Structural determination of bioactive ulvan extracted from green seaweeds ulva lactuca and ulva reticulata

rated from the supernatant by centrifugation then washed several times with ethanol and dried in a vacuum oven at 60ºC to a constant weight. The yield of ulvan was 7.2% and 6.4% calculated based on algae dried weight. The samples signed UR-H and UL-H. 2.1.3.3. Ulvan extraction by alkali: 20g dry algae per 400mL of 0.1N NaOH solution was heated for of 2h at 60ºC on a boiling-water bath and under continuous stirring. After ltration through a cotton cloth, the alkali extract was neutralized to pH=7, then centrifuged, and the liquid supernatant was ltered. The extract was concentrated to reduce initial volume in a rotary evaporator and precipitated with 4 vol. of absolute ethanol. The alcohol precipitate was separated from the supernatant by centrifugation then washed several times with ethanol and dried in a vacuum oven at 60ºC to a constant weight. The yield of ulvan was 5.1% and 4.1% calculated based on algae dried weight. The samples signed UR-K and UL-K. Purification of ulvan: The crude polysaccharides were dissolved in distilled water and the solution was passed through 10000 (Da) MWCO under the tap in 72h and precipitated with 4 vol. of absolute ethanol. The alcohol precipitate was separated from the supernatant by centrifugation then washed several times with ethanol and dried in a vacuum oven at 60ºC to a constant weight. 2.1.4. Evaluation of the biological activity of ulvan Bioactive experiments were examined for native and derivative ulvan. 6 2.1.4.1. Cytotoxic activity assays Three human cancer cell lines HepG2 (Hepatocellular carcinoma), MCF7 (human breast cancer), and Hela (cervical cancer) were used for the assays. Cytotoxic assays were performed according to a method developed by Monks et al. 2.1.4.2. Anticoagulant activity assays Anticoagulant assays were performed according to a method developed by Anderson et al. 2.1.4.3. Antimicrobial activity assays Antimicrobial assays were performed according to a method developed by Vanden và CS. 2.1.4.4. Antioxidant activity assays - Total antioxidant capacity was determined by the method of Prieto et al - Ferric reducing activity was determined according to the method of Zhu et al. 2.2. Structural determination of ulvan 2.2.1. Analyze the chemical components of ulvan - Sulfate content was determined according to the method of Dodgson et al. - Uronic acid content was determined by following the method of Bitter et al. - Neutral monosaccharide compositions were elucidated by the method of Bilan et al. 2.2.2. Gel Permeation Chromatography GPC: The weight average molecular mass and the number average molecular mass were elucidated on a HPLC Agilent 1100. 2.2.3. IR spectra: IR spectra was recorded on a FT-IR Affinity-1S SHIMADZU spectrometer. 2.2.4. NMR spectra: NMR spectra were recorded on Bruker ASCEND 500 in D2O solution using DSS as internal standard at 70ºC. 7 2.2.5. ESI-MS: ESI-MS experiments were performed on a LTQ Orbitrap Mass spectrometer. The analyses were carried out in negative mode. 2.2.6. SAXS: SAXS experiments were performed at BL19B2, SPring- 8, Hyogo, Japan. 2.2.7. SEM: SEM pictures were photographed on a Nova NaNoSEM 450 – FEI equipment. 