Glycobiology Advance Access originally published online on June 24, 2006
Glycobiology 2006 16(10):902-915; doi:10.1093/glycob/cwl018
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Glycolipids from the red alga Chondria armata (Kütz.) Okamura
National Institute of Oceanography, Dona Paula, Goa 403 004, India
1 To whom correspondence should be addressed; e-mail: solima{at}nio.org
Received on November 22, 2005; revised on June 12, 2006; accepted on June 12, 2006
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Abstract |
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Three distinct fractions containing polar glycolipids (PF1–3) were isolated from the chloroform soluble fraction of crude methanolic extract of red alga Chondria armata (Kütz.) Okamura on gel chromatography over Sephadex LH20. Their structure was elucidated by multidimensional nuclear magnetic resonance (NMR) techniques like 1H, 1H correlation spectroscopy (COSY), 1H, 1H total COSY (TOCSY), 1H, 13C heteronuclear multiple quantum coherence (HMQC), and 1H, 13C heteronuclear multiple bond correlation (HMBC) complemented by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. The coupling constant of the anomeric proton in 1H NMR spectrum and sign of rotation indicated an exclusive configuration of the sugar molecules in the glycerolipids. Major glycolipids were identified as (2R)-2-O-(5,8,11,14-eicosatetranoyl)-3-O-
-D-galactopyranosyl-sn-glycerol (GL2), its pentacetate (GL1), and (2R)-1-O-(palmitoyl)-2-O-(5,8,11, 14,17-eicosapentanoyl)-3-O-ß-D-galactopyranosyl-sn-glycerol (GL3). Each was methanolysed to give the same galactosylglycerol which on ESI-MS provided a pseudomolecular ion at m/z 309 representing deacylated glycolipid with the sodiated sugar moiety. Additionally, six minor glycolipids were also identified on the basis of ESI-MS. These include a 1,2-di-O-acyl-3-O-(acyl-6'-galactosyl)-glycerol (GL1a), sulfonoglycolipids 2-O-palmitoyl-3-O-(6'-sulfoquinovopyranosyl)-glycerol (GL2a) and its ethyl ether derivative (GL2b), 1-oleoyl-2-palmitoyl-3-O-galactosyl glycerol (GL3a), and 1,2-diacyl phosphatidyl glycerol (GL3b). GL1, GL1a, and GL2b are new to the literature. The novelty of the remaining identified compounds lies in the diversity of their fatty acid composition. Antimicrobial properties of these glycolipids against pathogens were evaluated. The yeast Candida albicans and the bacteria Klebsiella sp. were as sensitive as the standard Nystatin and antibiotic Streptomycin against PF3. Considerable activity was expressed by the same metabolite against the fungus Cryptococcus neoformans as compared to the control. Weak activity against the bacteria Shigella flexineri and Vibrio cholerae and the fungus Aspergillus fumigatus was also observed. Fraction PF2 was weakly active against some strains whereas all of them were resistant to its acetyl derivative PF1. Antimicrobial activity of glycolipids is being reported here for the first time. Key words: antimicrobial / Chondriaarmata / ESI-MS / glycolipids / NMR
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Glycoglycerolipids are glycolipids in which one or more saccharide residues are linked by a glycosyl linkage to a lipid moiety containing a glycerol residue. They constitute an important class of membrane lipids that are synthesized by both prokaryotic and eukaryotic organisms (Kates, 1990
). They are reported to exhibit diverse biological functions. There is currently considerable interest in both intracellular and extracellular glycolipids, especially galactosyl glycolipids as antitumor promoters in cancer chemoprevention (Colombo et al., 2001).
As a part of our systematic search for potentially useful biomedicinal agents of marine origin, we have been investigating metabolites of the red alga Chondria armata (Kütz.) Okamura, belonging to the family Rhodomelaceae. The methanolic extract of this alga exhibited 75% antiviral activity against SFV (Semiliki Forest Virus) (Kamat et al., 1992) and hypotensive activity (Naqvi et al., 1981). Earlier, we have reported pigment caulerpin, novel ester pentyl hentriacontanoate, fatty acids and sterols (Wahidulla, 1999, 2000a, 2000b), and novel polyethers, armatols (Ciavatta et al., 2001), from this alga. This article presents a full account of the structural elucidation of major galactosylglycerols identified as (2R)-2-O-(5,8,11,14-eicosatetranoyl)-3-O--D-galactopyranosyl-sn-glycerol (GL2), its pentacetate (GL1), and 2R-1-O-(palmitoyl)-2-O-(5,8,11,14,17-eicosapentanoyl)-3-O-ß-D-galactopyranosyl-sn-glycerol (GL3) from the same source and reports antimicrobial activity exhibited by GL2 and GL3 (Figure 1A).
