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Depolymerization of sulfated polysaccharides under hydrothermal conditions
Carbohydrate Research 384 (2014) 56–60
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Depolymerization of sulfated polysaccharides under hydrothermal conditions Minoru Morimoto a,⇑, Masaki Takatori b, Tetsuya Hayashi b, Daiki Mori b, Osamu Takashima c, Shinichi Yoshida d, Kimihiko Sato e, Hitoshi Kawamoto f, Jun-ichi Tamura g, Hironori Izawa b, Shinsuke Ifuku b, Hiroyuki Saimoto b a
Division of Instrumental Analysis, Research Center for Bioscience and Technology, Tottori University, Tottori 680-8550, Japan Division of Applied Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori 680-8552, Japan Scientific Crime Laboratory, Tottori Prefectural Police Headquarters, Tottori 680-0911, Japan d Tottori Institute of Industrial Technology, Tottori 689-1112, Japan e Koyo Chemical Co. Ltd, Kita-Ku, Osaka 530-0051, Japan f Marine Products Kimuraya Co. Ltd, Sakaiminato 684-0072, Japan g Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Tottori 680-8551, Japan b c
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 21 November 2013 Accepted 27 November 2013 Available online 4 December 2013 Keywords: Hydrothermal treatment Fucoidan Chondroitin sulfate Sulfated polysaccharide Depolymerization without desulfation
a b s t r a c t Fucoidan and chondroitin sulfate, which are well known sulfated polysaccharides, were depolymerized under hydrothermal conditions (120–180 °C, 5–60 min) as a method for the preparation of sulfated polysaccharides with controlled molecular weights. Fucoidan was easily depolymerized, and the change of the molecular weight values depended on the reaction temperature and time. The degree of sulfation and IR spectra of the depolymerized fucoidan did not change compared with those of untreated fucoidan at reaction temperatures below 140 °C. However, fucoidan was partially degraded during depolymerization above 160 °C. Nearly the same depolymerization was observed for chondroitin sulfate. These results indicate that hydrothermal treatment is applicable for the depolymerization of sulfated polysaccharides, and that low molecular weight products without desulfation and deformation of the initial glycan structures can be obtained under mild hydrothermal conditions. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Several sulfated polysaccharides are known to have various biological activities. Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, is mainly composed of fucose and sulfated fucose, but also contains small amounts of mannose, galactose, glucose, xylose, uronic acids, and acetylated derivatives of these sugars.1–4 The content of these carbohydrates varies with the seaweed species.5–7 Fucoidan shows various biological activities, such as anticoagulant, antithrombotic, antiviral, antiproliferative, antifertilization, and antitumor activities, and has been used in food preparation.1,2 Chondroitin sulfate (CS) is also a well-known sulfated polysaccharide composed of repeating disaccharide units (GalNAc, N-acetylgalactosamine and GlcA, glucuronic acid) and is an important structural component of cartilage in animal bodies. Based on the position of the sulfate groups in the disaccharide units, chondroitin sulfates are classified into several subtypes: CS-O, A, C, D, E, and K, each of which has a respective repeating disaccharide; GalNAc-GlcA, GalNAc4S-GlcA, GalNAc6S-GlcA, GalNAc4S-GlcA2S, ⇑ Corresponding author. Tel.: +81 857 31 5990; fax: +81 857 31 5464. E-mail address: [email protected] (M. Morimoto). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.11.017
GalNAc4S6S-GlcA, and GalNAc4S-GlcA3S (the numbers represent the sulfated positions).8 These polysaccharides, extracted mainly from bovine trachea, porcine skin and rib cartilage, and shark cartilage, are used as drugs and supplements for the treatment of degenerative joint diseases. Other sulfated polysaccharides, such as dermatan sulfate (chondroitin sulfate B), keratan sulfate, heparan sulfate, and heparin, are also found in animal tissues, and are used as biological materials and agents based on their specific biological activities. The biological activities of these sulfated polysaccharides depend on their molecular weights and the degree of sulfation.9–13 To investigate the proposed relationship between structure and activity, it is necessary to prepare the sulfated polysaccharides with controlled molecular weights and degrees of sulfation. Furthermore, several grams of each sample are required for the studies in vivo. In general, the preparation of polysaccharides with various molecular weights is achieved via hydrolysis using enzyme or acid. However, these two methods have disadvantages. The enzyme shows high substrate specificity which strongly restricts the use. The enzyme is normally expensive and is difficult to be removed after the reaction especially in the large scale. The acid, such as HCl and H2SO4,
M. Morimoto et al. / Carbohydrate Research 384 (2014) 56–60
is cheap but the sulfate groups on the polysaccharides are sometimes hydrolyzed during the reaction. Hydrothermal treatment has been used for depolymerization of polysaccharides such as cellulose,14 starch,15 guar gum,16 and alginate.17,18 Recently the extraction and degradation of fucoidan via hydrothermal treatment were reported.19 However, there was no mention on the desulfation of the degraded fucoidan. We have reported that various compounds bearing acetal and ketal groups are easily hydrolyzed in sub-critical water under hydrothermal conditions without enzyme and acid.20 Using hydrothermal treatment, chitosan, a polymer of glucosamine, was also depolymerized to produce low molecular weight products because the glycoside of polysaccharides is chemically equivalent to an acetal.21 Interestingly, the hydrothermal treatment is effective only for acetal in the presence of ester. This result suggested that sulfated polysaccharides could be depolymerized without the hydrolysis of the sulfate under hydrothermal conditions. In this study, we optimized hydrothermal conditions for the depolymerization without desulfation of fucoidan and chondroitin sulfate as a typical sulfated polysaccharide.
