Structural characteristics of carrageenans of red alga Mastocarpus pacificus from sea of Japan

Carbohydrate Polymers xxx (xxxx) xxxx

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Structural characteristics of carrageenans of red alga Mastocarpus pacificus from sea of Japan Anna O. Kravchenko*, Stanislav D. Anastyuk, Valery P. Glazunov, Ekaterina V. Sokolova, Vladimir V. Isakov, Irina M. Yermak G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100 Let Vladivostoku Prosp., 159, 690022, Vladivostok, Russian Federation

A R T I C LE I N FO

A B S T R A C T

Keywords: Carrageenan Kappa/iota-hybrid Carrageenan oligosaccharide 18 O-labeled MALDI-TOFMS ESIMS/MS

The sulfated polysaccharide from sterile alga Mastocarpus pacificus was investigated. Partial reductive hydrolysis and NMR spectroscopy showed that the extracted polysaccharides were only carrageenans. According to FT-IRand NMR spectroscopy this polysaccharide was a hybrid kappa/iota-carrageenan with a predominance of kappatype units. According to MALDI-TOFMS, oligosaccharide fragments obtained by mild acid hydrolysis had a polymerization degree of 1–9, while chains built up of galactose residues were up to 3. Tandem ESI mass spectrometry together with innovative 18O-labelling method showed that the polymer chain of the carrageenan included kappa-carrabiose, kappa-carratetraose, iota-carrabiose, hybrid kappa/iota oligosaccharide units and contained minor insertions of mu-carrageenan (the precursor of kappa-carrageenan). Parallel artificial membrane permeability assay shown that the studied carrageenan inhibited bile salts permeation through an artificial membrane imitating the gastrointestinal barrier by 50 % on average compared to negative control independent of incubation time. However, its action was less pronounced than the hindering ability of cholestyramine.

1. Introduction Carrageenans are water-soluble sulfated polysaccharides from red algae, consisting of alternating residues of 1,3-linked β-D-galactose (Gunits) and 1,4-linked α-D-galactose (D-units), which may be partially or completely in the form of 3,6-anhydro-derivative (DA-units). The hydroxyl groups of these monosaccharides can be sulfated, methylated, substituted by pyruvic acid residues or D-xylose (Craigie, 1990; Usov, 2011). Carrageenans differ from each other by the presence or absence of 3,6-anhydrogalactose in a 1,4-linked residue, as well as by the number and location of sulfate groups. The three commercially most important carrageenans are called kappa-, iota- and lambda-carrageenan, and the corresponding IUPAC-inspired names and letter codes are carrageenose 4′-sulfate (G4S-DA), carrageenose 2,4′-disulfate (G4SDA2S) and carrageenose 2,6,2′-trisulfate (G2S-D2S,6S). In addition to these three major carrageenan types, two other types, called mu- and nu-carrageenan (letter code G4S-D6S and G4S-D2S,6S, respectively), are often encountered in carrageenan samples and are the biological precursors of, respectively, kappa- and iota-carrageenans. Since natural carrageenan is a mixture of nonhomologous polysaccharides, the term disaccharide repeating unit refers to the idealised structure (Knutsen,

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Myslabodski, Larsen, & Usov, 1994; Rees, 1963; Van de Velde, Pereira, & Rollema, 2004; Yermak & Khotimchenko, 2003). Kappa- and iotacarrageenans are the main gel-forming polysaccharide fractions at low concentrations of some metal ion salts (KCl, CaCl2). These polysaccharides contain 1,4-linked 3,6-anhydro-α-D-galactose units which have 1C4 conformation that allows a helicoidal secondary structure, which is essential for the gel-forming properties. In contrast, lambdacarrageenan contains 1,4-linked α-D-galactose 2,6-disulfate which has 4 C1 conformation, so this polysaccharide is non-gelling at low salt concentrations (Campo, Kawano, Silva, & Carvalho, 2009; Chapman & Chapman, 1980; Piculell, 1995). Natural polysaccharides rarely correspond to regular structures containing one type of carrageenan. As a rule, they contain repetitive disaccharide units of several carrageenan types enclosed in a single polymer chain, forming so-called “hybrid” structures with a predominance of one or another type (Craigie, 1990; Knutsen et al., 1994). It has been established that the cystocarpic alga Stenogramme interrupta produces gelling iota/alpha-carrageenan (Cáceres, Carlucci, Damonte, Matsuhiro, & Zuñiga, 2000), the Far Eastern alga Tichocarpus crinitus – kappa/beta-carrageenan (Barabanova et al., 2005), and the Far Eastern cystocarpic alga Ahnfeltiopsis flabelliformis – iota/kappa-carrageenan (Kravchenko et al.,

Corresponding author. E-mail address: [email protected] (A.O. Kravchenko).

https://doi.org/10.1016/j.carbpol.2019.115518 Received 27 August 2019; Received in revised form 11 October 2019; Accepted 21 October 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Anna O. Kravchenko, et al., Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2019.115518

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2.2.1. Extraction of polysaccharides with water The algal residue was re-suspended in distilled water in the ratio of 1:60 (w/v), and the polysaccharides were extracted three times at 80 °C for 3 h in a boiling water bath with constant stirring. Three hot extracts were combined, centrifuged at 4000 rpm−1 to remove cell wall residues, filtered through a Vivaflow200 membrane (Sartorius, Germany) with a pore size of 100 kDa, concentrated on a rotary evaporator and the polysaccharides were precipitated with a triple volume of 96 % ethanol. The precipitate was centrifuged at 4000 rpm−1, dissolved in distilled water, concentrated on a rotary evaporator, and lyophilized. The polysaccharide was labelled as Mpw.

2016). Due to their unique physico-chemical properties, carrageenans are widely used in the food industry as thickeners, stabilizers and emulsifiers (McHugh, 2003), in biotechnology and in cosmetology in the manufacture of lotions, creams and shampoos (Cosenza, Navarro, Ponce, & Stortz, 2017). These polysaccharides have a broad spectrum of biological activity: antiviral (Buck et al., 2006; Talarico et al., 2004; Wang, Wang, & Guan, 2012), antitumour (Yuan, Song, Li, Li, & Liu, 2011; Zhou et al., 2005), immunomodulating (Campo et al., 2009; Yermak & Khotimchenko, 2003), antioxidant (Sokolova et al., 2011; Sun, Wang, Shi, & Ma, 2009) and anti-coagulant (Costa et al., 2010; Silva et al., 2010; Wijesekara, Pangestuti, & Kim, 2011). In addition, carrageenans are classed as dietary fibre and are able to reduce the reabsorption of bile salts in the small intestine, resulting in a decrease in cholesterol in the organism, which in turn reduces the risk of cardiovascular disease, currently one of the main causes of death in the world (Gunness & Gidley, 2010; Lahaye & Kaeffer, 1997). Physico-chemical and biological properties of the polysaccharides are in close relationship with their varied and complex chemical structure, which depends on a number of factors, one of which is the macrophyte species (Craigie, 1990; Yermak & Khotimchenko, 2003). Since new biomedical technologies are being developed using renewable and environmentally friendly biopolymers, the investigation of the structure of sulfated polysaccharides from previously unexplored algae and the establishment of the relationship between the structure of the polymer and its physico-chemical and biological properties are currently relevant. Algae of the genus Mastocarpus (family Phyllophoraceae) belong to the carrageenophytes. Currently, studies on the structure and properties of polysaccharides produced by algae of this genus are few and are limited to several scientific groups from Spain and Portugal (GomezOrdonez, Jimenez-Escrig, & Ruperez, 2014; Hillou et al., 2006). It has been established that Mastocarpus stellatus produces a hybrid kappa/ iota-carrageenan (Hillou et al., 2006). The alga Mastocarpus pacificus (Phyllophoraceae) grows in the Far Eastern Russian seas along the entire coast (Belous, Titlyanova, & Titlyanov, 2013). However, there is currently no information on the structure of the polysaccharide produced by the alga. At the same time, M. pacificus may be a new potential source of sulfated galactans, which makes study of the structure and properties of the polysaccharides from this alga relevant. This work was aimed at investigating the structural characteristics of the polysaccharide from the sterile form of the M. pacificus and its ability to affect permeability of bile salts through an artificial membrane imitating the gastrointestinal barrier.