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An effort to elucidate the structure of the additional related molecular species, with the same Rf values on thin layer chromatography (TLC) and were inseparable from the purified major glycolipids GL1–3, was made based on tandem mass spectrometry. The structures proposed for these minor constituents have also been incorporated.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Extraction and fractionation of glycolipids
Fresh alga was exhaustively treated with methanol and the chloroform soluble fraction on column chromatography gave, in order of polarity, fractions PF1–3, apparently homogenous on TLC, yielding purplish pink spots on spraying with methanolic sulfuric acid. Their 1H and 13C nuclear magnetic resonance (NMR) spectra (Table I) closely resembled those reported for galactosyl glycerolipids. Electrospray ionization mass spectrometry (ESI-MS) was useful to characterize molecular ions and the sequence of groups in the studied molecules and to distinguish lipid structures that gave similar NMR spectra.
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Structural characterisation of PF1
The purified PF1 gave protonated molecular ion [M + H]+ at m/z 751 in its ESI-MS spectrum (Figure 2A). The presence of spin systems corresponding to one hexose, glycerol and fatty acid was readily identified from the 1D and 2D homonuclear 1H correlation spectroscopy (COSY) NMR spectra. Thus, the 1HNMR spectrum (300 MHz, deuterated chloroform [CDCl3]) was in agreement with monogalactosyldiacylglycerol (MGDG), with the fatty acyl chain being evident by the presence of a triplet due to a terminal methyl at 
0.827, a broad methylene signal at 1.202 [(CH2)n] of aliphatic chain, multiplets at 2.268, 1.967, and 1.562 assigned to three methylenes linked , ß, and 
to the ester carbonyl functionality. A broad multiplet at 2.7 arises from allylic methylene protons and the olefenic methine protons were evident at 5.293. A sharp singlet at 2.12 was attributed to acetyl methyls.
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The presence of glycerol moiety was also confirmed by a heteronuclear multiple quantum coherence (HMQC) experiment, which showed two doublets arising from C-3 and C-1. The signals at 4.22 and 4.35 correspond to the substitution at C-1 ( 62.2) by an O-acyl group and the doublet at 3.56 and 3.96 was assigned to C-3 ( 68.2) of glycerol substituted by the -galactose residue. The glycerolipid structure was confirmed by the presence of a characteristic signal at 70.0/5.23 (C-2) having a distinct -shift to lower field for 13C and 1H nuclei when substituted by an O-acyl group, this being a fingerprint for glycolipids containing glycerol as alcohol rather than sphingosine (Sassaki et al., 1999).
1H-1H COSY, total COSY (TOCSY), and HMQC correlations allowed assignment of sugar carbons and protons (Table I). 1H-1H COSY and TOCSY correlation of the anomeric proton at 4.178 with the sn-3 protons at 3.56 and 3.96 established connectivity of the sugar moiety with the glycerol. The anomeric proton at 4.178 with a coupling constant of 2.1 Hz indicated -glycosidic configuration of the sugar linkage with the glycerol (Dabrowski et al., 1980).
Long-range heteronuclear multiple bond (HMBC) diagnostic correlations were observed between the ester carbonyls at 173.8 and 173.5 and C-1 and C-2 of glycerol, indicating the linkage. The stereochemistry at C-2 was assigned to be R by comparison of the coupling constant values between H-2/H-3a (J = 3.6 Hz) and H-2/H-3b (J = 6.0 Hz), respectively, with those of published data (Oshima et al., 1994; Murikami et al., 1995; Reshef et al., 1997). On the basis of the above data the major component of PF1 was identified as pentacetate of GL2 (GL1). The fragmentation observed in tandem mass spectrometry (MS/MS) spectrum of GL1 (Figure 2C) is well in agreement with the structure assigned. The pseudomolecular ion at m/z 751 generated a series of daughter ions at m/z 691, 631, 571, and 511, reflecting successive loss of four acetic acid molecules. The presence of a fifth acetyl group was evident from the elimination of yet another acetic acid molecule, yielding sodiated fragment at m/z 473. Alternately, the ion at m/z 473 might have originated, as diprotonated sodiated fragment ion, after the elimination of arachidonate ion. This ion on elimination of the fifth acetic acid molecule would lead to ion at m/z 413. This confirmed the presence of acetylated hexose linked to the glycerol moiety, with the latter being diesterified by acetic acid and eicosatetraenoic acid. The proposed structure of GL1 along with identified fragments is represented in Figure 2C.