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Figure 2. GPC chromatograms of fucoidan before and after hydrothermal treatment (Method A). (a) Untreated fucoidan, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
2. Results and discussion 2.1. Hydrothermal treatment of fucoidan Reaction conditions for the hydrothermal treatment are represented schematically in Figure 1. Typical GPC chromatograms of fucoidan obtained by Methods A and B are shown in Figures 2 and 3, respectively. The GPC peaks gradually shifted to longer retention times, which indicate that the molecular weight changed as a function of the reaction time and temperature. Multiple peaks in the chromatograms were observed at 160 and 180 °C (Fig. 2d and e, respectively). The Mw and DS values of fucoidan treated under various hydrothermal conditions are summarized in Table 1. Compared to the untreated fucoidan (Mw 320,000), the Mw values gradually reduced from 123,000 to 1800 along with increasing reaction temperature from 120 to 180 °C (entries 1, 2, 4, and 5 in Table 1). Similarly, depolymerization increased along with the increased reaction time (entries 6, 8, 11, 2, 14, and 15). On the other hand, the DS values were not altered by the hydrothermal conditions, except at reaction temperatures above 160 °C (entries 5 and 6). These results indicate that fucoidan was depolymerized without desulfation under mild conditions (below 140 °C). The DS values obtained above 160 °C were larger than 0.35 which
Figure 3. GPC chromatograms of fucoidan before and after hydrothermal treatment (Method B). (a) Untreated fucoidan, treated for (b) 5 min, (c) 10 min, (d) 15 min, (e) 40 min, and (f) 60 min at 140 °C.
was the value obtained at 120 and 140 °C. Because the DS value was calculated from the S/C ratio as determined via elemental analysis, an increase in the DS value suggests that either sulfation (increase in the S content) or decarbonization (decrease in the C content) reaction occurred above 160 °C. However, sulfation is
Figure 1. Schematic representation of the hydrothermal conditions. Method A: the sample solution was heated from room temperature to the setting temperature in 10 min (20 min for 180 °C), maintained for 20 min at the reaction temperatures (120–180 °C), and cooled to room temperature in 20 min (30 min for 180 °C). Method B: the sample solution was heated at 140 °C for 10 min, maintained for the reaction time (5–60 min) at 140 °C, and cooled to room temperature in 20 min.
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Table 1 Molecular weight and degree of sulfation of fucoidan before and after hydrothermal treatments
a b c
Entry
Method
Volume (mL)
Concentration (wt %)
Temperaturea (°C)
Time (min)
Mwb
Mw/Mn
DSc
Fucoidan 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
A A A A A B B B B B B B B B B B
80 80 80 80 80 80 80 80 80 800 80 800 800 80 80 80
1 1 5 1 1 1 5 1 5 1 1 1 5 1 1 5
120 140 140 160 180 140 140 140 140 140 140 140 140 140 140 140
20 20 20 20 20 5 5 10 10 10 15 15 15 40 60 60
320,000 123,000 18,000 19,000 3200 1800 75,000 74,000 36,000 36,000 51,000 26,000 33,000 32,000 8000 6000 6000
2.2 2.8 1.7 2.3 1.3 1.2 2.5 3.2 2.5 2.5 2.4 2.7 2.2 2.3 1.3 1.2 1.7
0.35 0.35 0.35 — 0.39 0.42 0.37 — 0.35 — — — — 0.33 0.36 0.36 —
Setting temperature. Determined by gel permeation chromatography. Determined by elemental analysis.
difficult to achieve under hydrothermal conditions. Thus, it seems reasonable to conclude that decarbonization took place. The Mw values were not affected for the samples with concentrations ranging from 1 to 5 wt % (entries 2 vs 3, 6 vs 7, 8 vs 9, 12 vs 13, and 15 vs 16 in Table 1) and were reproducible within ±15% for repeated runs. On the other hand, the Mw values obtained on the 800 mL scale in a large bottle were 30–40% greater than those run on an 80 mL scale in a small bottle (Entries 10 vs 8 and 12 vs 11). The difference could be due to the difference in heating rate of the sample solution. Since 800 mL of aqueous solution has a larger heat capacity, the actual heating rate of the 800 mL scale is expected to be lower than that of the temperature inside or that of the 80 mL scale. In order to obtain a constant Mw value, we must use the same-sized bottles for the hydrothermal treatment. The IR spectra of fucoidans treated at 120–180 °C for 20 min and the untreated sample exhibited characteristic peaks at 1730, 1630, 1240, 1030, and 840 cm1 corresponding to the C@O stretching band of the acetyl group, the COO antisymmetrical stretching band of the carboxylate group of glucuronic acid, the AS@O stretching band of the sulfate group, the C–O–C symmetrical stretching band of the carbohydrate skeleton, and the C–O–S stretching band of the sulfate group, respectively (Fig. 4). The IR spectra of the products obtained at 120 and 140 °C were nearly identical to that of the untreated fucoidan. On the other hand, the IR spectra of the products obtained at 160 and 180 °C showed spectral changes and a decrease in the peak intensity near 1030 cm1 (assigned to the carbohydrate skeleton) and 1630 cm1 (assigned to the carbonyl groups). These results support the fact that deformation and decarbonization occurred above 160 °C.
Figure 4. IR spectra of fucoidan before and after hydrothermal treatment (Method A). (a) Untreated fucoidan, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
Table 2 Molecular weight and degree of sulfation of chondroitin sulfate C before and after hydrothermal treatments Entry
Temperaturea (°C)
Chondroitin sulfate C 1 A 120 2 A 140 3 A 160 4 A 180 5 A 180 6 B 140 7 B 140 8 B 140 9 B 140
2.2. Hydrothermal treatment of chondroitin sulfate To confirm the applicability of this hydrothermal treatment for the depolymerization of sulfated polysaccharides, we subjected chondroitin sulfate to similar reaction conditions. The Mw and DS values of the products obtained from chondroitin sulfate treated under the same conditions as those applied to fucoidan are listed in Table 2. A similar dependence of depolymerization on the reaction temperature and time was observed. At mild reaction temperatures below 140 °C, the DS values did not vary from that of the untreated chondroitin sulfate. Multiple peaks were observed in the GPC chromatogram treated at 160 °C. The
Method
a b c
Time (min)
Mwb
Mw/Mn
DSc
20 20 20 20 20 5 10 40 60
80,000 73,000 44,000 3500 2800 1800 55,000 50,000 29,000 20,000
1.4 1.4 1.6 1.7 1.3 1.0 1.5 1.5 1.5 1.4
0.95 0.93 0.92 0.84 0.39 2.26 0.95 0.96 0.92 0.92
Setting temperature. Determined by gel permeation chromatography. Determined by elemental analysis.
