1.101.94
2.300.37
0.691.21
1.540.99
2.920.32
2.102.46
8.740.01
3.361.75
3.810.28
1.980.59
1.620.07
1.150.21
2.050.17
1.550.31
42.713.38

nd
2.230.58
7.281.75
2.500.03
nd
nd
2.340.69
nd
nd
10.820.61
5.960.03
5.860.47
2.411.12
4.132.67
1.850.99
6.343.14
4.590.82
66.313.21

0.362.96
2.260.29
7.021.11
1.700.83
4.350.29
0.790.03
1.190.07
1.290.73
0.640.02
12.652.55
4.270.19
5.640.35
3.140.12
1.790.08
0.950.07
6.560.94
3.230.13
57.833.85

C14:1
C15:1
C16:1
C17:1
C18:1n9t
C18:1n9c
C20:1
C22:1n9
C24:1
MUFA
Polysaturated
C18:2n6t
C18:2n6c
C20:2
C18:3n6
C18:3n3
C20:3n6
C20:4n6

20.586
22.934
25.107
27.124
28.492
29.080
34.117
38.197
41.360

1.500.11
2.540.02
3.910.93
2.671.02
1.410.33
0.930.05
1.690.19
0.850.22
2.790.60
18.290.36

1.020.13
2.320.17
2.642.12
1.410.75
1.080.97
0.860.23
1.900.23
0.520.61
1.150.31
12.901.51

1.190.06
2.000.09
2.451.02
1.730.11
1.280.09
0.490.04
1.610.03
0.300.11
1.200.06
12.250.88

29.418
30.909
31.526
33.242
35.216
36.815
37.635

4.141.34
4.881.01
4.270.93
5.990.73
5.151.13
3.300.21
6.700.53

4.461.11
3.091.42
3.861.39
4.400.77
3.452.32
2.380.44
5.860.21

4.170.21
3.142.41
3.230.83
4.380.31
3.393.65
2.370.02
5.610.05

44.000

3.640.64
38.074.11
20.161.43
14.270.25
118.545.31
2.850.27

2.770.72
30.273.98
15.401.67
12.100.13
93.575.01
3.250.02

3.391.01
29.683.72
15.082.04
11.210.31
76.282.96
2.900.07

C22:6n3
PUFA
C18 PUFA
C20 PUFA
UI
Ratio n-6/n-3

1024

J Appl Phycol (2014) 26:10191027

H

CO2Me
H
N

N
H
MeO2C

Search for the active ingredient was initiated by subjecting the

extracts for bioassay-guided separation. This approach
yielded an orange-red pigmented compound as the active
ingredient in the extracts investigated which was confirmed
to be caulerpin (Fig. 1). This active metabolite was utilized
to evaluate the dynamics of pro-inflammatory cytokine
activity.

Fig. 1 Structure of caulerpin isolated from all three Caulerpa species

organism with the lowest minimum inhibition concentration,

and the results are displayed in Table 3. The MIC value of
Caulerpa extracts against E. coli falls within the range of
136.50 to 360.50 mg mL1. C. lentillifera exhibited the lowest
MIC value against E. coli followed by C. racemosa var.
clavifera f. macrophysa and C. racemosa var. laetevirens.
The MIC values of the extracts against S. aureus were found
to be in the range of 125.25 to 375.75 mg mL1, while the
MIC value against Streptococcus sp. ranged from 275.25 to
450.75 mg mL1. The extracts were found to be less inhibiting
for Streptococcus sp. The MIC value against Salmonella sp.
falls in the range of 140.50 to 345.25 mg mL1. The antimicrobial activity of caulerpin was tested on the same organisms
with the result of MIC falling at constant concentration:
5.25 mg mL1 against E. coli, S. aureus, and Salmonella sp.
On the other hand, the MIC value of caulerpin against
Streptococcus sp. was higher15.50 mg mL1.

Effects of caulerpin on the production of pro-inflammatory

cytokines (TNF- and IL-6)
The effects of caulerpin on pro-inflammatory cytokines
(TNF- and IL-6) were investigated via LPS-activated macrophages (Table 4). The macrophage RAW 246.7 cells were
incubated in increasing concentration of caulerpin (25, 50, and
100 g L1). The quantity of cytokines secreted from the
cells was monitored by enzyme-linked immunosorbent assay.
It was clearly observed that caulerpin lowers the production of
TNF- and IL-6 in a dose-dependent manner. Comparatively,
the lowest effect of caulerpin was observed in IL-6 production. Based on this finding, it can be postulated that caulerpin
contributed to the anti-inflammatory effects of the Caulerpa
by decreasing NO and downregulating the expression of proinflammatory cytokines such as TNF- and IL-6 in LPSactivated macrophages.

