Summary of dissertation on Chemitry: Structural determination of bioactive ulvan extracted from green seaweeds Ulva lactuca and Ulva reticulata

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In this context, we have chosen the topic: “Structural determination of bioactive ulvan extracted from green seaweeds Ulva lactuca and Ulva reticulata”, the purpose of the research are to study the structure of bioactive polysaccharides from green seaweed, to improve the researches ofseaweed polysaccharidesand to extend the applied possibilities of Vietnamese seaweed species.

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Nội dung Text: Summary of dissertation on Chemitry: Structural determination of bioactive ulvan extracted from green seaweeds Ulva lactuca and Ulva reticulata

This research has been done at Graduate University of MINISTRY OF EDUCATION VIETNAM ACADEMY OF Science and Technology – Vietnam Academy of Science and AND TRAINING SCIENCE AND TECHNOLOGY Technology GRADUATE UNIVERSITY OF SCIENCE AND Supervisor 1: Asc.Prof.Dr Thanh Thi Thu Thuy TECHNOLOGY Supervisor 2: Asc.Prof.Dr Tran Thi Thanh Van ----------------------------- Reviewer 1: Reviewer 2: QUACH THI MINH THU Reviewer 3: STRUCTURAL DETERMINATION OF BIOACTIVE ULVAN The dissertation will be evaluated by the Scientific Council for doctoral EXTRACTED FROM GREEN SEAWEEDS ULVA LACTUCA dissertation at the Graduate University of Science and Technology - AND ULVA RETICULATA Vietnam Academy of Science and Technology in ... ... ', date ... month… 2017. Major : Theoretical Chemistry and Physical Chemistry Code: 62.44.01.19 SUMMARY OF DISSERTATION ON CHEMITRY The dissertation can be found at: Ha Noi – 2017 - Library of the Graduate University of Science and Technology - National Library of Vietnam
1 2 INTRODUCTION Recently, in Vietnam, polysaccharides from the red seaweed and 1. Necessary of the dissertation brown seaweed have been researched and obtained very good results, Historically, seaweeds are used as raw materials for many but up to now, polysaccharides from the green seaweed in general and industrial applications such as food, pharmaceutical and cosmetics ulvan from Ulva genus in particular is still pooly investigated. industry. Based on pigmentation, seaweeds can be classified into three In this context, we have chosen the topic: “Structural divisions as brown (Phaeophyte), red (Rhodophyte) or green algae determination of bioactive ulvan extracted from green seaweeds (Chlorophyte). Vietnam has a coastline of about 3000 km with the Ulva lactuca and Ulva reticulata”, the purpose of the research are to climate varying from subtropical in the northern part to tropical in the study the structure of bioactive polysaccharides from green seaweed, southern part of the country, which is a rich source of seaweed species. to improve the researches ofseaweed polysaccharidesand to extend the Green seaweeds are known as valuable sources to isolate applied possibilities of Vietnamese seaweed species. bioactive compounds such as lipid, protein, polysaccharide, peptide, 2. Research purposes of the dissertation carotenoid, phenolic and alkaloid compounds. Vietnam has a rich - Determination of structure of bioactive ulvan extracted from source of green seaweeds with 152 species, mostly the genera Ulva, green seaweeds Ulva lactuca and Ulva reticulata collected at Nha- Caulerpa, Chaetomorpha, Enteromorpha, in there Ulva genus has 69 Trang bay of Vietnam. species and the most popular are Ulva reticulata và Ulva lactuca. - Evaluation of the effects of chemically structural modification Ulvan are water-soluble sulfated polysaccharides extracted from of ulvan to its biological activities. Ulva and Enteromorpha green seaweed genus. Ulvan essentially 3. Research contents of the dissertation contain rhamnose, xylose, glucuronic acid, iduronic acid and sulfate - Collect two green seaweed species that belong to Ulva genus: groups. Chemical components, chemical structure and bioactivities of Ulva lactuca and Ulva reticulata. ulvan with respect to linkage and sulfate position may vary depending - Research extraction methods to obtain ulvan samples. on the algae species, place of cultivation and method of extraction. - Studying on chemical structure of ulvan which have high Unfortunately, ulvan from green seaweeds are structurally diverse and bioactivities. heterogeneous, which makes studies on their structures challenging and - Study the effects of chemical denaturation (sulfation and hinders their development as therapeutic agents. Therefore, the acetylation) to structure and bioactivities of ulvan. determination of chemical structure of ulvan is demanded combining 4. Structure of the dissertation by numerous methods in logically. On the other hand, many works The study consists of 129 typewritten pages with 16 tables and reported that there is a relationship between structure and biological 55 figures. For instance, 2 pages of Introduction, 35 pages of activities of polysaccharides, so it is very important to study the Overview, 14 pages of Experiment, 55 pages of Results and overall structure including chemical and conformational structures of Discussions, 3 pages of Conclusions and Recommendations, 2 pages of polysaccharides. publication list, and 18 pages of References.