peak shifted to the lowest molecular weight at 180 °C (Fig. 5). Some changes in the IR spectrum of chondroitin sulfate treated at 160 °C were observed at 600 cm1 and 1100 cm1 compared
M. Morimoto et al. / Carbohydrate Research 384 (2014) 56–60
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Figure 5. GPC chromatograms of chondroitin sulfate C before and after hydrothermal treatment (Method A). (a) Untreated chondroitin sulfate C, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
Figure 7. HPLC chromatograms of Chondroitinase ABC digested chondroitin sulfate C treated under hydrothermal conditions. (a) Untreated chondroitin sulfate C, treated at (b) 120 °C, (c) 160 °C, and (d) 180 °C for 20 min. The elution times of unsaturated disaccharides are determined by their standard samples.
conclusion that the depolymerization occurred only at lower reaction temperatures without deformation of the disaccharide units. 3. Conclusion
Figure 6. IR spectra of chondroitin sulfate C before and after hydrothermal treatment (Method A). (a) Untreated chondroitin sulfate C, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
with those of unreacted polysaccharide and the chondroitin sulfate treated below 140 °C (dashed circle in Figure 6). Furthermore, drastic spectral change was observed at 180 °C. Therefore, the depolymerization of chondroitin sulfate under hydrothermal conditions gave nearly the same results as that of fucoidan.
We have demonstrated the depolymerization of fucoidan and chondroitin sulfate under hydrothermal conditions without enzyme and acid. The Mw value was controlled by the reaction temperature and time. Under mild conditions, sulfated polysaccharides with controlled molecular weights but a constant degree of sulfation were obtained. This method can also be employed for the preparation of several grams of polysaccharide samples. We have, in fact, already investigated the effect on tumor-bearing mice of the oral administration of fucoidan samples prepared using this method.25 Furthermore, in addition to fucoidan and chondroitin sulfate, the hydrothermal treatment proposed in this study should be applicable to the depolymerization of various other sulfated polysaccharides.
2.3. Sulfation pattern of chondroitin sulfate 4. Experimental Both untreated and hydrothermally treated chondroitin sulfate were digested by chondroitinase ABC to unsaturated disaccharides22 that correspond to repeating disaccharides found in the polysaccharide. The unsaturated saccharides with different sulfation patterns were assigned by HPLC using authentic standards.23,24 The HPLC patterns of the unsaturated disaccharides obtained from enzymatically digested chondroitin sulfate treated under various hydrothermal conditions are shown in Figure 7. As expected, untreated chondroitin sulfate exhibited a main peak at 24 min, which was assigned to DDi-6S. Chondroitin sulfate treated at 140 °C gave nearly the same peak pattern and area. This result indicates that neither transfer of the sulfate group nor desulfation occurred under mild reaction conditions, whereas no disaccharide peak derived from chondroitin sulfate was observed in chromatograms treated at 160 and 180 °C. This finding also supports the
4.1. Materials Fucoidan powder extracted from Cladosiphon okamuranus Tokida was provided by Marine Products Kimuraya Co. Ltd (Sakaiminato, Japan). Analysis via inductively coupled plasmaatomic emission spectroscopy (ICP-AES) confirmed that the fucoidan existed as its sodium salt. Chondroitin sulfate (Chondroitin sulfate C sodium salt) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Chondroitinase ABC (EC 4.2.2.4), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)D-galactose (DDi-0S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4enepyranosyluronic acid)-6-O-sulfo-D-galactose (DDi-6S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid) -4-O-sulfo-D-galactose (DDi-4S), 2-acetamido-2-deoxy-3-O-(2-O-
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M. Morimoto et al. / Carbohydrate Research 384 (2014) 56–60
sulfo-b-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DDi-diSD), and 2-acetamido-2-deoxy-3-O-(2-O-sulfob-D-gluco4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose (DDi-triS) were obtained from Seikagaku Biobusiness Co. (Tokyo, Japan). The water used in this study was purified with a water purification system (Millipore Elix 3). All other chemicals were analytical grade and were used without further purification. 4.2. Hydrothermal treatment Fucoidan and chondroitin sulfate were each dissolved in purified water with gentle shaking, and the concentration was adjusted to 1 or 5 wt %. The aqueous solutions (80 mL or 800 mL) were then placed, respectively, in 100 mL volumetric bottles (56 / mm 100 mm) or 1000 mL volumetric bottles (101 /mm 225 mm) that were sealed using screw caps. The hydrothermal treatment of the samples was performed in an airtight autoclave (HTP-50/250, Hisaka Works Ltd, Osaka, Japan). Based on the temperature–time program, sample solution in the bottle was heated from room temperature to the setting temperature (reaction temperature) in 10–20 min, and the temperature was maintained for 5–60 min and then the solution was cooled to room temperature in 20–30 min. A schematic representation of these reaction conditions is shown in Figure 1. In Method A, the reaction temperature was set in 120–180 °C for a constant reaction time of 20 min. In Method B, the reaction time was set in 5–60 min at a constant reaction temperature of 140 °C. After completion of the hydrothermal treatment, the solutions were divided into two parts. One part was lyophilized without further workup to give a brown powder. The other was dialyzed for one week using a dialysis membrane (Spectra/Por CE dialysis tubing, MWCO: 500) against purified water to remove the eliminated sulfate salts, and then the solution was lyophilized. The dialyzed samples were used for elemental and IR analyses. 4.3. Molecular weight The molecular weight distributions of the sulfated polysaccharides were analyzed using gel permeation chromatography (GPC) on connecting GPC columns: TSKgel PWXL + TSKgel PWXL (Tosoh CO., Tokyo, Japan) or Shodex Asahipak GS-520HQ + GS320HQ + GS-220HQ (Shoko Co. Ltd, Tokyo, Japan). The mobile phase was 0.1 M NaNO3 at a flow rate of 0.5 mL/min at 40 °C. The lyophilized samples without dialysis were dissolved in the mobile phase (10 mg/mL) and injected (100 lL). GPC peaks were detected with a refractive index (RI) detector (SHIMADZU RID-10, Shimadzu Co., Kyoto, Japan). The weight-average molecular weight (Mw), the number-average molecular weight (Mn), and the polydispersity (Mw/Mn) were calculated using molecular weight calculation software connected to the HPLC integration system (HITACHI D-7000, Hitachi Co., Tokyo, Japan) and a pullulan standard kit (Shodex P-82, Shoko Co., Tokyo, Japan). 4.4. Degree of sulfation The degree of sulfation (DS) of sulfated polysaccharides was calculated via elemental analysis. For this calculation, we assumed that all of the carbohydrate residues of the sulfated polysaccharides used in this study were hexose. Based on this assumption, the DS values were calculated using the following formula:
Sð%Þ=Cð%Þ ¼ ð32:06 DSÞ=ð12:01 6Þ: For the elemental analyses, the dialyzed samples were used.
