MEDICINAL Medicinal Chemistry Research CHEMISTRY https://doi.org/10.1007/s00044-018-2143-7 RESEARCH ORIGINAL RESEARCH

Cytotoxic and anti-inflammatory compounds from Red Sea grass ciliatum

1,2 2 1 2 Reda F. Abdelhameed ● Amany K. Ibrahim ● Koji Yamada ● Safwat A. Ahmed

Received: 24 October 2017 / Accepted: 23 January 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Chemical investigation of the less polar fraction methylene dichloride–methanol extract of the Red Sea grass Thalassodendron ciliatum led to the isolation of a new phytoceramide molecular species TCC-1, along with four known compounds: 7β-hydroxy cholesterol (10), 7β-hydroxysitosterol (11), stigmasterol glucoside (12), and β-sitosterol glucoside (13). Phytosphingosines with 2-hydroxy fatty acid residues constituted the phytoceramide molecular species TCC-1. Further purification of TCC-1 afforded two new phytoceramides: TCC-1-5 (5) and TCC-1-7 (7) as well as the known ceramide TCC- 1-6 (6). All compounds are reported for the first time from this . The chemical structures of the isolated compounds were clarified on the basis of spectroscopic techniques including IR, NMR experiments, mass spectrometry, and chemical fi

1234567890();,: methods, in addition to comparison with literature data. All isolated compounds exhibited signi cant cytotoxicity against two human cell lines (Hep G2 and MCF-7). Moreover, compounds (10–13) have been found to possess significant anti- inflammatory activity in which compound (13) is the most potent.

Keywords Thalassodendron ciliatum ● Ceramide ● Cytotoxic compounds ● anti-inflammatory compounds

