Degradation of Eight Sulfated Polysaccharides Extracted from Red

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Characterization, Synthesis, and Modifications

Degradation of eight sulfated polysaccharides extracted from red and brown algae and its impact on structure and pharmacological activities Eric Lahrsen, Ann-Kathrin Schoenfeld, and Susanne Alban ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01113 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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ACS Biomaterials Science & Engineering

Degradation of eight sulfated polysaccharides extracted from red and brown algae and its impact on structure and pharmacological activities Eric Lahrsena, Ann-Kathrin Schoenfelda, Susanne Albanb,* *corresponding

author

aPharmaceutical bPharmaceutical

Institute, Kiel University, Gutenbergstraße 76, 24118 Kiel, Germany Institute, Kiel University, Gutenbergstraße 76, 24118 Kiel, Germany,

[email protected], Tel: +49-431-880-1135, Fax: +49-431-1352

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Degradation represents a strategy to improve the biopharmaceutical properties of native algae sulfated polysaccharides (SP) with high Mw. The aim of the study was to compare the degradability of four sulfated xylogalactans (SXG) and four fucose-rich sulfated polysaccharides (FRSP) extracted from red and brown algae, respectively, using three simple methods causing no desulfation as well as to examine the chemical and pharmacological changes of the resulting fractions. The achieved degradation proved to be dependent on the basic glycan structure of the SP. Treatment with hydrogen peroxide (3 %, 4 h, 50 °C) led to the most efficient degradation of both FRSP and SXG. The Mw decrease was associated with distinct reduction of the activities (complement inhibition (>) elastase inhibition > C1-INH potentiation) and resulted in a modified pharmacological profile. Despite their much lower degree of sulfation, some of the fractions with Mw < 15 kDa exhibited similar or even stronger activities than heparins, whereas they have only weak anticoagulant effects.

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Keywords algae polysaccharides; degradation; fucoidan; pharmacological activities; sulfated glycan; sulfated xylogalactan

Abbreviations C1-INH: C1 esterase inhibitor DS: degree of sulfation, i.e. mean number of sulfate groups per monosaccharide FRSP: fucose-rich sulfated polysaccharide(s) GC-MS: gas chromatography–mass spectrometry HT: hydrothermal degradation HP20: hydrogen peroxide degradation at 20 °C HP50: hydrogen peroxide degradation at 50 °C IC50: concentration for 50 % inhibition LMWH: low molecular weight heparin MALLS: multi angle laser light scattering MLev: calculated molecular mass by performing a colorimetric assay1 Mw: weight average molecular mass Mn: number average molecular mass NMR: nuclear magnetic resonance PMN-elastase: elastase from polymorphonuclear granulocytes SEC: size exclusion chromatography SP: sulfated polysaccharide(s) extracted from brown or red algae SXG: sulfated xylogalactan(s) UFH: unfractionated heparin VBS: veronal buffer saline

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1.

Introduction Algae are well-known for their content of sulfated polysaccharides (SP) as renewable

source of biocompounds useful for the development of new systems and devices for biomedical applications2 and additionally showing manifold pharmacological activities similar to heparins. Most intensely studied are the complex pharmacological proﬁles of several fucose-rich SP (FRSP), also referred to as fucoidans, from brown algae including antimetastatic, immunomodulatory, anti-inflammatory, antiviral, radical scavenging, and anticoagulant activities.3–10 Beside the interest on these SP as active substances, nanomedicine has meanwhile grasped these compounds to develop innovative therapeutic and diagnostic nanosystems.10,11 However, despite the increasing attention in research, there is no licensed medicinal product containing FRSP yet. So far, FRSP-containing products are only marketed as ingredients in food supplements and cosmetics.3,6,9,12,13 Red algae are the main source of commercially utilized sulfated polysaccharides at all. Representatives of many orders contain large amounts of sulfated galactans, i.e. either carrageenans or agarans, in their cell walls and intercellular matrix, which are widely used as hydrocolloids in food and for many other applications.14 In contrast to FRSP, these linear homogalactans have a masked regular structure, which basically consists of alternating 3linked -D-galactose units and 4-linked -D or -L galactose units, respectively. However, there are also red algae species producing other types of SP like xylogalactans, glucuronogalactans, and xylomannans.15 In contrast to carrageenans and agarans, these heteropolysaccharides are not utilized as hydrocolloids, but exhibit interesting pharmacological activities similar to FRSP, e.g. antiviral, antiproliferative, antiadhesive, anticoagulant and anticomplementary effects.14,16–20 Compared to brown algae, red algae usually have a lower content of phenolic compounds21 so that the extracted SP from red algae are usually less contaminated with such 4


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co-extracted compounds. Apart from the issue of reproducible quality,22–25 the high molecular weight of native algae SP represents another critical aspect for medical applications as it is associated with disadvantageous biopharmaceutical characteristics, which may even lead to undesired effects.26–28 Therefore, it seems reasonable to develop SP fractions with reduced size. For this, it is important to choose a method that is not associated with desulfation, as the degree of sulfation (DS) is important for the majority of pharmacological activities of SP. Another aspect is its applicability for the production of degraded SP in large scale. For that reason, the certainly promising use of specifically acting enzymes29 represents not yet an option. With hydrothermal treatment at 120° C and oxidative degradation by incubation with hydrogen peroxide (H2O2), respectively, we have recently chosen two simple approaches to degrade the commercially available FRSP from Sigma, a sulfated galactofucan from the brown alga Fucus vesiculosus28. Advantages of these two simple methods are that they can be performed without any special equipment and possibly toxic reagents as well as that they represent antimicrobial procedures. Both methods resulted in gradual degradation of the FRSP with a weight average molecular mass (Mw) of 38.2 kDa down to 4.91 kDa without concomitant desulfation. An additional benefit of the degradation with H2O2 turned out to be the elimination of co-extracted terpenoids like fucoxanthin or phenolic compounds like phlorotannins. Based on these results, we used these methods to degrade another high-molecular weight algae SP, namely the sulfated xylogalactan extracted from the red alga Delesseria sanguinea (D.s.-SP)24 with a Mw of 214 kDa. However, both hydrothermal treatment (for up to 90 minutes at 120 °C) and oxidative degradation (3 % H2O2, 4 hours at 20 °C) reduced the Mw of D.s.-SP only by at most 26 % and 24 %, respectively, whereas that of the FRSP had been

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reduced by 73 % and 66 %, respectively28. This observation led to the hypothesis that the glycan structure of SP may not only be important for their pharmacological activities30–32, but also for their sensitivity or stability, respectively, toward degradation. In this case, a degradation method established for a specific SP would not be generally applicable to another SP. Against this background, we performed the presented study on FRSP and sulfated xylogalactans (SXG) to answer the following questions: (1) Do FRSP and SXG in principle differ in their degradability using hydrothermal or H2O2 treatment? (2) Is the degradation of these SP associated with other structural changes than reduction of Mw? (3) What effects has the degradation on selected pharmacological activities of the SP? (4) Has the glycan structure any additional impact on the activities and their Mw-dependent decrease? Finally, (5) are there any SP among the tested FRSP and SXG that can be degraded down to Mw < 15 kDa and still exhibit pronounced pharmacological activities? Eight different algae SP were included in the study, i.e. four FRSP extracted from the brown algae Fucus vesiculosus (F.v.-SP), Saccharina latissima (S.l.-SP), Macrocystis pyrifera (M.p.-SP), and Undaria pinnatifida (U.p.-SP) and four sulfated xylogalactans (SXG) extracted from the red algae Delesseria sanguinea (D.s.-SP), Coccotylus truncatus (C.t.-SP), Phycodrys rubens (P.r.-SP), and Phyllophora pseudoceranoides (P.p.-SP). . After initial characterization of the SP, they were degraded using three of the methods recently applied for the degradation of FRSP from Sigma28, namely hydrothermal treatment for 90 min at 120 °C and degradation with 3 % H2O2 for 4 hours at 20 °C and 50 °C. The resulting fractions were chemically characterized and examined for three exemplary pharmacological activities of FRSP and SXG, i.e. PMN-elastase inhibitory activity, inhibition of complement activation and potentiation of C1 esterase inhibitor (C1-INH), an important endogenous inhibitor of the complement and contact system.20,30,33

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2.