2.2.2. Extraction of polysaccharides with alkali The algal residue was re-suspended in 0.6 % NaHCO3 (рH = 8) in the ratio of 1:60 (w/v), and the polysaccharides were extracted three times at 80 °C for 3 h in a boiling water bath with constant stirring. Three hot alkaline extracts were combined, centrifuged at 4000 rpm−1 to remove cell wall residues, filtered through a Vivaflow200 membrane (Sartorius, Germany) with a pore size of 100 kDa until the universal indicator shown pH = 5.5–6.0, concentrated on a rotary evaporator and the polysaccharides were precipitated with a triple volume of 96 % ethanol. The precipitate was centrifuged at 4000 rpm−1, dissolved in distilled water, concentrated on a rotary evaporator, and lyophilized. The polysaccharide was labelled as Mpb. 2.3. Fractionation of polysaccharides The separation of Mpw polysaccharides into gelling and non-gelling fractions was performed using KCl in various concentrations. For this, 1, 2 and 4 % KCl solutions were added to the hot 1 % polysaccharide solution with stirring and left at 4 °C for 12 h. Then the obtained gels were centrifuged at 9000 rpm−1 at 4 °C for 30 min and the supernatant was separated from the precipitate. The supernatant was dialyzed against distilled water, and the precipitate was dialyzed against 0.9 % NaCl, and then against water. The obtained fractions were concentrated on a rotary evaporator and lyophilized. In addition, sequential fractionation of 1 % polysaccharide solution with 0.5 % KCl and then 2 % CaCl2 was carried out. The resulting gel was separated from the liquid phase by centrifugation, and the resulting precipitate and the supernatant were dialyzed and lyophilized as described above. 2.4. Analysis of the polysaccharide gel For the analysis, 0.5 and 1.1 % solutions of the Mpw polysaccharide were prepared by heating to 60 °C. 0.03 M KCl was added to the 0.5 % polysaccharide solution and 0.1 M KCl to the 1.1 % solution with stirring. The samples were left at room temperature for 2 days. The obtained gels were then centrifuged at 10,000g for 2 h and the presence of phase separation was assessed visually. Commercial kappa- and iotacarrageenans (Sigma-Aldrich) were used as standards. As in the case with the polymer under study, 0.5 and 1.1 % solutions of individual polysaccharides, as well as their mixtures in equal amounts were prepared, 0.03 and 0.1 M KCl solutions were added and the samples were kept for 2 days at room temperature. After centrifugation, the phase separation was assessed visually.

2. Experimental 2.1. Algal material The attached form of the red alga Mastocarpus pacificus was collected in Troitsa Bay (Sea of Japan) in July 2016 at a depth of 0.5 m. The identification of algae, as well as the determination of the morphological and anatomical characteristics of macrophytes, was carried out using an electron microscope in the chemotaxonomy laboratory of PIBOC FEB RAS by Dr. Oksana Belous according to Perestenko (1994). The collected algae, represented by sterile plants, were washed with running water to remove epiphytes and soluble salts and frozen in sealed plastic bags.

2.5. Analytical methods Total reductive hydrolysis of the polysaccharides with 2 M trifluoroacetic acid (TFA) (100 °C, 4 h) with 4-methyl-morpholine borane was carried out and then aldononitrile acetates were obtained to determine the content of 3,6-anhydrogylactose (Usov & Elashvili, 1991). Other monosaccharides (galactose, glucose, xylose) were determined as alditol acetates. To obtain alditol acetates of these monosaccharides, the polysaccharide (5 mg) was hydrolyzed with 2 M TFA containing myo-inositol as an internal standard (1 mg mL−1) at 100 °C for 4 h.

2.2. Extraction of polysaccharides Frozen algae (5–7 g) were crushed, added to distilled water in a ratio of 1:60 (w/v), and low-molecular weight substances were extracted at a temperature of 20 °C for 12 h. The resulting extract was filtered and discarded. 2

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concentrated on a rotary evaporator, and lyophilized. The polysaccharide was labelled as Mpat.

After cooling, the hydrolyzate was evaporated three times with 3 mL of 96 % ethanol to remove excess TFA. Then 1 mL of distilled water and a small amount of NaBH4 were added and solution was left overnight at room temperature in the dark. The sample was evaporated to dryness with 200 μL of concentrated acetic acid, and three times with 3 mL of methanol to remove excess NaBH4. 1 mL of pyridine and 1 mL of acetic anhydride were added to the resulting alditol mixture and solution was heated in a closed flask at 100 °C for 1 h. The mixture was cooled to room temperature and evaporated to dryness three times with 3 mL of toluene to remove pyridine. The obtained alditol acetates were extracted with chloroform and evaporated to dryness. The obtained alditol and aldononitrile acetates of monosaccharides were analyzed by gas-liquid chromatography on a 6850 chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5MS capillary column (30 m × 0.25 mm, 5 % Phenyl Methyl Siloxane) and a flameionization detector. The analyses were carried out using a temperature gradient program from 150 to 230 °C; the rate of temperature change was 3 °C min−1. The sulfate ester content of polysaccharides was determined by the turbidimetric method (Dodgson & Price, 1962). The protein content in the polysaccharide was determined by Lowry’s method (Lowry, Rosebrough, Farr, & Randall, 1951).

2.9. Mild acid hydrolysis Two portions of the Mpw polysaccharide (5 mg) were placed in a vial for hydrolysis and one was dissolved in 1 mL of a 0.1 N solution of HCl in H216O and the other in 1 mL of a 0.1 N solution of HCl in H218O. Then both samples were heated at 60 °C for 4 h. Hydrolysis was stopped by the addition of some drops of 0.5 M NH4HCO3 solution to pH 8-9. The obtained oligosaccharides were stored at 4 °C and analyzed by mass-spectrometry. 2.10. Molecular weight measurement The viscosimetric molecular weight of the polysaccharide sample was calculated using the Mark-Kun-Houwink equation: | η | = KmMα, where | η | is the intrinsic viscosity, Km and α are empirical constants for kappa-carrageenan with values 3 × 10−3 and 0.95, respectively, at 25 °C in 0.1 M NaCl, according to the literature data for this polymersolvent system (Rochas, Rinaudo, & Landry, 1990). The viscosity of the polysaccharide solution (1–2 mg mL-1 in 0.1 M NaCl) was measured with a modified Ubellode viscometer (Design Bureau Puschino, Russia) with a capillary diameter of 0.3 mm at 25 °C, the time of accuracy being within ± 0.1 s. The intrinsic viscosity of the carrageenan sample was calculated by extrapolation of the dependence ln (η)rel/C to infinite dilution using the least squares method.