Introduction marine vascular flowering that are widely distributed along the coastal and estuarine regions of the world. They Ethnobotanical studies have continued to validate folkloric are unique in that they are often completely submerged in use of plants (marine and terrestrial) (Moghadamtousi et al. water. There are approximately 12 genera affording 2015) in phytomedicinal remedies (Rivera et al. 2005); 50 species of sea grasses. Seven genera are tropical, while (Sharma and Gupta 2015). Now, more than any time, in the the remaining five are almost confined to temperate waters history of phytochemistry research, the chemical basis of (Pollard and Greenway 1993). In many places, sea grasses most age-long folkloric cures is better understood. Drug cover extensive areas, which are usually known as sea grass development in contemporary times have benefitted from beds. The sea grass beds play a vital role in the marine these bodies of knowledge, accounting for more than 50% ecosystem since they act as shelter for a variety of other of drugs and other pharmaceutical preparations approved organisms and provide food for commercially important fish for human use today (Sharma and Gupta 2015) for the species (Howard et al. 1989). McMillan (McMillan et al. treatment and/or management of diseases. Sea grasses are 1980) reported that sea grass is a rich source of secondary metabolites, especially phenolics, which are known to have various biological activities. One such whose bioac- tive principles are under intensive research currently is Electronic supplementary material The online version of this article Thalassodendron ciliatum. T. ciliatum is a tropical sea grass (https://doi.org/10.1007/s00044-018-2143-7) contains supplementary fi material, which is available to authorized users. which can be classi ed as sub-tidal and not deep water bed- forming sea grass (Carruthers et al. 2002). It is widely * Safwat A. Ahmed distributed in the Red Sea, the tropical part of the [email protected] Indo–Pacific region, and the western Indian Ocean (Den and 1 Graduate School of Biomedical Sciences, Nagasaki University, Hartog 1970). Recently, its phenolic constituents (rutin, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan asebotin, 3-hydroxyasebotin, quercetin-3-O-beta-D-xylo- 2 Departments of Pharmacognosy, Faculty of Pharmacy, Suez Canal pyranoside, catechin, and caffeic acid) were isolated and University, Ismailia 41522, Egypt characterized (Hamdy et al. 2012) and assayed for cytotoxic Medicinal Chemistry Research response in HCT-116, HEPG, MCF-7, and HeLa human Plant material, collection, and identification cancer cell lines. Furthermore, three independent studies have established that the extract of the plant and its isolated The sea grass T. ciliatum (coll. no. SAA-41) was collected components (especially asebotin) have potent anti-viral from Sharm el sheikh at the Egyptian Red Sea, air-dried, activities (Mohammed et al. 2014); (Ibrahim et al. 2013). and stored at low temperature (−24 ˚C) until processed. The Anti-oxidant activities have also been reported due to the plant was identified by Dr Tarek Temraz, Marine Science presence of flavonoids. Department, Faculty of Science, Suez Canal University, A limited number of phytochemical studies are found on Ismailia, Egypt, A voucher specimen was deposited in the the isolation and structure elucidation of the chemical herbarium section of Pharmacognosy Department, Faculty constituents from the polar fractions of the Red Sea grass of Pharmacy, Suez Canal University, Ismailia, Egypt under Thalassodendron ciliatum and to the best of our knowledge registration number SAA-41. there is no chemical investigation for the non polar fraction till now, except for the diglyceride ester isolated by our Extraction and isolation group (Ibrahim et al. 2013). Therefore, in this study we have further expanded the chemical landscape of the Red T. ciliatum was dried (250 g dry weight), grounded, and Sea grass T. ciliatum with the isolation and spectral char- repeatedly extracted with a mixture of CH2Cl2/MeOH (1:1) acterization of phytosphingosine type ceramides and ster- (3 × 2 L) at room temperature. The combined extracts were oidal scaffolds for the first time. concentrated under vacuum to afford a dark green residue TC (30 g). The residue was chromatographed over silica gel column using CHCl3: MeOH: H2O (100:0:0 ~ 60:40:10) Material and methods gradient elution to give 11 sub-fractions (TC-1–TC-11) based on TLC analysis. Fraction TC-6 (0.64 g), was sub- General experimental procedures jected to SiO2 column eluting with a gradient of increasing MeOH in CHCl3 to afford 6 fractions (TC-6-1 ~ TC-6-6). 1H-NMR chemical shifts are expressed in δ values refer- Fraction TC-6-4 (114 mg) was chromatographed over ring to the solvent peak δH 7.19, 7.55, and 8.71 for Pyr- Sephadex LH-20 column and eluted with CHCl3: MeOH idine-d5, 7.26 for CDCl3, and coupling constants are (1:1) to yield 4 fractions (TC-6-4-1 ~ TC-6-4-4). Fraction expressed in Hz. 13C NMR chemical shifts are expressed TC-6-4-3 (56 mg) was successively re-chromatographed in δ values referring to the solvent peak δC 123.5, 135.5, over silica gel column using CHCl3: MeOH (9.5:0.5) iso- 1 13 and 149.9 for Pyridine-d5, and 77 for CDCl3. Hand C cratic elution to give a pure phytoceramide molecular spe- NMR spectra were recorded with a Unity plus 400 spec- cies TCC-1 (28 mg). TCC-1 (23 mg) was finally purified on trometer (Varian Inc., U.S.A.) operating at 400 MHz for semi-preparative HPLC (cosmosil 5C18, 100% MeOH) to 1H, and 100 MHz for 13C. Optical rotations were mea- afford TCC-1-1 (1) (0.3 mg), TCC-1-2 (2) (0.9 mg), TCC-1- sured with a JASCO DIP-370 digital polarimeter. Positive 3(3) (0.6 mg), TCC-1-4 (4) (0.8 mg), TCC-1-5 (5) (2.1 mg), ion FAB-MS spectra were recorded on JMS DX-303 TCC-1-6 (6) (6.4 mg), TCC-1-7 (7) (2.4 mg), TCC-1-8 (8) spectrometer (JEOL Ltd., Japan), using m-nitrobenzyl (1.3 mg), and TCC-1-9 (9) (1.0 mg) as pure phytocer- alcohol or Magic bullet as a matrix. Sephadex LH-20 amides. Fraction TC-4 (1.4 g), was further chromatographed (Pharmacia Fine Chemical Co.Ltd) and Wakogel C-300 over SiO2 column using CHCl3: MeOH (100:0 ~ 80:20) (Wako Pure Chemical Industries, Ltd, Japan, 45 ~ 75 μm) gradient elution to afford 10 fractions (TC-4-1 ~ TC-4-10). were used for column chromatography. Precoated silica Fraction TC-4-5 (320 mg) was chromatographed over gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and RP- Sephadex LH-20 column and eluted with CHCl3: MeOH 18 F254s plates (Merck) were used for thin-layer chro- (1:1) followed by silica gel column using n-hexane: EtOAc matography (TLC) analysis. IR spectra were obtained (70:30 ~ 50:50) to yield 6 fractions (TC-4-5-1 ~ TC-4-5-6). with JASCO FT/IR-410 spectrophotometers. The UV Fraction TC-4-5-3 (65 mg) was chromatographed over spectra were recorded on a double beam Shimadzu silica gel column using n-hexane: EtOAc (70:30) isocratic UV–visible spectrophotometer (model UV-1601 PC, elution followed by semi-preparative reversed-phase HPLC Kyoto City, Japan). Semi-preparative high-performance (cholester, 100% MeOH) to give 10 (2.5 mg) and 11 (2.2 liquid chromatography (HPLC) was performed on a cos- mg). Fraction TC-7 (0.6 g), was subjected to silica gel mosil 5C18–MS–II (150 × 4.6 mm) at a flow rate of 0.5 ml/ column using CHCl3: MeOH (100:0 ~ 90:10) gradient elu- min, and cholester column at a flow rate of 0.5 ml/min. tion to afford seven fractions (TC-7-1 ~ TC-7-7). Com- Both columns were equipped with a TOSOH RI-8020 pounds 12 (7.7 mg) and 13 (13.5 mg) were isolated after detector and a JASCO BIP-I HPLC pump. column chromatography over Sephadex LH-20 column and Medicinal Chemistry Research