Materials and Methods

2.1. Sulfated polysaccharides The four SXG were extracted from the red algae Delesseria sanguinea (Ceramiales, Delesseriaceae) (D.s.-SP), Coccotylus truncates (Gigartinales, Phyllophoraceae) (C.t.-SP), Phycodrys rubens (Ceramiales, Delesseriaceae) (P.r.-SP), and Phyllophora pseudoceranoides (Gigartinales, Phyllophoraceae) (P.p.-SP) as previously described for D.s.-SP.24 The four FRSP were from the brown algae Fucus vesiculosus (Fucales, Fucaceae) (F.v.SP., Sigma-Aldrich, Lot No SLBC4004V), Saccharina latissima (Laminariales, Laminariaceae) (S.l.-SP, extracted from alga material harvested in May 2011 from the Atlantic Ocean as previously described),25 Macrocystis pyrifera (Laminariales, Laminariaceae) (M.p.-SP, SigmaAldrich, Lot No SLBH6625V) and Undaria pinnatifida (Laminariales, Alariaceae) (U.p.-SP, Sigma-Aldrich, Lot No SLBH3106V).

2.2. Further materials Unfractionated heparin (UFH) and a low molecular weight heparin (LMWH) were used

as reference compounds in all the activity assays. The UFH isolated from porcine mucosa (200 IU/mg, Lot No 73508019, Novartis, Germany) has a Mw of 15.0 kDa (certificate of analysis)

and a degree of sulfation (DS) of 1.0 (elemental analysis). The LMWH enoxaparin (BfArM sample no. 11-07/08 05.06.2008) showed to have a Mw of 4.50 kDa and a DS of 1.0. Human pooled serum was obtained from healthy volunteers as previously described.33 If not mentioned otherwise, all reagents were bought from Sigma-Aldrich.

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2.3. Hydrothermal Degradation Aliquots of 50.0 mg of each of eight SPs were weighted out in Wheaton® vials and dissolved in 5.0 ml aqua bidest. The vials were placed in a heating block (Stuart Block Heater SBH200DC, Bibby Scientific Limited, United Kingdom) preheated to 120 °C. After heating for 10, 20, 30, 40, 60 and 90 minutes, respectively, the vials were cooled down at room temperature. The solutions were dialyzed for three days using Spectra Por® 6 Dialysis Membrane with a molecular weight cut-off of 1000 Da (Repligen Corporation, USA) and then lyophilized. Before and after dialysis, the pH value was measured and adjusted to 7.0 with 1.0 mol/l sodium hydroxide.

2.4. Degradation with hydrogen peroxide Aliquots of 50.0 mg of each of eight SPs were weighed out in 15 ml tubes (Sarstedt, Germany) and dissolved in aqueous 3 % H2O2. The tubes were incubated for 4 h at 20 °C. A second series of SP solutions was incubated for 4 h at 50 °C. Then, the samples were processed as described in 2.3.

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2.5. Chemical characterization 2.5.1. Molecular mass determination The weight average molecular mass Mw was analyzed by size exclusion chromatography (SEC) using a PL-GPC 50 Plus system with online multi-angle laser light scattering (MALLS) and refractive index (RI) detection as previously described.28

2.5.2. Uronic acids Uronic acids were quantified by a microplate assay using the reaction with 3Phenylphenol according to the method by Blumenkrantz and Asboe-Hansen.34

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2.5.3. Elemental analysis The contents of carbon, hydrogen, nitrogen and sulfur in the native and degraded SP samples were determined by elemental analysis as previously described.28 The sulfur content (%) was used to calculate the content of −SO3Na groups. The degree of sulfation (DS) respresents the number of sulfate groups per monosaccharide. By means of the nitrogen content (%), the content of total protein was estimated by multiplying by 6.25.

2.5.4. Monosaccharide composition by acetylation analysis The neutral monosaccharide composition of the SP samples was determined by converting the SPs into alditol acetate derivatives, which were then analyzed by gas liquid chromatography analysis as previously described.28

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2.5.5. Colorimetric determination of reducing glycan end groups The amount of reducing glycan end groups in the SP samples was determined by the color reaction with p-hydroxybenzoic acid hydrazide (PAHBAH) as previously described.1 To calculate the molar concentration (mol/l) of reducing glycan end groups in the SP samples, three different calibration solutions of L-fucose, D-galactose and D-xylose, that were roughly similar to the monosaccharide composition of the various SPs: (1) 92.2 % (% m/m) galactose and 7.8 % xylose, (2) 50.9 % galactose and 49.1 % fucose and (3) 87.9 % fucose and 12.1 % galactose. By means of the concentration of reducing glycan end groups (i.e. fucose / galactose / xylose equivalent conc. (mol/l)) in the SP sample, the molecular weight MLev was calculated by the formula: Equation 1

MLev =

monomer conc. in the sample (mol/l) x MMono (g/mol) fucose galactose/ xylose equivalent conc. (mol/l)

The monomer concentration in the SP sample (mol/l) was calculated by dividing the gravimetric concentration of the pure SP (i.e. without protein) (g/l) by MMono (g/mol), whereby MMono represents the mean monosaccharide molecular mass of the respective SP based on its results from acetylation analysis and elemental analysis as follows: Equation 2

MMono

(

)

fucose content x Mfucose + galactose content x Mgalactose + = - MH20 + xylose n mannose xylose content x M + man ose content x M (DS x MNaSO3)

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2.6. Activity assays The native SPs and the degraded were examined in the following activity assays: (1) Fluorigenic PMN-elastase activity assay (2) Hemolytic classical complement modulation assay (3) Chromogenic C1 esterase inhibitor assay (4) Coagulation assay: activated partial thromboplastin time (APTT). The details are described by Lahrsen et al.33 The final assay concentrations ranged from 0.125 to 125 µg/ml. If possible, the result values represent arithmetic means ± standard deviation (SD) of at least three testings on different days in duplicates. Concentrations for 50 % inhibition (IC50) are based on the final assay concentrations. The percentages of activity increases and decreases refer to the activities of the corresponding native SP.

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3.

Results and Discussion

3.1. Characteristics and activities of native sulfated xylogalactans and FRSP Based on the observation that the SXG from the red alga Delesseria sanguinea (D.s.SP) was considerably less degraded than FRSP from the brown alga Fucus vesiculosus (F.v.-SP), we intended to evaluate whether these structurally different algae SP principally differ in their degradability and thus the basic glycan structure plays a role. For this, we selected four SXG from different red algae species (i.e. Delesseria sanguinea, Coccotylus truncatus, Phycodrys rubens, and Phyllophora pseudoceranoides) and four FRSP from different brown algae species (i.e. Fucus vesiculosus, Saccharina latissima, Macrocystis pyrifera, and Undaria pinnatifida. The four SXG D.s.-SP, C.t.-SP, P.r.-SP, and P.p.-SP as well as the FRSP S.l.-SP were extracted using the corresponding established procedures.24,25

3.1.1. 3.1.2. Structural Characterization Table 1 and 2 show basic structural characteristics of the various SP as well as their mean values ± SD for the SXG and FRSP, respectively. To our knowledge, this is the first description of the four SXG in literature. The DS of the SXG ranges from 0.52 to 0.68, that of the FRSP from 0.34 to 0.70 (Table 1). Like the DS also the Mw range of the SXG is some smaller (100 – 214 kDa) than that of the FRSP (38.2 – 480 kDa), whereby S.l.-SP has the highest Mw, but the lowest DS (Table 2). The main monosaccharides of the SXG are galactose (76 – 88 %) and xylose (3.7 – 14 %), those of the FRSP fucose (50 – 83 %) and galactose (7.3 – 50 %) (Table 2). The uronic

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acid content of the SXG (3.7 – 4.1 %) is lower than that of the FRSP (4.2 – 9.9 %), whereas their protein content (6.9 – 11 % versus 0.0 – 13 %) is higher (Table 2). Table 1 Native SXG and FRSP: degree of sulfation (DS), weight average molecular mass Mw, inhibition of PMN-elastase activity (IC50), anticomplementary activity measured by inhibition of the complement-mediated hemolysis (IC50), potentiation of the C1s inhibition by C1-INH (%) and anticoagulant activity (doubling concentration (DC)) in the APTT. The individual values represent the mean ± SD (n ≥ 3). The calculated mean of each SP group and the corresponding minimum and maximum values are marked bold.