Nitric oxide inhibition of crude extracts

Discussion

The macrophage RAW 264.7 cells pretreated with three

Caulerpa extracts (50 g mL1) showed various degrees of
reduction in the production of nitric oxide (NO) after LPS
stimulation (Fig. 2). Extract of C. lentillifera was found to best
suppress the NO production, followed by C. racemosa var.
laetevirens and C. racemosa var. clavifera f. macrophysa.
The cytotoxic effects of Caulerpa extracts were not
manifested and confirmed by the absence of LDH release.
Hence, it is evident that the inhibitory effect on NO production
was not contributed by the cytotoxicity of the tested extracts.

Measureable differences in nutritional composition were evident among the three Caulerpa species investigated. It was
apparent that C. lentillifera has the best nutritional composition followed closely by C. racemosa var. clavifera f.
macrophysa and C. racemosa var. laetevirens. The ash content in this three Caulerpa species was found to be much
higher than that found spinach. High content in ash also
indicates the presence of diverse minerals (Matanjun et al.
2008). The protein content varies significantly between the
species ranging from 17.28 to 19.38 %. C. racemosa var.

Table 3 Antimicrobial activity of Caulerpa extracts and active ingredient, caulerpin against food pathogens
Test organism

Escherichia coli
Staphylococcus aureus
Streptococcus sp.
Salmonella sp.

Minimum inhibitory concentrations (MIC/mg mL1)

C. l

C. rc

C. rl

Caulerpin

136.500.85
125. 253.78
175. 250.23
140.500.55

245.252.11
225.50 0.45
450.751.09
275. 20 0.66

360.502.14
375.750.07
450. 250.42
345. 250.35

5.250.22
5.250.79
15.500.13
5.250.62

All values are reported in mean standard deviation; n=3

C. l, C. lentillifera; C. cr, C. racemosa var. clavifera f. microphysa; C. rl, C. racemosa var. laetevirens

J Appl Phycol (2014) 26:10191027

1025

Fig. 2 Effect of three Caulerpa

species crude extracts on
lipopolysaccharide (LPS)stimulated nitric oxide (NO) and
lactate dehydrogenase (LDH)
release in RAW 264.7 cells. (NO produced by LPS-induced
cells, - NO produced by three
Caulerpa species crude extracts
treated LPS-induced cells, LDH production in cells)

N=3

N=3
N=3

laetevirens was found to be higher in carbohydrate and moisture content. Among the three species of Caulerpa studied, C.
lentillifera and C. racemosa var. clavifera f. macrophysa had
a higher percentage of 18:3n3 and 18:2n6 as the dominant C18
PUFA content, whereas -linolenic acid as dominant PUFA is
only found in C. racemosa var. laetevirens. Taxonomic differences may also affect the quantitative characteristics of fatty
acids. Algae of the order Ulvales have been reported to contain
a high level of -linolenic acid (Blazina et al. 2009), while
algae from the genus Chaetomorpha were found to be rich in
linolenic acid but poor in -linolenic acid. However, the ratio of
omega-6 fatty acids to omega-3 fatty acids was found to be
highest in C. racemosa var. clavifera f. macrophysa (3.25)
followed by C. racemosa var. laetevirens (2.90) and C.
lentillifera (2.85). The World Health Organization (WHO)
currently recommends that the ratio of omega-6 to omega-3
fatty acids should not exceed 10 in the daily diet. Although
seaweeds are low in total lipids, their PUFA content is relatively
higher than those found in terrestrial vegetables (Matanjun et al.
2008; Kumar et al. 2011). Hence, the presence of classes of
PUFA and MUFA in the investigated species is beneficial to
health for human consumption.
As all green macrophytic algae are capable of synthesizing
long-chain C 20 PUFAs such as docosahexaenoic acid
(22:6n3), arachidonic acid (20:4n6), and dihomo--linolenic
acid (20:3n6), the presence of all these three PUFAs was
found to be less than 5 % in all the specimens studied.
These results of variations between different species of the
same genus were more likely due to the interspecific variations rather than the geographical and environmental conditions (Kumari et al. 2013). According to Kendel et al. (2013),
the environmental conditions, such as illumination, will influence their nutritional content and fatty acid ratio. Eventually,
differences in light tolerance will affect the degree of