Table 1 1H and 13C-NMR spectral data of TCC-1 (δ values in C D N)a eluted with CHCl3: MeOH (1:1) followed by semi- 5 5 preparative reversed-phase HPLC (cholester, 90% MeOH) Position TCC-1 from fraction TC-7-5 (66 mg). 1H 13C TCC-1: White amorphous powder. IR (KBr) cm−1: 3300 (hydroxyl), 1624, 1545 (amide). Positive ion FAB-MS m/z: NH 8.58 (1H, d, J = 8.9 Hz) + 678, 692, 706, 720, 734 [M + Na] series. 1H and 13C- 1a 4.43 (1H, dd, J = 10.8, 5.2 Hz) 62.0 (t) NMR: see Table 1. 1b 4.49 (1H, dd, J = 10.8, 4.6 Hz) Methanolysis of TCC-1: Hydrolysis was achieved by 2 5.12 (1H, m) 52.9 (d) heating TCC-1 (5 mg) with 5% HCl in MeOH (0.5 ml) at 3 4.35 (1H, m) 76.7 (d) 70 °C for 8 h in a sealed small-volume vial. The reaction 4 4.27 (1H, m) 73.0 (d) mixture was extracted with n-hexane, and the hexane layer 1′ 175.2 (s) was evaporated under vacuum till dryness to give a mixture 2′ 4.61 (1H, m) 72.4 (d) of FAM for 1H, 13C- NMR and mass analysis. –CH3 0.88 (m) 14.2 (q) EI mass analysis of fatty acid methyl esters (FAMEs) 22.4 (q) from TCC-1: The FAMEs mixture from TCC-1 showed a δ four molecular ion peak series at 370, 384, 398, 412 [M+] Spectra were acquired at 23 °C. Chemical shifts were given in (ppm) and referenced to internal solvent for C D N at 7.19 (δ ) and 123.5 indicating C-22, C-23, C-24, C-25 FAMEs. 5 5 H (δC) ppm Isolation of ceramides from TCC-1: The ceramide molecular species TCC-1 showed nine peaks in the reversed phase HPLC [column, cosmosil 5C18 (4.6 × 150 mm); sol- vent, MeOH; flow rate, 0.5 ml/min; RI detector]. With these (10) in the manuscript and the structure is as drawn in conditions, 23 mg of TCC-1 was separated to give nine Fig. 1, detailed spectral data is included in supplementary compounds: TCC-1-1 (0.3 mg, tR = 27.0 min), TCC-1-2 data). 1 (0.9 mg, tR = 29.0 min), TCC-1-3 (0.6 mg, tR = 32.0 min), 7β-hydroxysitosterol (11): White powder; H-NMR (400 TCC-1-4 (0.8 mg, tR = 34.0 min), TCC-1-5 (2.1 mg, tR = MHz, CDCl3) δ: 3.55 (1H, m, H-3), 5.29 (1H, brs, H-6), 37.0 min), TCC-1-6 (6.4 mg, tR = 41.0 min), TCC-1-7 (2.4 3.85 (1H, brs, H-7), 0.69 (3H, s, H-18), 1.05 (3H, s, H-19), mg, tR = 44.0 min), TCC-1-8 (1.3 mg, tR = 48.0 min), and 0.94 (3H, d, J = 6.5 Hz, H-21), 0.82 (3H, d, J = 6.5 Hz, H- TCC-1-9 (1.0 mg, tR = 57.0 min) 26), 0.82 (3H, d, J = 6.5 Hz, H-27), 0.86 (3H, t, J = 7.5 Hz, 13 TCC-1-1 (1): Amorphous powder, positive-ion FAB- H-29); C-NMR (100 MHz, CDCl3) (Henceforth, the MS: m/z 656 [M + H]+, 678 [M + Na]+. structure is referred to Cpd. (11) in the manuscript and the TCC-1-2 (2): Amorphous powder, positive-ion FAB- structure is as drawn in Fig. 1, detailed spectral data is MS: m/z 656 [M + H]+, 678 [M + Na]+. included in supplementary data). TCC-1-3 (3): Amorphous powder, positive-ion FAB- Stigmasterol glucoside (12): White powder; 1H-NMR + + MS: m/z 670 [M + H] , 692 [M + Na] . (400 MHz, CDCl3) δ: 4.06 (1H, m, α H-3), 5.33 (1H, brs, TCC-1-4 (4): Amorphous powder, positive-ion FAB- H-6), 0.65 (3H, s, H-18), 0.95 (3H, s, H-19), 1.09 (3H, d, J MS: m/z 670 [M + H]+, 692 [M + Na]+. = 6.5 Hz, H-21), 5.08 (1H, dd, J = 8.9, 15.1 Hz, H-22), TCC-1-5 (5): Amorphous powder, positive-ion FAB- 5.23 (1H, dd, J = 8.7, 15.1 Hz, H-23), 0.85 (3H, d, J = 6.5 + + MS: m/z 684 [M + H] , 706 [M + Na] ,[α]D =+8.4. Hz, H-26), 0.87 (3H, d, J = 6.5 Hz, H-27), 0.88 (3H, t, J = TCC-1-6 (6): Amorphous powder, positive-ion FAB- 8.7 Hz, H-29), 5.07 (1H, d, J = 7.2, H-1’); 13C-NMR (100 + + MS: m/z 684 [M + H] , 706 [M + Na] ,[α]D =+7.2. MHz, CDCl3) (Henceforth, the structure is referred TCC-1-7 (7): Amorphous powder, positive-ion FAB- to Cpd. (12) in the manuscript and the structure is as + + MS: m/z 698 [M + H] , 720 [M + Na] ,[α]D =+26.3. drawn in Fig. 1, detailed spectral data is included in TCC-1-8 (8): Amorphous powder, positive-ion FAB- supplementary data). MS: m/z 698 [M + H]+, 720 [M + Na]+. β-sitosterol glucoside (13): White powder; 1H-NMR TCC-1-9 (9): Amorphous powder, positive-ion FAB- (400 MHz, CDCl3) δ: 4.06 (1H, m, α H-3), 5.33 (1H, brs, MS: m/z 712 [M + H]+, 734 [M + Na]+. H-6), 0.65 (3H, s, H-18), 0.92 (3H, s, H-19), 97 (3H, d, J = 7β-hydroxy cholesterol (10): White powder; 1H-NMR 6.5 Hz, H-21), 0.85 (3H, d, J = 6.5 Hz, H-26), 0.87 (3H, d, (400 MHz, CDCl3) δ: 3.55 (1H, m, H-3), 5.29 (1H, brs, H- J = 6.5 Hz, H-27), 0.88 (3H, t, J = 7.5 Hz, H-29), 5.06 (1H, ’ 13 6), 3.84 (1H, brs, H-7), 0.69 (3H, s, H-18), 1.05 (3H, s, H- d, J = 7.2, H-1 ); C-NMR (100 MHz, CDCl3) (Hence- 19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.86 (3H, d, J = 6.5 Hz, forth, the structure is referred to Cpd. (13) in the manuscript H-26), 0.87 (3H, d, J = 6.5 Hz, H-27); 13C-NMR (100 and the structure is as drawn in Fig. 1, detailed spectral data MHz, CDCl3) (Henceforth, the structure is referred to Cpd. is included in supplementary data). Medicinal Chemistry Research