SXG D.s.-SP native C.t.-SP native P.r.-SP native P.p.-SP native Mean FUCOIDANS F.v.-SP native S.l.-SP native M.p.-SP native U.p.-SP native Mean

DS

Mw (kDa)

Elastase inhibition IC50 (µg/ml)

Complement inhibition IC50 (µg/ml)

C1-INH potentiation (%)

Anticoagulant activity DC (µg/ml)*

0.65 ± 0.02 0.52 ± 0.01 0.68 ± 0.01 0.53 ± 0.02 0.59

213.9 ± 27.6 127.6 ± 4.0 105.3 ± 2.5 100.4 ± 0.8 136.8

0.21 ± 0.03 0.51 ± 0.07 0.25 ± 0.06 0.50 ± 0.09 0.37

1.11 ± 0.27 2.60 ± 0.33 1.83 ± 0.07 2.56 ± 0.35 2.02

23.28 ± 3.45 14.26 ± 1.94 14.16 ± 1.75 17.58 ± 2.08 17.32

4.80 ± 0.05 6.55 ± 0.12 5.64 ± 0.08 7.04 ± 0.21 6.00

0.59 ± 0.01 0.34 ± 0.00 0.56 ± 0.03 0.70 ± 0.04 0.51

38.2 ± 1.4 480.7 ± 71.6 77.3 ± 17.1 126.6 ± 6.0 197.5

0.48 ± 0.08 0.61 ± 0.07 0.42 ± 0.06 0.26 ± 0.05 0.49

18.28 ± 3.94 6.80 ± 0.55 3.74 ± 0.57 2.76 ± 0.33 7.62

26.42 ± 3.06 50.42 ± 3.34 47.10 ± 3.72 23.14 ± 3.05 38.44

16.70 ± 0.33 8.75 ± 0.25 10.55 ± 0.11 6.12 ± 0.14 10.53

* the DC of UFH amounted to 0.77 ± 0.03 µg/ml

Table 2 Native SXG and FRSP: composition of neutral monosaccharides, contents of uronic acids and proteins. The individual values represent the mean ± SD (n ≥ 2). The calculated mean of each SP group and the corresponding minimum and maximum values are marked bold. Fucose (% mol/mol)

Galactose (% mol/mol)

Xylose (% mol/mol)

Mannose (% mol/mol)

Uronic acids (%)

Proteins (%)

Mean

0.72 ± 1.02 0.65 ± 0.92 0.58 ± 0.82 1.32 ± 0.04 0.82

75.52 ± 0.28 86.95 ± 0.08 83.18 ± 0.14 87.91 ± 0.36 83.39

14.27 ± 2.62 4.28 ± 0.33 11.27 ± 0.04 3.74 ± 0.15 8.41

3.03 ± 0.78 6.21 ± 0.62 2.10 ± 0.05 5.14 ± 0.05 4.12

3.96 ± 0.54 3.74 ± 1.29 4.08 ± 1.39 3.74 ± 0.84 3.88

7.24 ± 0.07 9.50 ± 0.23 6.89 ± 0.06 10.73 ± 0.18 9.34

Mean

83.07 ± 2.36 51.69 ± 1.13 79.30 ± 0.36 50.33 ± 0.93 63.49

7.30 ± 1.17 15.11 ± 2.43 13.28 ± 0.25 49.67 ± 0.93 20.93

6.52 ± 1.15 17.48 ± 0.35 4.23 ± 0.10 0.00 ± 0.00 8.39

2.01 ± 0.17 3.68 ± 0.32 2.30 ± 0.56 0.00 ± 0.00 3.90

7.07 ± 1.59 9.92 ± 2.43 7.82 ± 3.06 4.23 ± 1.81 7.24

0.27 ± 0.27 12.94 ± 1.28 5.86 ± 0.47 0.00 ± 0.00 5.72

SXG D.s.-SP native C.t.-SP native P.r.-SP native P.p.-SP native FRSP F.v.-SP native S.l.-SP native M.p.-SP native U.p.-SP native

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3.1.3. Pharmacological activities Previously, we showed that the used algae SP exhibit a multitude of effects relevant for both anti-inflammatory and antimetastatic activities including targets like chemokines, growth factors, adhesion molecules, complement factors and extracellular matrix degrading enzymes.20,28,30,33,39–42 The concert of multiple actions is thought to contribute to the in vivo anti-inflammatory and antimetastatic activity of these algae SP.5,14,33,43 In this study, all the SP as well as the degraded fractions were tested for three exemplary pharmacological activities: The inhibition of the PMN-elastase (elastase from polymorphonuclear leukocytes, elastase inhibition) was determined using a routinely used and standardized fluorigenic PMN-elastase assay. The anticomplementary activity (complement inhibition) was examined by means of the inhibition of hemolysis induced by classical complement activation. The potentiating effect on C1 esterase inhibitor (C1-INH, C1INH potentiation) was measured using a chromogenic substrate assay and the complement factor C1s as an important target enzyme of C1-INH. The three selected effects are of interest, inter alia, with regard to the search for new therapies of age-related macular degeneration (AMD).44,45 They could support another shown anti-AMD effect of fucoidan, namely the reduction of the expression and secretion of vascular endothelial growth factor in retinal pigment epithelium and reduction of angiogenesis in vitro.46 Moreover, the anticoagulant activity of the native SP was determined by the activated partial thromboplastin time (APTT) assay. The anticoagulant activity was one of the first activities known for algae SP and in the past, this was of interest in the context of search for alternatives to animal-derived heparin.27 It is meanwhile well-known that the pharmacological activities of SP are not only dependent on their DS and Mw, but also on their basic glycan structure, whereby a high DS 15



may compensate a low Mw and vice versa.20,30–32,38,47 Moreover, the importance of the various structural parameters for a specific activity may more or less vary.30,33 In addition, it has to be considered that algae SP, especially FRSP, are usually complex and heterogeneous molecule mixtures29,31,32,48 so that the observed activity only reflects the net effect of the mixture. For these reasons, it is delicate to compare the activities of the eight structurally very different algae SP. Nevertheless, the activity data of both the individual SP and the means of the SXG and FRSP demonstrate that the three activities differ in their dependence on the glycan structure (Table 1). The anticomplementary activity of the SXG (IC50 range: 1.1 - 2.6 µg/ml) is stronger than that of the FRSP (IC50 range: 2.8 - 18 µg/ml), whereas the latter are better C1-INH potentiators (potentiation range: 14 – 23 %) than the SXG (potentiation range: 23 – 50 %). In contrast, the elastase inhibition turned out to be rather independent of the glycan structure (IC50 mean ± SD: 0.37 ± 0.14 for SGX, 0.49 ± 0.15 for FRSP) and to be mainly determined by the DS of the SP. With increasing DS (0.34 - 0.70), the IC50 linearly decreased from 0.61 ± 0.07 µg/ml to 0.21 ± 0.03 µg/ml (y = -1.1063x + 1.0374, r = 0.89 (SXG, FRSP), r (SXG) = 0.97, r (FRSP) = 0.92; minor deviations from this order can be explained by respective Mw differences. The anticoagulant activity of the native algae SP (DC range 4.8 - 16.7 µg/ml) turned out to be 6.2 to 21.7 times weaker than that of UFH (DC 0.77 ± 0.03 µg/ml). The nonsignificant differences between the SXG (DC range: 4.6 – 7.0 µg/ml) and the FRSP (DC range: 6.1 – 16.7 µg/ml) can be explained by the corresponding DS and Mw. Degradation of F.v.-SP was recently shown to result in strong reduction of its anticoagulant activity. In relation to UFH, it amounted to only about 2.5 %, whereas the other activities ranged between 19.1 % and 66.7 %.33 Therefore, it was concluded that degradation makes the anticoagulant activity irrelevant.