There is a solitary reference in the literature on the identification of 2-O--D-galactopyranosyl glycerol hexacetate from Ruellia britoniana E. Leonard (Acanthaceae) (Ahmad et al., 1990). The acetylated galactoglycerolipid is being reported here for the first time from a marine source.
ESI-MS (Figure 2A) of PF1, though apparently homogenous on TLC, showed some heterogeneity by the presence of an additional related molecular species with m/z 1017. Based on the fragmentation pattern observed in MS/MS (Figure 2B) it was characterized as 1,2-di-O-acyl-3-O-(6-acylgalactosyl)-glycerol GL1a.
MS/MS studies of the [M + H]+ ion at m/z 1017 (Figure 2B) resulted in three major diagnostically important daughter ions at m/z 481, 735, and 761. The ions at m/z 735 and 761 reflect the neutral losses of the sn-1 and sn-2 substituents as free C18:1 and C16:0 carboxylic acid, respectively, supporting the presence of palmitic and oleic acyl moieties in the molecule. The intensity differences of these various ions indicated the position of the different fatty acid moieties, as the substituent position at sn-2 fragments comparatively easily (Murphy and Harrison, 1994). This leaves a mass for the core of the molecule of 481 amu. Such a mass can be explained by a substituted hexose connected to a glycerol backbone after elimination of fatty acyl groups from the protonated molecular ion [M + H]+. This is further supported by the presence of an additional fragment ion at m/z 441, which reflects the loss of acyl groups (C18:1 and C16:0) from the molecular ion along with the glycerol backbone, together corresponding to a total mass of 577 amu. Fragment ion at m/z 423 results from the cleavage between C1 of hexose and C-3 of glycerol. Cleavage of the molecule between C5 and C6 of the sugar leads to sodiated fragment at m/z 313 which corresponds to the third acyl substituent (C18:3) along with C-6 of sugar, which possibly seems to be galactose. The ion at m/z 295 results from the cleavage between C1 and C2 of glycerol. Furthermore, there were a number of fragments in the upper mass region at intervals of about 14 amu. These correspond to fragmentation along the fatty acid acyl chains. On the basis of this fragmentation pattern of the molecular species with the pseudomolecular ion at m/z 1017, we propose the structure of the molecule as being 1-oleoyl-2-palmitoyl-3-O-(linolenyl-6'-galactosyl)-glycerol (GL1a), which along with identified fragments is illustrated in Figure 2B.
Structural characterization of PF2
A similar approach was adopted for PF2, which showed physicochemical characteristics of glycolipids. Its NMR data differed from that of PF1 only by the absence of signals for the acetyl groups (Table I), indicating it to be a deacetylated derivative of GL1. This was further supported by its ESI-MS (in MeOH), which exhibited pseudomolecular ion [M + H]+ at m/z 541 consistent with the molecular formula of C29H49O9 (PF2). The MS/MS at m/z 541 (Figure 3D) showed peak at m/z 179 for loss of a sugar unit. Subsequent loss of the four water molecules from the hexose led to the base peak at m/z 107. The cleavage of the molecule between C-3 of glycerol and oxygen linking it to the hexose gives the fragment ion at m/z 343 with simultaneous elimination of water molecule. The sodiated ion at m/z 204 results from the attachment of two hydrogens to the hexose moiety. Fragmentation of the ester bond leads to the ion at m/z 239. Similarly, the fragment at m/z 223 could be explained as being formed by cleavage of C2–C3 bond of glycerol backbone and cleavage between the oxygen and carbonyl of carboxylate group. The ion at m/z 267 results from the addition of sodium to the fragment derived from the McLafferty rearrangement in the acyl moiety. Thus the structure of the major component from PF2 was established as GL2.
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The ESI-MS examination of PF2 (Figure 3A) when taken in a dilution solvent (as given under Materials and methods) showed additional peaks at m/z 601 and 629 corresponding to pseudomolecular ions of the Na salt (Na+ form) of sulfonoglycolipids [M–H + 2Na]+. An effort was made to elucidate their structure by tandem mass spectrometry of these molecular species.