2.6. Partial recovery hydrolysis A sample of Mpw polysaccharide (5 mg) and 40 mg of 4-methylmorpholine borane (Sigma-Aldrich, USA) were placed in a vial and hydrolyzed with 2 M TFA containing myo-inositol (1 mg mL−1) as an internal standard at 65 °C for 8 h. To remove excess TFA, the hydrolysate was evaporated three times with 3 mL of 96 % ethanol. Then 1 mL of a 6 % solution of hydroxylamine hydrochloride was added to the dry residue and the hydrolysate was heated at 100 °C for 1 h. After cooling, 0.5 mL of acetic anhydride was added and mixture was heated again at 100 °C for 1 h. After cooling, the mixture was re-evaporated three times with 3 mL of toluene. The obtained aldononitrile acetates were extracted with chloroform, evaporated to dryness and analyzed on a 6850 chromatograph (Agilent, Germany) equipped with an HP-5MS capillary column (30 m × 0.25 mm) with 5 % Phenyl Methyl Siloxane (Agilent, Germany), with a flame ionization detector, at a temperature of 175–290 °C (the rate of temperature change 10 °C min−1). Agarose (Sigma-Aldrich, USA) and kappa-carrageenan from Kappaphycus alvarezii (Sigma-Aldrich, USA) were used as standards for the production of aldononitrile acetates of agarobiose and carrabiose.

2.11. Fourier transform-infrared spectroscopy (FT-IR) IR spectra of the studied polysaccharides were recorded in films on a Vector 22 Fourier transform spectrophotometer Equinox 55 (Bruker, USA) taking 120 scans with 4 cm–1 resolution. For the sample preparation, compound (6 mg) was dissolved in H2O (1 mL) and heated at 37 °C on a polyethylene substrate until a dry film was produced. Then, the film was clamped between two NaCl plates and the IR spectra were recorded in the 4000-600 cm−1 region. The spectra were cut out in the region of 1900–700 cm−1, and the baseline was corrected for scattering. The spectra were normalized by the absorption of the monosaccharide ring skeleton at ∼1070 cm–1 (A1070 ≈ 1.0). The simulated spectrum was fitted to the experimental curve of the spectrum by the program Fitting curve. This spectrum was created by specifying the bans of individual component according to the number of observed maxima of the bands or shoulders in the experimental spectrum. If in some parts of the spectrum the coincidence of the simulated and experimental curves was unsatisfactory, in these areas the component was additionally specified. The simulated contour was fitted by varying the parameters of the individual components: purity of the maximum, intensity, half-width and shape (Lorentz, Gauss). The convergence between the experimental and simulated curves was better than RMS 0.004. The areas of the individual components were used to analyze the carrabiose units.

2.7. Desulfation of polysaccharides A sample of the polysaccharide (100 mg) was dissolved in 20 mL of distilled water and passed through a Q-2 column (1 × 5 cm) in the H+form. The eluate was collected into a flask with 0.5 mL of anhydrous pyridine (Sigma-Aldrich, USA). The column was further washed with 20 mL of distilled water and the resulting solution was evaporated to dryness. Then 18 mL of DMSO (absolute, anhydrous) (Panreac, Spain) and 2 mL of methanol (Vekton, Russia) were added to the residue. The solution was stirred until the polysaccharide was completely dissolved, after which it was transferred to a vial for hydrolysis and heated at 100 °C for 3 h. After cooling to room temperature, the sample was diluted with distilled water three times and dialyzed against distilled water. The dialyzed sample was concentrated on a rotary evaporator and lyophilized.

2.12. NMR spectroscopy Polysaccharide (3 mg) and its desulfated derivative (5 mg) were deuterium-exchanged twice in D2O (0.6 mL) by freeze-drying prior to being examined in a solution of 99.95 % D2O. One-dimensional 1H, 13C NMR and two-dimensional 1H-1H COSY and 1H-13C HSQC spectra of the samples were recorded with DRX-500 (125.75 MHz) spectrometer (Bruker, Germany) operating at 50 °C. Chemical shifts were described relative to acetone as internal standard (δC 31.45, δH 2.25). XWINNMR 1.2 software (Bruker, Germany) was used to acquire and process the NMR data.

2.8. Alkaline treatment of polysaccharide Mpw The isolated polysaccharide (100 mg) was dissolved in 20 mL of distilled water, 20 mg of NaBH4 was added and solution was kept at 25 °C for 12 h. Then, 60 mg of NaBH4 and 800 mg of NaOH were added and mixture was heated at 80 °C for 6 h. After cooling, it was neutralized with acetic acid to pH 5.5, dialyzed against distilled water, 3

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Table 1 Yield and chemical analysis of polysaccharides (PS) from M. pacificus. Sample

Mpw Mpb Mpat

PS yield, % of dried algal weight

31.5 47.3 –

Content, % of PS sample weight

Molar ratio AnGal:Gal:SO3Na

Gal

AnGal

Xyl

Glc

SO3Na

36.1 35.4 32.1

22.8 22.2 24.9

0.8 0.7 1.2

0.2 0.5 0.8

26.3 27.0 27.4

1.0:1.4:1.6 1.0:1.4:1.7 1.0:1.1:1.5

Note: Mpw – polysaccharide by water extraction, Mpb – polysaccharide by 0.6 % NaHCO3 extraction, Mpat – Mpw polysaccharide after 1 M NaOH treatment.

2.13. Mass-spectrometry

3. Results and discussion

Matrix-assisted laser desorption/ionization mass spectra (MALDIMS) were recorded with an Ultra Flex III MALDI-TOF/TOF mass spectrometer (Bruker, Germany) with a nitrogen laser (SmartBeam, 355 nm), reflector and potential LIFT tandem modes of operation. Instrument settings for negative-ion mode: accelerating voltage, 25 kV; laser power, ∼20–30 μJ; number of shots, 250; laser shot rate, 66 Hz. Sample preparation: aqueous solution of sample (1 μL) (5 mg mL−1) and 1 M matrix solution (1 μL, ethanol:H2O, 1:1) were mixed, introduced on a stainless steel target (1 μL) and air dried. Beta-carboline (non-harmane) was used as matrix. Electrospray ionization mass spectra (ESIMS) were recorded with a Maxis Impact LC Q-TOF mass spectrometer (Bruker, Germany). All spectra were acquired in the negative-ion mode, pre-calibrated with a standard ‘HP-mix’ (Agilent, USA) for negative-ion mode at default instrument settings. The samples were diluted with acetonitrile:H2O (1:1) to approx. 0.01 mg mL−1 and introduced into the mass spectrometer at flow rate of 5 μL min−1 using a syringe pump (KD Scientific, USA).

