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Structure and hypolipidaemic activity of fucoidan extracted from brown seaweed Sargassum henslowianum a

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Ho Duc Cuong , Thanh Thi Thu Thuy , Tran Thu Huong , Bui Minh c

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Ly & Tran Thi Thanh Van a

School of Chemical Engineering, Hanoi University of Science and Technology, Ha Noi, Viet Nam b

Institute of Chemistry, Vietnam Academy of Science and Technology, Ha Noi, Viet Nam c

Nhatrang Institute of Technology Research and Application, Vietnam Academy of Science and Technology, Nha Trang, Viet Nam Published online: 21 Aug 2014.

To cite this article: Ho Duc Cuong, Thanh Thi Thu Thuy, Tran Thu Huong, Bui Minh Ly & Tran Thi Thanh Van (2014): Structure and hypolipidaemic activity of fucoidan extracted from brown seaweed Sargassum henslowianum, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.948436 To link to this article: http://dx.doi.org/10.1080/14786419.2014.948436

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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.948436

Structure and hypolipidaemic activity of fucoidan extracted from brown seaweed Sargassum henslowianum Ho Duc Cuonga*, Thanh Thi Thu Thuyb, Tran Thu Huonga, Bui Minh Lyc and Tran Thi Thanh Vanc

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School of Chemical Engineering, Hanoi University of Science and Technology, Ha Noi, Viet Nam; Institute of Chemistry, Vietnam Academy of Science and Technology, Ha Noi, Viet Nam; cNhatrang Institute of Technology Research and Application, Vietnam Academy of Science and Technology, Nha Trang, Viet Nam b

(Received 18 February 2014; final version received 22 July 2014) The aim of this study is to elucidate the structure and investigate the hypolipidaemic activity of a fucoidan extracted from brown seaweed Sargassum henslowianum collected at Hai Van – Son Cha peninsula, Hue province, Vietnam by using tandem electrospray ionisation mass spectrometry. The results demonstrated that the fucoidan has a(1 ! 3)-linked L -fucopyranose backbone and sulphate groups occupied mostly at C-2, C-4 and sometimes at C-3 position of fucose residues. The results of in vivo bioactivity examination revealed that the fucoidan in the dose of 100 mg/kgP/day by oral administration helped decrease cholesterol, triglyceride and LDL-cholesterol levels on obese mice. Keywords: Fucoidan; brown seaweed Sargassum henslowianum; structure; hypolipidaemic activity

1. Introduction Fucoidans are sulphated polysaccharides derived from marine brown seaweed. They essentially contain fucose and sulphate groups and with some others, such as galactose, xylose, mannose and uronic acids. Fucoidan is made up of a-L -fucose units linked by (1 ! 4) and (1 ! 3) glycosidic bonds and sulphated at positions 2 and/or 3 and/or 4 (Chevolot et al. 2001; Li et al. 2008). This structural complexity, chemical composition of fucoidans may vary depending on the algae source and method of extraction. Fucoidans were reported to possess various biological effects in vitro and in vivo such as anti-inflammatory, anticoagulant, antithrombotic (Cumashi et al. 2007), antiviral including antiHIV (Lee et al. 2004; Trinchero et al. 2009), immunomodulatory (Raghavendran et al. 2011), antioxidant (Wang et al. 2010) and antitumour (Synytsya et al. 2010). The aim of this study is to elucidate the structure and investigate the hypolipidaemic activity of a new fucoidan extracted from brown seaweed Sargassum henslowianum collected at Hai Van – Son Cha peninsula, Hue province, Vietnam. It was reported (Ale et al. 2011) that an unfractionated fucoidan from S. henslowianum may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3, the sugar compositional analysis indicated that this fucoidan was mainly made up of fucose and minor amounts of other monosaccharides, mainly galactose and mannose. Recently, chemical structure of fucoidan has been identified using NMR or tandem electrospray ionisation mass

*Corresponding author. Email: [email protected] q 2014 Taylor & Francis

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Figure 1. Dominated structure of the fucoidan.

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spectrometry (ESI-MS) techniques (Bilan et al. 2004; Anastyuk et al. 2012). In our work, tandem ESI-MS was employed to determine the structure of the fucoidan.