Thus, MS/MS of the pseudomolecular ion at m/z 601 exhibited the most abundant product ions at m/z 519 and 497, which have a mass difference corresponding to likely loss of sulfono group (82 amu) as sulfonic acid (SO3H) and as sodium salt (SO3Na) respectively. The product ion observed at m/z 345 appears to have originated by the loss of fatty acyl side chain as corresponding acid (palmitic acid, C16:0). Cleavage between C-3 of glycerol and the oxygen at the anomeric carbon of hexose results in the simultaneous formation of the fragments at m/z 313 and 273. The later ion is formed with the loss of two hydrogens. The ion at m/z 273 loses one water molecule to yield the fragment at m/z 259. The ion at m/z 165 results from the elimination of sodium sulfonate group from the sulfonoquinovopyranosyl moiety and cleavage between C-3 of glycerol and the oxygen at the anomeric carbon with the attachment of three hydrogens. Subsequent elimination of three water molecules leads to the ion at m/z 111. Based on fragmentation pattern, the glycolipid with pseudomolecular ion [M–H + 2Na]+ at m/z 601 was characterized as 2-O-palmitoyl-3-O-(6'-sulfoquinovopyranosyl)-glycerol (GL2a). The proposed structure along with its identified fragments is shown in Figure 3C.
A similar fragmentation pattern (GL2a) observed for the sulfonoquinovosyl molecular species with pseudomolecular ion at m/z 629 led to the structure GL2b as represented in Figure 3B. From the fragmentation observed it is interesting to note that the difference of 28 amu observed between the two sulfonolipids is not because of the difference in the fatty acid chain length as expected but seems to be due to the ethoxy group at C-1 of glycerol. The presence of sulfono group is further reinforced by the presence of 13C NMR signal for CH2 attached to sulfur at 53.6 ppm, as an impurity in PL2. The galactosylglycerolipids MGDG and digalactosyldiacylglycerol (DGDG) are uncharged species while sulfoquinovosyl diacylglycerol (SQDG) is negatively charged at neutral pH. This explains their presence in admixture as sodiated adducts.
Structural characterization of PF3
The ESI-MS profile of PF3 is illustrated in Figure 4A. ESI-MS of the major component of this fraction was consistent with the sodiated molecular ion [M + Na]+at m/z 799 corresponding to the molecular formula of C45H76O10Na. Its 1H NMR and 13C NMR (Table I) closely resembled those of PF2 except that the 13C signals due to the unsaturation in the fatty acid moiety were more distinct. The tandem MS/MS spectrum of ion at m/z 799 is illustrated in Figure 4B and it represents GL3. The main fragmentation pathway corresponding to concomitant elimination of two fatty acyl moieties yielded ion at m/z 243 characteristic of MGDG (Welti et al., 2003). Ions reflecting neutral loss of eicosapentanoate and palmitate as free fatty acids are evident from the fragment ions at m/z 497 and m/z 543 respectively. Elimination of the palmitoyl acyl group as an acid from the pseudomolecular ion [M + H]+ leads to the ion at m/z 521. Additional ions are observed due to loss of three molecules of water from the sugar moiety, yielding protonated ion at m/z 109. Loss of two water molecules along with hydroxymethyl group from the sugar moiety produced ion at m/z 97 and loss of arachidonate ion results in ion at m/z 301. HMQC and TOCSY spectra of GL3 are represented in Figure 5A and B. As evident, the TOCSY spectrum is characteristic of glyceroglycolipid with the spin systems of glycerol, sugar and the constituent fatty acids.
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As evident from Figure 4A, PF3 though apparently homogenous on TLC, showed some heterogeneity in its mass as evidenced by the presence in the ESI-MS of several other ions of related molecular species besides the main component. The MS/MS of sodiated molecular ion [M + Na]+ at m/z 779 (Figure 4C) yielded fragments at m/z 497 and m/z 523, indicative of loss of C18:1 and C16:0 fatty acyl groups from the molecule as free fatty acids, respectively. The presence of galactosyl sugar moiety was evident from the ion at m/z 243. The intensity of the signals led to the placement of palmitic acid at sn-2 position (Blair, 1990; Waugh and Murphy, 1996; Hankin et al., 1997). Taken together, the structural analysis for the molecular species with ion at m/z 779 is consistent with 1-oleoyl-2-palmitoyl-3-O-galactosyl glycerol (GL3a).
The collision-induced dissociation (CID) daughter-ion spectrum of the molecular species at m/z 691 is illustrated in Figure 4D and it represents 1,2-diacyl phosphatidyl glycerol (GL3b). The main fragmentation pathway observed here is the formation of ion at m/z 413 originating from the loss of 278 amu, corresponding to the loss of C18:3 as free fatty acid. The ions at m/z 171 and m/z 189 are consistent with the cleavage at C12 of -linolenic acid as free acid and as ketene, respectively (Kim et al., 1999). The fragment at m/z 171 could also arise from phosphoglycerol moiety. The most intense ion at m/z 301 was attributed to the concomitant elimination of palmitoleoyl and phosphatidyl groups along with the glycerol backbone as depicted in Figure 4D or elimination of linoleic acid as sodium salt. The abundance of the ion at m/z 301 as compared to the ion at m/z 413 is consistent with the notion that neutral loss of the fatty acid at sn-2 is sterically more favorable than the analogous loss at sn-1 position (Blair, 1990; Waugh and Murphy, 1996; Hankin et al., 1997). Thus structure GL3b was proposed for the molecular species with [M + Na]+ ion at m/z 691.