3.1. Analysis of the polysaccharide composition after water and alkaline extractions It is known that the biosynthesis of polysaccharides in the cell wall of red algae is a multistage process. In the first stage, it is believed that the main chain, consisting of galactose residues, is formed. In the second stage, there are the sulfation of galactose residues and the introduction of other substituents. In the final stage, the sulfate at C-6 of 1,4-linked α-galactose is enzymatically eliminated and a 3,6-anhydrous cycle is formed (Craigie & Wong, 1979). Biosynthesis can be completed at any stage; therefore, the final product can be a polysaccharide with both regular and irregular structure, which contains both gelling polysaccharides kappa, iota, theta, and their biosynthetic precursors mu, nu, lambda, respectively, in the polymer chain (Chapman & Chapman, 1980). It is known that the sulfate at C-6 of 1,4-linked αgalactose is able to eliminate under alkaline conditions, and a cycle closes between the hydroxyl groups at C-3 and C-6 to form 3,6-anhydrogalactose. This feature is often used to improve the gel-forming properties of carrageenans, even at the stage of polysaccharide extraction (Campo et al., 2009). In this regard, the polysaccharides were extracted with water (Mpw) and 0.6 % NaHCO3 solution (Mpb) from the sterile form of alga Mastocarpus pacificus collected in Troitsa Bay (Sea of Japan) in July 2016. Polysaccharide yields and their composition are presented in Table 1. Extraction of polysaccharides with alkali led to a 1.5-fold increase in their yields compared to water extraction. This could be due to a better degradation of the algal cell wall under alkaline condition. This was also observed for the algae Chondrus crispus and Ahnfeltiopsis devoniensis (Azevedo, Torres, Sousa-Pinto, & Hilliou, 2015). Analysis of the monosaccharide composition showed that the major monosaccharides of the isolated polysaccharides were galactose and 3,6-anhydrogalactose, the contents of which in both samples were 35.4–36.1 and 22.2–22.8 %, respectively (Table 1). The content of sulfate groups was also identical. In addition, trace amounts of xylose and glucose were present in the polysaccharides (0.7–0.8 % and 0.2–0.5 %, respectively). Glucose is typically the structural unit of floridean starch which is α-(1→4)-D-glucan with branches at C-6 (Meeuse, Andries, & Wood, 1960). Xylose can be either a structural unit of the neutral polysaccharide – xylan, which is present in small amounts in all red algae, or it is a single monosaccharide residue replacing one of hydroxyl groups of the galactan (Craigie, 1990; Usov, 2011). The AnGal:Gal molar ratio (1.0:1.4) indicated that some of the α-galactose units of these polymers were in the non-cyclized form. The molecular weight of the Mpw polysaccharide measured by the viscometric method was 700 kDa. The protein content in the polysaccharide determined by Lowry’s method was 2.69 %. According to the partial reductive hydrolysis, polysaccharide from M. pacificus contained only disaccharide units of 4-O-β-D-galactopyranosyl-3,6-anhydro-D-galactose (carrabiose) without any disaccharide units of 4-O-βD-galactopyranosyl-3,6-anhydro-L-galactose (agarobiose) which allowed it to be classified as a carrageenan. FT-IR spectroscopy was used to acquire preliminary data on the structure of the polysaccharide from M. pacificus. The spectra obtained

2.14. Parallel artificial membrane permeability assay (PAMPA) Bile salts, U.S. Pharmacopeia Reference Standard (cat. No. 1071304), composed of sodium taurocholate 46.87 %, sodium glycocholate 30.82 %, sodium taurodeoxycholate 9.45 %, sodium glycodeoxycholate 5.95 %, sodium taurochenodeoxycholate 2.37 %, sodium glycochenodeoxycholate 1.67 %, sodium cholate 0.08 %, and other components below reportable levels 2.79 % was purchased from U.S. Pharmacopeia, Rockville, MD, USA. The MultiScreen-IP PAMPA filter plate and Transport Plates Multiscreen plate were from Millipore Corporation, Tullagreen, Carrigtwohill, County Cork, Ireland. The PAMPA experiment was performed according to the method described earlier (Bujard, Voirol, Carrupt, & Schappler, 2015; Kansy, Senner, & Gubernator, 1998). The assay was performed at a gradient pH 5.0 acetic buffer (donor) – 7.4 phosphate buffer saline with bovine serum albumin (100 μM) (acceptor) in a 96-well MultiScreen-IP PAMPA filter plate and Transport Plates Multiscreen plate. A filter plate was impregnated with 15 μL of a lipidic mixture of lecithin (0.333 g per 100 mL) and cholesterol (0.267 g per 100 mL) in hexane/hexadecane (95:5, v/v). After evaporation of all of the hexane, the filter plate was filled with 150 μL of 0.048 M acetic buffer (pH 5.0) containing the polysaccharides (0.75, 1.5 and 3 mg mL−1) and physiologic conjugated bile mixture (2 mM). The donor plate was then placed into the acceptor plate. The plate was covered and incubated at room temperature under shaking 100 rpm and permeation was evaluated after 60 and 150 min. The content of bile acids permeated into the acceptor compartment was analyzed by the Trinity Biotech bile acids kit (Product No. 450-A, Trinity Biotech plc, Brau, Co Wicklow, Ireland). Each sample was analyzed in triplicate.

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3.2. Analysis of the polysaccharide modified with alkali In addition to alkaline extraction, it is known another widely used method in the structural chemistry of red algal polysaccharides to transform the biosynthetic precursors of gelling carrageenans and to increase their gel-forming properties. This method involves the treatment of the polysaccharide with alkali in the presence of sodium borohydride to prevent the degradation of the polysaccharide (Navarro & Stortz, 2005; Piculell, 1995). In this regard, modification of the Mpw polysaccharide with 1 M NaOH was conducted according to the procedure presented in the experimental section. According to the results of chemical analysis, the main monosaccharides of the modified polysaccharide Mpat were galactose and 3,6-anhydrogalactose (Table 1). The content of galactose decreased and that of 3,6-anhydrogalactose increased by approximately 3 % compared to the original sample. The molar ratio of AnGal:Gal became 1:1.1, which indicated that α-galactose units in the non-cyclized form which presented in the original polymer were transformed in 3,6-anhydro-α-D-galactose at alkaline treatment (Table 1). The IR spectrum (Supplementary 1) of Mpat was identical to that of the original polysaccharide Mpw. At the same time, the A1230/A1070 absorption bands ratio of Mpat decreased to 0.83 in comparison to that of Mpw, which indicated a decrease in sulfate groups of 5 % after alkaline treatment. In addition, the intensity of the absorption band at 932 cm–1, corresponding to CeO vibrations of 3,6anhydrogalactose increased slightly in Mpat. Thus, biosynthetic precursors of kappa- and iota-carrageeenans were likely to be present in the polysaccharide, but in very small amounts.

Fig. 1. IR-spectra of the Mpw (A) and Mpb (B) polysaccharides from sterile form of M. pacificus.

were compared with those of known carrageenan structures. The IR spectra of the polysaccharides Mpw (Fig. 1A) and Mpb (Fig. 1B) were identical. An intense absorption band in the region of 1230 cm−1 in the IR spectra of both polysaccharides (Fig. 1) indicated the presence of a significant amount of sulfate groups (–S = O asymmetric vibration) (Pereira, Amado, Critchley, Van de Velde, & Ribeiro-Claro, 2009). To assess the differences in the content of sulfate groups in the Mpw and Mpb polysaccharides using IR spectroscopy, the ratio of the intensities of the absorption bands at 1230 cm−1 (stretching vibrations of all sulfate groups) and 1070 cm−1 (skeletal vibrations of the carbohydrate ring) were calculated. The ratios of the intensities of the absorption bands A1230/A1070 = 0.89 indicated that the content of sulfate groups in both polysaccharides was the same, which agreed well with the results of chemical analysis (Table 1). There were an absorption band at 932 cm−1 that characteristic of 3,6-anhydrogalactose (CeO vibration) (Stancioff & Stanley, 1969) and an absorption band at 848 cm−1 belonging to the secondary axial sulfate group at C-4 of the 1,3-linked βD-galactose residue in both spectra. This made it possible to assign the polysaccharides to the kappa-type of carrageenan (Rees et al., 1993). The presence of a weak intensity band at 805 cm−1, characteristic of the secondary axial sulfate group at C-2 of a 1,4-linked residue of 3,6anhydro-α-D-galactose, indicated the presence of disaccharide units of the iota-type (Prado-Fernandez, Rodriguez-Vazquez, Tojo, & Andrade, 2003). Thus, the use of alkaline extraction of the polysaccharides from the M. pacificus instead of water did not lead to significant changes in the polysaccharide composition. It may indicate either the absence of biosynthetic precursors or the fact that the polysaccharide has a dimensional structure that shields minor quantities of precursors and makes their sulfate groups inaccessible to alkali. Regardless of the extraction method, the obtained sulfated galactans were built primarily of kappacarrageenan units, and also contained iota-carrageenan units.