2.3. Sulfation and acetylation - Sulfated derivatives were performed according to a method by Lihong Fan et al. Determination of sulfate content by weight method. - Acetylated derivatives were performed according to a method by Xiao-xiao Liu et al. Determination of acetyl content by method of Luis.A. Chapter 3. RESULTS AND DISCUSSIONS 3.1. Sample selection 3.1.1. Results on the chemical components of ulvan Table 3.1. Chemical compositions of 6 ulvans Ulvans SO3Na (%w) Uronic acid (%w) Monosaccharide compositions ( % mol) Rha Gal Xyl Man Glu UR-N 17,6 22,5 1 0,03 0,06 0,01 0,06 UR-H 14,3 19,3 1 0,1 0,11 0,01 0,21 UR-K 13,2 19,8 1 0,1 0,14 0,03 0,14 UL-N 18,9 21,5 1 0,03 0,07 0,01 0,06 UL-H 15,1 18,7 1 0,01 0,10 0,01 0,06 UL-K 14,2 18,2 1 0,04 0,09 0,01 0,07 Results of chemical components of ulvan were summarized in Table 3.1. Like other ulvan, ulvan from U.Reticulta and U.lactuca are mainly composed of rhamnose, with variable contents of galactose and xylose, trace amounts of glucose and mannose. Sulfate content of the sulfated polysaccharide from two Ulva species is relatively lower (13- 8 19%) than that of sulfated polysaccharides from other resources (20- 30%) and uronic acid is higher (18-23%) than others (6.5-19%) calculated based on algae dried weight. These results are similar to with last researches with the other green seaweed species. 3.1.2. Results on the biological activities of ulvans 3.1.2.1. Antimicrobial activity Table 3.2. Antimicrobial activity results of 6 ulvans Microbe UR-H UR-N UR-K UL-H UL-N UL-K Control Gra m (-) E. coli ++ ++ - ++ +++ - +++ Pseudomonas aeruginosa - + - - + - +++ Vibrio haveyi - - - - - - +++ Gra m (+) Bacillus cereus - - - - ++ - +++ Streptococcus faecalis - - - - - +++ Enterobacter cloace - ++ - + ++ - +++ Staphylococcus aureus - - - - - - +++ 3.1.2.2. Cytotoxic activity Alkali extraction ulvan UL-K and UR-K were not expressed this activity. Table 3.3. Cytotoxic activity results of 4 ulvans % survival cell Concen trations (µg/ml) UR-H Concen trations (µg/ml) UR-N Concen trations (µg/ml) Ellipticine HepG 2 HeLa MCF- 7 HepG 2 HeLa MCF-7 100 0.00 0.00 0.00 100 0.00 0.00 0.00 Hep G2 HeL a MC F-7 20 42.26 50.30 44.72 20 50.34 36.29 48.74 4 70.18 69.05 76.84 4 84.22 83.05 89.66 10 1.33 3.31 5.9 3 0.8 83.24 90.44 85.45 0.8 94.90 96.44 95.91 IC50 (µg/ml) 49.10 ±1.56 47.75± 2.37 50.69 ±1.86 IC50 (µg/ml) 31.31 ±1.56 34.75 ±1.38 36.37± 1.73 2 29.1 4 21.8 7 28. 86 9 Concen trations (µg/ml) UL-H Concen trations (µg/ml) UL-N HepG 2 HeLa MCF- 7 HepG 2 HeLa MCF-7 0.4 49.4 2 48.8 9 48. 58 100 0.00 0.00 0.00 100 0.00 0.00 0.00 20 50.21 55.34 48.67 20 60.67 66.24 58.71 0.08 81.5 9 77.1 3 76. 92 4 71.46 73.58 79.45 4 78.13 77.0 67.89 0.8 92.42 94.26 95.53 0.8 89.30 88.0 87.41 IC50 (µg/ml) 0.50 0.34 0.4 5 IC50 (µg/ml) 40.74 ±2.21 44.25± 2.32 50.93 ±1.67 IC50 (µg/ml) 29.67 ±2.87 36.33 ±3.84 25.09± 1.36 3.1.2.3. Antioxidant activity Table 3.4. Antioxidant activity results of 6 ulvans Samples Ferric reducing activity (mgFe2+/g ulvan) Total antioxidant capacity (mg AcidAscobic/g ulvan) 1 UL-N 2.51 3.75 2 UL-H 1.93 2.60 3 UL-K 0.01 1.12 4 UR-N 4.86 3.96 5 UR-H 1.34 2.56 6 UR-K 0.22 0.86 Conclusions: The ulvan samples have been chosen to study chemical structure as following: - Ulvan was extracted by water from Ulva reticulata (signed: UR-N) - Ulvan was extracted by acid from Ulva reticulata (signed: UR-H) - Ulvan was extracted by water from Ulva lactuca (signed: UL-N) - Ulvan was extracted by acid from Ulva lactuca (signed: UL-H) 3.2. Determination of ulvan chemical structure Molecular weight Mw and molecular weight distribution Mw/Mn showed that like other native sulfated polysaccharides, ulvan from Ulva Reticulta and Ulva lactuca are polymers with high polydisperse with Mw/Mn = 2.19 – 5.16 and molecular weights are Mw = 8.1x104 - 3.47x105 g/mol which independent on extration conditions. 