4.5. IR analysis Infrared (IR) spectra of the dialyzed samples were measured on a Fourier transform IR spectrometer (SHIMADZU FTIR-8700, Shimadzu Co., Kyoto, Japan) in KBr pellets. 4.6. Sulfation pattern of chondroitin sulfate The sulfation patterns of the chondroitin sulfate after hydrothermal treatment were determined using the disaccharide method: enzymatic digestion of the chondroitin sulfates followed by high performance liquid chromatography (HPLC) analysis. The chondroitin sulfate (200 lg) was dissolved in a mixture of water (200 lL), a 0.01% (w/v) bovine serum albumin (BSA) solution (20 mL), and a 250 mM Tris–HCl buffer (40 mL). Chondroitinase ABC (5 U/mL, 100 lL) was then added to the solution, and the mixture was incubated at 37 °C for 8 h. The reaction solution was then heated in boiling water for 30 s to halt digestion. The disaccharides produced during the enzymatic digestion were then analyzed via HPLC on an HPLC 616 system (Waters Co., Milford, MA) equipped with an amide column (PA-03, YMC Co. Ltd, Kyoto, Japan) using an NaH2PO4 linear gradient from 16 mM to 800 mM over 60 min at a flow rate of 1.0 mL/min. For the analysis, 100 lL of the sample solution was injected and monitored at 232 nm. References 1. Berteau, O.; Mulloy, B. Glycobiology 2003, 13, 29–40. 2. Li, B.; Wei, X.; Zhao, R. Molecules 2008, 13, 1671–1695. 3. Tako, M.; Yegara, M.; Kawashima, Y.; Chinen, I.; Hongou, F. J. Appl. Glycosci. 1996, 43, 143–148. 4. Tako, M.; Yoza, E.; Thoma, S. Bot. Mat. 2000, 43, 393–398. 5. Duarte, M. E. R.; Cardoso, M. A.; Noseda, M. D.; Cerezo, A. S. Carbohydr. Res. 2001, 333, 281–293. 6. Bilan, M. I.; Grachev, A. A.; Ustuzhanina, N. E.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2004, 339, 511–517. 7. Bilan, M. I.; Grachev, A. A.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2006, 341, 238–245. 8. Sugahara, K.; Yamada, S. Trends Glycosci. Glycotechnol. 2000, 12, 321–349. 9. Puperez, P.; Ahrazem, O.; Leal, J. A. J. Agric. Food Chem. 2002, 50, 840–845. 10. Koyanagi, S.; Tanigawa, N.; Nakagawa, H.; Soeda, S.; Shimeno, H. Biochem. Pharmocol. 2003, 65, 173–179. 11. Lake, A. C.; Vassy, R.; Di Bendetto, M.; Lavigne, D.; Le Visage, C.; Perret, G. Y.; Letourneur, D. J. Biol. Chem. 2006, 281, 37844–37852. 12. Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; Al-Hakim, A.; Gunay, N. S.; Zhang, Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R. I.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 669–675. 13. Azofeifa, K.; Angulo, Y.; Lomonte, B. Toxicon 2008, 51, 373–380. 14. Sasaki, M.; Fang, Z.; Fukushima, Y.; Adschiri, T.; Arai, K. Ind. Eng. Chem. Res. 2000, 39, 2883–2890. 15. Nagamori, M.; Funazukuri, T. J. Chem. Technol. Biotechnol. 2004, 79, 229–233. 16. Miyazawa, T.; Funazukuri, T. Carbohydr. Res. 2006, 341, 870–877. 17. Holm, K.; Davidson, L.; Kristiansen, A.; Smidsrod, O. Carbohydr. Polym. 2008, 73, 656–664. 18. Aida, T. M.; Yamagata, T.; Watanabe, M.; Smith, R. L., Jr. Carbohydr. Polym. 2010, 80, 296–302. 19. Quitain, A. T.; Kai, T.; Sasaki, M.; Goto, M. Ind. Eng. Chem. Res. 2013, 52, 7940– 7946. 20. Sato, K.; Kishimoto, T.; Morimoto, M.; Saimoto, H.; Shigemasa, Y. Tetrahedron Lett. 2003, 44, 8623–8625. 21. Sato, K.; Saimoto, H.; Morimoto, M.; Shigemasa, Y. Sen-i Gakkaishi 2003, 59, 104–109. 22. Data sheet for Chondroitinase ABC, Seikagaku Biobusiness Co. 23. Toyoda, H.; Shinomiya, K.; Yamanashi, S.; Koshiishi, I.; Imanari, T. Anal. Sci. 1988, 4, 381–384. 24. Tamura, J.; Arima, K.; Imazu, A.; Tsutumishita, N.; Fujita, H.; Yamane, M.; Matusumi, Y. Biosci. Biotechnol. Biochem. 2009, 73, 1387–1391. 25. Azuma, K.; Ishihara, T.; Nakamoto, H.; Amaha, T.; Osaki, T.; Tsuka, T.; Imagawa, T.; Minami, S.; Takashima, O.; Ifuku, S.; Morimoto, M.; Saimoto, H.; Kawamoto, H.; Okamoto, Y. Mar. Drugs 2012, 10, 2337–2348.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Depolymerization of sulfated polysaccharides under hydrothermal conditions Minoru Morimoto a,⇑, Masaki Takatori b, Tetsuya Hayashi b, Daiki Mori b, Osamu Takashima c, Shinichi Yoshida d, Kimihiko Sato e, Hitoshi Kawamoto f, Jun-ichi Tamura g, Hironori Izawa b, Shinsuke Ifuku b, Hiroyuki Saimoto b a
Division of Instrumental Analysis, Research Center for Bioscience and Technology, Tottori University, Tottori 680-8550, Japan Division of Applied Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori 680-8552, Japan Scientific Crime Laboratory, Tottori Prefectural Police Headquarters, Tottori 680-0911, Japan d Tottori Institute of Industrial Technology, Tottori 689-1112, Japan e Koyo Chemical Co. Ltd, Kita-Ku, Osaka 530-0051, Japan f Marine Products Kimuraya Co. Ltd, Sakaiminato 684-0072, Japan g Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Tottori 680-8551, Japan b c
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 21 November 2013 Accepted 27 November 2013 Available online 4 December 2013 Keywords: Hydrothermal treatment Fucoidan Chondroitin sulfate Sulfated polysaccharide Depolymerization without desulfation