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3.2. Degradation of sulfated xylogalactans and fucoidans Hydrothermal treatment for 90 min at 120 °C (method abbreviated as HT) of the SXG D.s.-SP (Mw 214 kDa), C.t.-SP (Mw 128 kDa), P.r.-SP (Mw 105 kDa), and P.p.-SP (Mw 100 kDa) reduced the Mw to 157, 73.2, 82.1, and 87.7 kDa, respectively, and thus overall by only 26 ± 11 % in relation to the native Mw (Table 3, Figure 1). Degradation with 3 % H2O2 for 4 hours at 20 °C (method abbreviated as HP20) led to Mw of 164, 73.0, 68.9, and 79.2 kDa, respectively, which corresponds to a reduction by 30.5 ± 8.8 %. In contrast, the degradation with 3 % H2O2 at 50 °C (method abbreviated as HP50) was considerably more efficient with a reduction by 93.6 ± 3.4 % and resulted in Mw of 13.3, 3.40, 5.00, and 12.0 kDa, respectively. In case of the FRSP F.v.-SP (Mw 38.2 kDa), S.l.-SP (Mw 481 kDa), M.p.-SP (Mw 77.3 kDa), and U.p.-SP (Mw 127 kDa), HT caused the strongest Mw reduction (85 ± 12 %) and resulted in degraded fractions with Mw of 10.3, 128, 4.91, and 1.55 kDa, respectively (Table 3, Figure 1). HP20 led to fractions with Mw of 12.9, 205, 35.7, and 109 kDa, respectively, and thus a reduction by 48 ± 20 %, whereas HP50 was again more powerful (73 ± 12 %) and led to Mw of 9.12, 138, 8.25, and 54.4 kDa, respectively.

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Table 3 Characteristics of the degraded SXG and fucoidan fractions in relation to the native SP: yield after degradation and dialysis, degree of sulfation (DS) and DS increase (%), weight average molecular mass Mw and Mw decrease (%), and the polydispersity (calculated by Mw/Mn). The individual values represent the mean ± SD (n ≥ 3). The calculated mean of each SP (native and fractions) and the corresponding minimum and maximum values are marked bold.

SULFATED XYLOGALACTANS D.s.-SP native D.s.-SP hydrothermal D.s.-SP H2O2 3 %. 4 h. 20 °C D.s.-SP H2O2 3 %. 4 h. 50 °C Mean C.t.-SP native C.t.-SP hydrothermal C.t.-SP H2O2 3 %. 4 h. 20 °C C.t.-SP H2O2 3 %. 4 h. 50 °C Mean P.r.-SP native P.r.-SP hydrothermal P.r.-SP H2O2 3 %. 4 h. 20 °C P.r.-SP H2O2 3 %. 4 h. 50 °C Mean P.p.-SP native P.p.-SP hydrothermal P.p.-SP H2O2 3 %. 4 h. 20 °C P.p.-SP H2O2 3 %. 4 h. 50 °C Mean FUCOIDANS F.v.-SP native F.v.-SP hydrothermal F.v.-SP H2O2 3 %. 4 h. 20 °C F.v.-SP H2O2 3 %. 4 h. 50 °C Mean S.l.-SP native S.l.-SP hydrothermal S.l.-SP H2O2 3 %. 4 h. 20 °C S.l.-SP H2O2 3 %. 4 h. 50 °C Mean M.p.-SP native M.p.-SP hydrothermal M.p.-SP H2O2 3 %. 4 h. 20 °C M.p.-SP H2O2 3 %. 4 h. 50 °C Mean U.p.-SP native U.p.-SP hydrothermal U.p.-SP H2O2 3 %. 4 h. 20 °C U.p.-SP H2O2 3 %. 4 h. 50 °C Mean

DS increase (%)

Yield (%)

DS

82.9 80.8 92.4 85.4 82.8 80.6 65.4 76.3 98.4 93.7 69.5 87.2 89.2 82.6 83.5 85.1

0.65 ± 0.02 0.64 ± 0.02 0.75 ± 0.00 0.66 ± 0.01 0.67 0.52 ± 0.01 0.61 ± 0.00 0.58 ± 0.00 0.51 ± 0.01 0.55 0.68 ± 0.01 0.77 ± 0.00 0.85 ± 0.01 0.75 ± 0.01 0.76 0.53 ± 0.02 0.68 ± 0.00 0.67 ± 0.00 0.53 ± 0.00 0.60

75.7 86.0 69.8 77.2 88.6 91.0 92.2 90.6 75.1 87.8 77.6 80.2 63.4 83.8 87.2 78.1

0.59 ± 0.01 0.61 ± 0.02 0.63 ± 0.01 0.64 ± 0.00 0.62 0.34 ± 0.00 0.38 ± 0.00 0.42 ± 0.00 0.32 ± 0.00 0.36 0.56 ± 0.03 0.58 ± 0.01 0.67 ± 0.00 0.63 ± 0.00 0.61 0.70 ± 0.04 0.75 ± 0.00 0.83 ± 0.00 0.71 ± 0.04 0.75

18

-1.1 15.4 1.0 5.1 17.2 11.7 -0.8 9.4 12.6 24.6 10.6 15.9 29.2 26.5 0.1 18.6

2.3 6.0 7.1 5.1 12.7 24.2 -3.5 11.1 3.5 18.6 11.9 11.3 7.6 18.4 1.2 9.1


Mw (kDa) 213.9 ± 27.6 157.4 ± 2.3 163.5 ± 13.6 13.3 ± 0.4 137.0 127.6 ± 4.0 73.2 ±27.0 73.0 ± 23.3 3.4 ± 0.3 69.3 105.3 ± 2.5 82.1 ± 0.3 68.9 ± 4.8 5.0 ± 0.7 65.3 100.4 ± 0.8 87.7 ± 5.7 79.2 ± 2.1 12.0 ± 3.9 69.8 38.2 ± 1.4 10.3 ± 1.6 12.9 ± 1.6 9.1 ± 3.5 17.6 480.7 ± 71.6 128.1 ± 3.2 204.5 ± 20.1 138.3 ± 7.8 237.9 77.3 ± 17.1 4.9 ± 0.4 35.7 ± 5.9 8.3 ± 1.1 31.5 126.6 ± 6.0 1.6 ± 0.1 108.8 ± 8.8 54.4 ± 1.6 72.8

Mw decrease (%)

26.4 23.5 93.8 47.9 42.7 42.8 97.3 60.9 22.0 34.6 95.3 50.6 12.7 21.0 88.1 40.6

73.1 66.1 76.1 71.8 73.4 57.5 71.2 67.3 93.6 53.8 89.3 78.9 98.8 14.1 57.1 56.7

Polydispersity (Mw/Mn) 1.63 ± 0.34 1.79 ± 0.04 1.54 ± 0.24 1.57 ± 0.23 1.63 1.54 ± 0.30 1.61 ± 0.15 1.59 ± 0.01 1.23 ± 0.02 1.49 1.65 ± 0.07 2.00 ± 0.14 2.06 ± 0.25 1.48 ± 0.06 1.80 1.69 ± 0.13 1.46 ± 0.13 1.81 ± 0.15 1.58 ± 0.01 1.64 1.69 ± 0.08 1.63 ± 0.38 1.49 ± 0.14 1.35 ± 0.06 1.54 2.00 ± 0.16 2.31 ± 0.25 2.41 ± 0.31 2.38 ± 0.23 2.27 1.83 ± 0.14 1.23 ± 0.11 1.68 ± 0.16 1.51 ± 0.13 1.56 2.09 ± 0.18 1.05 ± 0.04 2.01 ± 0.16 2.05 ± 0.48 1.80

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Figure 1. Decrease of the molecular mass Mw of each SP fraction in relation to its native Mw (%) for SXG (on the left) and FRSP (on the right). The Mw was determined by SEC coupled with a MALLS detector (mean ± SD, n ≥ 3).