3 4 MAIN CONTENTS OF THE DISSERTATION Chapter 2. EXPERIMENT Chapter 1. OVERVIEW 2.1. Sample preparation This chapter includes overview of seaweed and sulfated 2.1.1. Seaweed collection and identification polysaccharide from seaweed, nutritional components and application Green seaweed Ulva reticulata and Ulva lactuca species were of seaweed, physicochemical properties, extraction, chemical harvested from Nhatrang sea of Vietnam in April 2014 and identified components, chemical structure and bioactivites of ulvan from Ulva by Dr Le Nhu Hau, Nhatrang Institute of Technology Research and species as following: Among the three main divisions of marine Application. A voucher specimen named UR-14 and UL-14 are macroalgae (Chlorophyta, Phaeophyta and Rhodophyta), marine green deposited in Nhatrang Institute of Technology Research and algae are valuable sources of structurally diverse bioactive compounds Application. and remain largely unexploited in nutraceutical and pharmaceutical 2.1.2. Analyze the chemical compositions of seaweeds areas. Green seaweeds are known to synthesize large quantities of Seaweed moisture, ash, protein, lipid and carbohydrate contents sulfated polysaccharides and are well established sources of these were estimated according to the method of AOAC – USA. particularly interesting molecules such as ulvan from Ulva and 2.1.3. Ulvan extraction and purification Enteromorpha, sulfated rhamnans from Monostroma, sulfated After study and reference many procedures and extractive arabinogalactans from Codium, sulfated galacotans from Caulerpa, and conditions reported by previous researcher, we could reveal 3 some special sulfated mannans from different species. These sulfated procedures to extract ulvan from two green seaweed species collected polysaccharides exhibit many beneficial biological activities such as as following: anticoagulant, antiviral, antioxidative, antitumor, immunomodulating, 2.1.3.1. Ulvan extraction by water: antihyperlipidemic and antihepatotoxic activities. Therefore, marine 20g dry algae per 400mL of water was heated for of 2h at 80– algae derived sulfated polysaccharides have great potential for further 90ºC on a boiling-water bath and under continuous stirring. After development as healthy food and medical products. ltration through a cotton cloth, the aqueous extracts were centrifuged, Ulvan are watersoluble sulfated polysaccharides extracted from and the liquid supernatant was ltered. The water extract was Ulva and Enteromorpha green seaweed genus. Ulvan essentially concentrated to reduce initial volume in a rotary evaporator. The water contain rhamnose, xylose, glucuronic acid, iduronic acid and sulfate extract was centrifuged, ltered and precipitated with 4 vol. of absolute groups. Chemical components, chemical structure and bioactivities of ethanol. The alcohol precipitate was separated from the supernatant by ulvan with respect to linkage and sulfate position may vary depending centrifugation then washed several times with ethanol and dried in a on the algae species, place of cultivation and method of extraction. This vacuum oven at 60ºC to a constant weight. The yield of ulvan was chapter also includes overview of the methods for structural 8.3% and 6.5% calculated based on algae dried weight. The samples determination of polysaccharide such as: GPC, IR spectroscopy, NMR signed UR-N and UL-N. spectroscopy, ESI-MS, Small angle X ray scattering SAXS. 2.1.3.2. Ulvan extraction by acid:
5 6 20g dry algae per 400mL of 0.05-0.1N HCl solution was heated 2.1.4.1. Cytotoxic activity assays for of 2h at 80º–90ºC on a boiling-water bath and under continuous Three human cancer cell lines HepG2 (Hepatocellular stirring. After ltration through a cotton cloth, the acid extract was carcinoma), MCF7 (human breast cancer), and Hela (cervical cancer) neutralized to pH=7, then centrifuged, and the liquid supernatant was were used for the assays. Cytotoxic assays were performed according ltered. The extract was concentrated to reduce initial volume in a to a method developed by Monks et al. rotary evaporator and precipitated with 4 vol. of absolute ethanol. The 2.1.4.2. Anticoagulant activity assays alcohol precipitate was separated from the supernatant by Anticoagulant assays were performed according to a method centrifugation then washed several times with ethanol and dried in a developed by Anderson et al. vacuum oven at 60ºC to a constant weight. The yield of ulvan was 2.1.4.3. Antimicrobial activity assays 7.2% and 6.4% calculated based on algae dried weight. The samples Antimicrobial assays were performed according to a method signed UR-H and UL-H. developed by Vanden và CS. 2.1.3.3. Ulvan extraction by alkali: 2.1.4.4. Antioxidant activity assays 20g dry algae per 400mL of 0.1N NaOH solution was heated for - Total antioxidant capacity was determined by the method of of 2h at 60ºC on a boiling-water bath and under continuous stirring. Prieto et al After ltration through a cotton cloth, the alkali extract was neutralized - Ferric reducing activity was determined according to the to pH=7, then centrifuged, and the liquid supernatant was ltered. The method of Zhu et al. extract was concentrated to reduce initial volume in a rotary evaporator 2.2. Structural determination of ulvan and precipitated with 4 vol. of absolute ethanol. The alcohol precipitate 2.2.1. Analyze the chemical components of ulvan was separated from the supernatant by centrifugation then washed - Sulfate content was determined according to the method of several times with ethanol and dried in a vacuum oven at 60ºC to a Dodgson et al. constant weight. The yield of ulvan was 5.1% and 4.1% calculated - Uronic acid content was determined by following the method of based on algae dried weight. The samples signed UR-K and UL-K. Bitter et al. Purification of ulvan: The crude polysaccharides were dissolved - Neutral monosaccharide compositions were elucidated by the in distilled water and the solution was passed through 10000 (Da) method of Bilan et al. MWCO under the tap in 72h and precipitated with 4 vol. of absolute 2.2.2. Gel Permeation Chromatography GPC: The weight average ethanol. The alcohol precipitate was separated from the supernatant by molecular mass and the number average molecular mass were centrifugation then washed several times with ethanol and dried in a elucidated on a HPLC Agilent 1100. vacuum oven at 60ºC to a constant weight. 2.2.3. IR spectra: IR spectra was recorded on a FT-IR Affinity-1S 2.1.4. Evaluation of the biological activity of ulvan SHIMADZU spectrometer. Bioactive experiments were examined for native and 2.2.4. NMR spectra: NMR spectra were recorded on Bruker ASCEND derivative ulvan. 500 in D2O solution using DSS as internal standard at 70ºC.
