Polysaccharides from Sargassum thunbergii: Monthly variations and anti-complement and anti-tumour activities

Accepted Manuscript Title: Polysaccharides from Sargassum thunbergii: Monthly variations and anti-complement and anti-tumour activities Authors: Weihua Jin, Ge Liu, Weihong Zhong, Chaomin Sun, Quanbin Zhang PII: DOI: Reference:

S0141-8130(17)31013-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.05.104 BIOMAC 7593

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International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

19-3-2017 8-5-2017 16-5-2017

Please cite this article as: Weihua Jin, Ge Liu, Weihong Zhong, Chaomin Sun, Quanbin Zhang, Polysaccharides from Sargassum thunbergii: Monthly variations and anti-complement and anti-tumour activities, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.05.104 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Polysaccharides from Sargassum thunbergii: Monthly variations and anti-complement and anti-tumour activities Weihua Jina, b, Ge Liub, c, Weihong Zhonga, Chaomin Sunb, d* and Quanbin Zhangb, d* a

College of Biotechnology and Bioengineering, Zhejiang University of Technology,

Hangzhou 310014, PR China b

Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese

Academy of Sciences, Qingdao 266071, PR China c

University of Chinese Academy of Sciences, Beijing 100049, PR China

d

Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for

Marine Science and Technology, Qingdao 266071, PR China *

Corresponding author. Tel.: +86 532 82898857; fax: +86 532 82898857.

E-mail address: [email protected] *

Corresponding author. Tel.: +86 532 82898703; fax: +86 532 82898703.

E-mail address: [email protected] Abstract: Monthly variations of polysaccharides from Sargassum thunbergii and their anti-complement and anti-tumour activities were investigated. It was observed that an increase in fucose and total sugar contents occurred during the growth period (from early April to mid-June), accompanied by a decrease in molar ratios of other monosaccharides to fucose. The highest yields were obtained from early July to early September, which was in accordance with the significant increase in molar ratio of glucose to fucose and decrease in molar ratio of other monosaccharides to fucose. And the above results suggested that S. Thunbergii synthesized large amount of

laminaran, the storage substance of brown algae, during the senescence period. However, sulfate contents were relatively stable in the life cycle of S. thunbergii. These results suggested that S. thunbergii synthesized complex sulfated heteropolysacchairdes during inactive period, while during other periods, it synthesized more sulfated galactofucan. All polysaccharides showed anti-complement activity, suggesting that the harvesting time did not influence the anti-complement activities. In the anti-tumour assay in vitro, the polysaccharides taken during the senescence period had much lower anti-tumor activity, suggesting that fucoidan, but not laminaran, determined the anti-tumor activities.Therefore, polysaccharides from S. thunbergii might have great potential in anti-complement and anti-tumor application.

Key words: Sargassum thunbergii; anti-complement activities; anti-tumour activities

1. Introduction Sargassum thunbergii is a brown alga that is widely distributed in the seas of Japan and China. This organism is usually used as fish bait for sea cucumber and abalone. In addition, S. thunbergii has been documented in China’s Marine Medicine Dictionary due to its ability to resolve hard lumps and diuresis detumescence. Research on S. thunbergii has focused mainly on polyphenols, phlorotannins, isopentadiene, quinone, isofucosterol, tetraprenyltoluquinols, monogalactosyl diacylglycerols, polyunsaturated fatty acids and crude extracts [1-11]. Polysaccharides from S. thunbergii are reported less often. Itoh et al. [12-13] and Zhuang et al. [14] found that polysaccharides (GIV-A and GIV-B) from S. thunbergii, consisting of fucose, uronic acid and sulfate, had anti-tumour activities. Jin et al. [15]

reported that a crude polysaccharide from S. thunbergii possessed neuroprotective and antioxidant activities. Yuan et al. [16] also reported that the polysaccharide (STP-II) from S. thunbergii had antioxidant and inhibitory activities in human colon cancer Caco-2 cells in vitro. In addition, the structure of STP-II was also elucidated [17]. There are many reviews [1, 18-30] regarding polysaccharides from brown algae. They carry out various biological activities, such as anticoagulant, antioxidant, anticancer, neuroprotective and immunomodulatory activities, among others. The structures of polysaccharides are complex. They mainly contain fucose and sulfate, accompanied by galactose, manose, xylose, glucuronic acid and others. Thus, it was difficult to obtain a distinct and reliable conclusion about the relationship between polysaccharide structure and activities. Many factors were considered [1, 21, 24, 29, 31-34], such as molecular weight, branching, sulfate group (content and location), method of administration, species, fractionations, extraction methods, monosaccharides, collection area and glycosidic linkages. In this study, 19 different polysaccharides from S. thunbergii were prepared to elucidate the relationship between harvesting time and anti-tumour and anti-complementary activities.