Fig. 1 Structure of the isolated OH OH O O TCC-1-2: m+n=17 compounds m TCC-1-1: m+n=17 m TCC-1-3: m+n=18 TCC-1-4: m+n=18 NH OH TCC-1-5: m=14 , n=5 NH OH TCC-1-6: m=13, n=6 TCC-1-7: m=13 , n=7 TCC-1-8: m+n=20 HO HO TCC-1-9: m+n=21 n n OH OH

HO OH HO OH 10 11

O O 12 13 O OH O OH

HO HO HO HO OH OH

In vitro evaluation of cytotoxic activity animals had a subplanter injection of 0.1 ml of 1% carra- geenan solution in saline, in the right hind paw and 0.1% of The cytotoxicity of the isolated compounds was measured saline in the left hind paw compared to Indomethacin by the Sulpho–Rhodamine-B (SRB) assay (Vichai and (20 mg/Kg) Kirtikara 2006). This was performed on different human Thickness of the right hind paw (mm) was measured cell lines: liver carcinoma cell line (Hep G2) and breast immediately before and 1, 2, 3 and 4 h post carrageenan carcinoma cell line (MCF-7), which were kindly provided injection with a micrometer caliber. by the National Cancer Institute (Kasr El Ainy Street, Cairo, The percentage of edema produced and that of edema Egypt). The cells were placed into 96-multiwell plates (104 inhibition due to drug administration were, respectively cells/well) for 24 h before treatment with the pure com- calculated as follows: pounds to allow attachment of cells to the plate’s wall. Cells were routinely cultured in Dulbecco’s modified Eagle’s Edema ¼ ðÞWt of right paw À Wt: of left paw medium. Different concentrations of the tested samples (0, Â100=Wt: of left paw 50, 100, 150 and 200 µg/ml in DMSO) were added to the %Edema inhibition ¼ ðÞÂMc À Mt 100=Mc cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the Where, Mc is the mean edema in control rats and Mt is the compounds under test for 48 h, at 37 °C and in atmosphere mean edema in drug-treated animals. of 5% CO2. After 48 h, the cells were fixed, washed and stained with SRB stain. Excess stain was washed with acetic The statistical comparison of the difference between the acid and the attached stain was recovered with Tris-EDTA control group and the treated sample was carried out using buffer. Color intensity was measured in an ELISA reader. two-way ANOVA followed by Duncan’s multiple range Moreover, the IC50 (the dose that reduces survival to 50%) test. Results are expressed as mean ± SE (n = 6). was calculated.