7 8 2.2.5. ESI-MS: ESI-MS experiments were performed on a LTQ 19%) than that of sulfated polysaccharides from other resources (20- Orbitrap Mass spectrometer. The analyses were carried out in negative 30%) and uronic acid is higher (18-23%) than others (6.5-19%) mode. calculated based on algae dried weight. These results are similar to with 2.2.6. SAXS: SAXS experiments were performed at BL19B2, SPring- last researches with the other green seaweed species. 8, Hyogo, Japan. 3.1.2. Results on the biological activities of ulvans 2.2.7. SEM: SEM pictures were photographed on a Nova NaNoSEM 3.1.2.1. Antimicrobial activity 450 – FEI equipment. Table 3.2. Antimicrobial activity results of 6 ulvans 2.3. Sulfation and acetylation Microbe UR-H UR-N UR-K UL-H UL-N UL-K Control - Sulfated derivatives were performed according to a method by Lihong Gra E. coli ++ ++ - ++ +++ - +++ Fan et al. Determination of sulfate content by weight method. m (-) Pseudomonas aeruginosa - + - - + - +++ - Acetylated derivatives were performed according to a method by Vibrio haveyi - - - - - - +++ Xiao-xiao Liu et al. Determination of acetyl content by method of Bacillus cereus - - - - ++ - +++ Luis.A. Gra Streptococcus faecalis - - - - - +++ m (+) Enterobacter cloace - ++ - + ++ - +++ Chapter 3. RESULTS AND DISCUSSIONS Staphylococcus aureus - - - - - - +++ 3.1. Sample selection 3.1.2.2. Cytotoxic activity 3.1.1. Results on the chemical components of ulvan Alkali extraction ulvan UL-K and UR-K were not expressed this Table 3.1. Chemical compositions of 6 ulvans activity. Table 3.3. Cytotoxic activity results of 4 ulvans SO3Na Uronic acid Monosaccharide compositions ( % mol) Ulvans (%w) (%w) Rha Gal Xyl Man Glu % survival cell UR-N 17,6 22,5 1 0,03 0,06 0,01 0,06 Concen UR-H Concen UR-N UR-H 14,3 19,3 1 0,1 0,11 0,01 0,21 trations trations Concen Ellipticine UR-K 13,2 19,8 1 0,1 0,14 0,03 0,14 (µg/ml) HepG HeLa MCF- (µg/ml) HepG HeLa MCF-7 trations UL-N 18,9 21,5 1 0,03 0,07 0,01 0,06 2 7 2 (µg/ml) UL-H 15,1 18,7 1 0,01 0,10 0,01 0,06 100 0.00 0.00 0.00 100 0.00 0.00 0.00 Hep HeL MC UL-K 14,2 18,2 1 0,04 0,09 0,01 0,07 20 42.26 50.30 44.72 20 50.34 36.29 48.74 G2 a F-7 Results of chemical components of ulvan were summarized in 4 70.18 69.05 76.84 4 84.22 83.05 89.66 5.9 10 1.33 3.31 Table 3.1. Like other ulvan, ulvan from U.Reticulta and U.lactuca are 0.8 83.24 90.44 85.45 0.8 94.90 96.44 95.91 3 mainly composed of rhamnose, with variable contents of galactose and IC50 49.10 47.75± 50.69 IC50 31.31 34.75 36.37± 29.1 21.8 28. xylose, trace amounts of glucose and mannose. Sulfate content of the (µg/ml) ±1.56 2.37 ±1.86 (µg/ml) ±1.56 ±1.38 1.73 2 4 7 86 sulfated polysaccharide from two Ulva species is relatively lower (13-
9 10 Concen UL-H Concen UL-N 3.2.1. Ulvan was extracted by acid from Ulva reticulata (UR-H) trations trations (µg/ml) (µg/ml) IR spectrum showed bands corresponding to a sulfated ester and HepG HeLa MCF- HepG HeLa MCF-7 49.4 48.8 48. 2 7 2 0.4 2 9 58 uronic acids. The signal at 848 cm-1 and 761 cm-1, might correspond to 100 0.00 0.00 0.00 100 0.00 0.00 0.00 the bending vibration of C-O-S of sulfate in axial and equatorial 20 50.21 55.34 48.67 20 60.67 66.24 58.71 81.5 77.1 76. position, respectively. The asymmetric stretching vibration of COO- 0.08 4 71.46 73.58 79.45 4 78.13 77.0 67.89 9 3 92 appears at 1624 cm-1. In addition, the band at 3315cm-1, owing to the 0.8 92.42 94.26 95.53 0.8 89.30 88.0 87.41 stretching vibration of OH bond, suggests the presence of hydrogen 0.4 IC50 40.74 44.25± 50.93 IC50 29.67 36.33 25.09± IC50 0.50 0.34 5 bonds in the molecules. The band at 2927 cm-1 corresponds to the (µg/ml) ±2.21 2.32 ±1.67 (µg/ml) ±2.87 ±3.84 1.36 (µg/ml) stretch vibration of C-H, while that at 1078 cm-1 signifies the 3.1.2.3. Antioxidant activity stretch vibration of CO and change angle vibration of OH. Table 3.4. Antioxidant activity results of 6 ulvans The 1H-NMR spectrum showed three anomeric proton signals at Ferric reducing activity Total antioxidant capacity 5.30, 5.20, and 4.65 ppm which were designated as A, B, and C, Samples 2+ (mgFe /g ulvan) (mg AcidAscobic/g ulvan) respectively. A broad signal at 1.17 ppm was assigned to the C-6 1 UL-N 2.51 3.75 methyl protons of rhamnopyranose and the signals in the range 3.4-4.3 2 UL-H 1.93 2.60 ppm were from the ring protons (Table 3.8). The 13C-NMR spectrum 3 UL-K 0.01 1.12 was indicative of complex polymers, containing signals in the 4 UR-N 4.86 3.96 resonance regions corresponding with anomeric carbons (94-102 ppm) 5 UR-H 1.34 2.56 and ring carbons (72-80 ppm) as well as C-CH3 signals (18.0 ppm) and 6 UR-K 0.22 0.86 a carboxyl signal at 167 ppm (Table 3.8). Four signals at 63.39, 63.32, Conclusions: The ulvan samples have been chosen to study chemical 63.12, and 63.06 ppm were from CH2 (C-6) groups of galactose and/or structure as following: glucose and/or CH2 (C-5) groups of xylose. The six 1H resonances from - Ulvan was extracted by water from Ulva reticulata (signed: UR-N) H-1 to H-6 of residue A were assigned from the cross peaks in the - Ulvan was extracted by acid from Ulva reticulata (signed: UR-H) COSY spectra. Based on the proton chemical shifts, the carbon - Ulvan was extracted by water from Ulva lactuca (signed: UL-N) chemical shifts (C-1 to C-6) were assigned from the HSQC spectra - Ulvan was extracted by acid from Ulva lactuca (signed: UL-H) (Figure 3.6). Both proton and carbon chemical shifts indicated that this 3.2. Determination of ulvan chemical structure glycosyl residue was typical of 6- deoxyhexopyranose, suggesting a Molecular weight Mw and molecular weight distribution Mw/Mn rhamnosyl moiety. The chemical shift of proton H-1(A) was 5.3 ppm, showed that like other native sulfated polysaccharides, ulvan from Ulva suggesting that residue A was α-linked. These results indicated that Reticulta and Ulva lactuca are polymers with high polydisperse with residue A might be α-rhamnose (Table 3.8). Furthermore, the Mw/Mn = 2.19 – 5.16 and molecular weights are Mw = 8.1x104 - downfield shift of the C-2 (79-80 ppm), C-3 (75-77 ppm), and C-4 (74- 3.47x105 g/mol which independent on extration conditions. 76 ppm) carbon signals with respect to the standard values for
11 12 rhamnose indicated that residue A might be linked by (1→2,3,4) From the peak of α-anomeric proton H-1(C) at 5.2 ppm in COSY glycosidic linkages and/or sulfated at C-2, C-3, and C-4 positions. It and HSQC spectrum, we can assign H-2(C) 3.6 ppm and C-2(C) at 77 also was confirmed by the correlations between H-1(A) and C-2(A), C- ppm, respectively. The downfield shift of the C-2 carbon signal with 3(A), C-4(A) in HMBC spectrum (Figure 3.7). The presence of some respect to the standard values for uronic acid indicated that residue C glycosidic linkage and positions of sulfate groups were indicated by the might be connected through (1→2) glycosidic linkages. Therefore split signals at H-1(A) and H-6(A). residue C might be α (1→2)-uronic acid (Table 3.8). Ulvan from ulva From the peak owing to the β-anomeric proton H-1(B) at green seaweeds essentially contains α-rhamnose, β-glucuronic acid, and 4.65 ppm in COSY and HSQC spectra, we can assign H-2(B) α-iduronic acid. We proposed residue B might be β (1→2)- glucuronic and H-3(B) at 3.2 and 3.8 ppm, and C-2(B) and C-3(B) at around acid and residue C might be α (1→2)-iduronic acid. The anomeric 77 and 74 ppm, respectively. The downfield shift of the C-2 carbons C-1(B) and C-1(C) were split into doublet, probably caused by carbon signal with respect to the standard values for uronic acid the linkage of B and C with two other sugars having α - and β - indicated that residue B might be connected through (1→2) conformation. The sequence of glycosyl residues was inferred from the glycosidic linkages. Therefore, residue B might be β (1→2)- HMBC spectrum. The HMBC spectrum revealed the inter-residue uronic acid (Table 3.8). 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 Figure 3.6. HSQC spectrum of UR-H Figure 3.7. HMBC spectrum of UR-H 375 were assigned to [RhaUroA]- and [RhaXylSO3]-, respectively. The Table 3.8. 1H and 13C-NMR chemical shifts of UR-H peaks at m/z 419 and 503 were assigned to the [RhaUroASO3]- and the 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 monosulfated trirhamnose [Rha3SO3]- ions, repsectively. The MS2 A 102- 79- 75-77 74-76 75-76 17-18 daughter ion at m/z 243 was fragmented (Figure 3.9). Three signals →2)--L-Rha 103 80 / 3.95 /3.85 /3.65 /1.17 from C-4 (m/z 183), C-3 (m/z 169), and C-2 (m/z 139) sulfation of α-L- →4)--L-Rha /5.30 /3.63 Rha residues were detected, indicating that the three positions C-2, C-3, B 98.52; 77.0 74.0 74.0 - 167 →2)--D-GlcA 98.39 /3.20 /3.75 / 3.85 and C-4 were sulfated. The peak at m/z 183, assigned to the major /4.65 fragment ion, the peak at m/z 139, and a minor fragment at m/z 169 C 94.70; 77.5 76.0 74.0 - 167 indicate that the rhamnosyl units of the ulvan are mainly sulfated at →2)--L-IdoA 94.51 /3.60 /3.95 /3.85 /5.20 position C-2 and C-4 and partly at C-3.