3.3. Fractionation of Mpw polysaccharides It is known that different types of carrageenans form gels when the salts of certain metals at low concentrations are added, showing a specificity to those salts. According to the literature, kappa-carrageenan has specificity for potassium ions; iota-carrageenan – for calcium ions (Chapman & Chapman, 1980; Piculell, 1995; Yermak & Khotimchenko, 2003). Sulfated galactans from red algae can either be a mixture of several types of carrageenans, or complex hybrid structures in which the polymer chain includes disaccharide units of various types of carrageenans (Craigie, 1990, Knutsen et al., 1994). In order to determine whether a polysaccharide isolated by water extraction is a mixture of kappa- and iota-carrageenans or a kappa/iota-hybrid, the polysaccharide sample was precipitated with 1, 2 and 4 % KCl. In addition, the polysaccharide was precipitated with 0.5 % KCl and the resulting gel was precipitated with 2 % CaCl2 according to the procedure described in the experimental section. The yield and composition of the obtained fractions were presented in Table 2. Fractionation of the polysaccharide with 1 and 2 % KCl led to a very small amount of non-gelling fractions (1.9 and 0.6 %, respectively), whereas the yield of gelling fraction ranged from 17.9 to 21.9 % by weight of dry algae depending on the KCl concentration. The polysaccharide passed completely into the gel state at 4 % KCl. The

Table 2 Yield and chemical analysis of polysaccharides (PS) from M. pacificus after fractionation with KCl and CaCl2. Salt concentration, %

1.0 % KCl 2.0 % KCl 4.0 % KCl 0.5 % KCl – 2.0 % CaCl2

Sample

g ng g ng g ng g ng

PS yield, % of dried algal weight

17.9 1.9 18.6 0.6 21.9 0.0 15.8 6.9

Content, % of PS sample weight

Molar ratio AnGal:Gal:SO3Na

S930/S805

S848/S805

2.1 0.8 1.9

Gal

AnGal

Xyl

Glc

SO3Na

37.6

23.9

0.7

–

27.0

1.0:1.4:1.6

31.6

24.2

0.5

–

26.6

1.0:1.2:1.5

2.4 1.1 1.1

33.2

27.1

0.4

–

27.7

1.0:1.1:1.4

1.0

1.2

32.8 29.0

25.6 21.5

0.4 1.2

– –

28.6 24.0

1.0:1.1:1.6 1.0:1.2:1.6

2.2 1.3

1.3 1.1

Note: g – gelling polysaccharide, ng – non-gelling polysaccharide. 5

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Fig. 2.

13

C NMR spectrum of Mpw polysaccharide.

carrageenans at various concentrations of KCl. It is believed that if the polysaccharide is a mixture of kappa- and iota-carrageenans, a 1.1 % solution of kappa-carrageenan will form a dense gel at 0.1 M KCl, while iota-carrageenan will be a sol. At the same time, during the precipitation of a 0.5 % polysaccharide solution with 0.03 M KCl, kappa-carrageenan is a weak gel with water syneresis, while iota-carrageenan is in the form of a solution. If the polysaccharide has a hybrid structure, including disaccharide units of kappa- and iota-carrageenans, their phase separation does not occur. In this regard, 0.5 and 1.1 % solutions of the standard samples of kappa- and iota-carrageenans (Sigma-Aldrich), mixtures of these polysaccharides in equal amounts and solutions of the polysaccharide from M. pacificus were prepared. KCl was added to give a 0.03 M solution to polysaccharides solutions with a lower concentration and 0.1 M KCl – to polysaccharides solutions with a higher concentration. These solutions were left for 2 days at room temperature. Over time, it was noted that kappa-carrageenan at lower salt concentration formed a weak gel, at a higher one – a dense gel. Iotacarrageenan at low salt concentration was in the form of a viscous solution, and at a high concentration – in the form of a weak gel. Mixing of kappa- and iota-carrageenans in equal amounts led to the formation of particles of iota-carrageenan sol in kappa-carrageenan gel, regardless of the salt concentration. In the case of the Mpw polysaccharide, no phase separation occurred, regardless of the salt. This fact reinforced the hypothesis of the hybrid structure of sulfated galactan under investigation.

yield of the gelling fraction was minimal when the polysaccharide was sequentially fractionated with 0.5 % KCl and 2 % CaCl2. According to the results of chemical analysis, the major monosaccharides in all the fractions obtained were galactose and 3,6-anhydrogalactose, the contents of which were 29.0–37.6 and 21.5–27.1 %, respectively. The lowest amount of these monosaccharides was observed in the fraction that did not form a gel with CaCl2 (Table 2). The highest galactose content was observed in the polysaccharide gelling with 1 % KCl, while 3,6-anhydrogalactose ones – in those with 4 % KCl. The content of sulfate groups varied from 24.0 to 28.6 %. However, despite the difference between samples regarding the amounts of galactose, 3,6-anhydrogalactose and sulfate groups, the molar ratio AnGal:Gal:SO3Na showed no significant difference in their composition (Table 2). The exception was the gelling polysaccharide at 1 % KCl, which had a higher AnGal:Gal ratio (1.0:1.4) due to the high galactose content, as in the original polysaccharide (Tables 1 and 2). In addition, all the samples studied contained trace amounts of xylose and did not contain glucose, which is the structural unit of floridean starch, the storage polysaccharide in these algae (Craigie, 1990). The IR spectra of the obtained polysaccharide fractions (Supplementary 2, 3, 4) and the original polysaccharide were similar. Both gelling and non-gelling polysaccharide fractions were represented mainly by kappa-carrageenan with insignificant numbers of iota-carrageenan units. The IR spectra of all fractions were decomposed into the individual components (not shown) and the ratios of the areas of the S930/S805 and S848/S805 absorption bands were calculated to estimate of the content of iota units in the studied polysaccharide fractions (Table 2). As can be seen from Table 2, the ratios of the S930/S805 and S848/S805 absorption band areas of polysaccharides gelling at various KCl concentrations decreased with increasing salt concentration, which was associated with an increase in the absorption band area at 805 cm−1, corresponding to the vibrations of the secondary axial sulfate group at C-2 of 1,4-linked residue of 3,6-anhydro-α-D-galactose iota-carrageenan. This fact indicated an increase in the content of iotatype disaccharide units. Comparison of polysaccharide fractions nongelling at 1 % KCl and 2 % CaCl2 with polysaccharides gelling in these salts showed a higher content of iota units in the non-gelling fractions, indicated by the decrease in the S930/S805 and S848/S805 absorption area ratios in the IR spectra of non-gelling fractions. Thus, the fractionation conditions used did not lead to the separation of kappa- and iota-carrageenans. This suggested that M. pacificus produced a complex hybrid carrageenan consisting of kappa- and iotatype units. The presence of a non-gelling fraction at 1 % KCl and its absence at higher salt concentrations may be due to the fact that the amount of KCl was insufficient for formation an ordered helical structure by all the polysaccharide molecules. To proof of the hybrid nature of these carrageenans, an experiment proposed by Hillou and co-authors was carried out (Hillou et al., 2006). This experiment is based on phase transitions of kappa- and iota-