These results confirm the hypothesis that the achieved degradation by a specific method is dependent on the basic glycan structure of the SP. The SXG showed to be quite resistant against HT and HP20, their Mw was, however, strongly reduced by HP50 (Figure 1 left). In contrast, FRSP showed to be degradable by all three methods, whereby HP20 was more or less inferior to both HT and HP50. But there were also pronounced differences of the degradability between the various FRSP, which differ structurally more between each other than the SXG (Figure 1 right). For example, the Mw of U.p.-SP was most extremely decreased by 99 % with HT and least efficiently by only 14 % with HP20, whereas the degradation of F.v.-SP showed similar results for all three methods. Overall, the results indicate that the optimal degradation method needs to be established for each individual SP. As previously reported,28 a clear advantage of degradation with H2O2 is the elimination of coextracted colored compounds. This purification effect is especially important for brown algae species rich in phenolic and terpenoid compounds. These compounds may have beneficial pharmacological effects,21,40,49 but own observations confirm that they are quite instable depending on the processing and storage conditions after extraction50 and may even reduce

19



its water solubility. Consequently, such compounds impair the so-called pharmaceutical quality of the SP, which is, however, a prerequisite for any medical application.

3.3. Chemical characterization of the fractions 3.3.1. Yield, polydispersity, degree of sulfation The yields of SP fractions after degradation, dialysis and lyophilization ranged from 63 to 98 %. They were only lower than 80 % for the seven fractions with Mw ≤ 10 kDa (mean 71 % vs. 87 % of the other fractions), which is probably due to more pronounced elimination of smaller fragments by dialysis (Table 3). The polydispersity (Mw/Mn) of the fractions did not increase, but indeed correlated with the molecular mass and, therefore, decreased with decreasing Mw (Table 3). The degradation processes did not reduce the degree of sulfation (DS). Instead, in most cases, the DS of the fractions increased by up to 29 %, which was most marked after HP20 (17.5 % ± 6.2 %) and less striking after HT and HP50 (6.4 % ± 8.5 %). It is conceivable that the cleavage of the glycan chains occurs preferably at low-sulfated domains resulting in fragments being so small that they are eliminated by dialysis. Accordingly, the preferred points of attack may be either low-sulfated side chains or fringe areas of the backbone. This is supported by the finding, that F.v.-SP lost many side chains as well as unsulfated fucose and other monosaccharides by degradation and purification.51 An explanation might be that cleavage at high-sulfated domains and endo-backbone degradation need more activation energy for two reasons: a) stronger sulfation increases ionic repulsion and b) steric hindrance by side chains. At higher reaction temperature, energy is sufficient for endo-backbone degradation and higher-sulfated domains resulting in fractions with DS similar to the native SP or only slightly increased. In line with these considerations, the DS increase of the degraded fractions could 20


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also be due to preferred degradation of low-sulfated or unbranched fractions of the native SP, which can be part of the often heterogeneously composed algae SP. As there is either no or only limited and partly inconsistent information about the composition and chemical structure of the selected algae SP,24,25,52–55 it is impossible to prove the above assumption.

21



3.3.2. Experiments on the cleavage sites of the degradation Nevertheless, to get any further information whether low-sulfated side chains and fringe regions and thus dialyzable fragments are really preferred cleavage sites, the following procedure was used: First, we calculated the molecular mass of the SP and their fractions based on the amount of reducing end groups (MLev) which were quantified using a colorimetric assay.1 By degradation, the MLev of a SP only decreases, if degradation produces new chains (and thus new reducing end groups) being too large for elimination by dialysis, whereas it remains constant, if all generated fragments are dialyzable. Consequently, the extent of the MLev decrease can be lower than that of the Mw. Based on this, we calculated the ratio of Mw decrease (%) / MLev decrease (%) for all the fractions. They were generally > 1.0 (except for 0.8 for P.p.-SP-HP50) and ranged from 1.3 to 6.7 for SXG and from 1.1 to 6.0 for FRSP. Plotting the ratio values against the Mw illustrates differences between the degradation methods as well as between SXG and FRSP (Figure 2). For both SXG and FRSP, the ratios of the HP20-fractions (3.4 ± 1.9) were considerably higher than those of the HT-fractions (1.50 ± 0.36). This suggests that HP20, the mildest degradation method associated with the strongest DS increase, preferentially cleaves the polysaccharide chains at sensitive sites such as side chains, fringe regions and low-sulfated domains, whereas HP50, the most powerful method, leads rather to random degradation. Concerning the HT-fractions, the ratios of the FRSP (1.52 ± 0.55) are lower than those of the HP20-fractions, whereas those of the SXG (3.6 ± 1.5) are in a similar range, which fits quite well with the respective extent of degradation.

22


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Figure 2. Comparison of the Mw of the SXG (on the left) and FRSP (on the right) fractions with the calculated ratio of Mw decrease (%) to MLev decrease (%). The fractions obtained with HP20 have the highest ratios, those resulting from HP50 have the lowest ones. The higher the ratio the lower was the extent of generation of additional, non-dialyzable molecules (i.e. reducing end groups) by the degradation.

23



3.3.3. Monosaccharide composition Like the native SP, all the fractions were subjected to acetylation analysis to determine their monosaccharide composition (Supporting Information Table S1). There were some notable changes (i.e. more than 20 % change of the content of a specific monosaccharide compared to the native SP), but the interpretation is difficult without knowing the composition and structure of the native SP. The SXG fractions, especially the HP20-fractions, had up to 114 % higher xylose contents, whereas the contents of mannose (2.1 - 6.2 % in the native SXG) were decreased and only increased in three of the HP20-fractions. Typical components of Delesseriaceae (D.s.SP and P.p.-SP) are sulfated xylogalactans from the agaran type,56 whereas the sulfated xylogalactans of Phyllophoraceae (C.t.-SP and P.r.-SP) belong to the carrageenan group,57 where it has to be considered that the DS and the degree of substitution of these galactans can vary and result in more or less heterogeneous mixtures.15,56,57 Further, it is known that many red algae additionally contain lower amounts of uncharged mannans and xylans, but also the presence of sulfated xylomannans was described.15 D.s.-SP was identified as an agaroid with a relatively high DS, containing side chains with terminal xylose and only a low content of anhydrogalactose.23,58 Ion exchange chromatography, however, revealed that D.s.SP consists not of a homogeneous high-sulfated fraction, but additionally contains lower charged portions (unpublished results). In agreement with the found DS increase, it can therefore be assumed that the reduced mannose content of the SXG fractions is due to degradation and elimination of unsulfated mannans and their increased xylose content results from the preferred degradation of lower-sulfated, less substituted galactans. The increase of both mannose and xylose in P.r-SP-HP20 and P.p.-SP-HP20 could be due to the presence of sulfated xylomannans, which are not degraded by the mild HP20 method. 24


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Among the FRSP, the altered ratios of fucose to galactose of the U.p.-SP fractions represented the most striking changes of the monosaccharide composition (Table S1). The FRSP from U.p.-SP stands out due to its composition of fucose and galactose at approximately equal amounts; its main fraction was found to be a highly branched fucogalactan with a galactan core and side chains consisting of fucans and other galactans.53,54,59 Whereas the native U.p.-SP had a molar ratio of fucose to galactose (fuc/gal ratio) of 1.01, it was reduced to 0.563 (35 % mol/mol fucose and 63 % mol/mol galactose) in the highly degraded HTfraction (Mw 1.55 kDa). The higher galactose content of the remaining oligosaccharides may be due to the galactan core. The fuc/gal ratio of the only modestly degraded HP20-fraction was inversely changed and amounted to 1.39 (58 % fucose and 42 % galactose). Since the HP20-fraction had a 19 % higher DS and 14 % less uronic acids compared to the native U.p.SP, it can be assumed that primarily the low-sulfated fraction of native U.p.-SP (18 %, DS 0.42) containing less galactose (14 %, fuc/gal 5.30), but much more uronic acid content (10 %),53 was degraded. Similar to U.p.-SP, the decreased fuc/gal ratios (HT: 2.93, HP20: 2.81; HP50: 2.62 vs. 3.42) of the S.l.-SP-fractions can be explained by the composition of the native S.l.-SP, which consists of a high-sulfated galactofucan and a lower sulfated heterogeneous mixture containing uronic acids and more galactose.25,55 Both the increase of the DS and the significantly reduced uronic acid contents support that again predominantly low-sulfated molecules were degraded. However, to prove all these interpretations of the changes of the monosaccharide composition, extensive further structural investigations including degradation experiments after fractionation of the native SP are required.

