Optimized and Standardized Isolation and Structural Characterization

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Biomacromolecules 2009, 10, 2998–3008

Optimized and Standardized Isolation and Structural Characterization of Anti-inflammatory Sulfated Polysaccharides from the Red Alga Delesseria sanguinea (Hudson) Lamouroux (Ceramiales, Delesseriaceae) Niels Gru¨newald and Susanne Alban* Department of Pharmaceutical Biology, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstrasse 76, D-24118 Kiel, Germany Received April 30, 2009; Revised Manuscript Received July 19, 2009

The red seaweed Delesseria sanguinea dominantly populates a large artificial reef in the southwestern Baltic Sea. It contains sulfated polysaccharides (SPs), which exhibit a pharmacological profile indicating anti-inflammatory and anti-skin aging potencies. To establish and optimize an extraction procedure for these SPs and to evaluate the influence of several parameters on their quality, 23 algae batches were harvested over the period of four years and extracted by different methods, resulting in 56 SP batches. Extraction with water at 85 °C proved to be superior and led to highly reproducible products with average yields of 11.6 ( 3.9%, reaching 18% in spring. Their quality was independent of generation form and vitality of the algae. The SPs were identified as sulfated branched xylogalactans (degree of sulfation 0.50 ( 0.08, mean Mr 142000). The coextraction of floridean starch turned out to be the only parameter causing any seasonal variability. However, by using water, this concerns solely the yields of the isolated products. Compared to NaOH extracts, the antielastase activity of H2O extracted SPs was about twice as strong (IC50 0.204 ( 0.024 µg/mL) and the batch to batch variability was much lower (CV 11.8 vs 28.6%). In conclusion, SPs from Delesseria sanguinea can be isolated in reproducibly high quality by using a specific extraction procedure. Thus, an important prerequisite for a potential economical utilization is fulfilled.

Introduction Marine seaweeds are well-known for their content of sulfated polysaccharides (SPs) showing manifold pharmacological activities similar to heparins. Most intensely studied are the complex pharmacological profiles of several fucoidans from brown algae including anticoagulant and antithrombotic as well as antiinflammatory, antiproliferative, antiadhesive, and antiviral effects.1-4 Also, red algae contain SPs other than carrageenans and agarans without gelling properties but with a comprehensive range of biological activities. For example, sulfated glucuronogalactans (SGG) isolated from the red alga Schizymenia dubyi have been shown to exhibit effects in vitro against several viruses to inhibit NSCLC-N6 tumor cell proliferation as well as to have anticoagulant and anticomplementary activities.5,6 Other examples of Herpes simplex virus inhibiting SPs from red algae are sulfated xylomannans from Scinaia hatei7 and sulfated xylogalactans from both Acanthophora spicifera8 and Nothogenia fastigiata.9 SPs from Asparagopsis armata displayed effects against human immunodeficiency virus (HIV-1).10 Due to the steadily increasing regulatory requirements on the pharmaceutical quality of new drug substances, especially for biologicals including plant and animal extracts, algae-derived SPs represent rather promising candidates for development of cosmetics, wellness products or food supplements. But also in these areas, there is currently a shift to higher quality standards and the suggested activities or health claims, respectively, have to be proven. * To whom correspondence should be addressed. Tel.: +49 431 880 1135. Fax: +49 431 880 1102. E-mail: [email protected].

As a part of the European Union operational program “Financial Instrument for Fisheries Guidance” (FIFG),11 a largescale artificial reef was established in the Baltic Sea close to Nienhagen (Germany) in 2003.12 The species colonization process of the reef elements resulted in a balanced, macroalgal dominated benthic community as described by Schygula.13 The perennial red alga Delesseria sanguinea (Hudson) J. V. Lamouroux 1813 (Ceramiales, Delesseriaceae; D.s.) turned out to be the dominant species with up to 80% of the algal biomass in the summer period. This cold water alga occurs widespread in subtidal regions of the entire eastern North Atlantic coast including the western Baltic Sea14 and is besides the salinity conditions mainly limited by its temperature optimum (growth between 2 and 20 °C, maximum growth at 15 °C, degeneration above 25 °C water temperature).15 Besides chlorophyll a and phycobiliproteins,16 as well as a seasonally varying content of sterol compounds,17 the γ-lactone delesserine,18 the phenolic metamorphosis inducer jacaranone,19 and the disaccharide trehalose20 have been described as ingredients of D.s. Already in 1938, Elsner et al. demonstrated the anticoagulant activity of D.s. cold water extracts.21 Later, Potin et al. could attribute this effect to sulfated heteropolysaccharides isolated from D.s. harvested at the Breton coast.22 Due to the continual availability of D.s. biomass and the promising pharmacological potential of SPs, a project was initiated to evaluate whether SPs from the Baltic Sea D.s. (D.s.-SPs) could be utilized economically. Thus, they were isolated, structurally analyzed, and examined for their biological activities, demonstrating an encouraging pharmacological profile. The nongelling D.s.-SPs were shown to have only moderate anticoagulant properties but to inhibit complement activation

10.1021/bm900501g CCC: $40.75  2009 American Chemical Society Published on Web 10/02/2009

Sulfated Polysaccharides from Delesseria sanguinea

and tumor cell adhesion to P-selectin as well as the extracellularmatrix-degrading enzymes polymorphonuclear neutrophil elastase (PMNE), hyaluronidase, and heparanase much stronger than heparin.23 In previous studies, spring has already been identified as the optimal harvest time by investigations on the impact of seasonal variations. But principally, algae collection is possible throughout the year, because there were no significant seasonal variations in relevant biological activities of the D.s.-SP.24 Thus, the aim of the present study was to establish and to optimize an extraction procedure to obtain D.s.-SPs in maximal yield but reproducibly high quality with constant biological activity. This interaction is an essential for the development of a potential product. Additionally, the structural composition should be characterized. The above-mentioned anticoagulant compounds from D.s. harvested at the Breton coast have already been described to consist of sulfated xylogalactans.22 However, it had to be verified to which extent these results are applicable in a reproducible way to D.s.-SPs from the tideless, brackish Baltic Sea as a rather exceptional habitat. A distinct morphodifferentiation was found between individuals from the Baltic Sea and those from the North Sea. Baltic Sea D.s. contrasts with North Sea D.s. by a twice less length-width ratio, stronger development of rhizoids, and a higher percentage of midribs, as described by Rietema.15 Further, the considerably differing salinity and temperature tolerances and growth optima have been regarded as genetic adaptations of distinct ecotypes. In the present study, the influence of two other algae materialrelated parameters, that is, the generation phase and the vitality of D.s. on structural composition and pharmacological activities of D.s.-SP should be investigated. The reproduction of the perennial D.s. follows a triphase diplohaplontic life cycle with an alternation of isomorphic phases as characteristic for Ceramiales where growth and reproduction periods are strictly reverse.25 Therefore, tetrasporophytes and carposporophytes were segregated from some D.s. batches and the isolated D.s.SPs were compared with those of phase mixtures. Regarding the aspect of vitality, sessile and torn individuals cumulated in swales and partially at the beginning of degeneration were contrasted. For a commercial utilization of the D.s.-SP, it is important to guarantee the constancy of their useful activities and thus of their quality with simultanous yields as high as possible. Since the used extraction method may substantially influence both yields and structural compositions,26 a procedure meeting both requirements had to be found. For this, 23 batches of D.s. fresh algae were harvested over the period of four years and extracted by different methods resulting in 56 D.s.-SPs batches. The isolated D.s.-SPs were structurally characterized and the inhibition of PMNE as a typical pharmacological marker effect of D.s.-SP23 was determined.