13 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 100.51/ 69.71/ 78.91/ 78.86/ 68.89/ 17.60/ →4)α-L-Rha3S 1→ 4.82 4.20 4.59 3.79 4.13 1.30 B 103.83/ 74.58/ 74.83/ 79.48/ 76.70/ 177.60 →2,4)β-D-GlcA 1→ 4.63 3.35 3.64 3.65 3.82 From obtained IR, NMR and MS results, we can conclusion that ulvan from green seaweed Ulva Reticulata collected at Nhatrang sea Figure 3.9. MS2 spectrum of [RhaSO3]- ion at m/z 243. obtained by water extraction has a backbone composed of disaccharide In conclusion, the major repeating dimeric sequences of ulvan [→4-β-D-GlcA-(1→4)-α-L-Rha3S-(1→] and branched point is at C-2 extracted from green seaweed Ulva reticulata collected at Nhatrang sea of glucuronic acid and the rhamnosyl units of the ulvan are sulfated at of Vietnam, consisted of β-D-GlcA(1→2)-α-L-Rha and α-L- position C-3. -3 IdoA(1→4)-α-L-Rha, and branched at O-2 of uronic acid. The Rha 7x10 Ulva reticulata Forsskål 1% -3 in water 6 in 0.5 M NaCl aq Ulva reticulata Forsskål 1% in water, Rgc=1.35nm 13 in 0.5 M NaCl aq, Rgc=1.37nm residues were sulfated at three positions, C-2, C-3, and C-4. The C -4 5 4 -5 ln(qI(q)) and 1H-NMR chemical shifts of the major signals in ulvan have been q I(q) 2 3 -6 2 attributed but those of xylose, galactose, and glucose present in the 1 -7 -8 polysaccharide remain to be identified. This is ulvan with new 0 0 1 2 q, nm -1 3 4 5 0 1 2 3 q 2 4 5 6 chemical structure as most of ulvan reported not have the linkage Figure 3.22. SAXS from UR-N ulvan 1% in water and in 0.5M NaCl: (1→2) in the main chains. Kratky plots and Guinier plots for cross-section 3.2.2. Ulvan was extracted by water from Ulva reticulata (UR-N) In order to find more structural information at molecular level of Chemical structure of ulvan extracted by water from green the ulvan, SAXS measurements have been carried out. Kratky [Left Fig seaweed Ulva reticulata were determined by IR, NMR, MS spectra as 3.22] and Guinier [Right Fig 3.22] plots for 1% UR-N ulvan in water UR-H ulvan. IR spectra of UR-N showed bands that are typical of and in 0.5M NaCl were the typical form for moleculars that was a rod- 1 1 1 -1 ulvan such as: 1595 cm , 1028 cm , 844 cm và 783 cm . The like confomational structure like to sulfate polysacchries extracted from complete NMR assignments are shown in Table 3.9. the other seaweeds such as carrageennan and fucoidan. From Kratky ESI-MS spectrum of UR-N shows fragment ion [M-H]- at m/z plots, the weak peak can be found in 0.5 nm-1 of q, due to the repulsive - 419, assinged to disaccharide [RhaUroASO3] . Monosulfated rhamnose electrostatic interaction. This means that this ulvan sample contains - - [RhaSO3] and uronic acid [UroA] are assigned corresponding with much amount of sulfate groups. An addition of saline, which produced peaks at m/z 243 and m/z 195. ESI-MS/MS spectrum of fragment ion the effect shielding against that force, made the peak disappear (L.Fig. m/z 243 shows a strong signal at m/z 169, this indicate that the 3.22). From Guinier plots (R.Fig. 3.22), the cross-sectional radius of rhamnosyl units of the ulvan are sulfated at position C-3. gyration Rgc can be estimated. The Rgc is 1.35-1.37nm, indicating
15 16 possibility of bulky side chains of the ulvan. This result is in agreement Obtained 1H and 13C-NMR, COSY, HSQC, HMBC analysis, the with the complex branching structure of the ulvan as elucidated by complete NMR assignments are shown in Table 3.10. 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 Figure 3.30. HSQC spectrum of UL-N calculated from the molecular model. Figure 3.31. HMBC spectrum of UL-N Hình 3.24. The molecular model of UR-N was constructed on the base Table 3.10. 1H and 13C-NMR chemical shifts of UL-N of chemical structure 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 100.27/ 77.92 73.82/ 71.87/ 61.20/ - →2)-α-D-Xyl-(1→ 5.36 3.65 3.95 3.82 3.80; 3.88 B 89.65; 79.21/ 71.74/ 76.88/ 68.40/ 17.12/ →2,4)-α-L-Rha-(1→ 101.50/ 4.06  3.71  3.82  4.0 1.31 4.90 C 100.40/ 69.46/ 78.68/ 78.68/ 68.65/ 17.35/ →4)-α-L-Rha3S-(1→ 4.82 4.23 4.61 3.80 4.13 1.31 D 103.65/ 74.40/ 74.58/ 79.40/ 76.88/ 177.76 Hình 3.25. Calculated scattering profiles (solid line) and observed →4)-β-D-GlcA-(1→ 4.64 3.35 3.65 3.68 3.81 SAXS (symbols) of UR-N. [UroA] - 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 [RhaSO 3]- such as: The signal at 848 cm-1 and 761 cm-1, might correspond with [XylUroA] - the bending vibration of C-O-S of sulfate in axial and equatorial [XylRhaSO 3] - [RhaUroA]- [UroARhaSO 3] - [Rha 3SO 3] - - position, respectively. The asymmetric stretching vibration of COO appears at 1599 cm 1, while that at 1026 cm 1 signifies the stretch Figure 3.32. ESI-MS spectrum of UL-N Figure 3.33. MS2 spectrum of [RhaSO3]- ion at m/z 243 vibration of glycoside linkage C-O-C.