Tandem MS scanning experiment of protonated molecular species at m/z 655 yielded the most prominent ion at m/z 301, reflecting the loss of 354 amu, which is probably due to the presence of a digalactosyl unit and a much less intense fragment at m/z 377 corresponding to the loss of palmitoyl group as sodium palmitate from the molecule. The relative abundance of the ions placed the palmitoyl group at sn-2 position. The spectrum is consistent with 3-digalactosyl-2-palmitoyl glycerol, represented in Figure 4E.
Methanolysis of PF1–3
To identify the acid substituents at C-1 and C-2 of component glycolipids of PF1–3, methanolysis was performed in anhydrous methanol with excess of Na2CO3. All the three fractions yielded the same glycoside, 3-O-D-galactopyranosyl-sn-glycerol, and methyl esters of corresponding fatty acids. The mixture of the reaction product was analyzed by ESI-MS in the positive ion mode. Thus, for example, the ESI-MS of PF3 gave pseudomolecular ions at m/z 183, 277, 309, 301, 334, and 389. Analysis of each of these ions by tandem MS established their identity. Thus the ion at m/z 183 corresponded to the attachment of two hydrogens to the sugar moiety [M + 2H]+. 3-O-D-galactopyranosyl glycerol as sodium adduct was observed at m/z 277. Deacylated glycolipid with the sodiated sugar moiety was evident as protonated molecular ion at m/z 309. The fragment at m/z 301 represented the presence of eicosapentanoate. Thus, the fragmentation observed in MS/MS of ion at m/z 309 (Figure 6), a fragment common as product of hydrolysis of PF1–3, is shown in Figure 6.
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To establish the nature of the sugar moiety as D-galactose, the glycolipids were subjected to acid hydrolysis and the compound identified by TLC with standard sugars as described in Materials and methods (Figure 1B). The optical rotation of the sugar obtained by hydrolysis was well in agreement with the values reported for D-galactose.
Antimicrobial activity of PF1–3
Bergsson et al. (2001) have studied the susceptibility of Candida albicans to several fatty acids and their 1-glycerides. They observed that capric acid, a 10-carbon saturated fatty acid, causes the fastest and most effective killing of all the three strains of C. albicans tested. Lauric acid, a 12-carbon saturated fatty acid, was the most active acid at lower concentrations. Subsequently, Frentzen et al. (2003) reported on the medium-chain fatty acids of 8–12 carbon atoms exhibiting antibacterial and antifungal properties, which are enhanced when these acids are esterified with glycerol. The same authors also state sucrose esters as being less effective in inhibiting the fungal growth. Based on these reports, it is expected that pathogens would be sensitive to glycolipids. This led us to evaluate the pure fractions PF1–3 of the present investigation, isolated and identified from the red alga C. armata, against different strains of pathogenic microorganisms, for antibacterial and antifungal activities and compare them with the commercially available antibiotics (Table II).
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As evident from Table II, all the bacteria and fungi tested were resistant to PF1 at the dose tested (65 µg/mL). PF2 showed mild inhibitory activity against the bacteria tested except Pseudomonas aeruginosa and Klebsiella pneumoniae, and at 250 µg/disc being also weakly active against the fungi Aspergillus fumigatus, Cryptococcus neoformans, Aspergillus niger, and Rhodotorula sp. PF3, at 130 µg/disc, was as effective as standard Nystatin and antibiotic Streptomycin, against the yeast C. albicans and bacteria Klebsiella sp.,- respectively. Considerable activity was also expressed by PF3 against the fungus C. neoformans, strain resistant to Nystatin. PF3 showed mild activity against the bacteria Shigella flexineri and V. cholerae and the fungus A. fumigatus. All the three compounds were ineffective against the multidrug-resistant strains tested.