Material and Methods Algae Material. In the years 2004-2007, fresh Delesseria sanguinea (Sea beech) algae material was collected from the sublittoral habitat of the large artificial reef near Nienhagen, Mecklenburg-Vorpommern, Germany (geographical position φ ) 54°10.50′ N; λ ) 11°56.60′ E), at a depth of about 12 m by scuba diving. In 2006, D.s. batches were harvested monthly from February to December. In general, collected sprouts were attached to an artificial hard substrate, and comparable torn sprouts were collected from seabed cavities. The algae were identified by Dr. Christof Schygula, Faculty of Science, Department of Marine Ecology, University of Rostock, Germany (voucher specimen

Biomacromolecules, Vol. 10, No. 11, 2009

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available). The raw material was manually purified from epiphytic and epizoic contaminants, in fact, primarily Mytilus edulis L., and stored in a biocide medium consisting of 80% (v/v) ethanol containing 4% (v/v) para-formaldehyde. To determine the dry mass of the algae, samples with different net weights (n ) 3) of each algae batch were weighed before and after freeze-drying. Extraction and Isolation. The extraction and isolation of the D.s.SPs were performed according to a modified and optimized procedure based on that described by Potin et al.22 The purified algae material was homogenized with a Braun PowerBlend and defatted by Soxhlet extraction with ethanol 96% (v/v) for 12 h. After evaporation of the ethanol, the material was extracted for 8 h with either demineralized water or aqueous 0.1 mol/L sodium hydroxide (NaOH) at 35, 60 or 85 °C under reflux conditions. In general, the defined ratio of the algae to the extracting agent of 75 g/L was used. The obtained raw extracts were centrifuged (10000 g, 30 min, 4 °C), the supernatants were adjusted to pH 7.4, and 8-10-fold concentrated by evaporation (Laborota 400 vacuum evaporator, Heidolph). To precipitate the D.s.SPs, iced 96% (v/v) ethanol was added resulting in a final ethanol concentration of 90% (v/v). After storing for 24 h at 4 °C followed by centrifugation (10000 g, 30 min, 4 °C), the sediments were dissolved in demineralized water. Finally, the solutions were exhaustively dialyzed against flowing demineralized water at 6 °C (Spectra Por dialysis membranes, Spectrum, MWCO 1000), adjusted to pH 7.4 with NaOH, and freeze-dried. Analytical Methods. Spectroscopic Data. Fourier transform infrared spectra of KBr discs (compactor Graseby Specvac 15.011, 9 bar, 2 min in vacuum) of D.s.-SP were recorded by a Perkin-Elmer 16PC FT-IR (eight scans at a resolution of 4 cm-1) scanning between 4000 cm-1 and 600 cm-1. Degree of Sulfation (DS). The DS (i.e., number of sulfate groups per monosaccharide) of the D.s.-SP was determined according to an European Pharmacopoeia method.27 D.s.-SP sodium salts were transformed into the free acid form by cation exchange with Amberlite IR120 (Fluka), which was titrated with 0.1 mol/L NaOH. The end point of titration was detected conductometrically using a SevenEasy conductivity meter (Mettler Toledo). Results were verified by elemental analysis (anal. carbon, hydrogen, nitrogen and sulfur measured by a HEKAtech CHNS Analyzer). Colorimetric Carbohydrate Analyses. Total carbohydrates were determined by the anthrone method28 calibrated with D-galactose. The 3,6-anhydrogalactose content was examined using the resorcinol reaction29 with agarose as the standard and D-galactose as the blank. Furthermore, uronic acids were quantified by reaction with mhydroxydiphenyl (Fluka) according to the method by Blumenkrantz and Asboe-Hansen30 as well as its modification by van den Hoogen et al.31 Both assays were calibrated with a mixture (1:1) of D-glucuronic acid and D-galacturonic acid. The absorptions (at wavelength of 625 nm for the anthrone method, at 485 nm for the resorcinol reaction, and at 520 nm for the uronic acid assay) were measured with a Cary 50 Scan spectrophotometer (Varian). A rapid test for the presence of amylose was performed by addition of 2% (v/v) Lugol’s solution 1% Ph. Eur. and measuring the absorption at 580 nm referring to a linear calibration with soluble starch (Merck). The presence of acetyl groups was checked by the absorption measurement after the formation of colored acetohydroxamic acid iron complexes with alkaline hydroxylammonium chloride and Fe(III) ions in perchloric acid according to McComb and McCready.32 Both assays were also read out with the Cary 50 Scan spectrophotometer (Varian). Total Proteins. Total proteins were quantified by means of various methods: First, the classical Lowry protein determination method with the Folin phenol reagent33 (Merck) whereby the assay was carried out in a microplate scale, further the Bradford assay modified by Zor and Zeliger34 converted as well into a microplate scale, finally a fluorescence microplate assay using ortho-phthalaldehyde (OPA; Fluoraldehyde Reagent Solution, Pierce).35 The absorption (OD (650 nm)), the ratio of absorption (OD (595 nm)/OD (465 nm)), and the fluorescence

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intensity (λex 370 nm, λem 475 nm), respectively, were measured with a microplate reader (POLARStar OPTIMA, BMG). For all the protein assays, bovine serum albumin (Sigma) was used as the standard; microplates were purchased from Nunc. Further protein determinations including enzymatic (PNGase) and chemical deglycosylations with trifluoromethanesulfonic acid (TFMS) followed by SDS-PAGE with both silver stain36 and fuchsine detection after periodic acid Schiff reaction (PAS-fuchsine) as well as an Edman degradation with conversion into phenylthiohydantoin amino acids (ATH-AAs) and their quantification by high performance liquid chromatography were performed by WITA GmbH, Teltow, Germany. Monosaccharide Composition. To determine the monomer composition, D.s.-SPs were hydrolyzed with 2 mol/L trifluoroacetic acid (TFA) for 1 h at 121 °C37 and, after evaporation of TFA, converted into alditol acetate derivatives (AAs) by reduction and acetylation.38 The AAs were separated by gas liquid chromatography (GLC) on an OPTIMA-225-0.25 µm fused silica capillary column (25 m × 0.25 mm i.d., film thickness 0.25 µm, Macherey-Nagel) using a HP 6890 gas chromatograph (Hewlett-Packard) with integrated flame ionization detector. The helium flow rate was 1 mL/min, the oven temperature was 180 °C for 5 min followed by an increase of 1 °C/min up to 210 °C held for 10 min, the temperature of the injector and detector was 250 and 240 °C, respectively. The AAs were identified by their retention times. For quantitative analysis, the D.s.-SP samples were supplemented with a defined amount of myo-inositol as the internal standard. The percentage of the respective AAs was calculated applying the software HP GC Chemstation, Rev. A.06.03 [509] (Hewlett-Packard). Monosaccharide Linkage. For glycosidic linkage analysis, the desulfated D.s.-SPs were transformed into partial methylated alditol acetates (PMAAs), which were analyzed by GLC-MS according to the method of Harris et al.39 In short, the D.s.-SPs were meythylated with dimsyl potassium and methyl iodide, hydrolyzed with 2 mol/L TFA, reduced with 0.5 mol/L sodium borohydride, and finally acetylated with acetanhydride, resulting in the corresponding PMAAs. These were separated and detected by GLC-MS using a OPTIMA-1701-0.25 µm fused silica capillary column (25 m × 0.25 mm i.d., film thickness 0.25 µm, Macherey-Nagel) and a Hewlett-Packard 5890 Series II gas chromatograph coupled with a Hewlett-Packard MS Engine 5898A (electron ionization 70 eV) with an injector temperature of 250 °C and a helium flow rate of 1 mL/min. The GLC oven temperature was 120 °C for 3 min, followed by an increase of 8 °C/min up to 170 °C, then one of 1 °C/min up to 200 °C, and finally one of 4 °C/min up to 250 °C, held for 28.25 min. The MS ion source and quadrupol temperature was 250 and 120 °C, respectively. PMAAs were identified by both their relative retention times in the total ion chromatogram and their mass spectra using a spectra bibliotheca as well as HP G 1034 C software for MS Chemstation (Hewlett-Packard). Chemical Modifications. Selected D.s-SP batches were chemically modified by following procedures: The obtained derivatives were then subjected to monosaccharide composition and monosaccharide linkage analysis, respectively. Desulfation. The native samples were transformed into the pyridinium salt form analogous to the determination of DS and desulfated in dimethyl sulfoxide-methanol (ratio 9:1, 7 h, 100 °C)40 followed by dialysis and freeze-drying. Reduction of uronic acids. The carboxyl groups of uronic acids were reduced to hydroxyl groups with sodium borodeuteride (Sigma) according to Taylor and Conrad.41 The pH value was adjusted to 4.75 by titration with 2 mol/L HCl using an autotitrator 719 S Titrino (Metrohm). N-Cyclohexyl-N′(2-morpholinoethyle)-carbodiimide-metho-p-toluene sulfonate was obtained from Sigma, sodium borodeuterite (98 atom % D) from Isotec. Smith degradation. The D.s.-SP samples were dissolved in 0.1 mol/L sodium periodate (Merck) and left in darkness for 48 h at room temperature. After degradation of remaining sodium periodate with equivalent volumes of ethylene glycol (Fluka) and 3 h of stirring, a surplus of NaBH4 (Sigma) was added, and the solution was left for