a b s t r a c t Fucoidan and chondroitin sulfate, which are well known sulfated polysaccharides, were depolymerized under hydrothermal conditions (120–180 °C, 5–60 min) as a method for the preparation of sulfated polysaccharides with controlled molecular weights. Fucoidan was easily depolymerized, and the change of the molecular weight values depended on the reaction temperature and time. The degree of sulfation and IR spectra of the depolymerized fucoidan did not change compared with those of untreated fucoidan at reaction temperatures below 140 °C. However, fucoidan was partially degraded during depolymerization above 160 °C. Nearly the same depolymerization was observed for chondroitin sulfate. These results indicate that hydrothermal treatment is applicable for the depolymerization of sulfated polysaccharides, and that low molecular weight products without desulfation and deformation of the initial glycan structures can be obtained under mild hydrothermal conditions. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Several sulfated polysaccharides are known to have various biological activities. Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, is mainly composed of fucose and sulfated fucose, but also contains small amounts of mannose, galactose, glucose, xylose, uronic acids, and acetylated derivatives of these sugars.1–4 The content of these carbohydrates varies with the seaweed species.5–7 Fucoidan shows various biological activities, such as anticoagulant, antithrombotic, antiviral, antiproliferative, antifertilization, and antitumor activities, and has been used in food preparation.1,2 Chondroitin sulfate (CS) is also a well-known sulfated polysaccharide composed of repeating disaccharide units (GalNAc, N-acetylgalactosamine and GlcA, glucuronic acid) and is an important structural component of cartilage in animal bodies. Based on the position of the sulfate groups in the disaccharide units, chondroitin sulfates are classified into several subtypes: CS-O, A, C, D, E, and K, each of which has a respective repeating disaccharide; GalNAc-GlcA, GalNAc4S-GlcA, GalNAc6S-GlcA, GalNAc4S-GlcA2S, ⇑ Corresponding author. Tel.: +81 857 31 5990; fax: +81 857 31 5464. E-mail address: [email protected] (M. Morimoto). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.11.017
GalNAc4S6S-GlcA, and GalNAc4S-GlcA3S (the numbers represent the sulfated positions).8 These polysaccharides, extracted mainly from bovine trachea, porcine skin and rib cartilage, and shark cartilage, are used as drugs and supplements for the treatment of degenerative joint diseases. Other sulfated polysaccharides, such as dermatan sulfate (chondroitin sulfate B), keratan sulfate, heparan sulfate, and heparin, are also found in animal tissues, and are used as biological materials and agents based on their specific biological activities. The biological activities of these sulfated polysaccharides depend on their molecular weights and the degree of sulfation.9–13 To investigate the proposed relationship between structure and activity, it is necessary to prepare the sulfated polysaccharides with controlled molecular weights and degrees of sulfation. Furthermore, several grams of each sample are required for the studies in vivo. In general, the preparation of polysaccharides with various molecular weights is achieved via hydrolysis using enzyme or acid. However, these two methods have disadvantages. The enzyme shows high substrate specificity which strongly restricts the use. The enzyme is normally expensive and is difficult to be removed after the reaction especially in the large scale. The acid, such as HCl and H2SO4,
M. Morimoto et al. / Carbohydrate Research 384 (2014) 56–60
is cheap but the sulfate groups on the polysaccharides are sometimes hydrolyzed during the reaction. Hydrothermal treatment has been used for depolymerization of polysaccharides such as cellulose,14 starch,15 guar gum,16 and alginate.17,18 Recently the extraction and degradation of fucoidan via hydrothermal treatment were reported.19 However, there was no mention on the desulfation of the degraded fucoidan. We have reported that various compounds bearing acetal and ketal groups are easily hydrolyzed in sub-critical water under hydrothermal conditions without enzyme and acid.20 Using hydrothermal treatment, chitosan, a polymer of glucosamine, was also depolymerized to produce low molecular weight products because the glycoside of polysaccharides is chemically equivalent to an acetal.21 Interestingly, the hydrothermal treatment is effective only for acetal in the presence of ester. This result suggested that sulfated polysaccharides could be depolymerized without the hydrolysis of the sulfate under hydrothermal conditions. In this study, we optimized hydrothermal conditions for the depolymerization without desulfation of fucoidan and chondroitin sulfate as a typical sulfated polysaccharide.
