Evaluation of Seasonal Variations of the Structure and Anti

Apr 8, 2009 - Delesseria sanguinea (Hudson) Lamouroux was shown to contain sulfated polysaccharides (D.s.-SP) with anti-inflammatory effects. This red...
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Biomacromolecules 2009, 10, 1155–1162

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Evaluation of Seasonal Variations of the Structure and Anti-inflammatory Activity of Sulfated Polysaccharides Extracted from the Red Alga Delesseria sanguinea (Hudson) Lamouroux (Ceramiales, Delesseriaceae) Niels Gru¨newald,† Inken Groth,† and Susanne Alban* Pharmaceutical Institute, University of Kiel, Gutenbergstrasse 76, D-24118 Kiel, Germany Received December 6, 2008; Revised Manuscript Received February 6, 2009

Delesseria sanguinea (Hudson) Lamouroux was shown to contain sulfated polysaccharides (D.s.-SP) with antiinflammatory effects. This red macroalga turned out to dominantly populate an artificial reef in the southwestern Baltic Sea. An important aspect for its economical utilization is to obtain D.s.-SP in a reproducible quality. The aim of the study was to evaluate the seasonal variability of D.s.-SP; for this, algae batches were harvested monthly over the period of one year. Structural and pharmacological characterization of the isolated D.s.-SP showed that an optimized and standardized aqueous extraction (85 °C) leads to reproducible products without any significant seasonal variations of their elastase and hyaluronidase inhibitory activities. Besides the yields (from 17.9% in April to 6.12% in September), only the glucose content of the D.s.-SP batches seasonally varied (April, 7.48% ( 1.00%, September, 22.8% ( 1.2%) due to certain coextraction of starch-like glucans. In principle, algae material can be harvested throughout the year, but the optimum harvesting time is in spring.

Introduction Marine organisms are a rich source for the isolation of various compounds as potential new candidates for pharmacological targets.1 Many investigations focus on lipophilic and alcoholic extracts but especially water-soluble compounds such as sulfated polysaccharides (SP) from algae are known to have promising anti-inflammatory activities.2 For example, the sulfated glucuronogalactans isolated from the red alga Schizymenia dubyi (SGG) were shown to have in Vitro effects against several viruses, to inhibit the proliferation of NSCLC-N6 tumor cells, and to exhibit anticoagulant and anticomplementary activities.3,4 For other SPs from red algae, antinociceptive effects have also been reported.5,6 A further well-described example is fucoidans produced by brown algae, which exhibit anticoagulant and antithrombotic effects as well as anti-inflammatory, antiproliferative, antiadhesive, and antiviral ones.7-9 However, despite these promising pharmacological effects, there has been no drug based on algae-derived SPs until now. One of the reasons might be the variability in both structural composition and pharmacological activities of these natural products.10,11 The quality and quantity of the SPs may be influenced by the exact species of the used algae and environmental factors of its habitat such as temperature, sea current, light exposure, salinity, food supply, epiphytes, and epizoans. Moreover, batch-to-batch variability results from insufficiently standardized extraction procedures. But nowadays, algae products are used more and more frequently as ingredients of cosmetics, food supplements, and wellness products. These product categories do not have to meet such strict quality requirements as medicinal products. Nevertheless, also within this field of modern life-style, high quality formulations gain in importance. Correspondingly, investigations in minimizing natural product variability are of interest. * Corresponding author. Tel. +49 431 880 1135. Fax: +49 431 880 1102. E-mail: [email protected]. † These authors contributed equally to this work.