Results indicate that acetylation inactivates the molecule and the activity is greatly influenced by the anomeric configuration of glycosidic linkage—compounds with ß configuration being more effective than the glycosides with configuration. Antimicrobial activity of glycoglycerolipids is being reported here for the first time.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Three major galactoglycerolipids have been isolated and identified, in the native form, from the red alga C. armata using NMR complemented with mass spectrometry. Six minor glycolipids have also been identified on the basis of electrospray ionization tandem MS/MS spectrometry alone. Methanolysis of the glycolipids yielded galactosylglycerol, which on ESI-MS provided a pseudomolecular ion at m/z 309 representing deacylated glycolipid with the sodiated sugar moiety. Recently, Shao et al. (2002)
reported the presence of a new sulfonoglycolipid, crassicaulisine, with palmitoyl and myrsitoyl as acyl groups, from the red alga belonging to the same genus Chondria crassicaulis. Acyl groups in PF1–3 were characterized as the corresponding acids or carboxylate ions (ESI-MS), and the principal components were arachidonic acid in PF1–2, palmitic acid, and eicosapentaenoic acid in PF3. There were minor components, which include C16:1, C18:1, and C18:3 acids.
It is of interest to note that polyunsaturated fatty acids eicosapentaenoic acid (EPA) and arachidonic acid (AA) are present in the alga in bound form as acyl substituents in galactosyl acyl glycerols. In agreement with previous reports, palmitic acid seems to be the major fatty acid in sulfonoglycolipids of marine algae. Contrary to the reports of Choi et al. (1999) in glycolipids of marine algae, the glycosidic linkage could be /ß and the sugar moiety is attached, mainly, to C-3 of sn-glycerol.
GL1a is the first example of the natural occurrence of acyl glycerol acylated at the sn-1, sn-2 and 6' positions. The presence of acyl glycerol acylated at the sn-1 and 6' positions of mannobiosyl is known from the bacteria Arthrobacter atrocyaneus and Microcoleus luteus (Bultel-Ponce et al., 1997; Niepel et al., 1997).
In recent years, glycoglycerolipid analogues have gained importance in cancer chemoprevention because of the promising inhibitory effect exhibited by them on tumor-promoting activity. The fatty acyl chain length, its position and the nature of sugar moiety influence the activity. Galactosyl glycerols are reported to be more potent than the corresponding glucosylglycerols with the same structural features (Colombo et al., 1996, 1999). The anomeric configuration does not seem to affect the activity (Colombo et al., 2000).
MGDGs, containing (7Z, 10Z)-hexadecadienoic acyl group, from the green alga Chlorella vulgaris are reported to exhibit anti-tumor promoting effect (Morimoto et al., 1995). SQDG from algae inhibits DNA-polymerase and HIV-reverse transcriptase (Gustafson et al., 1989; Loya et al., 1998; Ohta et al., 1998). It is well known that biological activity of marine macrophytes is related to the essential polyunsaturated fatty acids (PUFAs), which are the abundant components of macrophytic glycolipids (Khotimchenko, 1993; Goncharova et al., 2000; Sanina et al., 2000).
The red algae are reported to have high levels of polyunsaturated fatty acids, mainly EPA and AA (Khotimchenko and Svetashev, 1987), but the contents vary within the same genus. Chondria dasyphylla (Wood) Ag. is reported to have equal contents of EPA and AA whereas in Chondria decipiens EPA predominates (Khotimchenko and Vaskovsky, 1990). Further, in red algae PUFAs belonging to C20 series are reported to be mainly concentrated in MGDG (Sanina et al., 2004). This has in fact been observed in the present investigation, with EPA and AA being the constituent fatty acids of major glycolipids identified in PF1–3, and is well in agreement with our earlier communication on the fatty acids from the alga C. armata, where C20 acids were not detected as free fatty acids (Govenkar and Wahidulla, 1999).
Glycoglycerolipids occur widely and copiously in vascular plants (Van Hummel, 1975), certain green seaweeds (Arao and Yamada, 1989; Falsone et al., 1994; Mancini et al., 1998), cyanobacteria (Reshef et al., 1997), marine dinoflagellates (Oshima et al., 1994), and the freshwater alga C. vulgaris (Morimoto et al., 1995). As to the glycoglycerolipids of red algae, hydroxyeicosapentaenoyl galactosyl glycerols are known from the temperate red alga Gracilariopsis lemaneiformis (Jiang and Gerwick, 1990), and MGDG, DGDG, and SQDG are reported from Gracilaria verrucosa (Son, 1990), which is also known to contain sulfoquinovosylmonogalactosyl glycerol (SQMG) (GL2a). This SQMG is also reported to be a constituent of cyanobacterium Synechocystis PCC 6803 (Kim et al., 1999) and lichenized basidiomycetes, Dictyonema glabratum (Sassaki et al. 2001). 2-O--D-galactopyranosylglycerol is a metabolite of Laurencia pinnatifida (Aplin et al., 1967) and 2,3-dipalmitoyl sulfonoglycolipid has been identified in Laurencia pedicularioides and is reported to be the major glycolipid in red algae (Siddhanta et al., 1995). Recently, Shao et al. (2002) reported the presence of a new sulfonoglycolipid, crassicaulisine, in the red alga C. crassicaulis. Taxonomically, genus Laurencia and C. armata belong to the same family, Rhodomelaceae, but in the present investigation C. armata did not contain either of the glycolipids.