25



3.4. Pharmacological activities of the degraded fractions All the degraded SP fractions were tested for three exemplary activities to elucidate the impact of the three degradation methods on the pharmacological activities of the resulting SXG and FRSP fractions. Figure 3 presents the activities, i.e. IC50 (µg/ml) for elastase inhibition, IC50 (µg/ml) for complement inhibition and C1-INH potentiation (%), in dependence on the Mw. This overview illustrates the following trends: (1) All the three activities of the SP fractions decreased with decreasing Mw but to a different extent. (2) There are activity differences between the SXG and FRSP fractions. (3) The applied degradation method has an additional effect on the activities independent of the Mw.

26


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Figure 3. Pharmacological activities of the native SXG (on the left) and FRSP (on the right) and their degraded fractions in dependence on the molecular mass Mw. Mean ± SD (n ≥ 3)

27



3.4.1. Mw-dependent activity decrease and impact of the DS The complement inhibition showed the strongest decline with decreasing Mw and the CI-INH potentiation the weakest one as indicated by the trend lines of the individual activities (Figure 3), the mean activity decreases of the various SXG and FRSP fractions (Figure 5) as well as the mean activity decreases in dependence on the degradation method (Figure 6 right). Consequently, degradation results not in an uniform reduction of the activities, but modifies the pharmacological profile of the respective algae SP. Overall, the average activity reduction of the SXG and FRSP fractions, respectively, amounted to 20 % and 43 % for the elastase inhibition, 27 % and 47 % for the complement inhibition and 11 % and 26 % for the C1-INH potentiation. These mean activity reduction values have of course pronounced SD due to the distinct Mw loss, DS, and basic structure of the SP fractions. The elastase inhibition was quite stable down to a Mw of 35.7 kDa (IC50 0.38 µg/ml vs. lowest IC50 0.18 µg/ml), whereas the complement inhibition already decreased at higher Mw. There was generally a sharp activity reduction at Mw < 15 kDa (section 3.5). Since all three activities are also dependent on the DS, it has, however, to be considered that the Mw-dependent activity reduction was partly compensated by the DS increase of corresponding fractions (Table 4). Among the SXG fractions, the elastase and complement inhibition by some HT- and HP20-fractions (Mw > 68 kDa) were even stronger than those of the native SXG (e.g. IC50 of P.p.-SP-HT and -HP20 up to 50 % lower). In the FRSP group, this phenomenon was only observed with S.l.-SP-HT and -HP20, possibly due to the lower Mw of the other FRSP and fractions, respectively.

28


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3.4.2. Impact of the glycan structure on the activities Similar to the native SP, also the activities of the degraded fractions differed in their dependence on the glycan structure (Figure 3, Table 4). The SXG (native and fractions) had a stronger anticomplementary activity (IC50 13 µg/ml vs. 43 µg/ml), the FRSP (native and fractions) a stronger C1-INH potentiating effect (30 % vs. 16 %), although there were of course partly marked activity variations between the individual SP (Figure 4). In contrast, the elastase inhibiting

activity

turned

out

to

be

independent

on

the

glycan

structure

(average IC50 2.9 ± 2.5 µg/ml SXG vs. 2.1 ± 1.2 µg/ml FRSP). The different slopes of the two trend lines (Figure 3) suggest that the elastase inhibition by FRSP may be more robust toward a strong Mw reduction, but detailed analysis of the fractions with Mw < 15 kDa revealed that this is mainly due to DS differences, which so underlines the dependence of the elastase inhibition on the DS as already found for the native SP (Table 4). In contrast to its distinct influence on the three activities, the glycan structure turned out to have no influence on their Mw-dependent decrease. The apparently smaller activity loss of the SXG fractions (Figure 5) is just due to its smaller average Mw reduction (Figure 6 left). In detail, six of the FRSP fractions, but only four of the SGX fractions had a Mw < 15 kDa, which was associated with considerably reduced activities.

29



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Figure 4. Mean of the activities of the SXG and FRSP (native and fractions), i.e. elastase inhibition IC50 (µg/ml) on the left, complement inhibition IC50 (µg/ml) in the middle, and C1-INH potentiation (%) on the right. The corresponding range is shown in brackets.

Figure 5. Decreases of the pharmacological activities (%) of the SXG fractions (on the left) and FRSP fractions (on the right) in relation to the respective native SP. The activity decreases represent the average decrease of the three fractions each (for SD see Table 4). In addition, the degradation range and the respective average Mw decrease of the three fractions are indicated.

30


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3.4.3. Impact of the degradation method on the activities Due to the proven Mw dependence of the activities, the activity decrease logically correlated with the Mw decrease. Consequently, the most pronounced average activity decrease among the SXG was observed after HT degradation (66 %), among the FRSP after HP50 degradation (76 %) (Figure 6 right), whereby the partly marked differences between the three activities (Figure 7) as well as between the individual algae SP (Table 4) are here neglected.

Figure 6. Decreases of the Mw (on the left) and the pharmacological activities (on the right) of the SXG fractions and FRSP fractions in relation to the respective native SP in dependence on the degradation method (%). The activity decreases represent the average decrease of all SXG and FRSP fractions, respectively. The corresponding range is shown in brackets.

Remarkable are the SXG-HP20- and –HT-fractions with Mw reduced by 31 % and 26 % on average (Figure 6 left), respectively, but without any activity loss (-3.4 %) and even slightly improved activities (-15 %) (Figure 6 right). Similarly, the FRSP-HP20 fractions with Mw reduced by 48 % on average (Figure 6 left) exhibited activities only reduced by 30 % (Figure 6 right). Independent of the effect on each individual SP, the treatment of algae SP with 3 % hydrogen peroxide for 4 h at RT turned out as a procedure only moderately degrading, but rather purifying and “optimizing” the algae SP concerning their activities (Figure 8). The elimination of co-extracted impurities was already previously proven28 and the presented structural data suggest that preferentially lower sulfated polysaccharides or parts of the molecules, respectively, are degraded and eliminated. Depending on the MW of the native algae SP, its 31



degradability and the intended application, HP20 can be the preferred method. The F.v.-SP fractions represent a good example with F.v.-SP-HP20 having only a slightly higher Mw (12.9 kDa) than F.v.-SP-HT (10.3 kDa) and F.v.-SP-HP50 (9.12 kDa), but being much more active (Table 4). HP20 is particularly suitable as “optimizing” treatment for algae SP to be used as biomaterials for technological purposes such as nanoparticles and other carrier devices for drug substances, since high Mw is rather favorable here.

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Figure 7. Decreases of the pharmacological activities (%) of the SXG fractions (on the left) and FRSP (on the right) fractions in relation to the respective native SP in dependence on the degradation method. The activity decreases represent the average decrease of all SXG and FRSP fractions, respectively. The corresponding range is shown in brackets.

Figure 8. Mean of the activities of the SXG and FRSP fractions in dependence on the degradation method, i.e. elastase inhibition IC50 (µg/ml) on the left, complement inhibition IC50 (µg/ml) in the middle, and C1-INH potentiation (%) on the right. The corresponding range is shown in brackets.

33



Page 34 of 50

Table 4 Activities of the degraded SXG and fucoidan fractions in relation to the native SP: inhibition of PMN-elastase activity (IC50), anticomplementary activity measured by inhibition of the complement-mediated hemolysis (IC50), and potentiation of the C1s inhibition by C1-INH (%).The individual values represent the mean ± SD (n ≥ 3). The calculated mean of each SP (native and fractions) and the corresponding minimum and maximum values are marked bold.