Anti-inflammatory assay Results and discussion

Using the carrageenan-induced rat paw edema test, 36 male Chemistry albino mice divided into six groups (each of six animals) were used. They were administered one single oral dose of TCC-1 (28 mg) was obtained as a white amorphous solid, the tested sample and the reference drug in specific doses. showing a single spot on silica gel thin layer chromato- The negative control group received saline. 1 h later all the graphy (TLC). TCC-1 exhibited a characteristic absorption Medicinal Chemistry Research band for hydroxyl group (3300 cm−1), and amide absorp- The n-hexane layer afforded a mixture of FAMEs, which tion (1623, 1545 cm−1) in the IR spectrum. were subjected to 1H-NMR and EI-MS analysis. EI-MS 1 The H-NMR spectrum in C5D5N showed resonances of analysis of the FAMEs mixture showed the presence of four an amide proton doublet at δH 8.59 (1H, d, J = 8.9 Hz), components at m/z: 370, 384, 398, and 412, which were protons of a long methylene chain centered at δH 1.27, and characterized as FAM-1, FAM-2, FAM-3, and FAM-4 overlapped methyls at δH 0.86 indicating sphingolipid indicating C-22, C-23, C-24, and C-25 FAMES, respec- skeleton. The characteristic signals of 2-amino-1,3,4,2′- tively (supplementary figure S5). tetrol of the hydrocarbon chain were observed at δH 5.11 Furthermore, FAMEs mixture was thought to possess (1H, m, H-2), 4.61 (1H, m, H-2′), 4.49 (1H, m, H-1a), 4.43 normal type side chains, since the protons signals for the (1H, m, H-1b), 4.34 (1H, m, H-3), 4.28 (1H, m, H-4) in the terminal methyls in the 1H-NMR spectrum were observed at 1 H-NMR spectrum and at δC 52.9 (C-2), 72.4 (C-2′), 62.0 δH = 0.85 (3H, t, J = 8 Hz) (Ahmed et al. 2008). (C-1), 76.7 (C-3), 72.9 (C-4) in the 13C-NMR spectrum. In Rooted in the considerable interest and importance of ’ addition to amide carbonyl at δC 175.2 (C-1 ), a series of determining the molecular species composition of sphin- molecular ion peaks due to [M + Na]+ were observed in the golipids, isolation and structure elucidation of the ceramide positive ion FAB-MS spectrum at m/z: 678, 692, 706, 720, components in the molecular species TCC-1 was conducted. 734 (supplementary figure S3). Therefore, TCC-1 is regar- Through using reversed phase HPLC, TCC-1 was separated ded to be a molecular species of a phytosphingosine-type into nine peaks and they were recovered to yield nine ceramide possessing 2-hydroxy fatty acid groups. Further- fractions ranging from TCC-1-1 to TCC-1-9. On the basis more, TCC-1 is suggested to have normal and iso-type side of the molecular mass of TCC-1-1 m/z 678 [M + Na]+, chains (Yamada et al. 2001), due to the carbon signals for TCC-1-2 m/z 678 [M + Na]+, TCC-1-3 m/z 692 [M + Na]+, + + the terminal methyl groups which were observed at δC 14.2 TCC-1-4 m/z 692 [M + Na] , TCC-1-5 m/z 706 [M + Na] , (normal type), 22.7 (iso type) in the 13C-NMR spectrum TCC-1-6 m/z 706 [M + Na]+, TCC-1-7 m/z 720 [M + Na]+, (supplementary figure S2, table1). The structure of TCC-1 TCC-1-8 m/z 720 [M + Na]+, TCC-1-9 m/z 734 [M + Na]+, shown in Fig. 2 was characterized by comparison of its 13C- the structures of these compounds were determined as NMR spectral data with that of known ceramides (Ahmed shown in Fig. 1. et al. 2008); (Loukaci et al. 2000); (Krishna et al. 2004). The 1H-NMR spectra of TCC-1-1, TCC-1-3, TCC-1-5, The relative stereochemistry of the ceramide molecular and TCC-1-7 showed the characteristic signals of the species is suggested to be (2 S,3S,4R,2′R), since the terminal methyls and iso-methyls at δH = 0.85 (3H, t, J = 8 1 13 aforementioned H-NMR (H-2, H-3, H-4, H-2′) and C- Hz) and δH = 0.85 (6H, d, J = 8 Hz), respectively, while NMR signals (C-1, C-2, C-3, C-4, C-2′) were in good TCC-1-2, TCC-1-4, TCC-1-6, TCC-1-8, and TCC-1-9 agreement with those of phytosphingosine-type ceramide showed the signals of the terminal methyls groups at δH molecular species possessing (2 S,3S,4R,2′R) config- = 0.85 (6H, t, J = 8 Hz). The presence of the iso-methyl in urations (Chen et al. 2002); (Azuma et al. 2003); (Sandjo the long-chain base (LCB) of TCC-1-5, TCC-7 was further et al. 2008). confirmed by the characteristic carbon signals in their 13C- In order to determine the length and branching pattern NMR spectra at δC 22.7 (iso type). Further hydrolysis of of the long-chain bases and fatty acids, TCC-1 was sub- TCC-1-5, TCC-1-6, and TCC-1-7 followed by EI-MS jected to methanolysis with methanolic hydrochloric acid analysis of their FAMEs, afforded homogenous FAMEs m/z followed by 1H-NMR, 13C-NMR, and FAB-MS analysis. 412, 398, 398 [M]+, respectively indicating C-24, C-23, C- 23 FAMES. OH Taking into consideration the biosynthetic pathway of O // normal type phytosphigosines (Kolter and Sandhoff 1999); (Pruett et al. m // NH OH 2008) and by comparing the optical rotation values of TCC- + + + HO // // iso type 1-5, TCC-1-6, and TCC-1-7 [ 8.4, 7.2, 26.3] with ana- n logs reported in the literature (Sun et al. 2006), the proposed OH relative configuration of TCC-1 was confirmed. TCC-1 On the basis of the above findings and the molecular methanolysis followed by extraction with n-hexane + + and final analysis of the resulting FAMEs mass of TCC-1-5 m/z (706 [M + Na] , 684 [M + H] ), TCC-1-6 m/z (706 [M + Na]+, 684 [M + H]+), and TCC-1- + + OH 7 m/z (720 [M + Na] , 698 [M + H] ), the length of the O normal type hydrocarbon chain and the structure of TCC-1-5, TCC-1-6, m and TCC-1-7 were finally elucidated as shown in Fig. 1. OCH3 m = 11, 12, 13, 14 To the best of our knowledge, TCC-1-5 and TCC-1-7 Fig. 2 Structure of TCC-1 have been reported for the first time, while TCC-1-6, was Medicinal Chemistry Research previously isolated from the mycoparasitic fungi Tricho- derma koningii (Huang et al. 1995). The known compounds were identified through the analysis of the spectroscopic data and comparison of their β data with those in the literature, which are 7 -hydroxy Edema thickness (mm) cholesterol (10) (Roh et al. 2010), 7β-hydroxy sitosterol 6) =