Gru¨newald and Alban another 20 h at room temperature. Then, the preparation was neutralized with acetic acid, dialyzed and freeze-dried followed by a mild hydrolysis with 0.5 mol/L TFA at room temperature. Molecular Mass (Mr). The Mr of D.s.-SP was estimated by size exclusion chromatography (SEC) with online multiangle laser light scattering (MALLS) detection using a PL-GPC 50 Plus system with degasser and integrated differential refractive index detector (Polymer Laboratories), ancillary coupled with a miniDAWN MALLS detector (Wyatt). For separation of D.s.-SPs corresponding to their hydrodynamic volumes by SEC, a PL aquagel-OH Guard 8 µm precolumn followed by three PL aquagel-OH Mixed 8 µm (Polymer Laboratories) columns in series were used. Despite the deficiency of directly comparable polymer references, the additional MALLS detection allows the direct specification of the absolute Mr42 in comparison to the hydrodynamic volume. The samples (injection volume 100 µL) were dissolved with an ultrasonic bath and eluted with 0.1 mol/L NaNO3 (pH 8, containing 0.05% NaN3) at a flow rate of 0.7 mL/min, the column temperature was kept at 35 °C by a column oven. Absolute Mr values were calculated with ASTRA for Windows software version 4.70.07 (Wyatt). Pharmacological Methods. Test Compounds and Materials. Besides D.s.-SP, unfractionated heparin (UFH) from porcine mucosal origin (Lot No 73508019, Novartis) and the semisynthetic linear β-1,3-glucan sulfate PS3 (U.S. Patent No. US7008931-B2)43,44 were tested. The fluorescence or absorbance assays were read out by the microplate reader Polarstar Optima (BMG Labtech). ActiVated Partial Thromboplastin Time (APTT). The APTT was measured as “doubling concentration” (inhibitor concentration causing a prolongation of the coagulation time to twice the time of the negative control) with an Amelung-coagulometer KC 10 macro as previously described.23,24 Elastase Inhibition Assay. The PMNE inhibition was examined by the fluorigenic substrate microplate assay using human PMNE (EC 3.4.21.37, Calbiochem) and MeOSucc-Ala-Ala-Pro-Val-7-amido-4methylcoumarin (Bachem) as substrate solution as previously described.23,24 Statistical Analysis. All measurements were done in duplicate and repeated three times on different days (n ) 6). All data are presented as mean ( standard deviation (SD). The DC and IC50 values were calculated by nonlinear curve fitting using the program Sigma Plot 8.0. Statistical analysis was performed using Student’s t-test, p e 0.05 was considered as statistically significant.

Results Over the course of four years, overall 23 batches of D.s. were harvested and extracted using several modifications of the extraction procedure (Table 1) resulting in 56 fractions mainly consisting of sulfated polysaccharides (D.s.-SPs). In general, the batches (named regular algae batches (reg)) consisted of D.s. algae, which were sessile on hard substratum structures and were composed of unspecified mixtures of its generation phases as typical for the respective harvest time. To examine the influence of the generation phase on the obtained D.s.-SPs, separated carposporophytes (carp) and tetrasporophytes (tet) were extracted. These were segregated from D.s. batches harvested in spring when they are best distinguishable. To check any effect of the vitality of the algae, batches of torn individuals were collected from swale accumulations (torn). Over the course of time and the increasing number of extracted batches, the number of the applied different extraction procedures was successively reduced. This was decided on weighing respective yields and quality aspects of the isolated D.s.-SPs. Consequently, in some cases, mean results presented below are based on different numbers of values. Dry Mass Determination. The determination of the dry mass of drained D.s. material revealed an average of 17.5% ( 3.2%



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Table 1. Time of Harvest of D.s.-SP Batches and Applied Extraction Procedurea season

2004

eluent T (°C) Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

H2O 35

reg

60

reg

2005 NaOH

85

reg

35

reg

60

reg

2006

H2O 85

NaOH

60

85

60

85

reg reg, carp, tet

reg reg, carp, tet

reg reg, carp, tet

reg reg, carp, tet

reg

reg

reg

reg

reg

2007

H2O

NaOH

H2O

85

85

85

reg reg, torn reg, torn reg reg reg reg reg reg reg reg

reg reg, torn reg, torn reg reg reg reg reg reg reg reg

carp, tet reg, torn

a Regular algae batches (reg) means D.s. algae, which were composed of D.s. algae consisting of season-dependent mixtures of its generation phases and were fixed on hard substratum structures (total 56 batches). Torn individuals (torn) consisted also of mixed sex phases, but were cumulated in swales and partly at the beginning of degeneration. Further, algae harvested in both April 2005 and April 2007 were separated into carposporophytes (carp) and tetrasporophytes (tet). In January, no batch could be harvested because of inclement weather conditions. The extractions were performed with demineralized water (H2O) and 0.1 M NaOH (NaOH), respectively, at different temperatures.

