New fatty acids from the Red Sea sponge Mycale euplectellioides

Chemical investigation of the Red Sea sponge Mycale euplectellioides afforded two new compounds; hexacosa-(6Z,10Z)-dienoic acid methyl ester (1) and hexacosa-(6Z,10Z)-dienoic acid (2), along with two known compounds: icosa-(8Z,11Z)-dienoic acid methyl ester (3) and β-sitosterol (4). The structures were elucidated by the interpretation of their spectral data. The total methanol extract (TME) of the sponge exhibited potent antimicrobial activity against the different strains at a concentration of 100 mg/mL. All tested fractions did not exhibit any activity against Serratia marcescens and tested fungal strains. The TME and different fractions displayed anti-inflammatory and antipyretic activities at doses of 100 and 200 mg/kg compared with indomethacin (8 mg). The TME exhibited a remarkable hepato-protective effect in CCl4-induced liver damage compared with silymarin. Furthermore, compounds 1 and 2 displayed weak activity against A549 non-small cell lung cancer, the U373 glioblastoma and the PC-3 prostate cancer cell lines.


Introduction
The sea is an important source of new natural compounds (Skropeta 2008;Blunt et al. 2013). The Red Sea has high levels of marine biodiversity and great seasonal fluctuations of air and water temperatures. The marine environment is proven to be a rich source of natural products that have a wide variety of biological activities. During the last three decades, more than 15,000 natural products have been isolated from marine organisms. 1 Sponges have evolved a vast number of bioactive secondary metabolites to protect themselves against predation, overgrowth by other organisms and bacterial and fungal infections (Proksch et al. 2003;Schupp et al. 2009). Sponges of the genus Mycale are a rich source of bioactive natural compounds with diverse chemical structures. The mycalamides (triisoxazole-containing macrolides) (Perry et al. 1990), mycalolides (Fusetani et al. 1989), diterpenoid rotalins (Corriero et al. 1989), mycalisines (Kato et al. 1985), polybrominated C-15 acetogenins (Giordano et al. 1990), brominated isocoumarins (Fusetani et al. 1991) and norterpene cyclic peroxides (Capon & MacLeod 1987) are examples of compounds isolated from the genus Mycale. In this study, we report the isolation and structural elucidation of two new compounds (1 and 2), together with two known compounds (3 and 4) from the Red Sea sponge Mycale euplectellioides, which are reported here for the first time from the sponge ( Figure 1). The total methanol extract (TME), EtOAc and aqueous fractions were evaluated for their antimicrobial, anti-inflammatory, antipyretic and hepato-protective activities. The in vitro growth inhibitory activity of the isolated compounds against non-small cell lung cancer (NSCLC), glioblastoma (GBM) and PC-3 prostate cancer cell lines was evaluated.