3.4. Structural analysis of Mpw polysaccharide Analysis of the NMR spectrum of the polysaccharide confirmed the IR spectroscopy data (Fig. 1). Chemical shifts of signals in the 13C NMR spectrum were compared to those of known carrageenan structures (Kolender & Matulewicz, 2004; Miller & Blunt, 2000; Van de Velde, Knutsen, Usov, Rollema, & Cerezo, 2002). Four signals with different intensities at 92.5, 95.6, 102.6 and 102.8 ppm were observed in the anomeric area of the 13C NMR spectrum of the polysaccharide (Fig. 2), which indicated the presence of two types of disaccharide units. Poorly resolved signals at 102.6 and 102.8 ppm were the result of overlapping of C-1 signals of 1,3-linked β-D-galactose 4-sulfate of iota- (G4S’) and kappa-carrageenans (G4S), respectively (Usov & Shashkov, 1985). The signal at 95.6 ppm was assigned to C-1 of 1,4-linked 3,6-anhydro-α-Dgalactose (DA) of kappa-carrageenan, and the signal at 92.5 ppm was characteristic of the C-1 of 1,4-linked 3,6-anhydro-α-D-galactose of iotacarrageenan (DA2S) (Fig. 2). The 13C NMR spectrum in the upfield region was typical of kappa- and iota-types of carrageenans. The DEPT135 experiment revealed that there were two oxymethylene groups at 61.8 and 70.2 ppm. The latter was substituted, as seen from the values of its chemical shift. Thus, the polymer chain of the polysaccharide from M. pacificus consisted mainly of kappa-carrageenan disaccharide units and smaller 6

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Fig. 3.

13

C–1H HSQC spectrum of desulfated polysaccharide from M. pacificus.

two types of disaccharide units were present in the polysaccharide structure, which differed from each other only by the presence or absence of sulfate groups at C-4 of the galactopyranose residue. The correlation signals at 76.3/4.48 and 66.5/4.12 ppm corresponded to C4/H-4 sulfated (A) and unsulfated (C) 1,3-linked β-D-galactose, respectively. The presence of the sulfate group at C-4 1,3-linked β-D-galactose (4A) indicated that the signal in the 13C NMR spectrum was shifted to 9.8 ppm in the weak field compared to the signal at C-4 unsulfated 1,3-linked β-D-galactose (4C), which was consistent with literature data (Liao, Kraft, Munro, Craik, & Bacic, 1993; Miller & Blunt, 2000). Correlation signals at 61.7/3.7–3.8 ppm were assigned to the C6/H-6 both 1,3-linked A and C, while those at 70.1/3.62–4.20 were characteristic of C-6/H-6 of the two 1,4-linked 3,6-anhydro-α-D-galactose residues (B, D) (Fig. 3). Correlation signals at 78.5/4.62 and 77.5/4.66 ppm were present in the spectrum as the result of overlapping carbon signals of 4B, 4D and 5B, 5D, respectively, of kappa- and beta-carrageenans. According to the HSQC spectrum, the three signals in the region 70.1 ppm was a result of multiple carbon signals overlapping: 2C, 6B and 6D (Fig. 3, Table 3). The effect of glycosylation of the galactopyranose residues at C-3 indicated that the monosaccharide residues in the both disaccharide units had D-configuration, which was in good agreement with the results of the partial reductive hydrolysis and indicated the presence of carrabiose units only. The following structural changes can be noted at comparison of 13C NMR spectra of the original (Fig. 2) and desulfated (Supplementary 5) polysaccharides. The signal intensity at 95–96 ppm, corresponding to C1 of 1,4-linked 3,6-anhydro-α-D-galactose (DA) of kappa-carrageenan in the 13C NMR spectrum of the original sample (Fig. 2), decreased in the 13 C NMR spectrum of the desulfated sample (Supplementary 5). The signal at 92.5 ppm, characteristic of the C-1 of 1,4-linked 3,6-anhydroα-D-galactose of iota-carrageenan (DA2S), presented in the spectrum of the Mpw polysaccharide, completely disappeared in the desulfated sample. At the same time, an intense signal at 94.8 ppm, corresponding to C-1 of 1,4-linked 3,6-anhydro-α-D-galactose (DA’) of beta-carrageenan, appeared in the 13C NMR spectrum of the desulfated sample (Supplementary 5). In addition, a signal at 66.5 ppm, characteristic of the C-4 of unsulfated 1,3-linked β-D-galactose (G), presented in betacarrageenan, appeared in the spectrum of the desulfated polysaccharide. Thus, the desulfated polysaccharide was mainly beta-carrageenan and contained disaccharide units of kappa-carrageenan in small amounts. This was due to the fact that the kappa- and iota-carrageenan presented in the original polysaccharide were desulfated. The

quantities of iota-carrageenan. A similar structure has been defined for the polysaccharide from Mastocarpus stellatus (Hillou et al., 2006). Since the 13C NMR spectrum of the original polysaccharide contained many overlapping signals, which made it difficult to interpret and did not allow the discovery of minor amounts of possible biosynthetic precursors of kappa- and iota-carrageenans, the Mpw polysaccharide was desulfated, as described in the experimental section. It was believed that the desulfation process allowed simplification of the spectrum. Perhaps this would help to see the signals corresponding to desulfated precursors. It was shown by DEPT experiment that there were two oxymethylene groups at 61.7 and 70.1 ppm, the latter of which was substituted as seen from the values of its chemical shift. Two-dimensional COSY and HSQC (Fig. 3, Table 3) spectroscopy was used to assign the signals of monosaccharide residues protons and to carry out C/H correlation. There were the correlation signals C-1/H-1 at 94.8/5.06, 96.5/5.14, 102.9/4.57 and 103.1/4.61 ppm, corresponding to the four types of galactose residues in the anomeric area of heteronuclear 2D HSQC spectrum (Fig. 3, Table 3). The first two correlation signals corresponded to 1,4-linked residues of 3,6-anhydro-α-D-galactose (1D and 1B, respectively), and the second were characteristic of 1,3-linked β-Dgalactose (1C and 1A, respectively). It was shown from the spectra that Table 3 Chemical shift values of protons and carbons (ppm) in 1H and 13C NMR spectra of desulfated polysaccharide from M. pacificus. Carrageenan type

kappa beta

Carrageenan type kappa beta

MS residue

A B C D

1

H chemical shifts (ppm)

H-1

H-2

H-3

H-4

H-5

H-6

4.61 5.14 4.57 5.06

3.52 3.97 3.62 4.07

3.68 4.44 3.84 4.53

4.48 4.62 4.12 4.62

3.72 4.66 3.69 4.66

3.80-3.70 4.20-3.62 3.80-3.70 4.20-3.62

C-1 103.1 96.5 102.9 94.8

C-2 71.2 71.0 70.1 70.4

13

MS residue A B C D

C chemical shifts (ppm) C-3 C-4 C-5 81.3 76.3 75.6 79.1 78.5 77.5 80.7 66.5 76.0 79.7 78.5 77.5