(m/m; CV 18.2%) and was subject to a seasonal pattern with highest values in Oct and Nov (Oct 22.0 ( 0.6%, CV 2.83%; Nov 19.5 ( 1.2%, CV 6.14%) and a subsequent decline until April (14.2 ( 2.0%, CV 14.2%) followed by a permanent increase until autumn. These findings were coincident with the seasonal variation in morphology, thus, the mean coefficient of variation (CV) of the dry mass was with 18.2% relatively high.24 The dry masses of the D.s. batches from April 2005 (11.9 ( 0.39%) and 2006 (14.2 ( 0.6%) differed by 19.1% and those from Oct 2005 (21.8 ( 1.1%) and 2006 (22.2 ( 0.7%) by 1.56%. The CV of the three April batches, including 2007, amounted to 14.2%. Yield of Isolated D.s.-SPs. The yields of the nongelling SP fraction turned out to be dependent on both the extraction procedure and the harvest time of the algae. The first D.s. batch from Oct 2004 was extracted with both demineralized water (H2O/H2O-D.s.-SP) and 0.1 mol/L NaOH (NaOH/NaOHD.s.-SP) at three different temperatures resulting in the following yields (% w/w of dry mass): H2O: 0.96% (35 °C), 4.13% (60 °C), 7.86% (85 °C); and NaOH: 3.90% (35 °C), 10.2% (60 °C), 20.7% (85 °C). Thus, the yields increased with increasing temperature and were considerably higher with NaOH than with H2O. Because the yields of the 35 °C extracts were by far the lowest (Figure 1A) but their pharmacological activities were similar to those obtained at higher temperatures, extractions at 35 °C were not continued. The anticoagulant activities of the D.s.-SPs were similar indicating that no heat-mediated destruction occurred (data not shown). Nevertheless, for economic reasons and to limit coextraction of reserve glucans (see below), higher extraction temperatures than 85 °C were not used for the subsequent extractions. Parallel extractions of five further batches confirmed that an increase of the temperature from 60 to 85 °C at least doubles the amount of isolated D.s.-SP (Figure 1A), whereas at 60 °C the mean yields with H2O (5.60 ( 1.43%, CV 25.6%) and NaOH (5.75 ( 2.99%, CV 51.9%) were almost equal, the use of NaOH at 85 °C resulted in about 40% higher yields compared to H2O. Furthermore, the extractions of six batches of D.s. harvested between Oct 2004 and Oct 2005 revealed a pronounced variability in the yields of D.s.-SP. Thus, those from the four batches from March and April 2005 were considerably higher than those from the two harvested in Oct, especially when they were related to the dry mass instead of the drain weight. In addition, the yields of the H2O extracts from the April batches were similar to those of the NaOH extracts.

Figure 1. (A) Rates of yields based on dry mass in % (w/w) in dependence on various parameters. Values represent means ( SD (n ) 1 - 16 extraction processes; Table 1). Yields resulted from extraction with H2O (light gray bar) and 0.1 mol/L NaOH (dark gray bar) at different temperatures. The pair of bars marked with the asterisk represent the corresponding 85 °C values of the extraction with 60 °C. (B) Yields of regular batches extracted at 85 °C have been compared with carposporophytes (carp) and tetrasporophytes (tet). However, torn individuals have been compared with equivalent batches from the same time of harvest (reg).

These discrepant results prompted us to harvest D.s. monthly in 2006 and to extract the batches with both H2O and NaOH at 85 °C in parallel. The yields of H2O extracts averaged 11.6 ( 3.9%, (CV 33.5%) in relation to the dry mass; those of the NaOH were 40.7% higher, with 16.4 ( 4.0% (CV 24.5%; Figure 1A). Related to the drain weight, they amounted to 1.84 ( 0.55 (CV 30.1%) and 2.87 ( 0.78% (CV 27.2%), respectively. Thus, both eluents were associated with pronounced variability of the yields. However, the variations of the yields of H2O extracts revealed a strong seasonal dependence with the lowest yields (6.12% of dry mass) in autumn (Sep) and the highest (17.9%) in spring (March, April;26 Figure 2). In contrast, the yields of the NaOH varied independently of the harvesting time and were only in the second half of the year clearly higher as those of the water extracts. In comparison, the CV of the three April batches 2005, 2006, and 2007 extracted with H2O at 85 °C amounted to 20.8% and is therefore lower than that caused by annual variations. Comparing the yields, D.s.-SP obtained from carposporophytes and tetrasporophytes revealed no significant divergence (Figure 1B). Similarly, no difference was found between the yields from torn D.s. and regular batches harvested in parallel.

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Figure 2. Rates of yields of isolated D.s.-SPs as a function of the harvesting time and the used eluent. Full lines represent the yields related to dry mass obtained with H2O (black squares) and 0.1 mol/L NaOH (gray circles), respectively. Dotted lines represent the yields related to drain mass (H2O (open squares) and 0.1 mol/L NaOH (open circles)). The full lines refer to the left axis of ordinates and the dotted lines refer to the right axis. The presented values are based on one extraction process each. Table 2. Degree of Sulfation (DS) of D.s.-SP Obtained by Extraction with H2O and NaOH, Respectivelya class of D.s.-SP

DS of H2O extracts

DS of 0.1 M NaOH extracts

total 85 °C (n ) 16) 60 °C (n ) 4) 85 °C* (n ) 4) carp 85 °C (n ) 2) tet 85 °C (n ) 2) torn 85 °C (n ) 3) reg 85 °C (n ) 3)

0.50 ( 0.08 (CV ) 15.9%) 0.42 ( 0.03 (CV ) 6.73%) 0.42 ( 0.03 (CV ) 7.01%) 0.45 ( 0.12 (CV ) 27.0%) 0.43 ( 0.03 (CV ) 6.58%) 0.44 ( 0.11 (CV ) 24.9%) 0.44 ( 0.07 (CV ) 14.7%)

0.39 ( 0.14 (CV ) 35.2%) 0.23 ( 0.10 (CV ) 45.0%) 0.39 ( 0.15 (CV ) 39.7%) 0.56 ( 0.20 (CV ) 35.4%) 0.49 ( 0.11 (CV ) 21.9%) 0.49 ( 0.15 (CV ) 33.7%) 0.42 ( 0.03 (CV ) 6.30%)

a The data marked with an asterisk represent the DS of those D.s. batches extracted at both 60 and 85 °C in parallel. For the comparison between carposporophytes (carp) and tetrasporophytes (tet) as well as between torn and sessile individuals (reg), the corresponding D.s. batches (n ) number of batches) were harvested at the same time. Represented DS values are the mean ( SD, whereby the DS of each D.s.-SP was determined at least three times.

The D.s. batches for the latter comparison were from spring, the harvest time resulting in highest yields (March-May, Table 1). Spectroscopic Data. The infrared spectra of all the investigated samples showed strong bands attributed to SdO asymmetric and symmetric stretching vibrations of sulfate groups (ν ) 1254-1258 cm-1 and ν ) 1030-1046 cm-1, respectively) as well as C-O-S bending vibrations of equatorially 6-linked sulfate groups (ν ) 818-824 cm-1).45 In addition to the broad range of O-H stretching vibration bands (ν ) 3442-3454 cm-1), strong absorption bands induced by CdO stretching vibrations of carboxyl groups (ν ) 1646-1654 cm-1) were recorded, indicating the presence of uronic acids. Neither characteristic bands of axial sulfate groups in the range of ν ) 850 cm-1 nor bands between 930 cm-1 and 940 cm-1 representing 3,6-anhydrogalactose were detected.46 Determination of Degree of Sulfation (DS). The DS was chosen as the primary quality marker because there is a correlation between the DS of SPs and some of their pharmacological effects.47 With a mean value of 0.5 (i.e., 18.5% (w/ w) sulfate (-SO32-)), the DS of the D.s.-SP isolated with H2O at 85 °C was about 20% higher than that of those extracted with NaOH (Table 2). In the evaluation phase with D.s. batches from 2004 and 2005, the sulfate content of D.s.-SP extracted with H2O proved to be independent of the temperature (60 °C: 16.4 ( 0.9% vs 85 °C: 16.1 ( 1.3%; for DS, see Table 2). In contrast, the sulfate content of the corresponding NaOH extracts was generally lower and, in addition, strongly differed in dependence on the