Interestingly, palmitic acid has been found to be the most abundant fatty acid present in the sulfonoglycolipids of marine origin (Fusetani and Hashimoto, 1975; Araki et al., 1989; Gustafson et al., 1989; Son, 1990; Siddantha et al., 1991). The two sulfonoglycolipids of the present investigation provide yet another example of a glycolipid which contains palmitic acid as the only fatty acid component. Palmitic acid was described as having hemolytic activity in sea urchin eggs (Fusetani and Hashimoto, 1976) and was presumed to be playing a unique role in algal physiology (Fusetani and Hashimoto, 1975).
Sulfonoquinovosyl acyl glycerols, in particular compounds with C18 fatty acid on the glycerol moiety, may be clinically promising antitumor or immunosuppressive agents (Aoki et al., 2005).
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Biological material
The alga was collected during the low tides from coastal waters of Goa, west coast of India [15°51'N to 15°54'N and 73°51'E to 73°52'E], during the pre-monsoon periods. The alga, sample no. 1316, identified by Geeta Deshmukh, CIFE, Mumbai, has been deposited at NIO Repository and Taxonomic Center.
General procedures
Sephadex LH20 (Pharmacia) and silica gel (60–120 mesh) [Qualigens] were used for gel filtration and column chromatography, respectively. Precoated Kieselgel 60 F254 TLC plates (Merck) were used for analytical TLC. Compounds were visualized as purplish spots on spraying with 5% methanolic sulfuric acid followed by heating at 100oC. Solvent system for TLC I and II was light petrol/ethyl acetate (6:4) and (1:1), respectively, and TLC III was methanol: chloroform (5:95).
Mass spectrometry
Mass spectra were recorded, in the positive mode, on a QSTARXL MS/MS, Applied Biosystems, Switzerland, equipped with Analyst Software. The declustering potential and the collision energy were optimized for MS/MS experiments so as to cause fragmentation of the selected molecular ion species as evident by the appearance of fragment ions and decrease in the intensity of the molecular ion. ESI-MS was carried out by dissolving the compounds in methanol as solvent. ESI-MS of PF2 was taken in methanol, as well as dilution solvent.
Dilution solvent
It was prepared as follows: 15.4 mg of ammonium acetate was dissolved in 49.9 mL of water. To this solution was added a mixture of 49.9 mL of methanol, 0.1 mL of formic acid, and 0.1 mL of acetonitrile.
NMR
1H, 13C, COSY, HMQC, and HMBC experiments were recorded, in CDCl3, on a Bruker (Avance 300) spectrometer with TMS (tetramethylsilane) as internal standard.
Extraction and isolation of glycolipids
The red alga, C. armata (3.5 kg, dry weight) was cleaned and extracted thrice with methanol using a sonicator (15 min) at room temperature. The combined methanolic extracts were evaporated under reduced pressure at 37°C temperature to a certain minimum volume (
200 mL), and then partitioned into chloroform, n-butanol and water-soluble fractions.
The chloroform fraction (123 g) was fractionated, initially on a column of Sephadex LH20 with methanol (500 mL) as eluant collected in fractions of 20 mL each. The fractions obtained were examined by TLC (solvent : light petrol : ethylacetate, 1:1,v/v, spray: 5% methanolic sulfuric acid) and combined according to their profile. Fractions yielding purplish spots were then purified by repeated silica gel chromatography using petroleum ether (60–80°C) : ethyl acetate (1:1) to give PF1 (4 mg, Rf 0.52 in solvent I), and methanol : chloroform (2:98) yielded PF2, []D-16° (c 0.02, CHCl3, Rf 0.45 in solvent II; yield 13 mg). Further elution of the same column with methanol: chloroform (5:95) yielded PF3 []D-20° (c 0.02, CHCl3, Rf 0.175 in solvent III, yield 23 mg). Final purification was done on RP-18 column with methanol as eluant. As the neutral glycolipids yielded purplish pink spots with methanolic sulfuric acid, all the constituents, from chloroform soluble fraction, showing purplish pink spots on TLC were purified.