10 3.2.1. Ulvan was extracted by acid from Ulva reticulata (UR-H) IR spectrum showed bands corresponding to a sulfated ester and uronic acids. The signal at 848 cm-1 and 761 cm-1, might correspond to the bending vibration of C-O-S of sulfate in axial and equatorial position, respectively. The asymmetric stretching vibration of COO- appears at 1624 cm-1. In addition, the band at 3315cm-1, owing to the stretching vibration of OH bond, suggests the presence of hydrogen bonds in the molecules. The band at 2927 cm-1 corresponds to the stretch vibration of C-H, while that at 1078 cm-1 signifies the stretch vibration of CO and change angle vibration of OH. The 1H-NMR spectrum showed three anomeric proton signals at 5.30, 5.20, and 4.65 ppm which were designated as A, B, and C, respectively. A broad signal at 1.17 ppm was assigned to the C-6 methyl protons of rhamnopyranose and the signals in the range 3.4-4.3 ppm were from the ring protons (Table 3.8). The 13C-NMR spectrum was indicative of complex polymers, containing signals in the resonance regions corresponding with anomeric carbons (94-102 ppm) and ring carbons (72-80 ppm) as well as C-CH3 signals (18.0 ppm) and a carboxyl signal at 167 ppm (Table 3.8). Four signals at 63.39, 63.32, 63.12, and 63.06 ppm were from CH2 (C-6) groups of galactose and/or glucose and/or CH2 (C-5) groups of xylose. The six 1H resonances from H-1 to H-6 of residue A were assigned from the cross peaks in the COSY spectra. Based on the proton chemical shifts, the carbon chemical shifts (C-1 to C-6) were assigned from the HSQC spectra (Figure 3.6). Both proton and carbon chemical shifts indicated that this glycosyl residue was typical of 6- deoxyhexopyranose, suggesting a rhamnosyl moiety. The chemical shift of proton H-1(A) was 5.3 ppm, suggesting that residue A was α-linked. These results indicated that residue A might be α-rhamnose (Table 3.8). Furthermore, the downfield shift of the C-2 (79-80 ppm), C-3 (75-77 ppm), and C-4 (74- 76 ppm) carbon signals with respect to the standard values for 11 rhamnose indicated that residue A might be linked by (1→2,3,4) glycosidic linkages and/or sulfated at C-2, C-3, and C-4 positions. It also was confirmed by the correlations between H-1(A) and C-2(A), C- 3(A), C-4(A) in HMBC spectrum (Figure 3.7). The presence of some glycosidic linkage and positions of sulfate groups were indicated by the split signals at H-1(A) and H-6(A). From the peak owing to the β-anomeric proton H-1(B) at 4.65 ppm in COSY and HSQC spectra, we can assign H-2(B) and H-3(B) at 3.2 and 3.8 ppm, and C-2(B) and C-3(B) at around 77 and 74 ppm, respectively. The downfield shift of the C-2 carbon signal with respect to the standard values for uronic acid indicated that residue B might be connected through (1→2) glycosidic linkages. Therefore, residue B might be β (1→2)- uronic acid (Table 3.8). Figure 3.6. HSQC spectrum of UR-H Figure 3.7. HMBC spectrum of UR-H Table 3.8. 