Within the framework of the European Union “Financial Instrument for Fisheries Guidance” (FIFG),12 a large-scale artificial reef was established in the Baltic Sea close to Nienhagen (Germany) in 2003 in order to increase the economic value of the local marine resources.13 Meanwhile, the colonization of the reef elements has reached an ecological balance. The dominant species with up to 80% of the algal biomass in the summer period is the perennial red alga Delesseria sanguinea (Hudson) Lamouroux (Ceramiales, Delesseriaceae) (D.s.),14 which seems to be promising for product development. D.s. harvested from the North Atlantic coast was recognized to contain anticoagulant compounds already for a long time,15 which were later identified as SP (D.s.SP).16 Due to the dominant abundance of D.s. on the reef and the pharmacological potential of SP, a project was initiated to evaluate whether SP from Baltic Sea D.s. could be utilized economically. The D.s.-SP were isolated, structurally analyzed and examined for their biological activities demonstrating to have a promising pharmacological profile.17 The aim of the present study was to evaluate whether the algae material and the extractable D.s.-SP show seasonal variations concerning structural and pharmacological characteristics. So far, only few studies on seasonal variations of algaederived polyglycans have been published.10,18,19 In the case of D.s., growth and reproduction processes have been described to occur independently of each other,20 but to be regulated by various environmental or endogenous factors such as temperature and photoperiodic effects.21 With regard to these complex influences, the question arose as to whether the SPs of D.s. also vary over the year. Moreover, information was expected on any potential restrictions for the time of harvesting and on the optimal period to obtain the material, respectively, which is important to guarantee reproducible products. Over the period of one year, D.s. material was monthly harvested, extracted following an optimized standard procedure, and characterized. According to Gru¨newald et al. the D.s.-SPs consist of a galactan

10.1021/bm8014158 CCC: $40.75  2009 American Chemical Society Published on Web 04/08/2009

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backbone branched with xyl side chains, substituted with sulfate groups and containing low amounts of glucuronic acids.22 In addition, the anticoagulant effects in activated partial thromboplastin time (APTT), inhibition of polymorphonuclear neutrophil elastase (PMNE), and hyaluronidase as typical pharmacological effects of D.s.-SP17 were determined, since the reproducibility of useful effects might be most important for commercial utilization.

Materials and Methods Algae Material. In 2006, fresh Delesseria sanguinea (Sea beech) algae material was monthly 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 January, no batch could be harvested, since the rough weather did not allow diving. The algae were identified by Dr. Christof Schygula, Faculty of Science, Department of Marine Ecology, University of Rostock, Germany (voucher specimen available). The raw material was manually purified from epiphytes such as Phycodrys rubens (L.) Batters and epizoes such as Mytilus edulis L. and stored in a biocide medium consisting of ethanol 80% (v/v) 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.16 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 sodium hydroxide (NaOH) 0.1 mol/L at 85 °C under reflux conditions. In general, the defined ratio of the algae to the extracting agent of 750 g/L was used. The obtained raw extracts were centrifuged (10 000 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 ethanol 96% (v/v) was added resulting in a final ethanol concentration of 90% (v/v). After storing for 24 h at 4 °C followed by centrifugation (10 000 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 of 1000), adjusted to pH 7.4 with NaOH and freeze-dried.

Analytical Methods Degree of Sulfation (DS). The DS (i.e., number of sulfate groups per monosaccharide) of the D.s.-SPs was determined according to an European Pharmacopoeia method,23 first described by Casu et al.24 D.s.-SP sodium salts were transformed into the free acid form by cation exchange with Amberlite IR120 (Fluka), which was titrated with NaOH 0.1 mol/L. The end point of titration was detected by measuring the change of conductivity using a SevenEasy conductivity meter (Mettler Toledo). Results were verified both by elemental analysis (determined elements: carbon, hydrogen, nitrogen and sulfur) and Fourier transform infrared spectrometry (16PC FT-IR, Perkin-Elmer). Protein Content. Total proteins were quantified with the Bradford assay,25 modified by Zor and Zeliger,26 as well as by conversion into a microplate scale and further with a fluorescence microplate assay using ortho-phthalaldehyde (OPA) (Fluoraldehyde Reagent Solution, Pierce).27,28 The ratio of absorption (OD (595 nm)/OD (465 nm)) and the fluorescence intensity (λex 370 nm, λem 475 nm), respectively, were measured