2. Materials and methods Sargassum thunbergii was collected in Qingdao, China. The fresh seaweeds were sun dried. 2.1 Extraction of polysaccharides Crude polysaccharides were extracted from the decoloured algae residues by hot water. Briefly speaking, algae (100 g) were cut into pieces, then, decoloured with 85 % ethanol (2 L) for 24 h three times. Crude polysaccharides were extracted from the decoloured algae residues with hot

water (3 L) for 4 h. The extract solutions were filtered with celite and concentrated. Further elimination of alginate was achieved using 20 % ethanol with MgCl2 (0.05 mol/L). After removal of the alginate, the supernatant fluid was dialyzed with running water for 24 h and distilled water for 24 h. Finally, the dialysate was concentrated, and the crude polysaccharides were obtained using ethanol precipitation. 2.3 Compositional analysis Chemical compositions of crude polysaccharides were elucidated from the sulfated content, molar ratio of monsaccharides, fucose content, total sugar content and uronic acid (UA) content. The sulfated content was used to perform ion chromatography on a Shodex IC SI-52 4E column (4.0 × 250 mm) eluted with 3.6 mM Na2CO3 at a flow rate of 0.8 mL/min at 45°C. Calculation of the molar ratio of monosaccharide composition was followed as described by Zhang et al. [35]. Briefly, polysaccharides (10 mg/mL) were hydrolysed by trifluoroacetic acid (2 M) under a nitrogen atmosphere for 4 h at 110°C. Then, the hydrolysed mixture was neutralized to pH 7 with sodium hydroxide. Later, the mixture was converted into its 1-phenyl-3-methyl-5-pyrazolone derivatives and separated by HPLC chromatography on a YMC Pack ODS AQ column (4.6 × 250 mm). The content of fucose was determined according to the modified method [36], using fucose as a standard. UA was determined using a modified carbazole method [37], using glucuronic acid as a reference. Finally, the content of total sugar was determined according to the modified phenol-sulfuric acid method [38], using fucose as a standard. 2.4 Anti-complement activities The anti-complement activities of the polysaccharides were determined with the classical pathway using the methods described in previous studies [39]. For the classical pathway, various

dilutions (100 μL) of polysaccharides were mixed with 1:10 diluted normal human serum (NHS, obtained from healthy adult donors) (100 μL), GVB2+ (veronal buffer saline [VBS] containing 0.1 % gelatin, 0.5 mM Mg2+ and 0.15 mM Ca2+) (200 μL) and sensitized erythrocytes (EA) (200 μL). Then, the mixtures were incubated at 37°C for 30 min. The following assay controls were incubated under the same conditions: (1) 100 % lysis: EA (200 μL) in water (400 μL); (2) sample control: sample (100 μL) in GVB2+ (500 μL); (3) complement: 1:10-diluted NHS (100 μL) and EA (200 μL) in GVB2+ (300 μL); and (4) blank: EA (200 μL) in GVB2+ (400 μL). After incubation, the mixture was centrifuged (5000 rpm × 10 min) and the erythrocyte lysis was determined at 405 nm. Decreased lysis in the presence of the tested polysaccharides indicated anti-complement activities. All of the samples were dissolved in GVB2+. The percent inhibition was calculated using the following equation: inhibition of EA lysis (%) = (Acomplement – [Asample– Asample control]) / Acomplement × 100. 2.5 Anti-tumour activities Anti-tumour activities of polysaccharides from Sargassum thunbergii (STW) against human lung cancer A549 cell were determined. A 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium (MTT) assay was used to measure cell viability. Briefly, A549 cells were cultured in RPMI 1640 medium containing 10 % foetal bovine serum and penicillin-streptomycin (100 units/mL) in an atmosphere of 5 % CO2 at 37°C. The cells were then seeded in a 96-well plate at a density of 4 × 103 cells/well for 24 h. Subsequently, the cells were divided into the following three groups: (1) blank group which only contained medium for 24 h, (2) control group in which cells were added for 24 h and (3) experimental groups in which cells and polysaccharides at different concentrations (1, 0.5 and 0.25 mg/mL) were cultivated in medium for 24 h. After removal of the