1H and 13C-NMR chemical shifts of UR-H Monosaccharides C-1/H-1 C-2/H-2 C-3/H-3 C-4/H-4 C-5/H-5 C-6/H-6 A →2)--L-Rha →4)--L-Rha 102- 103 /5.30 79- 80 /3.63 75-77 / 3.95 74-76 /3.85 75-76 /3.65 17-18 /1.17 B →2)--D-GlcA 98.52; 98.39 /4.65 77.0 /3.20 74.0 /3.75 74.0 / 3.85 - 167 C →2)--L-IdoA 94.70; 94.51 /5.20 77.5 /3.60 76.0 /3.95 74.0 /3.85 - 167 12 From the peak of α-anomeric proton H-1(C) at 5.2 ppm in COSY and HSQC spectrum, we can assign H-2(C) 3.6 ppm and C-2(C) at 77 ppm, respectively. The downfield shift of the C-2 carbon signal with respect to the standard values for uronic acid indicated that residue C might be connected through (1→2) glycosidic linkages. Therefore residue C might be α (1→2)-uronic acid (Table 3.8). Ulvan from ulva green seaweeds essentially contains α-rhamnose, β-glucuronic acid, and α-iduronic acid. We proposed residue B might be β (1→2)- glucuronic acid and residue C might be α (1→2)-iduronic acid. The anomeric carbons C-1(B) and C-1(C) were split into doublet, probably caused by the linkage of B and C with two other sugars having α - and β - conformation. The sequence of glycosyl residues was inferred from the HMBC spectrum. The HMBC spectrum revealed the inter-residue correlations between H-1 of residue C and C-4 of residue A, and between H-1 of residue B and C-2 of residue A. Thus, the major repeating unit of ulvan is β-D-GlcA(1→2)-α-L-Rha and α-L- IdoA(1→4)-α-L-Rha and the branching point is at O-2 of uronic acid (Table 3.8). ESI-MS shows the mass spectrum with a major fragment at m/z 195 corresponding with deprotonated uronic acid, signal at m/z 243 was signed to monosulfated rhamnose [RhaSO3] -. Ions at m/z 339 and 375 were assigned to [RhaUroA]- and [RhaXylSO3] -, respectively. The peaks at m/z 419 and 503 were assigned to the [RhaUroASO3] - and the monosulfated trirhamnose [Rha3SO3] - ions, repsectively. The MS2 daughter ion at m/z 243 was fragmented (Figure 3.9). Three signals from C-4 (m/z 183), C-3 (m/z 169), and C-2 (m/z 139) sulfation of α-L- Rha residues were detected, indicating that the three positions C-2, C-3, and C-4 were sulfated. The peak at m/z 183, assigned to the major fragment ion, the peak at m/z 139, and a minor fragment at m/z 169 indicate that the rhamnosyl units of the ulvan are mainly sulfated at position C-2 and C-4 and partly at C-3. 13 Figure 3.9. MS2 spectrum of [RhaSO3] - ion at m/z 243. In conclusion, the major repeating dimeric sequences of ulvan extracted from green seaweed Ulva reticulata collected at Nhatrang sea of Vietnam, consisted of β-D-GlcA(1→2)-α-L-Rha and α-L- IdoA(1→4)-α-L-Rha, and branched at O-2 of uronic acid. The Rha residues were sulfated at three positions, C-2, C-3, and C-4. The 13C and 1H-NMR chemical shifts of the major signals in ulvan have been attributed but those of xylose, galactose, and glucose present in the polysaccharide remain to be identified. This is ulvan with new chemical structure as most of ulvan reported not have the linkage (1→2) in the main chains. 3.2.2. Ulvan was extracted by water from Ulva reticulata (UR-N) Chemical structure of ulvan extracted by water from green seaweed Ulva reticulata were determined by IR, NMR, MS spectra as UR-H ulvan. IR spectra of UR-N showed bands that are typical of ulvan such as: 1595 cm 1, 1028 cm 1, 844 cm 1 và 783 cm-1. The complete NMR assignments are shown in Table 3.9. ESI-MS spectrum of UR-N shows fragment ion [M-H]- at m/z 419, assinged to disaccharide [RhaUroASO3]-. Monosulfated rhamnose [RhaSO3] - and uronic acid [UroA]- are assigned corresponding with peaks at m/z 243 and m/z 195. ESI-MS/MS spectrum of fragment ion m/z 243 shows a strong signal at m/z 169, this indicate that the rhamnosyl units of the ulvan are sulfated at position C-3. 