DS

Mw (kDa)

Elastase inhibition IC50 (µg/ml)

Activity decrease (%)

Complement inhibition IC50 (µg/ml)


C1-INH potentiation (%)


SXG D.s.-SP D.s.-SP-HT D.s.-SP-HP20 D.s.-SP-HP50 Mean C.t.-SP C.t.-SP-HT C.t.-SP-HP20 C.t.-SP-HP50 Mean P.r.-SP P.r.-SP-HT P.r.-SP-HP20 P.r.-SP-HP50 Mean P.p.-SP P.p.-SP-HT P.p.-SP-HP20 P.p.-SP-HP50 Mean

0.65 0.64 0.75 0.66 0.67 0.52 0.61 0.58 0.51 0.55 0.68 0.77 0.85 0.75 0.76 0.53 0.68 0.67 0.53 0.60

213.9 157.4 163.5 13.3 137.0 127.6 73.2 73.0 3.4 69.3 105.3 82.1 68.9 5.0 65.3 100.4 87.7 79.2 12.0 69.8

0.21 ± 0.03 0.18 ± 0.02 0.21 ± 0.02 0.54 ± 0.04 0.29 0.51 ± 0.07 0.48 ± 0.07 0.52 ± 0.03 26.07 ± 1.95 6.90 0.25 ± 0.06 0.18 ± 0.01 0.23 ± 0.07 6.74 ± 0.78 1.85 0.50 ± 0.09 0.34 ± 0.07 0.50 ± 0.03 8.56 ± 0.92 2.47

-18.9 -1.6 60.8 13.4 -5.9 1.8 98 31.3 -39.0 -7.5 96.3 16.6 -45.5 1.2 94.2 16.6

1.11 ± 0.27 1.08 ± 0.20 1.02 ± 0.03 37.12 ± 10.83 10.08 2.60 ± 0.33 3.80 ± 0.51 3.05 ± 0.92 46.26 ± 6.08 13.93 1.83 ± 0.07 1.50 ± 0.16 2.33 ± 0.21 43.01 ± 1.85 12.17 2.56 ± 0.35 1.77 ± 0.15 1.70 ± 0.26 52.52 ± 3.56 14.64

-2.2 -9.1 97.0 28.6 31.5 14.6 94.4 46.8 -21.9 21.6 95.8 31.8 -44.1 -50.7 95.1 0.1

23.28 ± 3.45 25.87 ± 3.15 25.49 ± 2.30 19.73 ± 4.87 23.59 14.26 ± 1.94 14.54 ± 1.71 15.84 ± 1.56 6.49 ± 2.18 12.78 14.16 ± 1.75 15.44 ± 2.56 14.12 ± 5.51 10.93 ± 2.75 13.66 17.58 ± 2.08 18.80 ± 0.62 16.05 ± 6.01 3.28 ± 3.56 13.93

-11.1 -9.5 15.3 -1.8 -2.0 -11.1 54.5 13.8 -9.1 0.3 22.8 4.7 -6.9 8.7 81.4 27.7

FRSP F.v.-SP F.v.-SP-HT F.v.-SP-HP20 F.v.-SP-HP50

0.59 0.61 0.63 0.64

38.2 10.3 12.9 9.1

0.48 ± 0.08 4.27 ± 0.23 1.09 ± 0.06 5.03 ± 0.52

88.7 55.7 90.4

88.4 31.0 84.2

26.42 ± 3.06 11.09 ± 3.38 24.32 ± 3.57 16.94 ± 4.68

58.0 7.9 35.9

Mean S.l.-SP S.l.-SP-HT S.l.-SP-HP20 S.l.-SP-HP50 Mean M.p.-SP M.p.-SP-HT M.p.-SP-HP20 M.p.-SP-HP50 Mean U.p.-SP U.p.-SP-HT U.p.-SP-HP20 U.p.-SP-HP50

0.63 0.34 0.38 0.42 0.32 0.37 0.56 0.58 0.67 0.63 0.63 0.70 0.75 0.83 0.71

17.6 480.7 128.1 204.5 138.3 237.9 77.3 4.9 35.7 8.3 31.5 126.6 1.6 108.8 54.4

3.46 0.61 ± 0.07 0.54 ± 0.07 0.73 ± 0.10 0.80 ± 0.09 0.69 0.42 ± 0.06 8.19 ± 0.04 0.38 ± 0.05 0.71 ± 0.05 3.09 0.26 ± 0.05 3.20 ± 0.90 0.29 ± 0.01 0.38 ± 0.07

78.3 -12.7 7.3 14.5 3.0 94.8 -11.4 40.2 41.2 91.9 9.9 31.9

18.28 ± 3.94 106.41 ± 3.47 26.49 ± 2.33 115.61 ± 43.33 66.70 6.80 ± 0.55 6.97 ± 0.88 5.73 ± 0.95 7.22 ± 0.81 6.68 3.74 ± 0.57 140.73 ± 2.28 7.56 ± 0.66 10.84 ± 2.27 40.72 2.76 ± 0.33 225.99 ± 3.06 3.26 ± 0.93 4.88 ± 0.84

67.9 2.4 -13.7 9.7 -0.6 97.3 50.5 65.4 71.1 98.8 15.4 43.5

19.69 50.42 ± 3.34 42.27 ± 4.13 44.58 ± 2.19 42.24 ± 3.05 44.88 47.10 ± 3.72 6.62 ± 1.92 49.88 ± 2.36 42.02 ± 4.17 36.41 23.14 ± 3.05 5.08 ± 1.62 24.67 ± 6.12 23.01 ± 4.02

34.0 16.2 11.6 16.2 14.7 85.9 -5.9 10.8 30.3 78.0 -6.6 0.5

34


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Mean

0.76

72.8

1.29

44.6

59.22

52.6

18.97

24.0

3.5. Determination of the most suitable low-molecular mass fractions A goal of this study was to identify algae SP, which are degradable to a reasonable size but still exhibit pronounced activities. For this, SXG and FRSP fractions with MW < 15 kDa were considered, although all these small fractions displayed considerably reduced activities compared to the native SP. This MW limit was chosen, since SP with lower size have acceptable biopharmaceutical properties and since unfractionated heparin (UFH), still an indispensable anticoagulant drug and the best investigated SP, has an average Mw of 15 kDa. The activities of the fractions were compared with those of both UFH and the low-molecular weight heparin (LMWH) enoxaparin (Mw 4.5 kDa), the most widely used parenteral anticoagulant. The heparins were used as reference compounds, as they are known to exhibit a wide range of biological activities including pronounced anti-inflammatory effects.27 However, the strong anticoagulant effects of heparins and their bleeding risk make it impossible to clinically utilize these activities.60 Therefore, pharmacologically active sulfated glycans without relevant anticoagulant effects comparable to heparins are desirable. This was already shown for both SXG20 and FRSP.33,40 For the comparison with heparins, all four SXG fractions and four of the six FRSP fractions with Mw < 15 kDa were selected (Figure 9). M.p.-SP-HT (Mw 4.91 kDa) and F.v.-SPHT (Mw 10.3 kDa) were excluded as they were less active than M.p.-SP-HP50 (Mw 8.25 kDa) and F.v.-SP-HP20 (Mw 12.9 kDa) as well as F.v.-SP-HP50 (Mw 9.12 kDa), respectively. The Mw of the fractions ranged between 1.55 kDa (U.p.-SP-HT) and 13.3 kDa (D.s.-SP-HP50) and included thus two fractions even smaller than LMWH. The DS of the fractions, which ranged

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between 0.51 (C.t.-SP-HP50) and 0.75 (U. p.-SP-HT and P.r.-SP-HP50), were much lower than the DS of 1.0 of UFH and LMWH.