media, 20 µL of MTT (5 mg/mL) was added to each well. After 4 h of incubation, the supernatants were removed, and dimethyl sulfoxide (DMSO) (150 µL) was added. Next, the absorbance was measured at 490 nm and the inhibition rate was determined using the following equation: Cell Inhibition rate (%) = (A1−A0)/(Ac−A0)×100, where A0 was the absorbance of the blank, A1 was the absorbance in the presence of samples, and Ac was the absorbance of the control. 3. Results and discussion 3.1 Extraction of polysaccharides It is shown in Fig. 1 that yields of crude polysaccharides from S. thunbergii fluctuated between 0.5 % and 1.5 % from November to June. Then, they increased significantly and peaked at 3.42 % on July 15. Later, they decreased. Finally, they remained relatively constant between 0.5 % – 1.5 % from September to November. According to observations of the algal growth, S. thunbergii started to grow leaves on March 16. Later, it began to grow rapidly. On July 15, it had the largest size. Finally, there were no blades on September 15. According to the previous study [40], it was reported that the growth pattern of S. thunbergii (in Luyang Bay, Yantai, China) might be divided into four phases: inactive period (before March), growth period (from early April to mid-June), reproductive period (from mid-June to late-July), and senescence period (from late-July to September). Therefore, it was concluded that the changes in crude polysaccharide yields were related to the life cycle of the algae. In addition, the change in the life cycle of S. thunbergii might result in physiological and chemical changes. Thus, the amounts of total sugar, fucose (Fuc), uronic acid (UA) and sulfate were determined as shown in Fig. 2.

The sulfate content fluctuated between 7.66 % and 15.17 %. Compared to those of total sugar, Fuc and UA, the changes in sulfate content remained stable. However, a previous study [41] showed that the molar content of sulfate residues of fucan fraction C from Laminaria japonica increased from April to September, but then decreased steeply in October. It was also reported by Mak et al. [42] that the sulfate content of blade-derived polysaccharides from Undaria pinnatifida from farm 327 remained constant from July to September, while the sulfate content of polysaccharides from farm 106 increased significantly between July and September, and then significantly decreased in October. The trend of fucose content was similar to that of sulfate content from November to March, while it was not similar from April to August. Specifically, the fucose content fluctuated between 14.21 % and 24.19 % from November to March. Then, it increased significantly from March 16 to May 1. Later, it decreased slowly from May to October. It was observed that S. thunbergii began to grow leaves on March 16. Thus, it was speculated that the fucose content was significantly affected by the life cycle. Mak et al. [42] reported that the fucose content of bladed-derived polysaccharides from Undaria pinnatifida from farms 327 and 106 significantly decreased between July and October. However, Skriptsova et al. [43] found that the fucose content of sporophyll-derived polysaccharides from Undaria pinnatifida had no significant changes during sporogenesis. Polysaccharides from brown algae were composed mainly of fucose and sulfate with substantial amounts of other monosaccharides, such galactose, mannose and xylose. Sulfate mainly occurred with fucose, with little sulfated galactose, mannose or xylose. Combining the fucose and sulfate content results, it was determined that the molar ratio of sulfate to fucose

remained relatively stable, but decreased during the growth reproductive periods. Uronic acid content remained stable, between 11.74 % and 16 %, from November to June. Then, it decreased from 18 % on June 16 to 2.1 % on July 31. Later, it increased to 13.64 % on September 1. Finally, it kept stable at approximately 13 %. It was reported [42] that changes in uronic acid content in polysaccharides from U. pinnatifida might be linked to algal maturity. In addition, it was postulated that the decrease in UA from sporophyll-derived polysaccharides may be the result of maturation of U. pinnatifida, and the increase in UA from blade-derived polysaccharides may be associated with blade degradation. Based on observations of algal growth, uronic acid content was in accordance with the above speculation. With respect to the content of total sugar, the trend was similar to the fucose content from November to June. However, total sugar content peaked during the reproductive period. Table 1 shows the monthly changes in the molar ratios of monosaccharides of crude polysaccharides from S. thunbergii. It was reported [44-50] that in addition to sulfated fucan, polysaccharides from brown algae also contain sulfated fucogalactan, sulfated glucuronomannan, fucoglucuronan, xylan and galactan. The trend in the molar ratio of mannose to fucose was similar to that of glucuronic acid, indicating that crude polysaccharides from S. thunbergii might contain glucuronomannan and glucuronan. During the senescence period, crude polysaccharides contained the lowest mannose and glucuronic acid contents. The trend in the molar ratio of rhamnose to fucose was not considered because there was not enough content. The most obvious change was in the molar ratios of glucose to fucose. Compared to the molar ratios of galactose and xylose, the change in the molar ratios of glucose to fucose was just the opposite. Specifically, the molar ratios of glucose to fucose were the greatest from July to