Gru¨newald and Alban

extraction temperature (85 °C: 9.64 ( 2.95% vs 15.7 ( 2.9%). Further, extraction with NaOH revealed a twice as high batch to batch variability of the DS compared to that with water (Table 2). The slight differences in DS between the various H2OD.s.-SPs are due to seasonal effects,24 but the dependence on the time of harvest was more pronounced regarding the NaOH-D.s.-SPs (DS 0.49 ( 0.11 (Feb-June) versus DS 0.31 ( 0.11 (July-Dec)). Thus, NaOH led only in the advantageous time of harvest in spring to D.s.-SP with a DS similar to those of the H2O-D.s.-SPs. Regarding the influence of the generation phase, there were no significant differences in DS between the D.s.-SPs from carposporophytic and tetrasporophytic batches (Table 2). Likewise, the vitality of the D.s. had no influence as reflected by the DS of the D.s.-SPs from torn and regular batches. In general, the differences between the various NaOH-D.s.-SPs were higher but also not significant. Further Structural Characteristics. The determination of total carbohydrates by the anthrone reaction revealed a content of about 68% (H2O 85 °C) and 64% (NaOH 85 °C) in the D.s.-SP (Table 3). Although the structural differences between D.s.-SPs and the standard D-galactose were considered for the calculation (i.e., polysaccharide vs monosaccharide, molar masses of sodium sulfate, and sodium carboxyl groups of uronic acids), the values are assured to be too low. To the noncarbohydrates contained in D.s.-SPs contribute structurally bound water of crystallization and additionally adsorbed residual water. The latter amounted to about 5% as measured gravimetrically according to the European Pharmacopoeia. Another portion are proteins, which may explain the higher total carbohydrate content of H2O-D.s.-SPs compared to NaOH-D.s.-SPs (Table 3). Salts and any other low molecular weight impurities should have been minimized by dialysis. The still too low carbohydrate content may be related to the anthrone assay. The absorption maximum of the formed furfural derivatives varies in dependence on the type of monosaccharide, and many other compounds are known to interfere with the anthrone reaction of carbohydrates. The content of uronic acids amounted to 3.13 ( 0.82% in H2O-D.s.-SPs extracted at 85 °C and thus is about 50% higher than that in NaOH-D.s.-SPs (Table 3). The mol percentage of uronic acids in D.s.-SPs based on these data was estimated to be approximately 5% in the polysaccharide. By further analysis after their reduction, they were identified as glucuronic acids. Further, H2O-D.s.-SPs from 85 °C extraction were found to contain 4.05 ( 1.48% (w/w) 3,6-anhydrogalactose (Table 3). Considering the structural characteristics of the D.s.-SPs, this roughly corresponds to 7.4% (mol/mol) of the D.s.-SP monosaccharides and means that about one of nine galactose units is a 3,6-anhydrogalactose. Compared to H2O-D.s.-SPs, NaOHD.s.-SPs contained about 50% more 3,6-anhydrogalactose. This is suggested to result from formation of 3,6-anhydrogalactose during the alkaline extraction.48 Moreover, no acetyl groups were found in any D.s.-SP samples. Total Proteins. Being aware of known problems of the total protein determination we used various methods to estimate the protein contamination of D.s.-SP, that is, the Lowry assay, the Bradford assay modified by Zor and Zeliger, a fluorometric assay with o-phthalaldehyde (OPA assay), as well as protein estimations by both the nitrogen content determined by elemental analysis and SDS-PAGE combined with Edman degradation. According to the OPA assay, the total protein content of all the H2O-D.s.-SP batches extracted at 85 °C averaged 9.44 ( 1.68% (CV 17.8%). The modified Bradford assay revealed an about



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Table 3. Further Nonseasonal Dependent Structural Data Represent Means ( SD, Percent Values Refer to Weights (n ) 2-5 Measurements of Each D.s.-SP from 26 D.s. Batches) extractions conditions

total carbohydratesa (% w/w)

uronic acids (% w/w)

3,6-anhydrogalactose (% w/w)

total protein (% w/w)

H2O 85 °C NaOH 0.1 M 85 °C

68.2 ( 5.8 64.2 ( 12.9

3.13 ( 0.82 2.07 ( 0.97

4.05 ( 1.48 6.12 ( 2.81

9.44 ( 1.68b (6.15 ( 1.86c) 12.93 ( 3.53b (8.18 ( 2.53c)

a Total carbohydrates as found with the anthrone method using D-galactose as a reference. b Total protein determined by OPA assay. c Total protein determined by Bradford assay modified according to Zor and Zeliger.34

Table 4. Composition of Neutral Monosaccharides of D.s.-SPs According to GLC Analysis of the Corresponding Alditol Acetates of the D.s.-SPsa class of D.s.-SP

gal (% mol)

xyl (% mol)

glc (% mol)

man (% mol)

total H2O 85 °C (n ) 16) 0.1 M NaOH 85 °C (n ) 4) H2O 85 °C* (n ) 4) H2O 60 °C (n ) 2)b H2O 85 °C** (n ) 2) carp H2O 85 °C (n ) 1) tet H2O 85 °C (n ) 2)b torn H2O 85 °C (n ) 3)b reg H2O 85 °C (n ) 3)b

66.1 ( 5.1 62.7 ( 16.6 67.0 ( 5.2 66.4 ( 1.9 62.5 ( 2.1 66.0 69.3 ( 0.9 72.4 ( 2.6 71.9 ( 2.0

12.3 ( 2.5 9.5 ( 4.3 10.7 ( 2.6 12.2 ( 0.9 12.5 ( 2.4 15.8 15.3 ( 0.7 13.7 ( 2.0 14.3 ( 2.4

14.2 ( 5.6 23.8 ( 18.8 14.9 ( 5.5 10.6 ( 2.4 16.7 ( 4.5 8.85 7.79 ( 0.77 7.57 ( 1.47 7.33 ( 1.71

5.38 ( 1.27 2.80 ( 0.93 5.44 ( 1.22 5.75 ( 0.69 5.06 ( 1.56 7.11 5.42 ( 0.46 4.32 ( 1.27 4.53 ( 1.11

fuc (% mol) 1.98 ( 1.26 1.26 ( 0.99 1.90 ( 1.37 3.47 ( 0,81 2.15 ( 1.81 2.28 2.13 ( 0.25 2.02 ( 0.66 1.95 ( 0.20

a The data marked with one asterisk represent the corresponding H2O values of the NaOH data, and the data marked with two asterisks represent the corresponding 85 °C values for the 60 °C vs 85 °C comparison. Carposporophytes (carp) and tetrasporophytes (tet) are detailed to the table as well as torn individuals, which have been compared with equivalent batches from the same month (reg). Represented values are the mean (SD of repeated (2-4 times) analyses of the given number of batches (n ) 1-16). b Divergence from the mean values of the 16 H2O-D.s.-SPs are due to the harvest time of D.s. in Spring.

