Brown Algae (Phaeophyceae) from the Coast of Madagascar: preliminary Bioactivity Studies and Isolation of Natural Products

Eight species of brown algae (Phaeophyceae) from the coast of Madagascar have been investigated for their chemical constituents. Fucosterol (3) was obtained as the most abundant compound. The brown alga Sargassum ilicifolium was the source for the first isolation of the terpenoid C27-alcohol 1,1′,2-trinorsqualenol (1) from marine sources. From S. incisifolium we isolated the highly unsaturated glycolipid 1-O-palmitoyl-2-O-stearidonoyl-3-O-β-D-galactopyranosylglycerol (4) and we report the first full assignment of its 1H and 13C NMR data. Apo-9′-fucoxanthinone (8) along with 24-ketocholesterol (5), (22E)-3β-hydroxycholesta-5,22-dien-24-one (6), and saringosterol (7) were obtained from Turbinaria ornata. The crude extracts of all eight species of brown algae exhibited a pronounced antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Staphylococcus aureus, and Streptococcus pneumoniae.

diverse places at the coast of Madagascar [9]. Among others, brominated indols, A-ring contracted steroids and debilone have been isolated from these species. To date, only few representatives of brown algae from Madagascar have been investigated for their chemical constituents. For the brown algae Spatoglossum sp., Sargassum sp. 1, Sargassum sp. 4, Zonaria sp., Chnoospora sp., and Spatoglossum sp. Andriamanantoanina and Rinaudo identified alginate polymers which are composed of (1 ? 4)-b-D-mannuronic acid (M) and (1 ? 4)-a-L-guluronic acid (G) units [10,11]. They examined the influence of the block distribution (MM and GG) along the alginate chain on their gel forming ability under acidic conditions. Recently, Andriamanantoanina and coworkers investigated the polysaccharide fraction of Sargassum sp., Turbinaria sp., and Hormophysa sp. and identified gel forming alginates [12]. The present study aims at the investigation of the chemical constituents of the nonpolar fractions from brown algae collected at the coast of Madagascar.
In an antimicrobial agar diffusion test, the crude extract of S. ilicifolium was found to be very active against the Gram-negative bacteria Shigella boydii and Klebsiella oxytoca, the Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus, and the yeasts Candida membranaefaciens, Trichosporon mucoides, and Cryptococcus neoformans (Table 3). Significant activity was also observed against the Gram-negative bacterium Enterobacter cloacae and the Gram-positive bacterium Bacillus cereus. No activity was found against the Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Salmonella enteridis, and the yeast Candida albicans.

Sargassum incisifolium
The brown alga Sargassum incisifolium was extracted with ethyl acetate. The ethyl acetate extract was adsorbed on silica gel and subjected to flash chromatography with diethyl ether and then methanol. Purification of the diethyl ether fraction by column chromatography afforded fucosterol (3). The methanol fraction was subjected to normalphase column chromatography to give two fractions.  10 . According to ESI mass spectrometry minor amounts of several other monogalactosyldiacylglycerols bearing fatty acids with a lower degree of unsaturation are present in the mixture. This corresponds to findings of Marcolongo et al. who investigated the monogalactosyldiacylglycerol fraction isolated from the thermophilic blue-green alga Phormidium sp. ETS-05 and identified palmitic acid and stearidonic acid as main acyl components during their fatty acid analysis by GC [26].
The IR spectrum of 4 exhibited absorption bands corresponding to hydroxyl (3391 cm -1 ) and carboxylic ester (1738 cm -1 ) groups. The 1D and 2D NMR spectra of 4 displayed signals for a carbon chain bearing four methylene-interrupted double bonds, a saturated carbon chain, a sugar moiety, a glycerol unit, and two carbonyl groups. The sugar residue could be identified as b-D-galactose by comparison of the 13 C NMR shifts with those of various sugars listed in Ref. [27]    The second acyl chain proved to be fully saturated as all olefinic proton and carbon signals could be assigned to the stearidonoyl part. The chain length of 16 carbon atoms was deduced from the intensities of the methylene signals in the 1 H NMR spectrum and the molecular mass of 750 derived from the ESI mass spectrum. The proton signals of the first methylene unit at d  S2a). This result proved that the C16:0 acyl chain is attached at C-1 of the glycerol moiety.
Monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols are widely spread in nature particularly in chloroplast membranes [28][29][30][31]. Accordingly, they have also been found in brown algae. For example, Kim et al. isolated four monogalactosyldiacylglycerols from the brown alga S. thunbergii collected at the coastal areas of Korea [32]. A series of monogalactosyldiacylglycerols has been identified by Liu and coworkers in Sargassum horneri [33] (4) has been mentioned as a trace component in a mixture of monogalactolipids obtained from Scytonema sp. as derived from enzymatic hydrolysis, HRMS, and EI-MS fragmentation studies [34]. A monogalactosyldiacylglycerol (MGDG) fraction that should contain considerable amounts of glycolipid 4 according to their fatty acid analysis by GC has been isolated by the group of Marcolongo from the thermophile blue-green alga Phormidium sp. ETS-05 [26]. The presence of the stearidonoyl side chain, however, was not supported by their 1 H and 13 C NMR data. The MGDG fraction displayed antiinflammatory activity in croton-oil-induced ear edema and carrageenan-induced paw edema in mice [35], and on human articular cartilage [36,37]. Herein, we describe the first isolation of 1-O-palmitoyl-2-O-stearidonoyl-3-O-b-Dgalactopyranosylglycerol (4) from a brown alga (Sargassum incisifolium) with full assignment of the 1 H and 13 C NMR signals.
In the agar diffusion test, the crude extract of Sargassum incisifolium exhibited very strong antimicrobial activity against the Gram-positive bacteria S. pneumoniae, S. aureus, and B. cereus (Table 3). No activity was found against the Gram-negative bacteria, Enterobacter cloacae, Klebsiella oxytoca, E. coli, S. enteridis, and the yeast C. albicans. Moreover, the crude extract of Sargassum incisifolium shows antimalarial activity with an IC 50 value of 57.80 ± 1.91 lg/mL for inhibition of the FCM29 strain of Plasmodium falciparum.
The mixture of 24-ketocholesterol (5) and enone 6 was obtained as a colorless, amorphous powder. The two components could be detected by GC-MS (5/6 = 3:1). The respective EI mass spectra showed molecular ion peaks at m/z = 400 and at m/z = 398 which could be assigned to the molecular formulae C 27 (6), saringosterol (7), apo-9 0 -fucoxanthinone (8) side chain could be differentiated and assigned to 5 or 6, respectively. The most significant differences are the signals for Ref. [40,49]) confirmed the identity of compounds 5 and 6.
While numerous papers are available on the isolation of 5 from various kinds of organisms, enone 6 has been mentioned less frequently and has been found only in marine organisms. Saringosterol (7) was obtained as a colorless, amorphous solid. Beside the signals for the steroidal backbone including the methyl groups, the 1 [21]. The respective 13 C NMR shifts have been compared with those published by the groups of Ayyad [45] and Wang [50]. In general a good agreement has been observed. The signals for C-28 and C-29 reported by the group of Ayyad are about 4-5 ppm upfield shifted compared to the values of Wang and us. Saringosterol (7) has been isolated from diverse organisms, in particular brown algae (for examples see Ref. [42,43,[50][51][52][53][54]). A variety of bioactivities has been disclosed for this compound including antitrypanosomal activity [54], selective LXRb agonist activity (potential cholesterol reducing agent) [55], and inhibition of Mycobacterium tuberculosis growth [56].   (Table 4) [47]. Apo-9 0 -fucoxanthinone (8) was first reported as an oxidative degradation product of fucoxanthin [46,57]. The structure and the absolute configuration of 8 were established by synthesis and X-ray crystallographic analysis [58]. Apo-9 0 -fucoxanthinone (8) has been isolated, for example, from the cultured marine dinoflagellate Amphidinium sp. [46] and from the brown algae Scytosiphon lomentaria [59], S. thunbergii [60], and Cladostephus spongiosus f. verticillatus [61]. This is the first report on the isolation of 8 from T. ornata. It has been disclosed previously that apo-9 0 -fucoxanthinone (8) exhibits cytotoxicity against murine lymphoma L-1210 and human epidermoid carcinoma KB cells in vitro with IC 50 values of 0.29 and 0.24 lg/mL, respectively [47].