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with a microplate reader (POLARStar OPTIMA, BMG). For both assays, bovine serum albumin (Sigma) was used as standard. Colorimetric Carbohydrate Analyses. Total carbohydrates were determined by the anthrone method29 calibrated with D-galactose. The 3,6-anhydrogalactose content was examined using the resorcinol reaction30 with agarose as the standard and D-galactose as blank. Furthermore, uronic acids were detected after reaction with m-hydroxydiphenyl (Fluka) according to the method by Blumenkrantz and Asboe-Hansen31 as well as its modification by van den Hoogen et al.32 Both assays were calibrated with a mixture (1:1) of D-glucuronic acid and D-galacturonic acid. The absorptions (at 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). 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 (Polymer Laboratories) coupled with a miniDAWN MALLS detector (Wyatt). Despite the deficiency of directly comparable polymer references, this method allows the direct specification of the absolute Mr.33 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. The samples (injection volume 100 µL) were eluted with NaNO3 0.1 mol/L (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. The Mr values were calculated with ASTRA for Windows software version 4.70.07 (Wyatt). Monosaccharide Composition. To determine the monomer composition, D.s.-SP were hydrolyzed with trifluoroacetic acid (TFA) 2 mol/L at 121 °C34 and after evaporation of TFA converted into alditol acetate (AAs) derivatives by reduction and acetylation.35 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, and the temperature of injector and detector was 250 and 240 °C, respectively. The monosaccharides were identified by their retention times. For quantitative analysis, the D.s.-SP samples were supplemented with a defined amount of myo-inositol as internal standard. The percentage of the respective AA was calculated applying the software HP GC Chemstation, Rev. A.06.03 [509].

Pharmacological Methods Test Compounds. Besides D.s.-SP, two other SPs were tested in parallel: (1) Highly sulfated glycosaminoglycan unfractionated heparin (UFH) from porcine mucosal origin consists of alternating D-glucosamine and D-glucuronic acid or L-iduronic acid units with a total molar mass ranging from 5000 to 30000 and a DS of about 1.2 (Lot No. 73508019, a purity of g99% has been verified by the certificate of analysis by Biochemie GmbH) and was a kind gift from Novartis. (2) Semisynthetic PS3 is a linear β-1,3-glucan sulfate with a DS of 2.2, in which the primary OH-group in position 6 is completely sulfated, and the two secondary OH-groups in positions 2 and 4 are equally sulfated to about 60%. PS3 shows in ViVo anti-inflammatory activity

Seasonal variations of SPs from Delesseria sanguinea

(US Patent No. US7008931-B2, Mr ) 10 000, produced under GMP conditions, purity g99%) and was synthesized as previously described.36,37 Materials. Coagulation assays were performed using the Amelung-coagulometer KC10 macro, and microplates (Nunc) in fluorescence or absorbance assays were read out by the microplate reader Polarstar Optima (BMG Labtech). Activated Partial Thromboplastin Time. Pooled human platelet-poor plasma from at least 10 healthy volunteers, stored at -80 °C, was thawed at 37 °C and mixed thoroughly. An aliquot of 90 µL of plasma was added to 10 µL of sample (diluted in NaCl 0.9%). After 60 s of incubation at 37 °C, 100 µL of Pathromtin SL (Dade Behring) was added followed by another incubation time of 120 s. Finally, 100 µL of preheated (37 °C) CaCl2-solution (0.025 mol/L, Dade Behring) was added. The time until fibrin clot formation was recorded and the “doubling concentration” (DC ) inhibitor concentration causing a prolongation of the coagulation time to twice the time of the negative control) was determined.38,39 NaCl 0.9% without inhibitor served as a negative control. Elastase Inhibition Assay. For the elastase inhibition assay,40 human PMNE (EC 3.4.21.37, Calbiochem) was used. Tests were performed in black 96-well microplates. An aliquot of 25 µL of Tris-buffer (50 mM Tris, 155 mM NaCl, pH 8.3) was mixed with 25 µL of inhibitor (diluted in NaCl 0.9%) and 25 µL of PMNE (c ) 100 nM) in Na-acetate buffer (50 mM Na-acetate, 200 mM NaCl, 1% BSA, pH 5.5). After 5 min of incubation at 37 °C, 25 µL of substrate solution (MeOSucc-Ala-Ala-Pro-Val7-amido-4-methylcoumarin, Bachem), diluted in Tris-buffer (c ) 3 mM), was added. Fluorescence was measured after 5 min incubation time at 37 °C at λex 370 nm, λem 450 nm. The fluorescence values from blanks were subtracted from all other values. The resulting values were used to determine the concentration-dependent inhibition (%) in relation to the positive control (100%). Hyaluronidase Inhibition Assay. Tests were performed in 96-well-microplates. For the enzyme reaction, 25 µL of phosphate buffer (pH 5.0), 25 µL of inhibitor in 0.9% NaCl, 25 µL of hyaluronic acid in water (4 mg/mL), and 25 µL of bovine hyaluronidase (80 U/mL) (Sigma, EC 3.2.1.35) were incubated for 120 min at 37 °C. To cleave the terminal N-GlcNAc units from the resulting hyaluronan oligosaccharides, 10 µL of K2B4O7 buffer (pH 10.0) was added, and the covered microplate was incubated for 30 min at 105 °C. After cooling, 170 µL of 4-dimethylaminobenzaldehyde (2%) was added, followed by incubation for 30 min at 40 °C (Morgan-Elson reaction). The OD at 570 nm was measured versus an enzymefree blank. The absorbance values from blanks were subtracted from all other values. The resulting values were used to determine the concentration-dependent inhibition (%) in relation to the positive control (100%). 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, and p e 0.05 was considered as statistically significant.