unsa tu ratio n in glyc olip id pro ductions in algae

(Khotimchenko and Yakovleva 2005).
Members of the genus Caulerpa have been studied to
better understand the inherently available secondary metabolites. The compounds have exhibited interesting biological
activities as well as pharmaceutical importance (Vairappan
2004). Caulerpin, a cyclooctatetraene ring containing
orange-red pigment, is commonly present in most of the
species in the Caulerpaceae. According to Raub et al.
(1987), caulerpin plays an important role as a plant growth
promoter. The presence of caulerpin in a form of dimer of
indole-3-arcylic acid was found to behave more like indole
auxins in healthier growth of the species Caulerpa . Based on
the findings from this investigation, caulerpin also demonstrates antimicrobial effect against food pathogens. It
is not surprising that this bioactive compound is involved either in the chemical defense against herbivore
pressure or within the framework of interspecific competition (antifeedant and antifouling effects) in the marine environment (Dumay et al. 2002).
Bacterial infection leads to high rate of mortality in aquaculture organisms and human population. Infections caused

Table 4 Effects of caulerpin on the production of TNF- and IL-6

Treatment (g mL1)

TNF- (pg mL1)

IL-6 (pg mL1)

Control
LPS (1)
Caulerpin (25)
Caulerpin (50)
Caulerpin (100)

82.2411.42
1,204.21163.27
1,140.62107.35
846.5138.56
342.6313.76

8.760.85
425.521.50
246.8521.82
183.2810.84
94.728.94

All values are reported in meanstandard deviations; n =3

1026

by E. coli, S. aureus, and Salmonella sp. cause illnesses like

mastitis, abortion, upper respiratory complications, diarrhea,
and typhoid fever. As these bacterial strains are becoming
more resistant towards antibiotics, the necessity towards the
development of new drugs is on the rise (Kumar et al. 2011).
Many seaweed-derived bioactive and pharmacologically important compounds such as alginate, carrageen, and agar as
phycocolloids have been utilized in medicine and pharmacy
(Kandhasamy and Arunachalam 2008). Therefore, the approach was taken to evaluate the antibacterial potential of C.
lentillifera, C. racemosa var. clavifera f. macrophysa, and C.
racemosa var. laetevirens. Overall, C. lentillifera was found to
be more active against E. coli, S. aureus, Streptococcus sp.,
and Salmonella sp. with the MIC values ranging from 125.25
to 140.50 mg mL1. Specifically, caulerpin gave a MIC value
of 5.25 mg mL1 against E. coli, S. aureus, and Salmonella sp.,
and 15.50 mg mL1 against Streptococcus sp. Shanmugapriya
and coworkers (2008) reported that the bactericidal effect in
algae could be due to the presence of amino acids, terpenoids, phlorotannins, acrylic acid, phenolic compounds, halogenated ketones and alkanes, cyclic polysulfides, and fatty
acids.
Although the genus Caulerpa had been a subject of research for years, no reports have been documented pertaining
to its anti-inflammation activity. The anti-inflammation potential was studied using murine macrophage RAW 246.7 cells.
There was no cytotoxicity effect exhibited by the extracts of
the three different Caulerpa species and caulerpin on RAW
246.7 cells by using MTT assay. The absence of LDH release,
an indicator of cell toxicity and suppression of NO production,
confirmed the nontoxic nature of the Caulerpa extracts and
the pure form of the active ingredient. Caulerpin was also
found to inhibit the production of pro-inflammatory cytokines
TNF- and IL-6 in a dose-dependent manner. The bioactivity
of caulerpin could also be structure-related activity. The presence of two indole moiety bearing nitrogen functionality
could have given rise to the bioactive attribute. In an event
of inflammation, TNF- as a major pro-inflammatory cytokine releases macrophage in response, while IL-6 as an endogenous mediator will trigger LPS-induced fever. Therefore,
it is noted that caulerpin has the potential to be regarded as an
anti-inflammatory agent as it inhibits the production of IL-6
mediator with minimum concentration (Vairappan et al.
2013).
C. lentillifera was found to be a rich source of protein,
lipid, MUFAs, and PUFAs. The presence of dietary omega-3
PUFA in all three species is known to reduce heart disease risk
and decrease low-density lipoprotein (LDL) cholesterol. The
extracts were also found to inhibit common food pathogenic
bacterial strains at a minimum inhibition concentration level.
Meanwhile, caulerpin, one of the active ingredients isolated
from all the investigated Caulerpa species, showed significant
potential as an anti-inflammatory agent. Hence, the

J Appl Phycol (2014) 26:10191027

consumption of Caulerpa especially C. lentillifera as a functional food rich in elements of nutritional properties along
with antibacterial and anti-inflammation properties is suggested to be beneficial to health.

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