In the agar diffusion test the crude extract of T. ornata showed very strong antimicrobial activity against the Gram-negative bacteria Shigella boydii, Enterobacter cloacae, and Klebsiella oxytoca and the Gram-positive bacteria S. pneumoniae, S. aureus, and B. cereus (Table 3). An inhibiting activity was also detected against the yeasts Candida membranaefaciens, Trichosporon mucoides, and Cryptococcus neoformans.  (Table 3). The crude methanol extract of T. decurrens was very active against the Gram-negative bacterium Klebsiella oxytoca, the Gram-positive bacteria S. pneumoniae, S. aureus, and B. cereus, and against the yeasts C. membranaefaciens, Trichosporon mucoides, and Cryptococcus neoformans. Moreover, significant activity was observed against Enterobacter cloacae. Kumar et al. reported various antimicrobial and cytotoxic activities of different extracts of T. conoides (J. Agardh) Kutzing [62]. In our study, the crude methanol extract of T. conoides exhibited very strong activity against S. pneumoniae, S. aureus, and B. cereus. The crude methanol extract of H. cuneiformis, was very active against the Gram-positive bacteria S. pneumoniae and S. aureus and only slightly active against B. cereus.
Obviously, the crude methanol extracts of all species of brown algae investigated in the present study show a similar activity against various pathogenic microbes. In particular, in all cases a very strong activity against the Gram-positive bacteria S. pneumoniae and S. aureus was observed. This may be ascribed partly to the content of phytosterols, which have been isolated from all species of brown algae. The antibacterial activity of b-sitosterol, stigmasterol, and their acetates has been described earlier by Sharma [63]. Fucosterol (3) has been reported to show anti-oxidant and hepatoprotective activities in rats [64]. In addition, antihistaminic, anticholinergic, and antiviral activities have been described for fucosterol isolated from T. conoides (J. Agardh) Kutzing [65]. Saringosterol (7) has been identified as active principle in the extract of Lessonia nigrescens for the inhibition of Mycobacterium tuberculosis [56].

Extraction and Isolation
General: The fresh seaweed was washed under tap water, rinsed with distilled water, subsequently dried at 48-50°C using a universal drying oven (Binder FD 53, Germany), and then finely powdered in an Ultra Turrax Janke-Kunkel T25 S1 homogenizer (IKA, Germany) with a stitch of 1 mm. In all cases, except Sargassum incisifolium (extraction with ethyl acetate), we extracted the dried and crushed samples of the algae with methanol. When checking the methanol extract by TLC, we obtained different results concerning the polarity of the compounds. Depending on whether the compounds were less polar (best eluted with diethyl ether) or more polar (best eluted with dichloromethane), we performed a second extraction with either diethyl ether or dichloromethane, respectively. Following this procedure, we obtained the non-polar compounds of the methanol extract that were purified by column chromatography.
The crushed and dried material of S. ilicifolium (500 g) was extracted with methanol. After removal of the solvent in vacuo, the methanol extract (11 g) was further extracted with different solvents of increasing polarity, namely diethyl ether, dichloromethane, and ethyl acetate. The successive extractions were carried out under magnetic stirring at room temperature. After removal of the solvent in vacuo, the diethyl ether extract (1.6 g) was subjected to flash chromatography on silica gel using pentane-diethyl ether (3:2) as eluent. A fraction of 130 mg was obtained, which was further purified by column chromatography (eluent: pentane-diethyl ether, 10:1) followed by preparative TLC (pentane-diethyl ether 10:1) to afford 2.0 mg of fucosterol (3). The dichloromethane extract (3.0 g) was separated into two fractions by column chromatography on silica gel using pentane-diethyl ether (3:2) as eluent. Fraction 1 (118 mg) was subjected to another column chromatographic separation on silica gel using pentane-diethyl ether (3:2) as eluent to afford two subfractions (A and B). Subfraction A (76 mg) was further purified by column chromatography on silica gel using pentane-dichloromethane (1:1) as mobile phase to afford 10 mg of 1,1 0 ,2-trinorsqualenol (1). Subfraction B (15 mg) was further purified by column chromatography eluting with pentane-dichloromethane (9:1) to obtain 8.0 mg of stigmasta-5,28-dien-3b-ol (2). Fraction 2 (300 mg) was subjected to column chromatography on silica gel with pentane-diethyl ether (3:2) and further purified by a second column chromatographic separation with pentane-ethyl acetate (7:3) to give 3 mg of stigmasta-5,28-dien-3b-ol (2). The ethyl acetate extract (563 mg) was subjected to flash chromatography on silica gel to afford one main fraction (70 mg). Purification of this fraction by column chromatography on silica gel followed by preparative TLC afforded 3 mg of fucosterol (3).