2. Result and discussion 2.1. Structural characteristics of the fucoidan The results of yield and chemical analysis of fucoidan extracted from brown seaweed S. henslowianum is summarised in Table S1. The results of the chemical analysis indicated that the fucoidan analysed in this study contains fucose and glucose as main sugars, unlike other fucoidans present in algae belonging to the family Sargassaceae, which are mainly the highly sulphated galactofucans. Fucoidan was subjected to mild hydrolysis as described in Section 3 to obtain oligosaccharides, suitable for mass spectrometric investigation. Figure S1 shows the mass spectrum of the fucoidan with a major signal at m/z 243 corresponding to the deprotonated molecule [M 2 H]2 of monosulphated fucose [FucSO3]2. Ion at m/z 225 was assigned for dehydration of monosulphated fucose [FucSO3 2 H2O]2. Ion at m/z 389 was from a monosulphated difucose [Fuc2SO3]. Ion at m/z 491 was from disulphated difucose [Fuc2(SO3Na)2 2 2Na]2 and a signal at m/z 307 was assigned to doubly charged ion [M 2 2H]22 of disulphated trifucose [Fuc3(SO3Na)2 2 2Na]22. A minor signal at m/z 535 corresponds to anion [M 2 H]2 of a monosulphated trifucose [Fuc3SO3Na 2 Na]2. Two signals at m/z 405 and 507 could have been from [FucGlu(SO3Na) 2 Na]2 and [FucGlu (SO3Na)2 2 Na]2, respectively. The MS2 daughter ion at m/z 243 was fragmented (Figure S2). Tissot et al. (2006) reported the effect of the sulphate position on the fragmentation pattern. In the spectra, three signals from C-4 (m/z 183), C-3 (m/z 169) and C-2 (m/z 139) sulphation of a-L -fucopyranose (a-L -Fucp) residues were detected, indicating that three positions C-2, C-3 and C-4 were sulphated. The ion at m/z 139 was the major fragment, then 183 and a minor fragment at 169 indicating that the fucosyl units of the fucoidan are mainly sulphated at positions 2 and 4 and partly at C-3. This indicated that the fucoidan under study was sulphated at three positions with different portions, while most of the other fucoidans extracted from other resources were sulphated at one or two positions. The fragmentation pattern of [Fuc2SO3Na 2 Na]-ion at m/z 389 (Figure S3) indicated that the Y1 and C1 ions at m/z 243 from glycosidic bond breaking had strong intensity. The spectra contained an intensive ion at m/z 225 (B-type) which indicated sulphation at C-2 of non-reducing fucose residues. The 0,2A1 and 0,3A1 ions at m/z 183 and 153 were from cross-ring cleavage of the a-L -Fucp-4-OSO3- residues. To interpret the spectra, we used the result of Anastyuk et al. (2009) that the formation of characteristic 0,2A-/0,2X-ion required available proton at the C-3 hydroxyl group. Two other fragment ions at m/z 285 (fragment ions 0,2X1) and 345 (loss of CH3CHO). The dehydrated form of the precursor ion was also observed at m/z 371. Our spectra lack of the 0,2A2 ion at m/z 329, which were found upon ESI-MS/MS of the same [Fuc2SO3Na 2 Na]2 ion containing (1 ! 4)-linked a-L -Fucp-2-OSO3- residues of fucoidan from Ascophyllum nodosum (Marais & Joseleau 2001). It indicated that the fucoidan under study contained predominantly

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(1 ! 3)-linked disaccharides, similar to the fucoidan from Laminaria cichorioides (Anastyuk et al. 2010). On the contrary, the presence of a very intensive signal of 0,2X0 at m/z 139 in the spectra may be attributed to the (1 ! 4) linked difucoside sulphated at position 2 of the reducing residue [a-L -Fucp-(1 ! 4)-a-L -Fucp-2-OSO3]. Therefore, MS/MS fragmentation of the ion at m/z 389 corresponded to a mixture of four monosulphated difucose. The MS results obtained allowed us to conclude that the fucoidan under study has a(1 ! 3)-linked L -Fucp backbone. Mass-spectral fragmentation of partial hydrolysis products of the fucoidan is demonstrated in Scheme S1. From the above-mentioned data, the dominated structure of studied fucoidan was proposed as in Figure 1. 2.2. Hypolipidaemic activity result We followed the diets as mentioned in Section 3 to obtain obese animals. The results demonstrated that experimental mice which followed two different diets (low-fat diet (LFD) and high-fat diet (HFD)) gained different amounts of weight week by week. The mice in the HFD group gained weight faster than those in the LFD group after 21 days. After 35 days, the mice in the HFD group gained more weight, with p , 0.05. The results are summarised in Table S2. The changes in blood biochemical levels of the obese mice before and after the oral administration of fucoidan are presented in Table S3. Cholesterol level of the mice after drinking fucoidan and distilled water decreased 22.85% and 1.76%, respectively. Thus, cholesterol level of the mice after fucoidan administration decreased 21.09% compared with the HFD group, though it still increased 20% compared with the LFD group. Triglyceride and LDL-cholesterol levels of the mice after fucoidan administration decreased 6.35% and 18.74%, respectively, compared with the mice in the HFD group. Hence, oral administration of fucoidan (at the dose of 100 mg/kgP/day) isolated from S. henslowianum led to decrease in the levels of cholesterol, triglyceride and LDL-cholesterol in obese mice. The changes in body, liver and fat weights of the experimental mice are presented in Table S4. The body, liver and fat weights of the mice in the HFD group were higher than those in the LFD group (with p , 0.05). In the subgroup that drank fucoidan, the body weights of the mice were not different from those in the LFD group ( p . 0.05), while liver weights were different from those in the HFD group ( p , 0.05) and not different from those in the LFD group ( p . 0.05). Fat weights of the mice in the subgroup that drank fucoidan were different from those in the HFD group ( p , 0.05), although still higher than that in the LFD group ( p , 0.05). 3. Experimental 3.1. Material Brown seaweed S. henslowianum was collected at Hai Van – Son Cha peninsula, Hue province, Vietnam in April 2012. A voucher specimen named FSH has been deposited in the Nha Trang Institute of Technology Research and Application, Vietnam. 3.2. Extraction and purification of fucoidan The extraction was carried out following the method described previously (Bilan et al. 2002). The yield of fucoidan was 1.8% calculated based on the dried seaweed weight. 3.3. Chemical analysis Neutral monosaccharide compositions were elucidated by gas – liquid chromatography (Bilan et al. 2002). Alditol acetate derivative was prepared by hydrolysis of fucoidan sample in 2 M