Results By using a standardized extraction and isolation procedure, the variations between the D.s.-SP batches obtained from the monthly harvested seaweed should exclusively reflect environmental influences.

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Figure 1. Dry mass of drained fresh D.s. algae material in % (w/w) as a function of the harvest time. Values represent the mean ( SD (n ) 3), whereby the repeated measurements were performed with different net weights. The black horizontal line indicates the mean of all month values, and the gray bar is the SD.

Figure 2. Rate of yield after extraction with H2O at 85 °C based on dry mass in % (w/w) shown as a function of the harvest time. The black horizontal line indicates the mean of all month values, and the gray bar is the SD. The presented values are based on an entire extraction process each, which has not been repeated.

Morphological Variations. As expected, the fresh algae material from the defined habitat showed visually noticeable alterations of morphology over the course of a year. In the winter period, the algae were almost completely reduced to more greenish tubercular midrib parts and their basal fixture organ. In February/March, new reddish blades were generated and attained their maximal development in May. Consequently, the proportion of blades was highest in the early summer months. Because of degeneration of thalli starting in early autumn, in winter the algae mostly consisted of midribs and stipes, which had grown stronger during the summer months. A cover with the widespread epizoan Mytilus edulis L. was observed more or less during the whole vegetation period, and less contaminated individuals of D.s. were harvested in early spring. Afterward, the algae were covered with very small juvenile mussels over the whole period, which increased in size until winter. Variability of Dry Mass. The dry mass determination of D.s. individuals revealed an evident seasonal dependency (Figure 1). The lowest value of dry mass was detected in April (14.2% ( 0.6%) followed by a permanent increase resulting in a highest value in October (22.2% ( 0.7%) and then a subsequent decline till spring. Thus, the coefficient of variation (CV) of the dry mass over the year was, at 16.7%, relatively high. Yields of Isolated D.s.-SP. Like the algae dry mass itself, the dry mass derived yields of D.s.-SP obtained by extraction with demineralized water considerably varied seasonally (Figure 2). But the trend was contrary with the highest amounts in March/April (17.9%) and the smallest ones in autumn (September 6.12%) or winter, respectively. With a mean of 11.9% ( 4.4%, the yield of D.s.-SP turned out to be the parameter most dependent on the harvest time (CV 37.4%). The yields in

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Figure 3. DS determined by conductivity measurement during titration with 0.1 mol/L NaOH after converting D.s.-SP into the free acid form. Values represent the mean ( SD (n ) 3), verified by elemental analysis. The black horizontal line indicates the mean of all month values; the gray bar is the SD.