(11) (Roh et al. 2010), Stigmasterol glucoside (12) (Rai n et al. 2006), and β-sitosterol glucoside (13) (Rai et al. 2006). an ± SE ( Biological activity Paw diameter (mm) s multiple range test ’ Cytotoxic activity an suspension in saline. Thickness of the

The cytotoxic activity was evaluated via a two-stage pro-

cess, beginning with measurement of the sensitivity of all Edema thickness (mm) isolated compounds against two human cancer cell lines: liver carcinoma cell line (Hep G2), and breast carcinoma cell line (MCF-7) at a single dose of 100 μg/ml. The second stage was the evaluation of potential cytotoxicity at five

different concentrations (0, 50, 100, 150 and 200 µg/ml in Paw diameter (mm) DMSO), corresponding to the maximum percentage of inhibition achieved at the single dose experiment. The initial screening effect i.e., sensitivity test indicated that, Ceramide mixture TCC-1 as well as isolated sterols (10–13) displayed a significant inhibitory activity against breast carcinoma cell line (MCF-7) and liver carcinoma cell line Edema thickness (mm) (Hep G2). The inhibitory properties of these compounds are compared with the standard Cisplatin (Table 2). 0.06 0.3 3.2* ± 0.01 0.2 3.14* ± 0.01 0.14 + Anti-inflammatory activity Paw diameter (mm)

The anti-inflammatory activity (Table 3)of(10–13) was evaluated on carrageenan-induced rat hind paw edema model. Compound (13) (20 mg/kg) has been found to possess significant anti-inflammatory activity on the tested

experimental model. Edema thickness (mm)