C-6 61.7 70.1 61.6 70.1

Note: A – 1,3-linked β-D-galactose 4-sulfate, B – 1,4-linked 3,6-anhydro-α-Dgalactose of kappa-carrageenan, C – 1,3-linked β-D-galactose, D – 1,4-linked 3,6-anhydro-α-D-galactose of beta-carrageenan. 7

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investigations of sulfated hexoses (Minamisawa & Hirabayashi, 2005). Recently, we have employed a technique of autohydrolysis in H218O of fucoidans to obtain selectively labelled oligosaccharides, and also mild acid hydrolysis in H218O (Usoltseva et al., 2017) that allowed us to discover new fragmentation pathways and withdraw stale data on MS/ MS of fucooligosaccharides (Anastyuk, Shevchenko, Belokozova, & Dmitrenok, 2018). Herein, the carrageenan sample was subjected to mild acid hydrolysis in heavy oxygen water to obtain selectively labelled oligosaccharides for mass-spectrometric analysis by ESIMS/MS. Unfortunately, the whole mass spectrum of the oligosaccharide mixture was complex (Supplementary 7), since the ESIMS generated a set of multiply-charged ions. In this regard, MALDI-TOFMS had the advantage of generating only singly-charged ions with the use of nor-harmane as matrix. However, the precise MS/MS mode of the ESIMS instrument allowed the obtaining of some information about oligosaccharide structure. A fragment ion at m/z 138.97 of 2,4A-type ion along with a 0,2A-type ion at m/z 198.99 were present in the negative-ion ESIMS/MS of the monosulfated galactose (labelled by heavy oxygen, which gives a +2 Da mass shift) at m/z 261.01 (Supplementary 8) (Table 4, m/z 259.00, unlabelled hexose detected by MALDI-TOFMS), which suggested sulfation at C-4 of the galactose molecule. This fact confirmed the MSn data (Minamisawa & Hirabayashi, 2005) and recent data obtained on fucoidans (Anastyuk et al., 2018). The same conclusion was reached in the work on MS/MS of mono- and disaccharides derived from carrageenans (Goncalves, Ducatti, Grindley, Duarte, & Noseda, 2010). It should be mentioned that it is impossible to distinguish 2- and 4-sulfated saccharides without a label at the reducing end, because the fragment ion at m/z 138.97 could arise from both X- or A-type cleavages. The tandem ESIMS/MS of the iota-carrabiose (Table 4, m/z 505.00, unlabelled, as detected by MALDI-TOFMS) fragment, the ion at m/z 242.00 (Fig. 4) was more complex and more interesting. As observed by Yu et al. (2006), Y- and Z-type ions, which retain the negative charge at the reducing end, were less intense than C- and B-type ions. A fragment ion of C1-type at m/z 259.01 showed the highest intensity, suggesting that the sulfated galactose residue was located at the non-reducing end (Fig. 4, on the left). Again, fragment ions of 2,4A1- and 0,2A1-type at m/z 138.97 and 198.99, respectively, suggested sulfation at C-4 of the galactose residue on the non-reducing end. Another rare fragment ion for carrageenans of 0,2A-type was detected at m/z 343.02. A Z1-type ion at m/z 225.00 was detected at low intensity, because, probably, the sulfate group at position C-2 of 3,6-anhydrogalactose is unstable and tends to decompose with formation of the fragment ions from desulfation at m/z 385.04 and 403.04. The ion from desulfation of the galactose residue

presence of kappa-carrageenan in small amounts in the spectrum of the desulfated sample indicated that the desulfation was not fully completed. However, there were no signals in the spectrum of the desulfated sample, which could indicate the presence of the desulfated precursors of kappa- and iota-carrageenans. 3.5. Analysis of the mild acid hydrolysis products of the polysaccharide from M. pacificus by MALDI-TOFMS To enhance our knowledge of the structural features of the Mpw polysaccharide beyond the data obtained by spectroscopic methods, we have applied mild acid hydrolysis to obtain oligosaccharide fragments for mass-spectrometric analysis (Sun et al., 2015; Yang et al., 2009; Yu et al., 2002). Recently, we used this approach successfully to obtain data about the structural features of oligosaccharides released from hybrid kappa/beta-carrageenan from the red seaweeds Tichocarpus crinitus and Ahnfeltiopsis flabelliformis (Anastyuk et al., 2011; Byankina Barabanova et al., 2013; Kravchenko et al., 2014). Preliminary analysis of the oligosaccharide mixture after mild acid hydrolysis of Mpw polysaccharide (0.1 N HCl, 4 h, 60 °C) was performed by MALDI-TOFMS using nor-harmane as matrix (Antonopoulos et al., 2005). The feature of this matrix for the analysis of sulfated oligosaccharides is the in-source desulfation: excess sulfate groups are cleaved, and thus oligosaccharides with higher degree of polymerization could be observed. The MALDI-TOFMS analysis (Supplementary 6) showed (Table 4) that the oligosaccharide mixture was represented by partially desulfated fragments of, probably, kappa-, kappa/iota-carrageenans and their precursors, since the oligosaccharides were rich in galactose. The degree of sulfation did not exceed three. Thus, the MALDI-TOFMS of fragments released by mild acid hydrolysis allowed us to observe oligosaccharides with degree of polymerization (DP) 1–9 and degree of sulfation 1–3, while chains built up of galactose residues were observed up to DP = 3. 3.6. Analysis of the mild acid hydrolysis products of the polysaccharide from M. pacificus by ESIMS/MS obtained in heavy oxygen water (H218O) The negative-ion ESIMS/MS and MALDI-TOFMS are techniques that have proven abilities in the analysis of the structural features of complex anionic carbohydrates such as glucosaminoglycans, carrageenans and fucoidans (Harvey, 2011). However, these methods have both benefits and weaknesses and served as complementary tools to NMR technique. In 2005 it was reported that the selective labelling of the oxygen at the reducing OH-group of the saccharide molecules by 18O significantly improved the quality of the mass-spectrometric data and thus enhanced the abilities of the MS methods in structural

Table 4 Composition of the oligosaccharide mixture, obtained by mild acid hydrolysis (0.1 N HCl, 60 °C, 4 h) of a polysaccharide from the red seaweed M. pacificus as observed by MALDI-TOFMS using negative-ion mode of registration and nor-harmane as matrix. m/z

composition

m/z

composition

259.00 403.06 505.00 547.10 565.11 583.13 667.06 685.06 691.14 695.15 709.16 769.00 797.01 811.09 853.19 871.20

GalSO3− Gal-AnGal-SO3− Gal-AnGal-SO3Na-SO3− (Gal)2-AnGal-SO3− – H2O (Gal)2-AnGal-SO3− (Gal)3SO3− (Gal)2-AnGal-SO3Na-SO3− (Gal)3-SO3Na-SO3− (Gal)2-(AnGal)2-SO3− – H2O (Gal)3SO3− +112 Da (Gal)2-(AnGal)2-SO3− (Gal)2-AnGal-(SO3Na)2-SO3− (Gal)3-SO3Na-SO3− +112 Da (Gal)2-(AnGal)2-SO3Na-SO3− (Gal)3-(AnGal)2-SO3− – H2O (Gal)3-(AnGal)2-SO3−

889.21 921.19 973.14 1001.23 1015.24 1075.08 1103.16 1117.18 1177.29 1195.30 1279.23 1307.32 1409.28 1483.39