relation to the dry mass thus differed by a factor up to about 3.0 over the year. When the yields are calculated in relation to the drain weight, they of course varied less, but still by factor 2.1, i.e., between 1.27% (September) and 2.62% (April) (mean 2.02% ( 0.47%, CV 23.5%). Compared to the extraction with water, that with 0.1 mol/L NaOH resulted in about 50% higher yields with a mean of 17.4% ( 5.1% (CV 29.5%) of the dry mass and of 3.06% ( 0.84% (CV 27.5%) of the drain weight (data not shown). In contrast to the water extracts, the pronounced variations of the yields were independent of the harvesting time; the yields of the April and September batches differed only by factor 1.6 (19.2% versus 12.4%) and 0.9 (2.34% versus 2.57%), respectively. Variability of Degree of Sulfation. In contrast, there was no clear seasonal trend of DS (Figure 3), which is expressed by a moderate overall variability (CV 9.28%) with similar values over the course of the year (mean 0.547 ( 0.051, min 0.475, max 0.602). Except for an aberration in April (0.510 ( 0.014), only a slight decline was noticeable from August till November. The mean DS corresponds to a total amount of 20.0% ( 1.4% (w/w) of sulfate groups. Variability of Monosaccharide Composition. The monosaccharide composition of D.s.-SP was examined by conversion into their AA derivatives and GLC analysis. Galactose (gal) was identified as the major component with a mean content of 69.0% ( 5.4% (CV 7.8%). Moreover, xylose (xyl) (mean 10.5% ( 1.3%; CV 12.4%) and glucose (glc) (mean 13.5% ( 5.0%; CV 36.7%) as well as traces of mannose (mean 5.49% ( 1.11%; CV 20.3%) and fucose (mean 1.57% ( 0.31%; CV 19.8%) were detected. As obvious from Figure 4 A, especially the percentages of gal and glc were found to be dependent on the harvest time. Whereas the content of gal successively decreased from April to September (75.2% ( 0.2% to 59.0% ( 2.0%), that of glc increased (7.48% ( 1.00% to 22.8% ( 1.2%), followed by inverse trends from September until April. The seasonal course of the xyl contents was similar to that of gal. The content values of the minor components mannose and fucose could not be related to seasonal influences. The mean ratios of gal/xyl and gal/glc were 6.65 ( 1.00, CV 15.1% and 5.91 ( 2.49, CV 42.1%. The significantly higher CV of the gal/glc ratio is mainly due to a sharp decline between April and September (Figure 5). Since the increase of glc content occurred during the growth period, the striking seasonal disparity of gal and glc may not be due to a change of the composition of the SP but rather due to concomitant reserve glucans such as floridean starch.22 To

Figure 4. (A) Variations in D.s.-SP neutral monosaccharide composition of gal (–0–), glc (–[–), xyl (–9–), man (–3–) and fuc (--4--) as a function of the harvest time quantified by GLC analysis after conversion into AAs. (B) Variations in neutral monosaccharide composition independent of assumed concomitant reserve glucans. Values are recalculated referring to minimum content of glc in April. All values represent the mean ( SD (n ) 2-3) mass percentages of the total saccharide amount of each month. The black horizontal lines in B indicate the mean of all month values, and the gray bars are the SDs for each monosaccharide, respectively.

Figure 5. Ratio of gal (%)/xyl (%) -9-, and ratio of gal (%)/glc (%) --0-- as a function of the harvest time quantified by GLC analysis after conversion into AAs. In addition, the linear regression lines of the two curves are shown.

prove this hypothesis, the following calculation was performed: Assuming that the portion of glc above the minimum content of 7.48% ( 1.00% found in April is part of reserve glucans, the lowest amount in April has been set as the content for all investigated months. On this basis, the percentages of the other monosaccharides were recalculated. In this way, the significant seasonal variability of the gal content was nearly eliminated (mean 73.7% ( 2.0%, CV 2.7%) (Figure 4B). For xyl (mean 11.3% ( 1.4%; CV 12.7%), mannose (mean 5.93% ( 1.38%; CV 23.3%) and fucose (mean 1.68% ( 0.29%; CV 17.5%) no significant shift was recognized. During the development of the extraction procedure, 0.1 mol/L NaOH was also used as an extractant. Compared to water, NaOH led to higher yields, but these NaOH extracts were characterized by significantly higher batch-to-batch variability of all structural as well as pharmacological parameters. Figure

Seasonal variations of SPs from Delesseria sanguinea

Figure 6. Effects of different extractants on seasonal variations in D.s.-SP. Batches of June and December were extracted with demineralized water and NaOH 0.1 mol/L, respectively. (A) Effects on neutral monosaccharide composition (fucose (dashed column), mannose (striped column), xyl (light shaded column), gal (white column) and glc (dark shaded column)). The mass % values of the total saccharide amount (mean ( SD of several acetylation procedures, n ) 2-3) have been quantified by GLC analysis after conversion into AAs. (B) Effects on elastase inhibitory potency (IC50, mean ( SD, n ) 6).