35% mean content (Table 3), but the values for the individual batches correlated with those determined by the OPA assay. The results of the Lowry assay were similar to those of the Bradford assay, but this assay turned out to be unsuitable for quality control due to large interassay variations. Based on the nitrogen content of H2O-D.s.-SPs batches (1.52 ( 0.32%), the protein content calculated by multiplication with 6.25 was 9.48 ( 2.00%, which sorts well with data of the OPA assay. Finally, SDS-PAGE revealed two protein fractions in D.s.-SPs in the Mr range of 15000-20000, whereby the proteins are not linked with the carbohydrates (data not shown). Combining these findings with the results of the Edman degradation, the protein content was estimated to be certainly not higher than 9%. Consequently, the OPA assay with the lowest inter- and intraassay variability was proven to be most suitable for routine analysis. Concerning the protein content in dependence on the extraction method, the NaOH-D.s.-SP batches had an about 37% higher protein content (12.9 ( 3.5%) and with a CV of 27.3% also a higher batch-to-batch variability than H2O-D.s.-SPs, whereby again the content according to the Bradford assay was about 37% lower (Table 3). Remarkably, extractions at 60 °C led to NaOH-D.s.-SPs even more contaminated with protein, whereas the contamination of the H2O-D.s.-SPs did not significantly differ from those isolated at 85 °C (D.s.-SPs extracted at 60 °C: OPA assay H2O 10.3 ( 1.6%; NaOH 16.4 ( 5.9%; Bradford assay H2O 5.38 ( 1.73%; NaOH 13.7 ( 8.8%; Lowry assay H2O 9.72 ( 3.46%; NaOH 19.1 ( 8.2%). Monosaccharide Composition. The monosaccharide composition of the D.s.-SPs was analyzed by conversion of the D.s.-SPs into their AAs, which were separated and quantified by GLC. Galactose (gal) was found to account for two-thirds of the neutral monosaccharides of D.s.-SPs. As further important components, xylose (xyl) and glucose (glc) were identified, minor ones were mannose (man) and fucose (fuc) (Table 4). In H2O-D.s.-SPs extracted at 85 °C, the content of galactose ranged between 56.2 and 75.8% mol, that from xylose between 6.21 and 17.3% mol. However, as recently demonstrated by

Gru¨newald et al.,24 these apparent variations were solely caused by the glucose content, which was subject to considerable seasonal variations (7.32-22.3% mol). By subtracting the season-dependent varying surplus of glucose, the percentage of the other monosacchrides was found to remain nearly constant. Regarding representative NaOH-D.s.-SPs batches, the percentage of the glc turned out to be on average 60% higher and to still stronger vary in dependence on the harvesting time. For example, NaOH-D.s.-SPs isolated from D.s. harvested in June consisted of 74.2 ( 1.2% mol gal, 13.9 ( 1.0% mol xyl and 8.65 ( 0.01% mol glc, those from the Dec batch contained 44.6 ( 0.3% mol gal, 9.44 ( 0.55% mol xyl, and 41.4 ( 1.1% mol glc. The percentage of glc in the corresponding H2O-D.s.-SPs amounted to 12.4 ( 2.5% mol glc (June) and 14.1 ( 0.2% mol glc (Dec), respectively. According to linkage analyses after desulfation and conversion into their corresponding PMAAs, the D.s.-SPs are composed of a galactan backbone consisting of 3-linked and 4-linked galactose units which is branched with pyranosidic terminal xylose, galactose and also glucuronic acid. Further, low amounts of xylose linked in positions 2 or 4 and also 3 were detected. The uronic acids have been identified as glucuronic acids, since after uronic acid reduction the content of glucose in the neutral monosaccharide composition increased to an extent similar to that determined with the Blumenkrantz assay. The glucose contained in D.s.-SPs is present as pyranosidic 4-linked glucose, 4,6-linked glucose, and terminal glucose as detected by linkage analyses. Further, treatment of D.s.-SPs with R-amylase, which selectively hydrolyses R-1,4-linked glucans, followed by dialysis, did not reduce the glucose content. Finally, D.s.-SPs did not develop blue color with iodine/potassium iodide, the reagent for simple detection of amylose. From the methylation analysis a mean degree of branching of about 0.06 ( 0.01 was calculated. All these data confirm the hypothesis reported in literature that floridean starch is medium branched with structure similarities in between amylopectin and glycogen.49

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Figure 3. Light scattering (black square) and refractive index (gray square) profiles from SEC analysis of two exemplary H2O- and NaOHD.s.-SP batches, i.e., one from D.s. harvested in July 2006 and extracted with water at 85 °C (A) and one from D.s. harvested in Dec 2006 and extracted with 0.1 mol/L NaOH at 85 °C (B).

Figure 4. Yields and quality of D.s.-SPs in dependence on various extraction conditions: H2O (light gray bar) and 0.1 mol/L NaOH (dark gray bar) batches extracted at 60 and 85 °C. (A) Rates of D.s.-SP yields based on dry mass in % (w/w). (B) Degree of sulfation (DS) determined after converting D.s.-SPs into the free acid form by conductimetry during titration with 0.1 mol/L NaOH. Values represent the mean ( SD (n ) 4-16), verified by calculation of elemental analysis results. (C) Elastase inhibitory activity determined in a chromogenic substrate assay (IC50, mean ( SD, n ) 4-16). The presented data (A-C) are based on 4 H2O- and NaOH-D.s.-SP batches, each extracted at 60 °C, and 16 H2O- and NaOH-D.s.-SP batches extracted at 85 °C each. The DS and elastase inhibitory activity of each batch was determined at least 3 times on different days.

Molecular Mass (Mr). To evaluate the Mr distribution of the D.s.-SPs, they were separated by their hydrodynamic volumes by means of SEC with MALLS detection. H2O-D.s.-SPs and NaOH-D.s.-SPs revealed to be constituted of one main fraction with a mean Mr of 142000 ( 28000 (mean ( SD of 16 batches) and 148000 ( 29000 (mean ( SD of 8 batches), respectively. The overall characteristic Mr range of D.s.-SPs was found to be 93000-318000. However, in some cases, the main fraction was found to have a higher Mr despite ultrasonic treatment of the sample solution. As exemplary shown in Figure 3B, that of the NaOHD.s.-SPs from D.s. harvested in Dec 2006 had a Mr of 399000, followed by a minor peak at 23 mL with a Mr of 195000 and thus approximately half this size. As macromolecules are known to tend to agglomerate, this finding is suggested to be due to measuring both the two monomer complexes and the single monomers. The peak at about 26 mL with a Mr of about 28000, which was consistently very intensive in the RI spectrum of NaOH-D.s.-SPs samples, was attributed to floridean starch and potentially proteins. The further peak at about 29 mL is due to the inorganic salt containing solvent. The fraction in the range between 20000 and 30000 was found in all the D.s.-SP batches, but the peak intensity varied seasonally. The good correlation between the peak intensity and the glucose content is considered a broad hint that the fraction consists of floridean starch.24 The molecular mass polydispersity (Mw/Mn) of the main fraction of H2O-D.s.-SPs ranged from 1.04 to 1.84 with a mean of 1.30 ( 0.22, whereas the main fraction of NaOH-D.s.-SPs ranged between 1.01 and 1.62 with a mean of 1.32 ( 0.20.