Table 2 IC values [µM] of isolated compounds and cisplatin against 0.04 0.4 3.3* 50 + different human cell lines; HepG2 and MCF-7 < 0.05 Paw diameter (mm) p Sample Human cell lines against carrageenan induced paw edema in rats HepG2 IC50 [µM] MCF-7 IC50 [µM]

TCC-1 23.2 ± 0.20 29.6 ± 0.15 10-13 Compound 10 25.4 ± 0.38 18.6 ± 0.72 3.22 ± 0.12 3.68* ± 0.22 0.46 3.54* ± 0.13 0.32 3.43* ± 0.10 0.21 3.4* ± 0.07 0.18 3.50 ± 0.11 4.19* ± 0.12 0.69 3.95* ± 0.11 0.45 3.90* ± 0.09 0.4 3.80* ± 0.06 0.30 3.45 ± 0.11 4.17* ± 0.21 0.72 4.00* ± 0.04 0.55 3.95* ± 0.07 0.5 3.90* ± 0.04 0.45 (mm) 3.10 ± 0.11 3.63* ± 0.22 0.53 3.55* ± 0.11 0.45 3.50* ± 0.05 0.4 3.32* ± 0.03 0.22 Compound 11 27.4 ± 0.65 22.5 ± 0.63 Compound 12 24.9 ± 0.23 19.8 ± 0.43 13 20 mg/ 20 mg/ 20 mg/ Compound 38.3 ± 0.45 28.3 ± 0.48 20 mg/ 13 12 11 Cisplatin 21.3 ± 0.4 15.3 ± 0.1 10 Effect of compounds cantly different from zero time at

Each data point represents the mean ± SD of three independent fi experiments (significant differences at p<0.05) right hind paw (mm) was measured immediately before and 1, 2, 3 and 4 h post carrageenan injection with a micrometer caliber. Results are expressed as me Drugs were orally administered 1 h prior to carrageenan injection. Edema was induced in the rat right hind paw by S.C. injection of 0.1 ml of 1% carrageen * Signi The statistical comparison of difference between the control group and the treated groups was carried out using two-way ANOVA followed by Duncan kg Indomethacin 20 mg/kg 3.00 ± 0.09 3.4* kg Compound Table 3 kg Compound Time (hour)Groups Zero Paw diameter 10 11 12 13 kg Compound NA Not active ControlCompound 3.61 ± 0.09 4.93 ± 0.07 1.32 4.03 ± 0.13* 1.42 4.13 ± 0.13* 1.52 4.27 ± 0.8 1.66 Medicinal Chemistry Research