6A exemplarily represents the monosaccharide composition of aqueous and alkaline extracts from June and December. In contrast to the comparable composition of the two aqueous fractions, that of the NaOH extracts significantly differed, especially in their gal and glc content, which were nearly equal in December. In addition, the DS values were 0.598 ( 0.010 in June and 0.588 ( 0.026 in December for water extracts of D.s.-SP versus 0.615 ( 0.021 in June and 0.277 ( 0.012 in December for NaOH extracts. These distinctly reduced DS values are in line with high glc contents, indicating the mentioned presence of coextracted unsulfated reserve glucans.17 These structural characteristics agree with the findings in the elastase inhibition assay, where the high glc content and low DS are associated with a considerably lower elastase inhibitory potency: IC50 (NaOH Dec) 0.480 ( 0.003, IC50 (NaOH Jun) 0.212 ( 0.0108, IC50 (H2O Dec) 0.211 ( 0.009, IC50 (H2O Jun) 0.164 ( 0.007 (Figure 6B). Further elucidated analytical parameters showed an extent of variations as expected for biomacromolecules, but these could not be related to the time of harvesting (Table 1). Contamination with protein was moderate, but strongly dependent on the applied method, as exemplarily shown by the Bradford and OPA data. In general, extraction with NaOH resulted in much higher batch-to-batch variability, which was, however, engendered not only by seasonal influences but also by the stronger and less specific extraction force of NaOH (data not shown). The sizeexclusion/light scattering chromatograms of all NaOH batches indicated additional peaks with higher retention volumes besides the major fraction of D.s.-SP. These peaks are assumed to represent floridean starch as their size (mean Mr ) 23 ( 9 × 103) correlated with the glc content of the D.s.-SP batches. Variability of Pharmacological Activities. The seasonal variability of the pharmacological activities was investigated by three exemplary activities identified for D.s.-SP.17 The inhibitory potency against elastase and hyaluronidase is supposed to be of interest with regard to a potential use of D.s.SP, whereas the anticoagulant activity is included as a wellknown effect of SP in general. The anticoagulant activity in the global coagulation assay APTT (expressed as DC ) inhibitor concentration causing a prolongation of the coagulation time to twice the time of the negative control) of the D.s.-SP was much lower than that of

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UFH (DC ) 1.13 µg/mL ( 0.01 µg/mL) and in most cases also lower than that of PS3 (DC ) 4.40 µg/mL ( 0.07 µg/mL) with an annual average of 6.40 µg/mL ( 1.05 µg/mL (CV 16.4%). The anticoagulant activity expressed in units amounted to 190 IU/mg for UFH and, on average, to 34.6 IU/mg for the D.s.-SP batches. As a function of harvest time, a certain seasonal variation of the D.s.-SP’s anticoagulant activity was observed. The DC of the D.s.-SP batches from February to June were somewhat lower (i.e., higher activity), whereas those from August to December were somewhat higher than the mean DC (Figure 7). The elastase inhibition was determined using a chromogenic substrate assay. The antielastase activity of the D.s.-SP batches revealed an annual average of 0.194 µg/mL ( 0.017 and was thus significantly better than that of UFH (IC50 ) 0.236 µg/mL ( 0.011 µg/mL). Compared with PS3, the D.s.-SP batches were even as active as this proven anti-inflammatory compound (IC50 ) 0.163 µg/mL ( 0.004 µg/mL). With a CV of 8.97% the activities of the different D.s.-SP revealed no relevant variation depending on the season. The autumn batches were only slightly less active than the spring batches (Figure 8). As examined by a two-step microplate assay, hyaluronidase was inhibited more efficiently by D.s.-SP than by UFH or PS3, which showed similar activities (UFH: 9.00 µg/mL ( 2.08 µg/ mL, PS3: 8.68 µg/mL ( 3.09 µg/mL, annual average of D.s.SP: 3.42 µg/mL ( 0.59 µg/mL). The relatively high CV of 17.25% of the activity of the D.s.-SP batches is attributable to the assay system rather than being seasonally caused (Figure 9), as the assay procedure includes long incubation times at high temperatures, provoking evaporation.