14 Table 3.9. 1H and 13C-NMR chemical shifts of UR-N Monosaccharides C-1/H-1 C-2/H-2 C-3/H-3 C-4/H-4 C-5/H-5 C-6/H-6 A →4)α-L-Rha3S 1→ 100.51/ 4.82 69.71/ 4.20 78.91/ 4.59 78.86/ 3.79 68.89/ 4.13 17.60/ 1.30 B →2,4)β-D-GlcA 1→ 103.83/ 4.63 74.58/ 3.35 74.83/ 3.64 79.48/ 3.65 76.70/ 3.82 177.60 From obtained IR, NMR and MS results, we can conclusion that ulvan from green seaweed Ulva Reticulata collected at Nhatrang sea obtained by water extraction has a backbone composed of disaccharide [→4-β-D-GlcA-(1→4)-α-L-Rha3S-(1→] and branched point is at C-2 of glucuronic acid and the rhamnosyl units of the ulvan are sulfated at position C-3. 7x10 -3 6 5 4 3 2 1 0 q2 I(q ) 543210 q, nm -1 Ulva reticulata Forsskål 1% in water in 0.5 M NaCl aq -8 -7 -6 -5 -4 -3 ln (q I(q )) 6543210 q 2 Ulva reticulata Forsskål 1% in water, Rgc=1.35nm in 0.5 M NaCl aq, Rgc=1.37nm Figure 3.22. SAXS from UR-N ulvan 1% in water and in 0.5M NaCl: Kratky plots and Guinier plots for cross-section In order to find more structural information at molecular level of the ulvan, SAXS measurements have been carried out. Kratky [Left Fig 3.22] and Guinier [Right Fig 3.22] plots for 1% UR-N ulvan in water and in 0.5M NaCl were the typical form for moleculars that was a rod- like confomational structure like to sulfate polysacchries extracted from the other seaweeds such as carrageennan and fucoidan. From Kratky plots, the weak peak can be found in 0.5 nm-1 of q, due to the repulsive electrostatic interaction. This means that this ulvan sample contains much amount of sulfate groups. An addition of saline, which produced the effect shielding against that force, made the peak disappear (L.Fig. 3.22). From Guinier plots (R.Fig. 3.22), the cross-sectional radius of gyration Rgc can be estimated. The Rgc is 1.35-1.37nm, indicating 15 possibility of bulky side chains of the ulvan. This result is in agreement with the complex branching structure of the ulvan as elucidated by NMR and MS methods. To understand the mode of interaction better, the molecular model of the UR-N is constructed on the base of chemical structure. The caculated scattering profile was compared with respective observed SAXS profile in Figure 3.24 and 3.25. The results showed that the observed SAXS scattering profile from gel was fitted with that calculated from the molecular model. Hình 3.24. The molecular model of UR-N was constructed on the base of chemical structure Hình 3.25. Calculated scattering profiles (solid line) and observed SAXS (symbols) of UR-N. 3.2.3. Ulvan was extracted by water from Ulva lactuca (UL-N) IR spectrum of UL-N showed bands that are typical of ulvan such as: The signal at 848 cm-1 and 761 cm-1, might correspond with the bending vibration of C-O-S of sulfate in axial and equatorial position, respectively. The asymmetric stretching vibration of COO- appears at 1599 cm 1, while that at 1026 cm 1 signifies the stretch vibration of glycoside linkage C-O-C. 16 Obtained 1H and 13C-NMR, COSY, HSQC, HMBC analysis, the complete NMR assignments are shown in Table 3.10. Figure 3.30. HSQC spectrum of UL-N Figure 3.31. HMBC spectrum of UL-N Table 3.10. 