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CF3COOH (TFA), 8 h at 1008C and analysed by using 17AAFM, GC-FID, Shimadzu, Japan. Uronic acid content was determined by following the carbazole method (Bitter & Muir 1962) using D -glucuronic acid as a standard. Sulphate content was estimated using gelatin/BaCl2 method (Dodgson 1961) after hydrolysis of fucoidan in 2 M TFA as described earlier.

3.4. Partial acid hydrolysis

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Partial acid hydrolysis of the fucoidan was carried out using trifluoroacetic acid (0.75 M, 1 h, 60oC), and the solution was evaporated several times with methanol.

3.5. Electrospray ionisation mass spectrometry ESI-MS experiments were performed on a Xevo TQ MS (Waters Corporation, Manchester, UK). The analyses were carried out in negative mode. Dried fucoidan sample was diluted in 1:1 MeOH – water and introduced into the mass spectrometer.

3.6. Hypolipidaemic activity 3.6.1. Biochemical assay A total of 30 albino house mice of BALB/c strain with weights ranging from 20 to 25 g were raised in the Animal Section of the Institute of Biological Technology, Vietnam Academy of Science and Technology. The mice were divided into two groups: 10 mice in group 1 and 20 mice in group 2. Their diets including LFD (for group 1) and HFD (for group 2) were based on the formula described previously (Han et al. 2000; Guo et al. 2009) (Table S5). Foods were made fresh every day and mice were fed depending on their appetite. They were weighed once a week to track their weight gain. After 5 weeks, mice that gained the most weight from group 2 were divided into two subgroups: 10 mice, to drink distilled water in subgroup 1 and 10 mice, to drink aqueous solution of fucoidan with the dosage of 100 mg/kgP/day in subgroup 2. The mice in two subgroups drank either fucoidan or water and followed an LFD for 4 weeks. After that, they were weighed, and their blood was collected to obtain blood serum and then cholesterol, LDLcholesterol and triglyceride levels were determined. The mice were killed by pulling on their neck bones, and quickly dissected to obtain their livers. The weights of liver and fat were weighed.

3.6.2. Statistical analysis The values were expressed as mean ^ SD for 10 animals in each group. All data were processed using Student’s t-test and one-way analysis of variance (one-way ANOVA). Statistical differences were considered to be significant at p # 0.05. 4. Conclusion Fucoidan extracted from brown seaweed S. henslowianum collected at Hai Van – Son Cha peninsula, Hue province, Vietnam has a(1 ! 3)-linked L -fucopyranose backbone and sulphate groups occupied mostly at C-2, C-4 and sometimes at C-3 position of fucose residues. Fucoidan administered at a dose of 100 mg/kgP/day has lowered cholesterol, triglyceride and LDLcholesterol levels in obese mice. However, to obtain a definite conclusion on its functions in preventing obesity and lowering blood lipid level, we need more research on dosage, testing time and other biochemical evaluations.

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Supplementary material Supplementary materials relating to this article are available online, alongside Scheme 1, Figures S1 –S3 and Tables S1 –S5. Funding This work was financially supported by the Ministry of Education and Training, Vietnam [grant number B2012-01-31].

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Structure and hypolipidaemic activity of fucoidan extracted from brown seaweed Sargassum henslowianum.

The aim of this study is to elucidate the structure and investigate the hypolipidaemic activity of a fucoidan extracted from brown seaweed Sargassum h...
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