57
Figure 2. GPC chromatograms of fucoidan before and after hydrothermal treatment (Method A). (a) Untreated fucoidan, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
2. Results and discussion 2.1. Hydrothermal treatment of fucoidan Reaction conditions for the hydrothermal treatment are represented schematically in Figure 1. Typical GPC chromatograms of fucoidan obtained by Methods A and B are shown in Figures 2 and 3, respectively. The GPC peaks gradually shifted to longer retention times, which indicate that the molecular weight changed as a function of the reaction time and temperature. Multiple peaks in the chromatograms were observed at 160 and 180 °C (Fig. 2d and e, respectively). The Mw and DS values of fucoidan treated under various hydrothermal conditions are summarized in Table 1. Compared to the untreated fucoidan (Mw 320,000), the Mw values gradually reduced from 123,000 to 1800 along with increasing reaction temperature from 120 to 180 °C (entries 1, 2, 4, and 5 in Table 1). Similarly, depolymerization increased along with the increased reaction time (entries 6, 8, 11, 2, 14, and 15). On the other hand, the DS values were not altered by the hydrothermal conditions, except at reaction temperatures above 160 °C (entries 5 and 6). These results indicate that fucoidan was depolymerized without desulfation under mild conditions (below 140 °C). The DS values obtained above 160 °C were larger than 0.35 which
Figure 3. GPC chromatograms of fucoidan before and after hydrothermal treatment (Method B). (a) Untreated fucoidan, treated for (b) 5 min, (c) 10 min, (d) 15 min, (e) 40 min, and (f) 60 min at 140 °C.
was the value obtained at 120 and 140 °C. Because the DS value was calculated from the S/C ratio as determined via elemental analysis, an increase in the DS value suggests that either sulfation (increase in the S content) or decarbonization (decrease in the C content) reaction occurred above 160 °C. However, sulfation is
Figure 1. Schematic representation of the hydrothermal conditions. Method A: the sample solution was heated from room temperature to the setting temperature in 10 min (20 min for 180 °C), maintained for 20 min at the reaction temperatures (120–180 °C), and cooled to room temperature in 20 min (30 min for 180 °C). Method B: the sample solution was heated at 140 °C for 10 min, maintained for the reaction time (5–60 min) at 140 °C, and cooled to room temperature in 20 min.
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Table 1 Molecular weight and degree of sulfation of fucoidan before and after hydrothermal treatments
a b c
Entry
Method
Volume (mL)
Concentration (wt %)
Temperaturea (°C)
Time (min)
Mwb
Mw/Mn
DSc
Fucoidan 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
A A A A A B B B B B B B B B B B
80 80 80 80 80 80 80 80 80 800 80 800 800 80 80 80
1 1 5 1 1 1 5 1 5 1 1 1 5 1 1 5
120 140 140 160 180 140 140 140 140 140 140 140 140 140 140 140
20 20 20 20 20 5 5 10 10 10 15 15 15 40 60 60
320,000 123,000 18,000 19,000 3200 1800 75,000 74,000 36,000 36,000 51,000 26,000 33,000 32,000 8000 6000 6000
2.2 2.8 1.7 2.3 1.3 1.2 2.5 3.2 2.5 2.5 2.4 2.7 2.2 2.3 1.3 1.2 1.7
0.35 0.35 0.35 — 0.39 0.42 0.37 — 0.35 — — — — 0.33 0.36 0.36 —
Setting temperature. Determined by gel permeation chromatography. Determined by elemental analysis.
difficult to achieve under hydrothermal conditions. Thus, it seems reasonable to conclude that decarbonization took place. The Mw values were not affected for the samples with concentrations ranging from 1 to 5 wt % (entries 2 vs 3, 6 vs 7, 8 vs 9, 12 vs 13, and 15 vs 16 in Table 1) and were reproducible within ±15% for repeated runs. On the other hand, the Mw values obtained on the 800 mL scale in a large bottle were 30–40% greater than those run on an 80 mL scale in a small bottle (Entries 10 vs 8 and 12 vs 11). The difference could be due to the difference in heating rate of the sample solution. Since 800 mL of aqueous solution has a larger heat capacity, the actual heating rate of the 800 mL scale is expected to be lower than that of the temperature inside or that of the 80 mL scale. In order to obtain a constant Mw value, we must use the same-sized bottles for the hydrothermal treatment. The IR spectra of fucoidans treated at 120–180 °C for 20 min and the untreated sample exhibited characteristic peaks at 1730, 1630, 1240, 1030, and 840 cm1 corresponding to the C@O stretching band of the acetyl group, the COO antisymmetrical stretching band of the carboxylate group of glucuronic acid, the AS@O stretching band of the sulfate group, the C–O–C symmetrical stretching band of the carbohydrate skeleton, and the C–O–S stretching band of the sulfate group, respectively (Fig. 4). The IR spectra of the products obtained at 120 and 140 °C were nearly identical to that of the untreated fucoidan. On the other hand, the IR spectra of the products obtained at 160 and 180 °C showed spectral changes and a decrease in the peak intensity near 1030 cm1 (assigned to the carbohydrate skeleton) and 1630 cm1 (assigned to the carbonyl groups). These results support the fact that deformation and decarbonization occurred above 160 °C.
Figure 4. IR spectra of fucoidan before and after hydrothermal treatment (Method A). (a) Untreated fucoidan, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
Table 2 Molecular weight and degree of sulfation of chondroitin sulfate C before and after hydrothermal treatments Entry
Temperaturea (°C)
Chondroitin sulfate C 1 A 120 2 A 140 3 A 160 4 A 180 5 A 180 6 B 140 7 B 140 8 B 140 9 B 140
2.2. Hydrothermal treatment of chondroitin sulfate To confirm the applicability of this hydrothermal treatment for the depolymerization of sulfated polysaccharides, we subjected chondroitin sulfate to similar reaction conditions. The Mw and DS values of the products obtained from chondroitin sulfate treated under the same conditions as those applied to fucoidan are listed in Table 2. A similar dependence of depolymerization on the reaction temperature and time was observed. At mild reaction temperatures below 140 °C, the DS values did not vary from that of the untreated chondroitin sulfate. Multiple peaks were observed in the GPC chromatogram treated at 160 °C. The
Method
a b c
Time (min)
Mwb
Mw/Mn
DSc
20 20 20 20 20 5 10 40 60
80,000 73,000 44,000 3500 2800 1800 55,000 50,000 29,000 20,000
1.4 1.4 1.6 1.7 1.3 1.0 1.5 1.5 1.5 1.4
0.95 0.93 0.92 0.84 0.39 2.26 0.95 0.96 0.92 0.92
Setting temperature. Determined by gel permeation chromatography. Determined by elemental analysis.