September. It was reported [41, 46, 51] that glucose in crude polysaccharides is derived from laminaran, which is a storage glucan for the maturation of alga. The molar ratios of galactose to fucose decreased gradually from November to June and increased slowly from July to September. In other words, the molar ratios of galactose to fucose were the lowest during maturation. This phenomenon also occurred in the molar ratios of xylose to fucose. It was shown that C. costata synthesized a low molecular weight (20-300 kDa) sulfated and acetylated galactofucan in adult seaweed, accompanied by a small amount of laminaran. However, during other life stages, it may synthesize a high molecular weight (200-800 kDa) low sulfated heterofucan [51]. Later, it was reported [52] that polysaccharides from vegetative alga had heterogeneous monosaccharide compositions. Thus, it was concluded that the chemical compositions were influenced by the life cycle of S. thunbergii. Specifically, during the reproductive period, S. thunbergii synthesized sulfated galactofucan and laminaran, accompanied by fewer other sugars. However, it synthesized heteropolysaccharides, including glucuronomannan and fucoglucuronan during other periods. The IR spectrum of a sample (Jul 15) is shown in Fig. 3. The IR spectra of other crude fucoidans were similar to each other. Thus, it was concluded that the crude polysaccharides obtained at different times had the same functional groups, indicating that the functional groups were not affected by the season of harvest. The intense and broad band at 1251 cm−1 was attributed to asymmetric O=S=O stretching vibration of sulfate esters with some contribution from COH, CC and CO vibrations. According to previous studies [53-54], it was reported that the band at 830 cm−1 was assigned to the C-O-S bending vibration of sulfate substituents at the equatorial

C-2 position, suggesting that the sulfation patterns were mainly at C-2. In addition, the absence of a band at 845-850 cm-1, which was attributed to sulfate groups at the axial C-4 position, indicated that there was no or less sulfation at C-4. Anti-complement activities were assessed in the classical pathway as shown in Fig. 4. The complement groups displayed 98.95 ± 1.02 % activation in the classical pathway. The anti-complement activities of the polysaccharides showed dosage-dependent responses. However, all polysaccharides reached a plateau of anti-complement activity at the approximately 10 μg/mL, indicating that the harvesting time of fucoidans did not influence their activities. In a previous study [55], it was reported that sulfated fucan and/or sulfated galactofucan had good anti-complement activities, which might explain why the harvesting time of polysaccharides did not influence the activities. The active components of all polysaccharides were sulfated fucan or sulfated galactofucan, which may have kept stable because the sulfate content remained stable. It was reported [56-58] that sulfate plays an important role in anti-complement activities. Thus, it was concluded that the harvesting time of polysaccharides did not influence their anti-complement activities. Anti-tumour activities of polysaccharides were assessed as shown in Fig. 5. The anti-tumour activities of polysaccharides showed dosage-dependent responses and all polysaccharides reached a plateau at 1000 μg/mL, indicating that harvesting time did not influence activities at the concentration of 1000 μg/mL. However, it is interesting to note that samples (Jul 31, Aug 15 and Sep1), which were collected during the senescence period, showed different patterns at the concentrations of 250 and 500 μg/mL. A sample taken Jul 31 had the lowest activities, followed by samples taken Aug 15 and Sep 1 at the concentrations of 250 and 500 μg/mL. Specifically, at the

concentration of 250 μg/mL, the Jul 31 sample had approximately 27 % inhibition, while samples from Aug 15 and Sep 1 had approximately 50 % inhibition. Still other samples had approximately 80 % inhibition. At the concentration of 500 μg/mL, sample on Jul 31 had approximately 50 % inhibition, while other samples had approximately 90 % inhibition. Further studies are needed to explain this phenomenon. Therefore, it was concluded that the anti-tumor activities depended on the harvesting time and concentration of polysaccharides.

4 Conclusions This study showed that S. thunbergii had an increase in crude polysaccharides yields from June to August, which may be related to the maturation of the algae. Hence, it could be collected in July to August to get the highest amount of crude polysaccharides. The growth period had the highest fucose content, while the senescence period had the lowest UA content. The sulfate content kept relatively stable. Analysis of the molar ratios of monosaccharides indicated that the senescence period had the lowest molar ratios of all monosaccharides tested, except fucose and glucose, suggesting that it had sulfated fucan/galactofucan and laminaran. During other periods, in addition to sulfated fucan/galactofucan and laminaran, polysaccharides from S. thunbergii had glucuronomannan

and

fucoglucuronan.