The crushed material of Sargassum incisifolium (80 g) was exhaustively extracted with ethyl acetate to afford 2.0 g of a crude product. The ethyl acetate extract (2.0 g) was subjected to flash chromatography on silica gel using diethyl ether and subsequently methanol as eluents. The fraction eluting with diethyl ether (1.0 g) was separated by column chromatography on silica gel using pentane-diethyl ether (3:1) to afford a main fraction of 310 mg which was further purified by column chromatography using pentane-diethyl ether (7:3) as eluent to afford 180 mg of fucosterol (3). The fraction eluting with methanol (1.0 g) was subjected to flash chromatography on silica gel with ethyl acetate-methanol (10:1) to obtain 500 mg of a product mixture. Subsequent column chromatography on silica gel with ethyl acetate as eluent afforded two fractions of 40 mg (first fraction) and 70 mg (second fraction). Both fractions were subjected to column chromatography on silica gel using ethyl acetate-methanol (20:1) as mobile phase. The first fraction (20 mg) was further purified by preparative HPLC (column: Vydac 208TP1030, reversedphase C8, 30  The crushed material of T. ornata (680 g) was extracted with methanol. The methanol extract was concentrated under reduced pressure and the residue (17 g) was further extracted with dichloromethane. After removal of the solvent in vacuo, the residue (9.0 g) was subjected to flash chromatography on silica gel using dichloromethane as eluent. The resulting mixture (260 mg) was further purified by column chromatography on silica gel eluting with pentane-diethyl ether (3:2) to obtain four fractions. Fraction 1 (19 mg) was identified as fucosterol (3). Fraction 2 (5.0 mg) showed one spot on the TLC but was identified as a mixture of the two steroids 5 and 6 in a ratio of about 3:1 according to GC-MS and 1 H NMR spectroscopy. Fraction 3 (8.0 mg) was identified as saringosterol (7) and fraction 4 (19 mg) as apo-9 0 -fucoxanthinone (8).
The crushed and dried S. polycystum (3.4 g) was extracted with methanol. The organic extract was evaporated to dryness and a dark oily residue (85 mg) was obtained. The crude extract (85 mg) was then further extracted with dichloromethane and purified by column chromatography on silica gel using pentane-ethyl acetate (10:1) as mobile phase to afford 12 mg of fucosterol (3).
The crushed and dried plant material of Sargassum sp. (S. sect. Binderiana) (16 g) was minced and extracted exhaustively with methanol. After filtration, the organic extract was evaporated to dryness, and a dark oily residue was obtained. The crude extract was further extracted with diethyl ether. The diethyl ether extract (900 mg) was subjected to flash chromatography on silica gel using pentane-diethyl ether (3:2) to give two fractions. Fraction 1 contained 2-(tert-butyl)-4-chloro-5-methylphenol as an artifact and was disposed. Fraction 2 (78 mg), was separated by column chromatography on silica gel using pentane-ethyl acetate (9:1) to give 6.0 mg of a mixture of fucosterol (3) and b-sitosterol in a ratio of 1:1.3.
The crushed material of T. decurrens (12 g) was repeatedly extracted with methanol and the combined extracts were concentrated under reduced pressure. The crude extract (300 mg) was subjected to column chromatography on silica gel with a mixture of pentane-diethyl ether (4:1) to give 19 mg of a product mixture which was further purified by a second column chromatography using the same conditions to afford 13 mg of fucosterol (3).