peak shifted to the lowest molecular weight at 180 °C (Fig. 5). Some changes in the IR spectrum of chondroitin sulfate treated at 160 °C were observed at 600 cm1 and 1100 cm1 compared
M. Morimoto et al. / Carbohydrate Research 384 (2014) 56–60
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Figure 5. GPC chromatograms of chondroitin sulfate C before and after hydrothermal treatment (Method A). (a) Untreated chondroitin sulfate C, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
Figure 7. HPLC chromatograms of Chondroitinase ABC digested chondroitin sulfate C treated under hydrothermal conditions. (a) Untreated chondroitin sulfate C, treated at (b) 120 °C, (c) 160 °C, and (d) 180 °C for 20 min. The elution times of unsaturated disaccharides are determined by their standard samples.
conclusion that the depolymerization occurred only at lower reaction temperatures without deformation of the disaccharide units. 3. Conclusion
Figure 6. IR spectra of chondroitin sulfate C before and after hydrothermal treatment (Method A). (a) Untreated chondroitin sulfate C, treated at (b) 120 °C, (c) 140 °C, (d) 160 °C, and (e) 180 °C for 20 min.
with those of unreacted polysaccharide and the chondroitin sulfate treated below 140 °C (dashed circle in Figure 6). Furthermore, drastic spectral change was observed at 180 °C. Therefore, the depolymerization of chondroitin sulfate under hydrothermal conditions gave nearly the same results as that of fucoidan.
We have demonstrated the depolymerization of fucoidan and chondroitin sulfate under hydrothermal conditions without enzyme and acid. The Mw value was controlled by the reaction temperature and time. Under mild conditions, sulfated polysaccharides with controlled molecular weights but a constant degree of sulfation were obtained. This method can also be employed for the preparation of several grams of polysaccharide samples. We have, in fact, already investigated the effect on tumor-bearing mice of the oral administration of fucoidan samples prepared using this method.25 Furthermore, in addition to fucoidan and chondroitin sulfate, the hydrothermal treatment proposed in this study should be applicable to the depolymerization of various other sulfated polysaccharides.
2.3. Sulfation pattern of chondroitin sulfate 4. Experimental Both untreated and hydrothermally treated chondroitin sulfate were digested by chondroitinase ABC to unsaturated disaccharides22 that correspond to repeating disaccharides found in the polysaccharide. The unsaturated saccharides with different sulfation patterns were assigned by HPLC using authentic standards.23,24 The HPLC patterns of the unsaturated disaccharides obtained from enzymatically digested chondroitin sulfate treated under various hydrothermal conditions are shown in Figure 7. As expected, untreated chondroitin sulfate exhibited a main peak at 24 min, which was assigned to DDi-6S. Chondroitin sulfate treated at 140 °C gave nearly the same peak pattern and area. This result indicates that neither transfer of the sulfate group nor desulfation occurred under mild reaction conditions, whereas no disaccharide peak derived from chondroitin sulfate was observed in chromatograms treated at 160 and 180 °C. This finding also supports the
4.1. Materials Fucoidan powder extracted from Cladosiphon okamuranus Tokida was provided by Marine Products Kimuraya Co. Ltd (Sakaiminato, Japan). Analysis via inductively coupled plasmaatomic emission spectroscopy (ICP-AES) confirmed that the fucoidan existed as its sodium salt. Chondroitin sulfate (Chondroitin sulfate C sodium salt) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Chondroitinase ABC (EC 4.2.2.4), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)D-galactose (DDi-0S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4enepyranosyluronic acid)-6-O-sulfo-D-galactose (DDi-6S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid) -4-O-sulfo-D-galactose (DDi-4S), 2-acetamido-2-deoxy-3-O-(2-O-
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sulfo-b-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DDi-diSD), and 2-acetamido-2-deoxy-3-O-(2-O-sulfob-D-gluco4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose (DDi-triS) were obtained from Seikagaku Biobusiness Co. (Tokyo, Japan). The water used in this study was purified with a water purification system (Millipore Elix 3). All other chemicals were analytical grade and were used without further purification. 4.2. Hydrothermal treatment Fucoidan and chondroitin sulfate were each dissolved in purified water with gentle shaking, and the concentration was adjusted to 1 or 5 wt %. The aqueous solutions (80 mL or 800 mL) were then placed, respectively, in 100 mL volumetric bottles (56 / mm 100 mm) or 1000 mL volumetric bottles (101 /mm 225 mm) that were sealed using screw caps. The hydrothermal treatment of the samples was performed in an airtight autoclave (HTP-50/250, Hisaka Works Ltd, Osaka, Japan). Based on the temperature–time program, sample solution in the bottle was heated from room temperature to the setting temperature (reaction temperature) in 10–20 min, and the temperature was maintained for 5–60 min and then the solution was cooled to room temperature in 20–30 min. A schematic representation of these reaction conditions is shown in Figure 1. In Method A, the reaction temperature was set in 120–180 °C for a constant reaction time of 20 min. In Method B, the reaction time was set in 5–60 min at a constant reaction temperature of 140 °C. After completion of the hydrothermal treatment, the solutions were divided into two parts. One part was lyophilized without further workup to give a brown powder. The other was dialyzed for one week using a dialysis membrane (Spectra/Por CE dialysis tubing, MWCO: 500) against purified water to remove the eliminated sulfate salts, and then the solution was lyophilized. The dialyzed samples were used for elemental and IR analyses. 4.3. Molecular weight The molecular weight distributions of the sulfated polysaccharides were analyzed using gel permeation chromatography (GPC) on connecting GPC columns: TSKgel PWXL + TSKgel PWXL (Tosoh CO., Tokyo, Japan) or Shodex Asahipak GS-520HQ + GS320HQ + GS-220HQ (Shoko Co. Ltd, Tokyo, Japan). The mobile phase was 0.1 M NaNO3 at a flow rate of 0.5 mL/min at 40 °C. The lyophilized samples without dialysis were dissolved in the mobile phase (10 mg/mL) and injected (100 lL). GPC peaks were detected with a refractive index (RI) detector (SHIMADZU RID-10, Shimadzu Co., Kyoto, Japan). The weight-average molecular weight (Mw), the number-average molecular weight (Mn), and the polydispersity (Mw/Mn) were calculated using molecular weight calculation software connected to the HPLC integration system (HITACHI D-7000, Hitachi Co., Tokyo, Japan) and a pullulan standard kit (Shodex P-82, Shoko Co., Tokyo, Japan). 4.4. Degree of sulfation The degree of sulfation (DS) of sulfated polysaccharides was calculated via elemental analysis. For this calculation, we assumed that all of the carbohydrate residues of the sulfated polysaccharides used in this study were hexose. Based on this assumption, the DS values were calculated using the following formula:
Sð%Þ=Cð%Þ ¼ ð32:06 DSÞ=ð12:01 6Þ: For the elemental analyses, the dialyzed samples were used.