Figure 9. Activities for degraded fractions with a Mw < 15 kDa, UFH and LMWH. Illustrated is the IC50 (µg/ml) for the inhibition of PMN-elastase and inhibition of complement-mediated hemolysis (smaller is better). The potentiation of C1-INH is illustrated in % (higher is better). Mean ± SD (n ≥ 3)

The sorting of the SP by their Mw (Figure 9) as well as by their DS (not shown) indicate that neither the Mw nor the DS are sufficient to explain the individual activities. Further, the ranking of the SP according to their potency turned out to be different for the three activities. For elastase inhibition, the IC50 range of the fractions amounted to 0.54 - 26 µg/ml, i.e. UFH was most active (IC50 0.29 ± 0.04 µg/ml), but D.s.-SP-HP50 (DS 0.75, 13.3 kDa, IC50 0.54 µg/ml), M.p.-SP-HP50 (DS 0.63, 8.25 kDa, IC50 0.71 µg/ml) and F.v.-SP-HP20 (DS 0.63, 12.9 kDa, IC50 1.09 µg/ml) were more active than LMWH (IC50 1.55 ± 0.08 µg/ml). Concerning complement inhibition, the IC50 of the fractions ranged from 10.8 µg/ml to 226 µg/ml. Here, M.p.-SP-HP50 (DS 0.63, 8.25 kDa, IC50 10.8 µg/ml) was superior to both UFH (IC50 15.0 ± 3.3 µg/ml) and LMWH (IC50 68 ± 16 µg/ml) and, notably, only F.v.-SP-HP50 (DS 0.64, 9.12 kDa) and the oligosaccharide fraction U.p.-SP-HT (DS 0.75, 1.55 kDa) were less active than LMWH. Regarding the C1-INH potentiation, the fractions improved the inhibition of C1-INH by 3.3 % up to 42 %, whereby M.p.-SP-HP50 (DS 0.63, 8.25 kDa, 42 %) was again at least as active as 36


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UFH (38.5 ± 5.2 %) and more active than LMWH (24.5 ± 3.2 %), and F.v.-SP-HP20 (DS 0.63, 12.9 kDa, 24.3 ± 3.6 %) was as active as LMWH. Worth mentioning is the oligosaccharide fraction U.p.-SP-HT (DS 0.75, 1.55 kDa) with its pronounced elastase inhibitory activity (IC50 3.2 µg/ml) representing the third best one among the small fractions, whereas its two other activities were only weak. In summary, despite their lower DS, several algae SP fractions with Mw similar to heparins turned out to be superior to heparins concerning the exemplarily tested activities, whereby they exhibit only marginal anticoagulants effects. The FRSP fraction M.p.-SP-HP50 (DS 0.63, 8.25 kDa) emerged as number one among all the tested SP, followed by F.v.-SP-HP20 (DS 0.63, 12.9 kDa) and the SXG fraction D.s.-SP-HP50 (DS 0.66, 13.3 kDa). These three fractions were obtained by treatment with hydrogen peroxide and are thereby additionally purified from co-extracted impurities. They are worth to be further investigated both chemically and pharmacologically. That should include IEC fractionation by ion exchange chromatography to determine the heterogeneity of composition and to identify and isolate the most active fractions. The latter should be subjected to detailed structure elucidation (branching, sulfation pattern, other substituents, etc.) using NMR methods and GC-MS analysis. In this way, more precise structure-activity relationships will be obtained. They are worth to be further investigated including fractionation to identify the most active fractions and testing of further activities.

37



4.

Conclusions As native algae SP usually have high Mw, which is associated with unfavorable

biopharmaceutical properties and possibly undesired effects, degradation may represent a strategy to make algae SP more suitable for medical applications. In this study, we degraded four sulfated xylogalactans (SXG) and four fucoidans (FRSP) extracted from red and brown algae, respectively, using three simple methods, which are applicable for the production of SP fractions in large scale: hydrothermal treatment for 90 min at 120 °C (HT) and degradation with 3 % H2O2 for 4 hours at 20 °C (HP20) and 50 °C (HP50). The resulting fractions were chemically characterized by means of some basic structural parameters and tested for three exemplary activities to elucidate the effects of the degradation on other structural parameters and their pharmacological profile. The results confirmed the hypothesis that the achieved degradation by a specific method is dependent on the basic glycan structure of the SP. The SXG showed to be quite resistant against HT and HP20, their Mw was, however, strongly reduced by HP50. In contrast, FRSP turned out to be degradable by all three methods, whereby HP20 was more or less inferior to both HT and HP50. The degradation processes was not associated with significant desulfation. Instead, in most cases, the DS of the fractions increased by up to 29 %, which was most marked after HP20. The DS increase and further analyses led to the assumption that HP20, the mildest degradation method, preferentially cleaves polysaccharide chains at sensitive sites such as side chains, fringe regions and low-sulfated domains or lower-sulfated fractions in case of heterogeneously composed algae SP, whereas HP50, the most powerful method, leads rather to random degradation. As treatment with hydrogen peroxide had already been shown to

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additionally eliminate co-extracted impurities,28 HP20 can also be considered as a procedure to purify and “optimize” algae SP, especially when it is intended to use them as biomaterials. The pharmacological activities of the SP fractions generally decreased with decreasing Mw. Notable is that less strongly degraded fractions with increased DS showed partly even increased activities by up to 50 %, which underlines the importance of the DS for the activities of SP. The extent of the activity reduction, however, differed between the three activities so that degradation led to a modified pharmacological profile. The complement inhibition showed the strongest decline with decreasing Mw (27 % for SXG and 47 % for FRSP) and the C1-INH potentiation the weakest one (11 % for SXG and 26 % for FRSP); the elastase inhibition was reduced by 20 % for SXG and 43 % for FRSP, but was quite stable down to a Mw of 35.7 kDa. In spite of this Mw-dependent shift of the activity profile, the activities of the degraded SXG and FRSP fractions showed the same dependence on their basic glycan structure as the native SP. The SXG (native and fractions) exhibited stronger anticomplementary activity (IC50 13 µg/ml vs. 47 µg/ml), whereas the FRSP (native and fractions) displayed a stronger C1-INH potentiating effect (30 % vs. 16 %). In contrast, the elastase inhibiting activity turned out to be independent on the glycan structure (IC50 2.8 µg/ml SXG vs. 2.1 µg/ml FRSP), but strongly dependent on the DS. One of the aims of this study was to identify algae SP, which are degradable to a reasonable size but still exhibit pronounced activities. For this, UFH (Mw 15.0 kDa) and a LMWH (Mw 4.50 kDa), widely used anticoagulants and well-known for their additional pronounced anti-inflammatory activity, served as reference compounds. Despite their much lower DS, some of the SXG and FRSP fractions with Mw < 15 kDa turned out to exhibit similar or even stronger activities than the heparins, whereas they have only weak anticoagulant effects. The

39



FRSP fraction M.p.-SP-HP50 (DS 0.63, 8.25 kDa) emerged as number one among all the tested SP, followed by F.v.-SP-HP20 (DS 0.63, 12.9 kDa) and the SXG fraction D.s.-SP-HP50 (DS 0.75, 13.3 kDa, IC

50

37 µg/ml). All three fractions were obtained by treatment with hydrogen

peroxide and are thus additionally purified from co-extracted impurities. Overall, the treatment with hydrogen peroxide at room temperature or at 50 °C, respectively, proved to be a simple method for the degradation of algae SP having the additional advantage to improve their quality as well as to represent an antimicrobial treatment.

5.

Acknowledgements We thank Juliane Grimm for the extraction of the sulfated xylogalactans.

6.

Supporting Information

Table S1: Monosaccharide composition, content of proteins, and uronic acids for native SXG and FRSP and their degraded fractions

7.

Funding This research was not supported by any specific grant from funding agencies in the

public, commercial, or not-for-profit sectors.

8.

Declaration of interest All authors declare that they have no actual or potential conflict of interest.

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9.

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For Table of Contents Use Only Title: Degradation of eight sulfated polysaccharides extracted from red and brown algae and its impact on structure and pharmacological activities Authors: Eric Lahrsen, Ann-Kathrin Schoenfeld, Susanne Alban

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Degradation of Eight Sulfated Polysaccharides Extracted from Red

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