Methanolysis of glycolipids (PF1–3)
PF1–3, 2 mg each, were dissolved in anhydrous methanol (1 mL), and an excess of sodium carbonate was added. The solution was stirred at room temperature overnight, filtered, and the solvent evaporated. The residue was analysed by ESI-MS in methanol. Tandem mass was taken at collision energy between 30 and 35 eV.
Acid hydrolysis of glycolipids (PF1–3)
Each fraction (4–8 mg) in 5 mL of 2% H2SO4 in methanol was refluxed for 3 h. This was followed by the addition of 4 mL of water to the reaction mixture. Methanol was removed in vacuo and the aqueous solution extracted with chloroform and then neutralized with Barium hydroxide. Precipitated barium sulfate was filtered through celite, water removed in vacuo, and the residue dissolved in 1 mL of water. TLC (butanol:acetic acid: water; 5:1:4) showed a single major spot identical with D-galactose. The NMR data do not distinguish between L and D forms of the glycosyl moieties. The D form of the monosaccharide dominates in living organisms (Hauksson et al., 1995), and the only occurrence of L galactose is in agar-agar (Mathews and van Hold, 1990).
For confirmation of configuration of sugar residue, PF1–were hydrolyzed with 2M TFA (trifloroacetic acid) at 110°C for 3 h, following concentration to dryness under stream of nitrogen. The product was then filtered through Sephadex G-10 (Pharmacia) using MeOH:H2O (1:1) as the mobile phase. Fractions (5 mL) were collected and monitored on TLC plates using butanol: acetic acid: water (5:1:4) as the solvent system for development. Rf value of the sugar thus obtained was equivalent to the standard D-galactose (Figure 1B). Fractions containing sugar (galactose) were combined, concentrated on a rotavapor, and their optical rotation was measured. It was found to be (+)150° [Lit.(+)150.7°, (Takahashi and Ono, 1973)] in case of hexose from PF1–2 and +52°(literature: (+)52.8°) for sugar from PF3. These results indicated that all the three samples yielded D-galactose having alpha configuration in PF1–2 and beta configuration for sugar in PF3.
Antibacterial assays
Antibacterial activity was determined against six Gram-negative bacteria (Escherichia coli, P. aeruginosa, Salmonella typhii, S. flexineri, K. pneumoniae and V. cholerae) and one Gram-positive bacteria (Staphylococcus aureus) using the paper disk assay method (El-Masry et al., 2000). The sterile paper disk of 6 mm diameter impregnated with 65 µg/disk of PF1 and 130 µg/disk of PF2 were placed on agar plates containing the tested microorganisms. In all cases, the concentration was approximately 1.2 x 108 CFU/µL. The impregnated disks were placed on the medium suitably spaced apart and the plates were incubated at 37°C for 24 h. Disk of Streptomycin (10 µg/mL) was used as a positive control. The diameter (mm) of the growth inhibition halos caused by the sample was examined.
Antifungal assay
Antifungal activity was determined against A. fumigatus, Fusarium sp., C. neoformans, A. niger, Rhodotorula sp., Nocardia sp., and C. albicans using the paper disk assay method as previously described in the antibacterial assay. The sterile disk was impregnated with the compound (65 µg/disk of PF1 and 130 µg/disk of PF2). The inoculum concentration was 0.5 x 103–2.8 x 103 CFU/mL. Nystatin (100 µg/disk) was used as positive control. The plates were incubated at 24°C for 18 h. The diameter (mm) of growth inhibition halos caused by the compound was examined.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The authors thank Dr S. R. Shetye, Director, National Institute of Oceanography, for his keen interest in the work. One of the authors, A. Al-Fadhli, thanks the Director for permitting to work at NIO and for providing all the facilities. Financial support by the Department of Ocean Development under the project ‘Development of Potential Drugs from the seas around India’ is gratefully acknowledged.
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Abbreviations |
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AA, arachidonic acid; CDCl3, deuterated chloroform; COSY, 1H, 1H correlation spectroscopy; DGDG, digalactosyldiacylglycerol; EPA, eicosapentaenoic acid; ESI-MS, electrospray ionization mass spectrometry; GL1–3, glycolipids 1–3; HMBC, 1H, 13C heteronuclear multiple bond correlation; HMQC, 1H, 13C heteronuclear multiple quantum coherence; MGDG, monogalactosyldiacylglycerol; MS/MS, tandem mass spectrometry; NMR, nuclear magnetic resonance; PF1–3, polar fractions 1–3; SQDG, sulfoquinovosyldiacylglycerol; SQMG, sulfoquinovosylmonogalactosyl glycerol; TLC, thin layer chromatography; TOCSY, 1H, 1H total COSY
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References |
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