4.5. IR analysis Infrared (IR) spectra of the dialyzed samples were measured on a Fourier transform IR spectrometer (SHIMADZU FTIR-8700, Shimadzu Co., Kyoto, Japan) in KBr pellets. 4.6. Sulfation pattern of chondroitin sulfate The sulfation patterns of the chondroitin sulfate after hydrothermal treatment were determined using the disaccharide method: enzymatic digestion of the chondroitin sulfates followed by high performance liquid chromatography (HPLC) analysis. The chondroitin sulfate (200 lg) was dissolved in a mixture of water (200 lL), a 0.01% (w/v) bovine serum albumin (BSA) solution (20 mL), and a 250 mM Tris–HCl buffer (40 mL). Chondroitinase ABC (5 U/mL, 100 lL) was then added to the solution, and the mixture was incubated at 37 °C for 8 h. The reaction solution was then heated in boiling water for 30 s to halt digestion. The disaccharides produced during the enzymatic digestion were then analyzed via HPLC on an HPLC 616 system (Waters Co., Milford, MA) equipped with an amide column (PA-03, YMC Co. Ltd, Kyoto, Japan) using an NaH2PO4 linear gradient from 16 mM to 800 mM over 60 min at a flow rate of 1.0 mL/min. For the analysis, 100 lL of the sample solution was injected and monitored at 232 nm. References 1. Berteau, O.; Mulloy, B. Glycobiology 2003, 13, 29–40. 2. Li, B.; Wei, X.; Zhao, R. Molecules 2008, 13, 1671–1695. 3. Tako, M.; Yegara, M.; Kawashima, Y.; Chinen, I.; Hongou, F. J. Appl. Glycosci. 1996, 43, 143–148. 4. Tako, M.; Yoza, E.; Thoma, S. Bot. Mat. 2000, 43, 393–398. 5. Duarte, M. E. R.; Cardoso, M. A.; Noseda, M. D.; Cerezo, A. S. Carbohydr. Res. 2001, 333, 281–293. 6. Bilan, M. I.; Grachev, A. A.; Ustuzhanina, N. E.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2004, 339, 511–517. 7. Bilan, M. I.; Grachev, A. A.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2006, 341, 238–245. 8. Sugahara, K.; Yamada, S. Trends Glycosci. Glycotechnol. 2000, 12, 321–349. 9. Puperez, P.; Ahrazem, O.; Leal, J. A. J. Agric. Food Chem. 2002, 50, 840–845. 10. Koyanagi, S.; Tanigawa, N.; Nakagawa, H.; Soeda, S.; Shimeno, H. Biochem. Pharmocol. 2003, 65, 173–179. 11. Lake, A. C.; Vassy, R.; Di Bendetto, M.; Lavigne, D.; Le Visage, C.; Perret, G. Y.; Letourneur, D. J. Biol. Chem. 2006, 281, 37844–37852. 12. Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; Al-Hakim, A.; Gunay, N. S.; Zhang, Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R. I.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 669–675. 13. Azofeifa, K.; Angulo, Y.; Lomonte, B. Toxicon 2008, 51, 373–380. 14. Sasaki, M.; Fang, Z.; Fukushima, Y.; Adschiri, T.; Arai, K. Ind. Eng. Chem. Res. 2000, 39, 2883–2890. 15. Nagamori, M.; Funazukuri, T. J. Chem. Technol. Biotechnol. 2004, 79, 229–233. 16. Miyazawa, T.; Funazukuri, T. Carbohydr. Res. 2006, 341, 870–877. 17. Holm, K.; Davidson, L.; Kristiansen, A.; Smidsrod, O. Carbohydr. Polym. 2008, 73, 656–664. 18. Aida, T. M.; Yamagata, T.; Watanabe, M.; Smith, R. L., Jr. Carbohydr. Polym. 2010, 80, 296–302. 19. Quitain, A. T.; Kai, T.; Sasaki, M.; Goto, M. Ind. Eng. Chem. Res. 2013, 52, 7940– 7946. 20. Sato, K.; Kishimoto, T.; Morimoto, M.; Saimoto, H.; Shigemasa, Y. Tetrahedron Lett. 2003, 44, 8623–8625. 21. Sato, K.; Saimoto, H.; Morimoto, M.; Shigemasa, Y. Sen-i Gakkaishi 2003, 59, 104–109. 22. Data sheet for Chondroitinase ABC, Seikagaku Biobusiness Co. 23. Toyoda, H.; Shinomiya, K.; Yamanashi, S.; Koshiishi, I.; Imanari, T. Anal. Sci. 1988, 4, 381–384. 24. Tamura, J.; Arima, K.; Imazu, A.; Tsutumishita, N.; Fujita, H.; Yamane, M.; Matusumi, Y. Biosci. Biotechnol. Biochem. 2009, 73, 1387–1391. 25. Azuma, K.; Ishihara, T.; Nakamoto, H.; Amaha, T.; Osaki, T.; Tsuka, T.; Imagawa, T.; Minami, S.; Takashima, O.; Ifuku, S.; Morimoto, M.; Saimoto, H.; Kawamoto, H.; Okamoto, Y. Mar. Drugs 2012, 10, 2337–2348.