Conclusion Huang Q, Tezuka Y, Hatanaka Y, Kikuchi T, Nishi A, Tubaki K (1995) Studies on metabolites of mycoparasitic fungi. III. New In the current study, phytochemical investigation of the sesquiterpene alcohol from Trichoderma koningii. Chem Pharm Bull 43:1035–1038 secondary metabolites isolated from the crude extract of Ibrahim A, Youssef A, Arafa A, Foad R, Radwan M, Ross S, Has- the Red Sea Thalassodendron ciliatum, led to the sanean H, Ahmed S (2013) Anti-H5N1 virus new diglyceride isolation of a new phytoceramide molecular species TCC-1, ester from the Red Sea grass Thallasodendron ciliatum. Nat Prod β Res 27:1625–1632 along with four known compounds: 7 -hydroxy cholesterol, — β β Kolter T, Sandhoff K (1999) Sphingolipids their metabolic pathways 7 -hydroxysitosterol, stigmasterol glucoside, and -sitos- and the pathobiochemistry of neurodegenerative diseases. Angew terol glucoside. Chem Int Ed 38:1532–1568 The cytotoxic and the anti-inflammatory activities of Krishna N, Muralidhar P, Kumar M, Rao D, Rao C (2004) New 10–13 Sphingosines from a Gorgonian, Pseudopterogorgia australiensis TCC-1 as well as isolated sterols ( ) were evaluated. – 10–13 fi Ridley, of the Indian Ocean. J Nat Prod 67:1423 1425 TCC-1 and the isolated sterols ( ) showed a signi cant Loukaci A, Bultel-Poncé V, Longeon A, Guyot M (2000) New lipids inhibitory activity against breast carcinoma cell line (MCF- from the tunicate Cystodytes cf. dellechiajei, as PLA2 Inhibitors. 7) and liver carcinoma cell line (Hep G2), while compound J Nat Prod 63:799–802 (13) (20 mg/kg) displayed a significant anti-inflammatory McMillan C, Zapata O, Escobar L (1980) Sulphated phenolic com- pounds in . Aquat Bot 8:267–278 activity on the tested experimental model. Moghadamtousi S, Nikzad S, Abdul Kadir H, Abubakar S, Zandi K (2015) Potential antiviral agents from marine fungi: an overview. Acknowledgements We are grateful to Mr. M. Inada and Dr. N. Tsuda Mar Drugs 13:4520–4538 of the Scientific Support Section of Joint Research Center, Nagasaki Mohammed M, Hamdy A, El-Fiky N, Mettwally W, El-Beih A, University, for 1H-NMR, 13C-NMR and MS measurements. This work Kobayashi N (2014) Anti-influenza A virus activity of a new was supported in part by a Grant-in-Aid for Scientific Research No. dihydrochalcone diglycoside isolated from the Egyptian seagrass 23590008 and No. 26460124 from the Japan Society for the Promo- Thalassodendron ciliatum (Forsk.) den Hartog. Nat Prod Res tion of Science and Takeda Science Foundation, which is gratefully 28:377–382 acknowledged. Pollard C, Greenway M (1993) Photosynthetic characteristics of sea- grasses (Cymodocea serrulata, Thalassia hemprichii and Zostera Compliance with ethical standards capricornia) in a low-light environment, with a comparison of leaf-marking and lacunal-gas measurements of productivity. Aust J Mar Freshw Res 4:127–139 Conflict of interest The authors declare that they have no conflict of Pruett S, Bushnev A, Hagedorn K, Adiga M, Haynes C, Sullards M, interest. Liotta D, Merrill A (2008) Thematic review series: sphingolipids. Biodiversity of sphingoid bases (“sphingosines”) and related References amino alcohols. J Lipid Res 49:1621–1639 Rai N, Adhikari B, Paudel A, Masuda K, Mckelvey R, Manandhar M fl Ahmed S, Khalifa S, Hamann M (2008) Antiepileptic ceramides from (2006) Phytochemical constituents of the owers of Sarcococca – the Red Sea sponge Negombata corticata. J Nat Prod 71:513–515 coriacea of Nepalese origin. J Nepal Chem Soc 21:1 7 Azuma H, Takao R, Niiro H, Shikata K, Tamagaki S, Tachibana T, Rivera D, Obon C, Inocencio C, Heinrich M, Verde A, Fajardo J, Ogino K (2003) Total syntheses of symbioramide derivatives Liorach R (2005) The ethnobotanical study of local Mediterra- from L-Serine and their antileukemic activities. J Org Chem nean food plants as medicinal resources in southern Spain. J – 68:2790–2797 Physiol Pharmacol 56:97 114 Carruthers B, Dennison C, Longstaff J, Waycott M, Abal G, Roh E, Jin Q, Jin H, Shin J, Choi E, Moon Y, Woo E (2010) Structural McKenzie J, Long L (2002) Seagrass habitats of northeast Aus- implication in cytotoxic effects of sterols from Sellaginella – tralia: models of key processes and controls. Bull Mar Sci tamariscina. Arch Pharm Res 33:1347 1353 71:1153–1169 Sandjo L, Hannewald P, Yemloul M, Kirsch G, Ngadjui B (2008) Chen X, Wu Y, Chen D (2002) Structure determination and synthesis Triumfettamide and Triumfettoside Ic, two ceramides and other of a new cerebroside isolated from the traditional Chinese med- secondary metabolites from the stems of wild Triumfetta cordi- – icine Typhonium giganteum Engl. Tetrahedron Lett folia A. Rich. (Tiliaceae). Helv Chim Acta 91:1326 1335 43:3529–3532 Sharma S, Gupta R (2015) Drug development from natural resource: a – Den Hartog C (1970) The Seagrasses of the world. North Holland systematic approach. Mini Rev Med Chem 15:52 57 Publishers, Amsterdam, p 275 (1970) Sun Y, Xu Y, Liu K, Hua H, Zhu H, Pei Y (2006) Gracilarioside and Hamdy A, Mettwally W, Abou El Fotouh M, Rodriguez B, El-Dewany gracilamides from the red alga Gracilaria asiatica. J Nat Prod – A, El-Toumy S, Hussein A (2012) Bioactive phenolic compounds 69:1488 1491 from the Egyptian Red Sea Seagrass Thalassodendron ciliatum.Z Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for – Naturforsch 67C:291–296 cytotoxic screening. Nat Protoc 1:1112 1116 Howard K, Edgar J, Hutchings A (1989) Faunal Assemblages of Yamada K, Matsubara R, Kaneko M, Miyamoto T, Higuchi R (2001) Seagrass Beds. In: Larkum AWD, McComb AJ, Shepherd SA Constituents of Holothuroidea. 10.1 Isolation and structure of a (eds) Biology of seagrasses: a treatise on the biology of sea- biologically active ganglioside molecular species from the Sea grasses with special reference to the Australian region. Elsevier, Cucumber Holothuria leucospilota. Chem Pharm Bull – Amsterdam, pp 539–564 49:447 452