Additionally,

anti-complement

activities

of

polysaccharides were determined. It was shown that harvesting time did not influence anti-complement activities. Therefore, polysaccharides from S. thunbergii might be good candidates for suppressing complement activation. Finally, anti-tumour activities were also examined. It was shown that harvesting time and the concentration of polysaccharides influenced anti-tumour activities.

Acknowledgements This study was supported by the National Natural Science Foundation of China (41506165), Shandong Provincial Natural Science Foundation, China (ZR2014DQ024), Science and Technology program of applied basic research projects of Qingdao Municipal (15-9-1-74-jch), the Ocean Public Welfare Scientific Research Project, and the State Oceanic Administration of the People’s Republic of China (No. 201405040).

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Figure captions: Fig. 1 The relationship between harvesting time and yields of crude polysaccharides from S. thunbergii. Fig. 2 The relationship between harvesting time and Fuc, UA and sulfate contents of crude polysaccharides from S. thunbergii. Fig. 3 IR spectrum of a sample (Jul 15). Fig. 4 Inhibition of the classical pathway-mediated haemolysis of EA in 1:10-diluted NHS in the presence of increasing amounts of polysaccharides from S. thunbergii. The results are expressed as percent inhibition of haemolysis. Data represent the means from three determinations +/- S.E.M. Fig. 5 Anti-tumor activities of polysaccharides from Sargassum thunbergii (STW) against human lung cancer A549 cell. The results are expressed as percent inhibition of cell viability. Data represent the means from three determinations +/- S.E.M.

Figures

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Tables Table 1 Monthly change in molar ratios of monosaccharides of crude polysaccharides from S. thunbergii. Samples

Man

Rha

Monosaccharides (molar ratio) Glc A Glc Gal

Xyl

Fuc

Oct 31

0.40±0.09**

0.09±0.02**

0.31±0.08**

0.29±0.10**

0.54±0.05**

0.53±0.19**

1

Nov 15

0.36±0.05**

0.03±0.01

0.38±0.07**

0.31±0.12**

0.51±0.09**

0.50±0.09**

1

Dec 1

0.38±0.08**

0.02±0.01

0.39±0.10**

0.22±0.06**

0.42±0.17*

0.74±0.19**

1

Dec 15

0.40±0.03**

0.08±0.02**

0.41±0.04**

0.20±0.06**

0.42±0.04*

0.61±0.14**

1

Dec 31

0.32±0.11**

0.01±0.00

0.29±0.05**

0.32±0.09**

0.48±0.07**

0.55±0.07**

1

Jan 15

0.27±0.10**

0.10±0.03**

0.23±0.06**

0.13±0.03**

0.45±0.03*

0.43±0.16**

1

Feb 23

0.29±0.11**

0.08±0.02

0.30±0.08**

0.18±0.10**

0.43±0.06*

0.48±0.12**

1

Mar 16

0.19±0.07*

0.07±0.03**

0.14±0.04

0.17±0.09**

0.38±0.10

0.39±0.13**

1

Apr 15

0.20±0.05*

0.11±0.04**

0.15±0.02

0.15±0.03**

0.36±0.01

0.36±0.10*

1

May 1

0.16±0.05

0.05±0.03

0.18±0.03

0.05±0.02**

0.25±0.03

0.34±0.07*

1

May 16

0.15±0.02

0.08±0.02**

0.12±0.01

0.07±0.03**

0.28±0.08

0.31±0.02*

1

Jun 3

0.17±0.03

0.05±0.01

0.16±0.03

0.04±0.01**

0.19±0.05

0.35±0.04*

1

Jun 16

0.15±0.02

0.06±0.01*

0.12±0.02

0.28±0.05*

0.28±0.13

0.27±0.07

1

Jul 15

0.07±0.02

0.01±0.01

0.09±0.05

1.24±0.22

0.26±0.07

0.12±0.06

1

Jul 31

0.06±0.03

0.01±0.01

0.06±0.01

1.46±0.19**

0.27±0.15

0.10±0.09

1

Aug 15

0.09±0.01

0.02±0.01

0.06±0.03

2.27±0.31**

0.27±0.11

0.13±0.11

1

Sep 1

0.20±0.05**

0.04±0.02

0.26±0.08**

0.64±0.19**

0.35±0.10

0.31±0.13*

1

Sep 15

0.32±0.10**

0.06±0.01*

0.33±0.12**

0.36±0.16**

0.44±0.08*

0.43±0.07**

1

Oct 2

0.37±0.11**

0.15±0.06**

0.32±0.09**

0.30±0.10**

0.45±0.12*

0.58±0.10**

1

Values are depicted as means ± SD (n=3). * P<0.05 compared to the Sample (Jul 15). ** P<0.01 compared to the Sample (Jul 15).