Pharmacological Effects. Among the currently reported pharmacological activities of D.s.-SPs,23 their elastase inhibitory potency was selected to additionally evaluate the influence of algae material- and extraction process-related aspects on an application-oriented parameter. Moreover, the characteristics of the applied validated chromogenic substrate assay predestine it for the routine qualitiy control of D.s.-SPs.50 The antielastase activity of H2O-D.s.-SPs revealed an IC50 average of 0.204 ( 0.024 µg/mL (CV ) 11.8%, n ) 17). Thus, it was in between that of UFH (IC50 ) 0.236 ( 0.011 µg/mL) and that of the β-1,3-glucan sulfate PS3 (IC50 ) 0.163 ( 0.004 µg/mL), a semisynthetic compound with proven in vivo antiinflammatory activity.43,44 Examination of the elastase inhibitory activity in dependence on the extraction procedure confirmed that water is superior to 0.1 M NaOH as eluent for D.s.. The activity of NaOHD.s.-SPs extracted at 85 °C (IC50 ) 0.339 ( 0.097 µg/mL, CV ) 28.6%) as well as of those produced at 60 °C (IC50 ) 0.555 ( 0.130 µg/mL, CV ) 23.4%) was significantly lower than that of the corresponding H2O-D.s.-SPs batches (Figure 4C). Furthermore, the respective batch to batch variability was considerably higher like as observed for the analytical data. Regarding the extraction temperature, the H2O-D.s.-SP batches obtained at 60 °C (IC50 ) 0.290 ( 0.025 µg/mL, CV ) 8.62%) showed inhibitory potencies similar to those of the six corresponding 85 °C extracts (IC50 ) 0.260 ( 0.027 µg/mL), whereas NaOH-D.s.-SPs significantly differed with 60 °C extracts (IC50 ) 0.555 ( 0.082 µg/mL, CV ) 28.6%) being still less active than the 85 °C extracts (IC50 ) 0.339 ( 0.097 µg/mL). Altogether, the antielastase activities of the various

Sulfated Polysaccharides from Delesseria sanguinea Table 5. Reproducibility of the H2O-D.s.-SPs Extracted at 85 °C from Five D.s. Batches Harvested in April Three Consecutive Years Regarding their Degree of Sulfation (DS), their Anti-Elastase Activitiy (IC50 µg/mL), and Their Anticoagulant Activitiy in the APTTa batches

DS

elastase inhibition (IC50 µg/mL)

APTT (DC µg/mL)

April 2005 April 2006 April 2006 torn April 2007 carp April 2007 tet mean ( SD

0.44 ( 0.03 0.51 ( 0.01 0.44 ( 0.01 0.46 ( 0.01 0.50 ( 0.01 0.47 ( 0.04

0.234 ( 0.007 0.196 ( 0.007 0.231 ( 0.011 0.226 ( 0.005 0.206 ( 0.006 0.219 ( 0.017

6.11 4.77 5.06 7.11 5.89 5.79 ( 0.83

a The values represent means ( SD from at least three measurements on different days (n ) 6).

D.s.-SPs correlated with their respective DS (Figure 4B,C, Table 2). Considering the analytical findings (see above) and the pharmacological potencies in combination with the D.s.-SP yields (Figure 4A), the extraction with H2O at 85 °C turned out to be the most suitable procedure for the isolation of high quality SP from D.s. Beyond that, comparison of H2O-D.s.-SPs isolated from five D.s. batches harvested in April 2005-2007 revealed also a low year-to-year variability (CV 7.64%). In the context of the evaluation of the pharmacological key activities of all the D.s.-SP batches, it was shown that the standardized aqueous extraction procedure at 85 °C leads to well reproducible products without any marked variations of the tested inhibitory activities.23 With a CV of 12.0%, the antielastase activities of H2O-D.s.-SPs did not show any relevant variation in dependence on the harvesting time of D.s.. Solely, the APTT activity was slightly impaired by increasing amounts of coextracted floridean starch in autumn.24 But regarding the period with the lowest reserve glucan content in spring, there was no significant batch to batch variability in APTT (CV 14.3%) and elastase inhibitory activity (7.76%) as demonstrated by D.s.-SPs isolated from five D.s. batches harvested in April of three consecutive years (Table 5).

Discussion In the context of the present study, a large number of single extraction and isolation processes were performed. The aim was to develop an optimized standard procedure, since in general optimization of the extraction method reduces the variability of the product.24 The use of NaOH as an extractant results in higher yields but only due to a less specific extraction with enhanced coextraction of concomitant reserve polysaccharides. This leads to a distinct but not only season-dependent batch to batch variability and reduced pharmacological activities. In comparison, the best reproducibility as well as the highest pharmacological potencies were achieved by the water extraction at 85 °C. This extraction method was superior to that with water at 60 °C concerning the yields, which amounted on average 11.6 ( 3.9% related to dry mass, but revealed a little higher glucose content representing the contamination with reserve glucans. Therefore, no boiling water was used within this work. Consequently, increasing the yield by the raise of the extraction temperature conflicts with a concurrently enhancement of the floridean starch coextraction. Nevertheless, the D.s.-SPs extracted at 85 °C were shown to have a DS as well as activities equivalent to those extracted at 60 °C. The variability of the yields was in an appropriate range for natural biopolymers


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showing no critical variations in dependence on season and year of D.s. harvest. According to our experience, there is a good correlation between the structural characteristic DS of SPs and their pharmacological effects.23 Thus, both the DS and the antielastase-activity are well suitable as quality markers and can be applied for the establishment and the routine quality control of an industrial production of D.s.-SPs. The DS can be determined by conductimetry, IR spectroscopy, or by the calculation from elemental analyses, whereby the latter allows the simultaneous determination of protein contamination. The anthrone method is characterized by relatively high inter- and intra-assay variations of the measurements and thus considered unsuitable for the quality control of D.s.-SPs. The improvement of the degree of purity by SEC-fractionation or enzymatic treatment with suitable glucoamylases might be suitable for an economical optimization of the D.s.-SPs production. Further on, the application of another well preserving extraction method such as percolation is a possible alternative to enhance the extraction process. Repetitive extraction of the algae material could further be applied because we found an increase of the yield by about 50% in that way. In contrast to various other red algae SPs, those isolated from D.s. were not gelling but well soluble up to a concentration of about 20 g/L, at high concentrations the solutions became increasingly viscous. Performed structural investigations revealed that the D.s.-SPs consist of high molecular mass branched xylogalactan structures. They are substituted with sulfate groups by a mean degree of 0.5 ( 0.08, that is, on average one sulfate group is linked to every second monomer. An equivalent DS has been described for SPs from Delesseria serrulata Harvey before.51 According to IR spectral analyses of sulfated galactans45,46 D.s.-SPs show no sulfate groups in position 4 but they are rather predominantly linked equatorially to position 6. These findings are in accordance with the work of Miller who postulated a backbone composition of alternating 1,3-β-D-galp and 1,4-R-L-galp for SPs occurring in the members of the family Delesseriaceae as characterized by 13C NMR analyses.52 The xylose of the D.s.-SPs showed to be present mainly as terminal sugar. Linkage analyses suggest that it occurs in side chains linked to the galactan backbone with a degree of branching of about 0.15-0.2. However, further studies are needed to elucidate the detailed molecular structure of the D.s.-SPs. Moreover, approximately one of nine galactose units in the D.s.-SPs is converted into the 3,6-anhydro form. This ratio of 3,6-anhydrogalactose is relatively low in comparison to carrageenans and agarans.46,53 This finding as well as the branched structure may be reasonable for the advantageous non gelling properties of D.s.-SPs. The higher content of 3,6-anhydrogalactose found in NaOH batches is most probably induced by the extraction process since alkaline treatment at high temperatures is a practice to produce anhydrosaccharides from saccharides with sulfate groups in position 6.48 Almost all of the glucose present in D.s.-SPs constitutes the seasonal varying amounts of concomitant floridean starch with a Mr of about 25000 to 30000.24 Linkage analysis as well as the amylose assay with iodine/potassium iodide and an R-amylase treatment consistently indicated that the 1,4-glucan in D.s.-SPs is branched in position 6 by a mean degree of 0.06. Reserve polysaccharides in Rhodophyta have formerly been defined as floridean starch since they were found to differ from that of higher plants by the absence of amylose and a structural composition similar to amylopectin or glycogen.49 The starch in D.s.-SPs represents now a further example substantiating the