17 18 ESI-MS/MS spectrum of fragment ion m/z 243 shows two obtained ulvans which are the same compositions in the main chains, signals at m/z 183 và m/z 169, this indicate that the rhamnosyl units of and difference of the branches, only. 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 Figure 3.37. HSQC spectrum of UL-H Figure 3.38. HMBC spectrum of UL-H 1 13 repeating units including GlcA- (1 → 2)-Xyl and GlcA-(1 → 2)-Rha Table 3.11. H and C-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 also occurred in the ulvan. The complete NMR assignments are shown D 102.45/ 71.62/ 80.80/ 80.70/ 70.79/ ~19.56/ in Table 3.10. Like this, ulvan obtained by water extraction from 4)-L-Rha3S 1 4.82 4.22 4.58 3.78 4.11 ~1.3 Vietnam green seaweed Ulva lactuca species which we are studying to E 105.69/ 76.44/ 76.72/ 81.52/ 78.49/ 1)-D-GlcA 4 4.65 3.35 3.60 3.65 3.86 have chemical structure the same as previous researches in the main D’ ~103.6/ 71.62/ 81.34/ ~74.10/ ~73.2/ ~19.48/ chains, difference in the branches and multiform in minor units. This 1)-L-Rha 3(4) 4.89 4.22 4.05 3.86 3.71 ~1.29 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: 1 The signals at 850 cm and 786 cm-1 might correspond with the bending vibration of C-O-S of sulfate in axial and equatorial position, 1 Figure 3.39. MS2 spectrum of [RhaSO3]- ion at m/z 243 respectively. The signal at 1014 cm is typical for C-O bond ESI-MS spectrum of UL-H shows fragment ion [M-H]- at m/z containing O-H group. The asymmetric stretching vibration of COO- 419, assinged to disaccharide [RhaUroASO3]-. Monosulfate rhamnose 1 group in uronic acid appears at 1599 cm . [RhaSO3]- and uronic acid [UroA]- are assigned corresponding with Obtained 1H and 13 C-NMR, COSY, HSQC, HMBC analysis of peaks at m/z 243 and m/z 195. MS2 spectrum of [RhaSO3]- ion at m/z UL-N ulvan, the complete NMR assignments are shown in Table 3.11, 243 (Fig. 3.39) shows: strong signal at m/z 169 and weak signal at m/z we could conclude that ulvan isolated by acid extraction from green 139, this indicate that the rhamnosyl units of the ulvan are maily seaweed Ulva lactuca mainly composed of disaccharide [→4)-β-D- sulfated at position C-3 and party at C-2, respectively. This results are GlcA-(1→4)-α-L-Rha3S-(1→] and branched at C-3 position of in agreement with the results obtained by IR and NMR analysis. rhamnose residues. In comparison with last reports about ulvan from In conclution, UL-H ulvan from green seaweed Ulva lactuca Ulva lactuca indicated that ulvan isolated by different extractions collected at Nhatrang sea obtained by acid extraction has a backbone
19 20 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 Figure 3.42. HSQC spectrum of UR-N (a) and UR-S (b) important role in the bioactivities of polysaccharides, especially Evaluating relation of structure - anticoagulant activity anticoagulant activity. On the other hand, acetylation could increase Surface structure of native ulvan and derivatives were antioxidant or antimicrobial activity. We synthesized some sulfated and photograph by SEM. Those pictures showed that there were obviously acetyled derivatives from native UR-N to aim studying the strutural differences in surface structure between native ulvan UR-N and modifications as well as ulvan biological activities. derivatives UR-S. UR-N moleculars are composed block that is flat 3.3.1. Effect of sulfation 13 surface structure while UR-S moleculars are seemly composed small IR và C- và HSQC - NMR spectra were used to examine the crytal blocks with definite conformation. structural change of derivatives UR-S. Obtained IR spectral analysis of Cytotoxic, antioxidant, antimicrobial and anticoagulant activities UR-S indicate that the signal at ~850 cm-1 is typical for sulfate group at assay were tested for sulfated derivatives and UR-N native ulvan axial position increased, at the same time the signal at ~3261-3370 cm-1 13 sample. Results revealed that anticoagulant activity was significant is typical for O-H group decreased; C-NMR spectrum shows that a increase while