The crude extract of T. conoides was obtained after extraction of the crushed material (40 g) with methanol. After removal of the solvent, the residue (960 mg) was extracted with dichloromethane at room temperature. The dichloromethane extract (890 mg) was subjected to flash chromatography on silica gel with diethyl ether. The diethyl ether fraction (190 mg) was separated by column chromatography on silica gel using pentane-diethyl ether (4:1) as eluent to provide 47 mg of a product mixture. Another purification step by column chromatography on silica gel with pentane-diethyl ether (4:1) as mobile phase afforded 21 mg of fucosterol (3).
The dried plant material (9.4 g) of H. cuneiformis was minced and extracted exhaustively with methanol. After evaporation of the solvent, a dark oily residue (84 mg) was obtained. The crude extract (84 mg) was then repeatedly extracted with diethyl ether, and the combined diethyl ether extracts were concentrated under reduced pressure to give a residue of 65 mg. The diethyl ether extract (65 mg) was separated by column chromatography on silica gel using a mixture of pentane-ethyl acetate (4:1) as eluent to afford 1 mg of fucosterol (3).

Spectroscopic Characterization
General: Optical rotations were determined on a Perkin Elmer 341 polarimeter at a wavelength of 589 nm (sodium D line) using a 1.0-decimeter cell with a total volume of 1.0 mL. UV spectra were measured on a Perkin Elmer Lambda 25 UV-Vis spectrometer. Fluorescence spectra were measured on a Varian Cary Eclipse. IR spectra were recorded on a Thermo Nicolet Avatar 360 E. S. P. FT-IR spectrometer using the ATR technique (attenuated total reflectance). NMR spectra were recorded on a Bruker AVANCE III 600 spectrometer. The chemical shifts d are reported in ppm using the solvent signal as internal standard. Assignment of the 1 H NMR and 13 C NMR signals was achieved using the following 2D NMR experiments: COSY, HSQC, HMBC, NOESY, and HSQC-TOCSY. The mass spectra were measured by GC-MS coupling with an Agilent Technologies 6890 N GC system equipped with a 5973 N Mass Selective Detector (electron impact, 70 eV). ESI-MS were recorded on a Bruker-Esquire mass spectrometer with an ion trap detector; positive and negative ions were detected. Thin layer chromatography was added via a pipette onto a sterile antibiotic filter disc of 6 mm diameter and oven dried at 40-50°C. The discs were placed on Müller-Hinton agar plates which had been inoculated with the microorganisms mentioned above. The plates were incubated for 24 h at 37°C for the bacteria and for 48 h at 25°C for the yeast. The diameters of the inhibition zones generated around the discs were measured (Ø in mm). The tests were performed in triplicate and the mean values were determined. Methanol, used to dissolve the extracts, was checked for the absence of antimicrobial activity. The diameters of the halos of inhibition can be rationalized on a qualitative basis as follows: Ø \ 7 mm: inactive, 7 mm B Ø \ 8 mm: slightly active, 8 mm B Ø \ 9 mm: significantly active, Ø C 9 mm: very active.
Antimalaria test: The antiplasmodial activity against the FCM29 strain of Plasmodium falciparum was determined by using the microfluorimetric assay previously reported [70]. The result is given as an IC 50 value in lg/mL.

Conclusions
We present one of the first extensive studies on the nonpolar chemical constituents of brown algae from the coast of Madagascar. Our results confirm the early reports that fucosterol (3) is the major sterol of brown algae (Phaeophyceae) [14]. Moreover, the sterols stigmasta-5,28-dien-3b-ol (2), 24-ketocholesterol (5), (22E)-3b-hydroxycholesta-5,22-dien-24-one (6), and saringosterol (7) have been isolated from S. ilicifolium and T. ornata, respectively. The allenic norterpenoid apo-9 0 -fucoxanthinone (8) was obtained from T. ornata. For the first time, 1,1 0 ,2trinorsqualenol (1) has been obtained as a pure compound from natural sources and moreover, its isolation from a marine organism (S. ilicifolium) is unprecedented. The glycolipid 1-O-palmitoyl-2-O-stearidonoyl-3-O-b-Dgalactopyranosylglycerol (4) was obtained from the brown alga Sargassum incisifolium and for the first time, we report the complete assignment of its 1 H NMR and 13 C NMR data. Antimicrobial tests of the crude extracts of the various brown algae revealed a strong activity against Gram-positive bacteria, in particular against S. aureus and Streptococcus pneumonia, which may ascribed to the content of phytosterols.