Discussion In the present study, the seasonal variations of the red alga D.s. from the Baltic Sea and especially of its isolated D.s.-SPs were examined by monthly harvesting and extracting algae material over the period of one year. Referring to pharmacologically active natural compounds and biomacromolecules from algae, not much is known about seasonal influences on their total content and their composition.10,18,19 As already described by Molenaar et al.,20 the morphology of the algae is subject to changes over the year, including an increase of dry mass from spring to autumn by about 50%. In part, this is due to the increasing portion of more dense, solid midrib material and inversely reduced portion of water-rich, thin blades composed of monocell layers. Another reason is the increasing generation of reserve polysaccharides during the summer months, which is well-known for algae growing in the Northern hemisphere. In the case of D.s., Turner and Evans detected accumulation of assimilates in lower, thickened locations of midribs and stipes in late summer via 14C-labeling.41 Both the changed morphology and the reserve polysaccharides had an effect on the yields and the composition of the isolated D.s.-SPs. Even though representing single determinations, the yields over the year show a definite and systematic trend. The yields continuously decreased from spring to autumn, in total by 66%. Thus, the increased dry mass mainly consisted of water-insoluble material such as cellulose fibers, and the structure of the rigid midribs and basal stipes material may impede the extraction of SP, respectively. Compared to the spring batches, the D.s.-SP batches extracted in autumn contained up to 15% more glc (April 7.48% ( 1.00% versus September 22.8% ( 1.2%) indicating coextraction of reserve glucans such as floridean starch, which has been described as

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Table 1. Further Non-Seasonal-Dependent Structural Characteristics of D.s.-SPa total carbohydratesb (% m/m)

uronic acids (% m/m)

3,6-anhydrogalactose (% m/m)

total protein (% m/m)

80.9 ( 5.0

3.06 ( 0.8

3.48 ( 1.11

8.98 ( 1.54 5.65 ( 1.29 c

Mr (× 103) d

139 ( 26 (93 - 318e)

a Data represent means ( SD of 11 batches. b Total carbohydrates as measured by the anthrone method using D-galactose as a reference. c Total protein determined by OPA assay. d Total protein determined by Bradford assay modified according to Zor and Zeliger.26 e Characteristic Mr range.

Figure 7. Anticoagulant activity of UFH (black column), PS3 (white column), and D.s.-SP (gray column) in APTT expressed as the DC. D.s.-SP results are shown as a function of the harvest time. The black horizontal line indicates the mean of all month values; the gray bar is the SD.

Figure 8. IC50 values of UFH (black column), PS3 (white column), and D.s.-SP (gray column) in elastase inhibition. D.s.-SP results are shown as a function of the harvest time. The black horizontal line indicates the mean of all month values; the gray bar is the SD.

Figure 9. IC50 values of UFH (black column), PS3 (white column), and D.s.-SP (gray column) in hyaluronidase inhibition. D.s.-SP-results are shown as a function of the harvest time. The black horizontal line indicates the mean of all month values; the gray bar is the SD.

the typical amylose-free assimilation product of Rhodophytae.42 This was confirmed by performing the extraction of all the D.s. batches additionally with 0.1 mol/L NaOH, which is a better solvent for starch and other sparingly soluble polysaccharides than water. The yields of these D.s.-SP fractions were shown

to be independent of harvesting time and were, particularly in autumn, significantly higher than those obtained with water. As shown by quantitative monosaccharide analysis, this gain was primarily due to coextraction of glucans. A high glc content in D.s.-SP was associated with reduced elastase inhibitory activity (Figure 6). Therefore, the coextracted polysaccharides virtually “dilute” the biological activities of D.s.-SP and have to be considered as undesired contaminants. Since water obviously allows a more selective extraction of the SP, demineralized water instead of NaOH was selected for the standard D.s.-SP extraction procedure.22 The results of the seasonal analysis enable one to eliminate the apparent disadvantage of lower yields obtained by aqueous extraction. The yields achievable with D.s. harvested between March and July are relatively high and similar to that of the corresponding NaOH extracts. Although the D.s.-SP batches obtained with water show a lower variability of both structural parameters and biological activities than the NaOH extracts,22 the monosaccharide analysis suggests that especially those extracted from algae harvested in autumn are contaminated with other glucans (Figure 4). Accordingly, the DS of these D.s.-SP batches was found to be up to 10% lower, and their anticoagulant activity was slightly reduced. In contrast, neither the elastase nor the hyaluronidase inhibitory potency showed any evident seasonal variability. By using the DS and the elastase activity as analytical markers for the pharmaceutical quality, the batch-to-batch variability of D.s.SP obtained by hot-water extraction (85 °C) of Baltic Sea D.s. growing on an artificial reef is within the range accepted by the European Medicine Agency (EMEA) for herbal medicinal products.43 If the harvest of D.s. would be limited to the time between February and July, the variability could be further decreased and the quality increased by reducing the contamination with glucans. The glc content of D.s.-SP ranged from 7.48% up to 22.8%. By regarding the content above 7.48% as reserve polysaccharides, the monosaccharide composition of the D.s.SP remains similar over the year (Figure 4B). However, further investigations are needed to clarify whether glc is present exclusively as a contaminant of D.s.-SP or also as a linked component of the SP, which has been identified as a branched xylogalactan so far.16,22 Nevertheless, the results on the seasonal variability of D.s.-SPs suggest that they are consistently produced by the algae year-round despite all other seasonal (morphologic and metabolic) changes and that they can be isolated in reproducible quality by an optimized and standardized procedure and by using algae material of a defined species growing at a defined place. In contrast, many other algae-derived SP with promising pharmacological activities have been described,5,7,9,44-46 but have only rarely been tested for their reproducibility. The structural composition and activities of fucoidans were found to vary extremely as a function of the source of the algae material,7 as well as those of SGG as a function of the season of harvesting.10 This disadvantage might be one reason why fucoidans have not been examined in a clinical study so far. On the other hand, because of the existing heterogeneity and polydispersity of algal-derived SPs, no direct comparison of the pharmacological potency is possible.