Results and discussion
Compound 1 was isolated as an oily substance. The gas chromatograph (GC)-mass spectrometer (MS) of 1 revealed a molecular ion peak at m/z 406 [M] þ , which is consistent with a formula of C 27 H 50 O 2 . The 1 H NMR spectrum exhibited four olefinic protons at d H 5.36 (1H, m, H-6), 5.35 (1H, m, H-7), 5.34 (1H, m, H-10) and 5.33 (1H, m, H-11) consistent with two olefinic double bonds in 1. It also revealed characteristic signals for terminal CH 3 protons of fatty acid at d H 0.87 (3H, t, J ¼ 6.8 Hz), broad signals at d H 1.25-2.29 for methylene protons and one methoxy at d H 3.66 suggesting the fatty acid methyl ester nature of 1. The 13 C NMR and HSQC spectral data revealed the presence of 27 carbon signals consisting of an ester carbonyl at d C 174.3 (C-1), 4 olefinic carbons at d C 130.5 (C-6), 130.4 (C-7), 128.9 (C-10) and 128.8 (C-11), 1 methoxy at d C 51.4, 20 methylenes between 22.7 and 34.1 ppm and a methyl carbon at d C 14.1. The HMBC spectrum ( Figure S11, see Supplementary materials) clearly pinpointed correlations of H-5 to C-6 and C-7, H-7 to C-5, H-9 to C-11, H-10 and H-11 to C-9, which established the position of the double bonds between C-5/C-6 and C-10/C-11 and further confirmed by the fragment ion peaks at m/z 291 (4%) and 141 (46%) for C-6/C-7 double bond and m/z 182 (5%) and 169 (6%) for C-10/C-11 double bond. The methyl ester nature was confirmed by the HMBC cross-peak between the methoxy group and C-1 (d C 178.3) (Pouchert & Behnke 1993). In addition, cross-peaks of H-2 to C-1 (d C 178.3) and C-3 (d C 24.7) were observed. The geometry of the double bonds was deduced to be Z based on coupling constant values and by comparing with the literature (Hsu & Turk 2008;Elkhayat et al. 2012). Accordingly, the structure of 1 was assigned as hexacosa-(6Z,10Z)-dienoic acid methyl ester and considered as a new natural product. Compound 2 was isolated as white crystals. Its GC -MS spectrum revealed a molecular ion peak at m/z 392 [M] þ , that is compatible with a molecular formula of C 26 H 48 O 2 , implying 3 degrees of unsaturation. Compound 2 is 14 mass units less than 1. The NMR spectral data of 2 were quite similar to those of 1, but the signals associated with the methoxy group (d H 3.66/d C 51.4) were not present. The position of double bond was established by the COSY and HMBC correlations and confirmed by the fragmentation pattern in GC -MS spectrum (Pouchert & Behnke 1993;Hsu & Turk 2008). The fragment peak at m/z 127 and 168 (12%), 224 confirmed the position of double bonds between C-6/C-7 and C-10/C-11, respectively. On the basis of these findings, 2 was assigned as hexacosa-(6Z,10Z)-dienoic acid and considered as a new compound.
To evaluate the biological activities of the different extracts of the Red Sea M. euplectellioides, the extracts were evaluated in a panel of different biological screens including antimicrobial, anti-inflammatory, antipyretic and hepato-protective. In addition, the pure compounds were evaluated for their cancer growth inhibitory activity against three cell lines. In all screens, positive as well as negative controls were used. The antimicrobial results (Table 1) revealed that the TME was the most active fraction against all bacterial strains at a concentration of 100 mg/mL, while EtOAc fraction exhibited moderate activity against Bacillus cereus at all concentrations and Staphylococcus aureus at a concentration of 25 mg/mL. The aqueous fraction exhibited high activity against Pseudomonas aeruginosa (concentration 50 mg/ mL). All tested fractions did not exhibit any activity against Serratia marcescens and all fungal strains. The TME and different fractions reduced the carrageenan-induced oedema with maximum effects being obtained after 4 h at concentrations of 100 and 200 mg/kg compared with indomethacin ( Table 2). The tested extracts exhibited antipyretic activity at doses of 100 and 200 mg/kg for each. They control the hyperthermia for 4 h without decrease in activity compared with reference compound indomethacin (8 mg/kg) ( Table 3). The decrease in CCl 4induced elevated enzyme levels and serum bilirubin in animals after treatment with the TME revealed that it has a remarkable hepato-protective effect in CCl 4 -induced liver damage compared with silymarin. This may be due to the maintenance of structural integrity of hepatocytic cell membrane or repair the damage of live cells (Table 4). The isolated compounds  from the M. euplectellioides were tested for in vitro growth inhibitory activity. Compounds 1 and 2 displayed weak activity against all cancer cell lines (Table 5).
In conclusion, this study resulted in the identification of four compounds from the Red Sea marine sponge M. euplectellioides. Compounds 1 and 2 are new natural compounds, while 3 and 4 were reported here for the first time from the sponge. The TME, EtOAc and aqueous fractions exhibited antimicrobial, anti-inflammatory, antipyretic and hepatoprotective activities. Compounds 1 and 2 exhibited weak cytotoxic activity.