cytotoxic, antioxidant, antimicrobial activities were not new signal at δ77,2 ppm is C-2 of rhamnose residue, this proved that significant change. sulfation was at C-2 position of rhamnose residue; HSQC-NMR The degree of sulfation (DS) of polysaccharides is an important spectrum of UR-S derivatives were some new signals that shift to the parameter for analyzing the bioactivities. To aim studying the effection downfield in comparision with HSQC spectrum of native ulvan of sulfate content on anticoagulant activity, sulfated derivatives with extracted by water (Fig. 3.42), the interactive signal C-1/H-1 of different sulfate contents were synthesized and evaluated anticoagulant rhamnose residue was separated and at one there were two new signals activity. The anticoagulant activity results are shown in Table 3.12, expressing the interaction between C-2/H2 at δ80,0/3,38 ppm and C- Aspirin was used as controled agent. 3/H3 at δ76,5/3,67 ppm of glucuronic acid, the signal C-2 of rhamnose Table 3.12. Anticoagulant activity results of UR-N và UR-S residue increased. Therefore, there were three positions including C-2 Samples UR-N UR-S1 UR-S2 UR-S3 UR-S4 UR-S5 Aspirin of rhamnose and C-2, C-3 of glucuronic acid partly sulfated. In DS (%) 17.60 21.25 25.68 30.42 33.21 35.96 Clotting conclusion, the results obtained by NMR analysis are in agreement 10 25 40 45 30 37 48 time (phút) with the results obtained by IR analysis and last studies. The results of The results were reported that the anticoagulant ability of ulvan spectral analysis affirmed increasing of sulfate content for UR-S sulfated derivatives increased significantly as well as native ulvan. samples. However, there was not dependent of anticoagulant ability on sulfate content by rule. It was explained that there are some other factors to
21 22 concern anticoagulant ability of ulvan such as molercular weight, CONCLUSION sulfate group position…Previous researches explained that the We have obtained the following results: anticoagulant activity of sulfated polysaccharide inceased due to the 1. 6 samples of ulvan were extracted from green seaweed Ulva raise of negatively charged group and it is ability to unite to positively lactuca and Ulva reticulata species collected at Nha-Trang bay of charged group of coagulable inhibiter, therefore to increase Vietnam by water, acid and alkali extrations. They were anticoagulant activity. determined the chemical compositions and evaluated biological 3.3.2. Effect of acetylation activities. The results showed that 06 isolated ulvan were very The structural change of UR-N after acetylation was examine by various chemical compositions and biological activities that were 1 13 H-NMR and C-NMR spectra. dependent on their extracted methods. Among them, ulvan 1 13 H-NMR and C-NMR of UR-Ac were shown in Fig 3.44. Those isolated by water were the highest sulfate content, uronic acid 1 indicated that there is a new peak at δ2.007 ppm ( H-NMR) and at and rhamnose which showed the best biological activities. 13 δ22.5 ppm ( C-NMR) which express to be present of acetyl group O- 2. Using physical methods such as GPC, IR, NMR and MS, we CH3. Like this, by NMR spectra analysis we could conclude that the studied successfully on chemical structure of 4 ulvan samples UR-N sample was acetyled. extracted from 2 green seaweed species, as the following results: - Ulvan was extracted by acid from Ulva reticulata Figure 3.44. 1H-NMR spectrum a) and 13C-NMR spectrum b) of UR-Ac Sulfate group at C-2, C-3 and C-4 position of Rha. SEM picture showed peviously the differences in surface This is a new ulvan, as most of ulvans contained 1→4 linkage in structure between UR-N and UR-Ac samples. Flat surface structure of main chains, but this ulvan is 1→2 linkage in main chain. UR-N became softer with holes and big hollows - Ulvan was ecctracted by water from Ulva reticulata Evaluating relation of structure - antioxidant Cytotoxic, antioxidant, antimicrobial and anticoagulant activities assay were tested for acetylated derivative - UR-Ac and native ulvan Sulfate group at C-3 position of Rha. sample - UR-N. Results indicated that were not significant change in all - Ulvan was extracted by acid from Ulva lactuca of these activities. It was explained that when acetylation, ulvan was attacted hydrophobic acetyled group, so ulvan was more difficult to dissolve in water and hence it was disadvantage for biochemical reaction.