Seasonal variations of SPs from Delesseria sanguinea

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 a increasing demand on algae-derived products. For example, D.s. algae material meanwhile has reached a high price on the European market. It is used in cosmetic formulations for the enhancement of microcirculation.47 Further, a fixed combination containing D.s. among other ingredients has recently been patented for applications to prevent the formation of stretch marks.48 However, not much has been published on any activities of its ingredients apart from the anticoagulant activity of its SPs.16 Recently, D.s.-SPs were found to inhibit several targets that play a role in anti-inflammatory and antimetastatic processes as well as in processes of skin aging.17 The extracellular-matrixdegrading enzymes PMNE and hyaluronidase are expressed by both inflammatory and tumor cells and thus enable these cells to pass basal membranes and to migrate through tissues.49-52 This is essential for inflammatory cells to extravasate and reach the center of inflammation, but is also important for tumor growth and metastasis. Depending on the target, different structural aspects are decisive for the particular inhibitory potency. Whereas the anticoagulant and elastase inhibiting activity depend on both DS and Mr, for inhibition of hyaluronidase, a high Mr is more important than a high DS.17 Furthermore, branched glycan sulfates such as D.s.-SPs were shown to be stronger anticoagulants than linear ones with similar DS and Mr values.53 This promising pharmacological profile offers a perspective for the interaction with further targets. SPs isolated from marine algae are also known to have a broad antiviral activity.10,54 For example, the sulfated xylogalactans from Nothogenia fastigiata inhibited the replication of herpes simplex virus (HSV-1).55 Consequently, a formulation containing D.s.-SP for the topical treatment of herpes labialis is conceivable. In summary, D.s.-SP would be a promising candidate for further development, especially when they are produced in a specified quality by standardizing both the source of the algae material and the isolation procedure. Our results show that, by using a standardized extraction procedure with hot water, D.s.-SP can be obtained in reproducible quality rather independently on the season. In principle, D.s. can be harvested throughout the year, yet the optimum harvesting time is, however, in spring, as then the yields of D.s.SP turned out to be the highest, and their contamination with floridean starch was the lowest. Further advantages of this period are also the low contamination with Mytilus edulis (i.e., before spatfall of Mytilus-larvae) and epiphytes as well as good conditions for diving. Nevertheless, annual climatic variations may have an impact on the ecosystem of the reef including the growth of D.s. biomass and the abundance of epiphytes, epizoans, and their predators. The data were collected for D.s. growing in a special habitat, i.e., the artificial reef in the brackish southwestern Baltic Sea. The reduced salinity and the limited occurrence of substratum are responsible for a restricted number and distribution of species in the Baltic Sea.56 But the reef structures obviously provided good conditions for the dominant settlement of D.s., a clear advantage concerning any economical use. Acknowledgment. This study is 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 and Verbraucherschutz des Landes Mecklenburg-Vorpommern (Germany)

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from January 2005 until December 2008. We are also grateful to Dr. C. Schygula and T. Mohr for diving at any weather conditions to collect the algae material.

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