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hypothesis of a special type of reserve glucans in Rhodophyta. In addition, we could show that it also differs from amylopectin and glycogen, because with a Mr of about 25000-30000 it has a considerably lower Mr and a degree of branching lying in between. The increasing accumulation of reserve polysaccharides in D.s. over the course of the summer months fits to the observation of Turner and Evans who detected accumulation of starch granules containing cells in lower, thickened locations of midribs and stipes in late summer via 14C labeling.54 Our findings on the structural composition of D.s.-SPs are basically in accordance with the structure determination of SPs from the atlantic ecotype of D.s. as published by Potin et al. However, these SPs extracted with water had a higher content of sulfate groups (28.1-37.7% of total weight, molar ratio of 0.59-0.74 sulfate groups per neutral monosaccharide) compared to the D.s.-SPs with a mean DS of 0.50 ( 0.08 (i.e., 18.5% (w/w) sulfate (-SO32-)).22 On the contrary, their anticoagulant effects determined as DC in the APTT assay (4.3-11.0 µg/ mL) were similar to those of the D.s.-SPs and not stronger. Because the anticoagulant activity of SPs, which does not differ concerning any other structural parameter, was shown to increase with increasing DS,47 the question arises whether there are any other structural differences between the D.s.-SPs and the SPs isolated from atlantic D.s., which compensate the higher DS of the latter. But this could only be clarified by a direct comparison of their structural characteristics and their activities. Both Potin et al.22 and we detected mannose or mannitol, respectively, as component of SPs from D.s., but so far it could not be elucidated how it is linked to the polysaccharides. The β-1,3-glucan laminarin is known to contain terminal mannitol, but the Mr of the D.s.-SPs seems to be too high to explain the portion of about 5% of the neutral monosaccharides. For other Ceramiales species, mannitol has been postulated to contribute to osmoregulation.55 However, free mannitol would have been separated due to several steps of the standardized extraction and isolation procedure, that is, defatting with ethanol 80% (v/ v), precipitation, and dialysis. For red algae, also other low molecular mass compounds have been described as osmolytes inducible by osmotic stress, for example, the heteroside digeneaside (R-D-galp)-(1f6)-(β-D-galp)-1-glycerol))56 in the genus Hypoglossum (Delesseriaceae). For D.s., also the occurrence of the diglucan trehalose has been reported years ago.20 Because trehalose is regarded as hydration factor and thus of interest for cosmetic applications, we could not find any trace of it in the ethanolic extract, the supernatant from the ethanol precipitation as well as the dialysate. Recent studies have restricted the synthesis of trehalose to bacteria, fungi, nematodes, and crustaceans but excepted algae and plants.57 Consequently, the trehalose formerly identified in D.s. may originate from any contaminant of the algae. As recently presented, the D.s.-SPs exhibit several pharmacological effects indicating an anti-inflammatory potency and thought to be important to retard the processes of skin aging.23 The pharmacological profile of D.s.-SPs in comparison to heparins is clearly shifted from anticoagulation to this field of activities. Their only moderate anticoagulant potency and thus a suggested reduced risk of bleeding is clearly related to the lack of the specific antithrombin binding pentasaccharide sequence. This is the prime example demonstrating that the pharmacological activities of SPs are not only due to unspecific ionic interactions but dependent on the individual structure.47,58 Accordingly, the activity profile of the D.s.-SPs, composed of branched xylogalactans with a relatively low DS, but a high Mr significantly differed from that of PS3, a highly sulfated, linear


β-1,3-glucan, by exhibiting strong inhibitory activity on both complement activation and hyaluronidase.23 Due to their pharmacological profile, the D.s.-SPs represent a promising candidate for further product development. Regarding this, another important prerequisite is fulfilled, since the D.s.-SPs can be obtained from D.s. growing at the artificial reef in a reproducible quality, whereby the low degree of contaminations with other species is considered an advantage over D.s. from North Sea or Atlantic. In fact, many other algae-derived SPs with promising pharmacological activities have been described,1,3,59 but studies on their reproducibility are extremely rare. On the contrary, considering the options of an economical use, rather disadvantageous properties have been reported for these SPs with fucoidans showing extreme diversity depending on the source of the algae material1 and SGG strongly varying in dependence on the season of harvesting.60 In the case of the D.s.-SPs, the reproducibility is assumed to be based on several facts: First, they are obtained from one exactly defined species of macroalgae. Second, this D.s. is growing at a defined place, whereby both the absence of tides in the Baltic Sea as well as the restricted number and distribution of species due to the low salinity14 are advantageous. Finally, the D.s.-SPs are isolated by an optimized procedure balancing natural seasonal variations, for example, the content on starch. Given the current requirements on drug candidates by the regulatory authorities, natural SPs have generally only minor chances to be approved as medicinal products. But, in other fields, there is an increasing demand on algae-derived products for the use in cosmetics and wellness therapies. D.s. algae material meanwhile has reached a high price on the European market because it is used in cosmetic formulations, for example, for the enhancement of microcirculation.61 In addition to their already identified pharmacological activities, D.s.-SPs may have a perspective for interactions with further targets. Thus, SPs isolated from marine algae are also known to have a broad antiviral activity.62,63 For example, the sulfated xylogalactans from Nothogenia fastigiata inhibited the replication of Herpes simplex viruses (HSV-1).9 Consequently, a formulation containing D.s.-SPs for the topical treatment of herpes labialis is conceivable. According to our experience, the manual purification and removal of Mytilus edulis is necessary until now. Investigations of extracts obtained from D.s. strongly contaminated with blue mussels demonstrated an increased protein content. Keith et al. found that Mytilus merely consist of 0.3-0.5% (w/w) soluble proteins composed of six fractions with a Mr in the range of 5000-200000. Moreover, neither N-acetyl-glucosamine nor glucosamine, which may potentially interfere with the pharmacological targets, were detected.64 In summary, D.s.-SPs can be obtained from D.s. growing at the artificial reef in the Baltic Sea close to Nienhagen in reproducible quality. The annual average of yield amounts to 11.6 ( 3.9% related to the dry mass of D.s. with a maximum of about 18% in spring. The good reproducibility of both their structural composition and their pharmacological activities is achieved by using a standardized extraction procedure with hot water and turned out to be independent of the harvest time of the D.s., its generation form, and its vitality. The pharmacological profile together with the specified quality and the attainable yields qualify the D.s.-SPs for the development of a product and, thus, D.s. from the artificial reef for an economical utilization.


Acknowledgment. This study was a part of the national German project “Unterwasserhabitate Nienhagen” at the southwestern Baltic Sea and financially supported by the EU (FIFG) and the Ministerium fu¨r Landwirtschaft, Umweltschutz und Verbraucherschutz Mecklenburg-Vorpommern (Germany) from January 2005 until December 2008. We are grateful to Dr. I. Groth for routinely performing the APTT and elastase inhibition assay as well as to Dr. U. Girreser for the performance of the mass spectrometry. To Dr. C. Schygula and T. Mohr we are indebted for the collection of the algae material by scuba diving in any weather condition and for its classification. Supporting Information Available. Fourrier transform infrared spectrum of a D.s.-SP batch harvested in March 2006 and extracted with H2O at 85 °C. This material is available free of charge via the Internet at http://pubs.acs.org.

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