23 24 Sulfate group at C-3 position of Rha. SUGGESTIONS - Ulvan was extracted by water from Ulva lactuca 1. Chemical struture of ulvan in particular and polysaccharide in general are very complecated, furthermore there is a relationship between structure of ulvan and their biological activities, this relation have not been cleared up to now, and need to be further studied. Sulfate group at C-3 and C-4 position of Rha. 2. In Vietnam, there are other green seaweed species with different The obtained results of this thesis once again confirmed, the structural characteristics and useful biological activity that need to chemical structural dependence of ulvan on the geographical be further researched in order to be able to use the seaweed location where the seaweeds grow and the method of extracting source most effectively. them from seaweeds. 3. Conformatinal structure of ulvan extracted by water from Ulva THE NEW CONTRIBUTIONS OF THE DISSERTATION reticulata UR-N was determinated, the result indicated that this is possibility of rod-like and bulky side chains of the ulvan. 1. It is the first time the chemical and conformational structure of 4. The molecular modelling of ulvan was constructed that based on ulvans extracted from Ulva reticulata and Ulvan lactuca species the chemical structure, compared the theoretical scattering curve collected in Nha Trang bay of Vietnam were determined by using from the molecular structure model with the experiment of SAXS model physical methods such as GPC, IR, NMR, tandem ESI-MS measurements to confirm the chemical structure. This is a method and SAXS. that contributes to solving the problem of studying complex natural 2. A new structure of ulvan have been found out, comparing to polymers. published studies in the world. 5. Effection of chemically structural modification of ulvan 3. The molecular modelling of ulvan was constructed that based on the extracted by water from Ulva reticulata to its surface structure chemical structure, compared the theoretical scattering curve from and bioactivities was studied. The results showed that: the molecular structure model with the experiment of SAXS  The sulfation changed in surface structure and were not measurements to confirm the chemical structure. This is a method significant change cytotoxic, antioxidant, antimicrobial that contributes to solving the problem of studying complex natural activities of ulvan. Meanwhile, anticoagulant activity was polymers. significant increase, however, there was not dependent of 4. The effect chemically structural modification of ulvan on its anticoagulant ability on sulfate content by rule. biological activity was studied, initially. This is a new research  The acetylation significantly effected surface structure but comparing to the same field studies in the country. didn’t increase exprimental biological activities of ulvan.
PUBLISHED WORKS OF THE DISSERTATION 1. Q.T.M Thu, Đ.V Lương, T.T.T Vân, B.M Lý và T.T.T Thủy (2014). Ulvan từ rong lục ulva reticulata: chiết tách, thành phần hóa học và hoạt tính sinh học. Tạp chí Hóa học, 52 (6A), 158-162. 2. Q.T.M Thu, N.T Nu, Đ.V Luong, B.M Ly, T.T.T Van and T.T.T Thuy (2015). Structural characterization of ulvan from green seaweed Ulva reticulata. Vietnam Journal of Chemistry, 53 (6e1,2), 386-390. 3. Q.T.M Thu, T.H Bang, N.T Nu, Đ.V Luong, B.M Ly, T.T.T Van and T.T.T Thuy (2015). Structural Determination of Ulvan from Green Seaweed Ulva reticulata Collected at Central Coast of Vietnam. Chemistry Letters, 44 (6), 788-790. 4. T.T.T Thuy, Q.T.M Thu, T.T.T Van, D.V Luong and N.T Tai (2015). Ulvan from green seaweed Ulva reticulata: Extraction and structure. The 13th Japan – Vietnam Joint Seminar, 29-30 October 2015 Kyoto, Japan, 23. 5. T.T.T Thuy, T.M.T Quach, T.N Nguyen, D.Vu Luong, B.M Ly and T.T.V Tran (2016). Structure and cytotoxic activity of ulvan extracted from green seaweed Ulva lactuca. International Journal of Biological Macromolecules 93, 695-703. 6. T.T.T Thuy, Q.T.M Thu, N.T Nu, D.V Luong, N.T Tai, T.T.T Van (2016). Structure and biological activity of sulfated polysaccharide extracted from green seaweed Ulva lactuca. The 14th Japan-Vietnam Joint Seminar, Hanoi 22th September 2016, 21. 7. Q.T.M Thu, N.T Nụ, Đ.V Lương, N.T Tài, T.H Bằng, T.T.T Vân, H.Đ Cường, T.T.T Thủy (2016). Nghiên cứu cấu trúc và hoạt tính chống oxy hóa của ulvan chiết tách từ rong lục Ulva lactuca. Tạp chí Hóa học, 54 (6e2), 75-78. 8. Q.T.M Thu, Đ.V Luong, N.T Nu, T.T.T Van, B.M Ly and T.T.T Thuy (2016). Effect of sulfation on the structure and anticoagulant activity of ulvan extracted from green seaweed Ulva reticulata. Vietnam Journal of Science and Technology, 54 (2C), 373-379. 9. Thanh T T T, Quach T M T, Nguyen T N, Dang V L, Tran T T V, Bui M L (2017). Structure and cytotoxicity of the green seaweed originated ulvan. The 17th Asian Chemical Congress, 23-28 July 2017 Melbourne Australia, 332. 10. T.T.V Tran, H.B Truong, N.H.V Tran, T.M.T Quach, T.N Nguyen, M.L Bui, Yoshiaki Yuguchi & T.T.T Thanh (2017). Structure, conformation in aqueous solution and antimicrobial activity of ulvan extracted from green seaweed Ulva reticulata. Natural Product Research, DOI: 10.1080/14786419.2017.1408098.