FATTY ACID COMPOSITION AND PROSTAGLANDIN CONTENT OF THE RED SEAWEED Gracilaria sp. FROM INDONESIA

High content of polyunsaturated fatty acids (PUFAs) such as arachidonic and eicosapentaenoic acids are typical for the red alga. Analysis of fatty acid composition and prostaglandin content was conducted in the red alga Gracilaria sp. from Indonesia. Total lipid of the alga was extracted with CHCl3-MeOH (2:1, v/v). Analysis of the fatty acids composition was performed on gas chromatography (GC) equipped with omega wax column (30 m x 0,32 mm i.d., Supelco, PA, USA) and analysis of prostaglandins were carried out by HPLC on ODS column (Mightysil RP-18 GP, 250 mm x 4.6 mm, 5 μm). The content of fatty acids high for were palmitic acid (50%) and arachidonic acid (26.9%), whereas prostaglandin E2 was identified and found lower concentration (44.2 μg/gram total lipid).

The red alga G. verrucosa, which is intensively cultured in coastal areas of shrimp and fish shrimp ponds in Indonesia, has luck of information in detail regarding its lipid bioactive contents. Although there were some species of Gracilaria have been published in detail about their lipid bioactive contents those collected from Japanese Waters, such as G. vermiculophylla (Itabashi et al., 2006), G. gigas (Hsu et al., 2007), and G. asiatica (Sajiki, 1996). Another species is G. chilensis (Lion et al., 2006) collected from Chile, which contained different lipids bioactive from other Gracilaria species.
The aim of study was to evaluate fatty acids and eicosanoid composition of Indonesian red alga G. verrucosa.

Se awee d
The red seaweed Gracilaria sp. was collected from shrimp ponds located in Bone District, South Sulawesi. A part of the alga was extracted at Department of Aquaculture, Pangkep State Polytechnic of Agriculture. Another part of the alga was freezed for further extraction at Bioanalytical Chemistry Laboratory, Hokkaido University, Japan

Lipid Extraction
Each Gracilaria sp. sample was cut into pieces (3-5 mm) and homogenized for 5 min. at room temperature. Lipids was extracted by soaking the homogenate overnight in CHCl 3 / MeOH/H 2 O (2:1:0.8 by vol.). After filtration, the solvent will removed at 25 o C under reduced pressure using a rotary evaporator, and then the residual lipids was made up to a known concentration with CHCl 3 /MeOH (2:1, v/v) and stored at -30 o C until use.

Analysis of FFA Composition
FFAs were converted to methyl esters by heating at 95 o C for 1 h in 5% HCl/MeOH (Christie, 2003). Analysis of the fatty acid methyl esters was carried out using a Shimadzu GC-14A gas chromatograph (Shimadzu) equipped with an Omegawax 320 column (30 m x 0.32 mm i.d., Supelco, PA, USA). Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The split ratio was 1:50. The column temperature was maintained at 160 o C for 17 min, then elevated to 230 o C at a ramp rate of 5 o C/min. The final temperature was kept for 30 min. The injector and flame-ionization detector (FID) temperatures were set at 240 o C. Peaks were monitored on a Chromatopac C-R6A (Shimadzu) and identified by comparing retention data of the known fatty acids from some marine organisms including seaweeds (Takagi et al., 1985;Takagi et al., 1986).

Identification of Prostaglandins
For identification of eicosanoid compounds, LC/MS was used. Total lipids was subjected to LC/MS equipped with a Mightysil column, RP-18 GP (250 mm x 4.6 mm, 5 µm). Identification of eicosanoid compounds was conducted by comparing their mass spectra with those of authentic standard. For complete identification of the eicosanoid compounds, co-chromatography using authentic compounds was employed.

Determination of Prostaglandin Contents
The alga was finely sliced and placed in 100 mL-bottle. To which 30 mL of ethyl acetate and 150 µL of 1 M HCl in methanol was added. The mixtures were shaken for 5 min. and then centrifuged at 2,500 rpm for 5 min. at 20 o C. The supernatants were pipetted and evaporated under reduced pressure using a rotary evaporator at 25 o C. This procedure was repeated once more. The residue was dissolved in 200 µL of methanol as test solution. For determination of PGs, the test solution (5 µL) was subjected to HPLC (Hitachi Ltd, Tokyo, Japan) on a Mightysil column, RP-18 GP (250 mm x 4.6 mm, 5 µm). Determination of PGs was carried out by comparing the peak of PGs extracted from samples and authentic standards of PG. The contents of PGs in the samples was calculated from standard curve of the PG standards.

HPLC and LC Conditions
HPLC was performed at 40 o C using a gradient elution from acetonitrile/water (40:60, v/v) containing 0.02% acetic acid (solvent A) to 100% acetonitrile (solvent B). The mobile phase system is as follows: 0-20 min.

Prostaglandin
In the HPLC chromatograms (Figure 2), one of peaks was identified as prostaglandin E 2 (PGE 2 ). The identification was conducted by comparing the chromatograms with HPLC chromatogram of prostaglandin standards (Figure 3). This prostaglandin is also found abundantly in many species of the red algae, such as G. gigas (Hsu et al., 2007), G. asiatica (Sajiki et al., 1998), G. vermiculophylla (Illijas, 2008).
Biosynthesis pathway of prostaglandin in seaweed is still unclear. However, it was found that prostaglandin is formed from oxidation of arachidonic acid, which is likely involve cyclooxygenase as catalyst, because prostaglandin formation decreased as addition of aspirin, an anti cyclooxygenase compound, was conducted to reaction mixtures of G. vermiculophylla extract and free arachidonic acid ( Figure 4) (Illijas, 2008).
Prostaglandin function is also still unclear in the seaweed. However, several result of researches showed that formation of prostaglandins occurred when the seaweed was physically treated (Nakajima et al., 1998) so that the prostaglandins also known as secondary metabolites. The prostaglandins have also been found to be produced along with other eicosanoid compounds when the red alga Chondrus crispus was incubated with pathogen extract (Bouarab et al., 2004;Gaquerel et al., 2007). In mammals and human, prostaglandins play an important role as hormone, which co ntro l se ve ral kinds o f me tabo lism (Samuelsson, 1975).

CONCLUSION
Arachidonic acid, the dominant polyunsaturated fatty acid found in the seaweed, is precursor of synthesis of prostaglandin E 2 , the only eicosanoic compound could be identified in this study.