General experimental procedures
Melting points were obtained in an Electrothermal 9100 Digital Melting Point (Electrothermal Engineering Ltd, Essex, England). IR was measured with a Shimadzu Infrared-400

Sponge material
The sponge, M. euplectellioides (Keller, 1889), was collected at depth 7 -10 m off Hurghada. It forms upright hollow lobes. The voucher specimen measures 10 cm high, 6 -8 cm in diameter, with a spinose macerated surface. Live sponge is red in colour and fades on preservation in 70% EtOH. The skeleton consists of a wide-meshed, loosely anastomosing, open network of spongin fibres, which are quite variable in thickness (50 -500 mm) and enclose a variable core of 10-70 spicules in cross-section. Spicules are thin subtylostyles with wide axial lumen measuring, 180-230 mm £ 1-2 mm. Microseleres were not found, but presumably these were washed out with the copious mucous produced by this sponge when collected (as known for this species). The voucher specimen is registered in the collections of the Netherlands Centre of Biodiversity Naturalis under number ZMA Por. 16626. Another voucher specimen has been deposited at the Red Sea Invertebrates Collection at the Department of Pharmacognosy of Suez Canal University under the code no. DY-13.

Pharmacological studies
3.6.1. Animals Adult male albino rats (120-150 g body weight) were used. All animal procedures were conducted in accordance with the internationally accepted principles for laboratory animals' use and care as found in the European Community Guidelines and Institutional Ethical Committee Approval was obtained. The study protocol was approved by the Animal Ethical Committee of Assiut University. The animals were housed under standardised environmental conditions in the pre-clinical Animal House, Pharmacology Department, Faculty of Medicine, Assuit University. The animals were fed with standard diet and had free access to water. They were maintained at 24-288C, 60-70% relative humidity, 12 h day and night cycle for 1 week to acclimatise to the environmental conditions.

Anti-inflammatory activity
The anti-inflammatory activity was evaluated in adult albino rats by carrageenan-induced rat hind paw oedema method according to the published procedures (Winter et al. 1962;Adams et al. 1968;Ali et al. 2013). The percentage of oedema inhibition (% of change) was calculated (Table 2).

Antipyretic activity
The antipyretic activity was screened in adult albino rats by using yeast-induced hyperpyrexia as previously described (Ibrahim, Mohamed & Al-Musayeib 2012;Ali et al. 2013). Rectal temperature of each rat was recorded after 1, 2, 3 and 4 h from administration of tested fractions.

Hepato-protective activity
The hepato-protective activity was determined as previously outlined (Bergmeyer & Bernt 1974;Bosma 1988;Mohamed et al. 2009). The animals were divided into four groups each of six animals.
Group I: Normal control received distilled water (1 mL/kg) daily for 5 days and olive oil (1 mL/kg, intraperitoneal) on days 2 and 3. Group II: CCl 4 control received distilled water (1 mL/kg) daily for 5 days and CCl 4 :olive oil (1:1, 1 mL/kg, intraperitoneal) on days 2 and 3. Group III: Treated with silymarin orally through intragastric feeding tube at dose of 50 mg/kg. Groups IV: Treated with TME orally through intragastric feeding tube at dose of 100 mg/kg.
3.6.4.1. Biochemical estimations. The rats were sacrificed on the sixth day and blood was collected from orbital sinus in plain tubes. The serum was obtained by centrifugation and serum samples were taken for biochemical assays; namely glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. The data were expressed as mean^SE (n ¼ 6). Results were analysed statistically by oneway ANOVA followed by comparison using Prism software (Graph Pab. Ver. 3.0). 3.6.5. Cytotoxicity study Cancer growth inhibitory activity was examined against the A549 NSCLC, U373 GBM and PC-3 (prostate cancer) cell lines as described earlier (Mijatovic et al. 2008;Ibrahim, Mohamed, Shaala, et al. 2012).

Conclusion
Two new and two known compounds were isolated from the Red Sea sponge Mycale euplectellioides. The structures were elucidated by the different spectral data. The antimicrobial, anti-inflammatory, antipyretic, and hepato-protective activities of the total methanolic extract and fractions were evaluated. Furthermore, compounds 1 and 2 displayed weak cytotoxic activity.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1 -S11.