(Gal)4-AnGal-SO3− not identified (Gal)3-(AnGal)2-SO3Na-SO3− (Gal)4-AnGal-SO3− +112 Da (Gal)3-(AnGal)3-SO3− (Gal)3-(AnGal)2-(SO3Na)2-SO3− (Gal)4-AnGal-SO3Na-SO3− +112 Da (Gal)3-(AnGal)3-SO3Na-SO3− (Gal)4-(AnGal)3-SO3− (Gal)5-(AnGal)2-SO3− (Gal)4-(AnGal)3-SO3Na-SO3− (Gal)5-(AnGal)2-SO3− +112 Da (Gal)5-(AnGal)2-SO3Na-SO3− +112 Da (Gal)5-(AnGal)4-SO3−

8

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Fig. 4. Negative-ion mode ESIMS/MS of the [GalAnGal(SO3Na)2 – 2Na]2− ion at m/z 242.00.

assigned by the presence of the characteristic isotope pattern (data not shown). The location of the 6-sulfated galactose residue was also confirmed by the presence of a 3,5A1-type ion at m/z 152.98 (Goncalves et al., 2010; Minamisawa & Hirabayashi, 2005). Other ions were assigned as suggested by Yu et al. (2006), but our method did not block the mobile proton (essential for cross-ring cleavages (Tissot, Salpin, Martinez, Gaigeot, & Daniel, 2006; Zaia, Miller, Seymour, & Costello, 2007)) at the reducing while giving the same m/z shift (+2 Da) as in the methods suggested by Yu et al. (2006), where the reducing end was converted into an alditol form by reduction. Thus, two variants of the selected ion were suggested:

was distinguished because of the +2 shift at m/z 387.04. Thus, two structural variants of the polysaccharide fragments could be suggested - [A2S-G4S – 2Na]2– (minor) and [G4S-A2S – 2Na]2–. The next informative ESIMS/MS (Fig. 5) of the ion [Gal3AnGal2(SO3Na)4 – 4Na]4– at m/z 277.51 (two desulfated variants in MALDI-TOFMS, Table 4) was evidence of the existence of the precursor of kappa- or iota-carrabiose (one could not exclude that the non-reducing Gal residue could be also sulfated at C-2, which was lost during acid hydrolysis) by the presence of the corresponding fragment ion of B2-type at m/z 241.00. Although this fragment ion overlaps with the B1type ion at the same m/z, the charge state of the B2 fragment ion is

Fig. 5. Negative-ion mode ESIMS/MS of the [Gal3AnGal2(SO3Na)4 – 4Na]4– at m/z 277.51. 9

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4. Conclusions The sulfated polysaccharides from a sterile form of red alga M. pacificus obtained by water (Mpw) and alkaline (Mpb) extractions were carrageenans with galactose and 3,6-anhydrogalactose as the main monosaccharides. The molar ratio AnGal:Gal of these polymers was 1.0:1.4 that indicated to the presence of biosynthetic precursors with non-cyclized α-D-galactose. The treatment of the Mpw polysaccharide with 1 M alkali led to an increase of 3,6-anhydro-α-D-galactose amount (the AnGal:Gal molar ratio was 1.0:1.1). According to partial reductive hydrolysis, FT-IR- and NMR spectroscopies, the Mpw polysaccharide consisted of carrabiose units only and represented a hybrid kappa/iotacarrageenan with a predominance of kappa-type units. The use of MALDI-TOFMS of fragments released by mild acid hydrolysis allowed us to observe oligosaccharides with DP 1–9, while chains built up of galactose residues were observed up to DP = 3. Tandem ESI mass spectrometry together with innovative isotope labelling allowed us to detect that the polymer chain of the Mpw polysaccharide, in addition to the main kappa-carrabiose, contained iota-carrabiose, hybrid kappa/ iota fragments and minor mu-carrageenan insertions (the precursor of kappa-carrageenan). The ability of carrageenan to affect permeability of bile salts through an artificial membrane imitating the gastrointestinal barrier was studied by PAMPA. It was shown that the polysaccharide inhibited bile salts permeation by 50 % on average compared to negative control, independent of incubation time. However, its action was less pronounced than the hindering ability of cholestyramine.

Fig. 6. Time and concentration-dependent penetration of bile salts through a parallel artificial membrane (PAMPA) loaded with mixture of hexane/hexadecane, cholesterol, and phosphatidylcholine in the presence of kappa/iota– control (vehicle) carrageenan from M. pacificus, and cholestyramine (CA). – 0.75 mg mL−1, final value; – 1.5 mg mL−1, final value; – 3 mg mL−1, final value. The results are expressed as % change in BS concentration in an acceptor compartment relative to the vehicle control (100 %).

[D6S-G4S-A-G4S-A2S – 4Na]4– and [G4S-A2S-G4S-A-G4S – 4Na]4–. Again, the ESIMS/MS confirmed the presence of iota-carrabiose insertion. Using the same approach, the following oligosaccharides were characterized by ESIMS/MS: [G4S-A-G4S – 2Na]2– at m/z 323.03 and [G4S-A-G4S-A-G4S – 3Na]3– ion at m/z 343.70 this being parts of kappa-carrageenan blocks (Supplementary 9, 10).

Acknowledgments 3.7. Ability of Mpw polysaccharide to influence bile salts permeability The authors are grateful to a researcher at the Laboratory of Enzyme Chemistry of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS Dr. Roza V. Usoltseva for consultation in decoding of the 2D NMR spectra.

The consumption of marine products has been and is increasingly gaining considerable attention and special focus has been attributed to edible seaweeds (Roohinejad et al., 2017). Not least of all health benefits of marine marcoalgae is associated with algal food fibres (Dousip et al., 2014; Kilpatrick, 1999; Lahaye & Kaeffer, 1997). Dietary fibres of red algae and in particularly carrageenans have been revealed to be able to affect cholesterol metabolism and normalize lipid profile indices in vivo (Panlasigui, Baello, Dimatangal, & Dumelod, 2003; Sokolova et al., 2014). It has been suggested that one of the main biological mechanisms explaining the hypochlesterolemic effects of dietary fiber is prevention of bile salts (BS) reabsorption from the small intestine and their release with feces (Gunness & Gidley, 2010). In this connection the ability of Mpw carrageenan to affect permeability of BS through an artificial membrane imitating the gastrointestinal barrier was studied by parallel artificial membrane permeability assay (PAMPA) (Fig. 6). The ability of the carrageenan to permeate the BS through the membrane has been correlated to vehicle control (100 %) and cholestyramine was used as positive control. The BS dynamics through the PAMPA membrane in the presence of the investigated sample were determined after 60 and 150 min incubation. The fact that we researched the BS permeation in dynamics with such short time intervals corresponded to very short time of BS presence in the small intestine due to vigorous enterohepatic cycling. In humans, the main pool of BS (2–4 g) undergoes recycling between the liver and intestine six to ten times each day (de Aguiar Vallim, Tarling, & Edwards, 2013). Our results showed that the Mpw polysaccharide was able to inhibit BS permeation by 50 % on average compared to negative control, independent of incubation time. However, its action was less pronounced than the hindering ability of cholestyramine. It should be noted that according to literature data red seaweed Mastocarpus stellatus intake significantly decreased the total cholesterol level in healthy rats (Gómez-Ordóñez, Jiménez-Escrig, & Rupérez, 2014). Another study elucidated that this red alga contains kappa/iota-hybrid carrageenans (Hillou et al., 2006). Thus, our data are in good agreement with published data.

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