1H and 13C-NMR chemical shifts of UL-N Monosaccharides C-1/H-1 C-2/H-2 C-3/H-3 C-4/H-4 C-5/H-5 C-6/H-6 A →2)-α-D-Xyl-(1→ 100.27/ 5.36 77.92 3.65 73.82/ 3.95 71.87/ 3.82 61.20/ 3.80; 3.88 - B →2,4)-α-L-Rha-(1→ 89.65; 101.50/ 4.90 79.21/ 4.06 71.74/  3.71 76.88/  3.82 68.40/  4.0 17.12/ 1.31 C →4)-α-L-Rha3S-(1→ 100.40/ 4.82 69.46/ 4.23 78.68/ 4.61 78.68/ 3.80 68.65/ 4.13 17.35/ 1.31 D →4)-β-D-GlcA-(1→ 103.65/ 4.64 74.40/ 3.35 74.58/ 3.65 79.40/ 3.68 76.88/ 3.81 177.76 [UroA]- [XylUroA]- [RhaUroA]- [RhaSO3] - [XylRhaSO3] - [UroARhaSO3] - [Rha3SO3] - Figure 3.32. ESI-MS spectrum of UL-N Figure 3.33. MS2 spectrum of [RhaSO3] - ion at m/z 243 17 ESI-MS/MS spectrum of fragment ion m/z 243 shows two signals at m/z 183 và m/z 169, this indicate that the rhamnosyl units of the ulvan are sulfated at position C-4 and C-3, respectively. Those results obtained by IR, NMR and MS analysis confirmed that ulvan from green seaweed Ulva lactuca collected at Nhatrang sea obtained by water extraction has a backbone composed of main disaccharide A3s [→4-β-D-GlcA-(1→4)-α-L-Rha3S-(1→] and branched at C-2 position of rhamnose residues and other minor repeating units including GlcA- (1 → 2)-Xyl and GlcA-(1 → 2)-Rha also occurred in the ulvan. The complete NMR assignments are shown in Table 3.10. Like this, ulvan obtained by water extraction from Vietnam green seaweed Ulva lactuca species which we are studying to have chemical structure the same as previous researches in the main chains, difference in the branches and multiform in minor units. This results proved that the structural complexity of ulvans; it may be derived from the differences in seaweed species, extraction method and place of cultivation. 3.2.4. Ulvan was extracted by acid from Ulva lactuca (UL-H) IR spectrum of UL-H shows the typical signals of ulvan such as: The signals at 850 cm 1 and 786 cm-1 might correspond with the bending vibration of C-O-S of sulfate in axial and equatorial position, respectively. The signal at 1014 cm 1 is typical for C-O bond containing O-H group. The asymmetric stretching vibration of COO- group in uronic acid appears at 1599 cm 1. Obtained 1H and 13C-NMR, COSY, HSQC, HMBC analysis of UL-N ulvan, the complete NMR assignments are shown in Table 3.11, we could conclude that ulvan isolated by acid extraction from green seaweed Ulva lactuca mainly composed of disaccharide [→4)-β-D- GlcA-(1→4)-α-L-Rha3S-(1→] and branched at C-3 position of rhamnose residues. In comparison with last reports about ulvan from Ulva lactuca indicated that ulvan isolated by different extractions 18 obtained ulvans which are the same compositions in the main chains, and difference of the branches, only. Figure 3.37. HSQC spectrum of UL-H Figure 3.38. HMBC spectrum of UL-H Table 3.11. 1H and 13C-NMR chemical shifts of UL-H Monosaccharides C-1/H-1 C-2/H-2 C-3/H-3 C-4/H-4 C-5/H-5 C-6/H-6 D 4)-L-Rha3S 1 102.45/ 4.82 71.62/ 4.22 80.80/ 4.58 80.70/ 3.78 70.79/ 4.11 ~19.56/ ~1.3 E 1)-D-GlcA 4 105.69/ 4.65 76.44/ 3.35 76.72/ 3.60 81.52/ 3.65 78.49/ 3.86 D’ 1)-L-Rha 3(4) ~103.6/ 4.89 71.62/ 4.22 81.34/ 4.05 ~74.10/ 3.86 ~73.2/ 3.71 ~19.48/ ~1.29 Figure 3.39. MS2 spectrum of [RhaSO3] - ion at m/z 243 ESI-MS spectrum of UL-H shows fragment ion [M-H]- at m/z 419, assinged to disaccharide [RhaUroASO3]-. Monosulfate rhamnose [RhaSO3] - and uronic acid [UroA]- are assigned corresponding with peaks at m/z 243 and m/z 195. MS2 spectrum of [RhaSO3] - ion at m/z 243 (Fig. 3.39) shows: strong signal at m/z 169 and weak signal at m/z 139, this indicate that the rhamnosyl units of the ulvan are maily sulfated at position C-3 and party at C-2, respectively. This results are in agreement with the results obtained by IR and NMR analysis. In conclution, UL-H ulvan from green seaweed Ulva lactuca collected at Nhatrang sea obtained by acid extraction has a backbone 19 composed of main disaccharide ulvan [→4)β-D-GlcA-(1→4)α-L- Rha3S-(1→] and branched at C-3 position of rhamnose residues. 3.3. Study the effects of sulfation and acetylation on biological activities of ulvan. Many previous reports reveal that the sulfate group plays an important role in the bioactivities of polysaccharides, especially anticoagulant activity. On the other hand, acetylation could increase antioxidant or antimicrobial activity. We synthesized some sulfated and acetyled derivatives from native UR-N to aim studying the strutural modifications as well as ulvan biological activities. 3.3.1. Effect of sulfation IR và 13C- và HSQC - NMR spectra were used to examine the structural change of derivatives UR-S. Obtained IR spectral analysis of UR-S indicate that the signal at ~850 cm-1 is typical for sulfate group at axial position increased, at the same time the signal at ~3261-3370 cm-1 is typical for O-H group decreased; 13C-NMR spectrum shows that a new signal at δ77,2 ppm is C-2 of rhamnose residue, this proved that sulfation was at C-2 position of rhamnose residue; HSQC-NMR spectrum of UR-S derivatives were some new signals that shift to the downfield in comparision with HSQC spectrum of native ulvan extracted by water (Fig. 3.42), the interactive signal C-1/H-1 of rhamnose residue was separated and at one there were two new signals expressing the interaction between C-2/H2 at δ80,0/3,38 ppm and C- 3/H3 at δ76,5/3,67 ppm of glucuronic acid, the signal C-2 of rhamnose residue increased. Therefore, there were three positions including C-2 of rhamnose and C-2, C-3 of glucuronic acid partly sulfated. In conclusion, the results obtained by NMR analysis are in agreement with the results obtained by IR analysis and last studies. The results of spectral analysis affirmed increasing of sulfate content for UR-S samples. 20 Figure 3.42. HSQC spectrum of UR-N (a) and UR-S (b) Evaluating relation of structure - anticoagulant activity Surface structure of native ulvan and derivatives were photograph by SEM. Those pictures showed that there were obviously differences in surface structure between native ulvan UR-N and derivatives UR-S. UR-N moleculars are composed block that is flat surface structure while UR-S moleculars are seemly composed small crytal blocks with definite conformation. Cytotoxic, antioxidant, antimicrobial and anticoagulant activities assay were tested for sulfated derivatives and UR-N native ulvan sample. Results revealed that anticoagulant activity was significant increase while cytotoxic, antioxidant, antimicrobial activities were not significant change. The degree of sulfation (DS) of polysaccharides is an important parameter for analyzing the bioactivities. To aim studying the effection of sulfate content on anticoagulant activity, sulfated derivatives with different sulfate contents were synthesized and evalu