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MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY Pham Thu Hue RESEARCH ON COMPOSITION AND BIOLOGICAL ACTIVITIES OF LIPIDS IN SOME SEAWEED SPECIES IN VIETNAM Major: Code: Chemistry of Natural Compounds 44 01 17 SUMMARY OF DOCTORAL THESIS IN CHEMISTRY Hanoi - 2021 The thesis was completed at: Graduate University of Science and Technology Vietnam Academy of Science and Technology Supervisor 1: Assoc Prof Dr Tran Thi Thanh Van Supervisor 2: Dr Le Tat Thanh Opponent 1: Opponent 2: Opponent 3: The thesis will be defended at an institutional-level Thesis Committee, meeting at Graduate University of Science and Technology - Vietnam Academy of Science and Technology at …… hour……., on day … month … year 2021 The thesis is available at: - Library of the Graduate University of Science and Technology - National Library of Vietnam INTRODUCTION Rationale of the doctoral thesis Research on chemistry of natural compounds from marine organisms, including seaweeds, is an important research direction in the 21st century With a total of about 1000 species found in Vietnamese waters, seaweeds have become an important economic object of our country In addition to researches which have created many valuable products from seaweeds such as alginate, carrageenan, fucoidan, etc., in recent years, much attention has been paid to seaweed lipids because of their effects on prevention and treatment of diseases of the joints, heart and brain Polyunsaturated fatty acids such as arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and polar lipids in seaweed such as monogalactosyldiacylglycerol (MGDG), sulfoquinovosyldiacylglycerol (SQDG) have shown antitumor and anti-inflammatory properties, and effects of inhibition of DNA polymerase and HIV Currently, with modern research equipment such as highperformance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS), world science has made great progress in the determination of polar lipid molecular species without the need to isolate each individual compound In the world, only a few seaweed species have been studied and published in regard to the polar lipid molecular form In Vietnam, seaweed lipids have only been studied biochemically in the traditional way Approaching the modern research direction on polar lipids, contributing to creating a scientific basis for effective use of seaweed resources and linking the publication of new scientific data with the affirmation of Vietnam’s sovereignty over seas and islands and territorial integrity, we carried out the thesis topic “Research on Composition and Biological Activities of Lipids in some Seaweed Species in Vietnam” Research objectives The objectives of the thesis are to approach modern research direction, build a basic survey data on seaweeds in Vietnam, support the field of seaweed taxonomy, and learn about the correlation between biodiversity and chemical diversity The thesis also aims to create a scientific basis to orient the conservation, cultivation and effective use of seaweed resources, as well as linking the publication of new research findings with the affirmation of Vietnam’s sovereignty over seas and islands and territorial integrity Research content of the thesis Study and examination of lipids and fatty acids in 60 samples of 50 seaweed species belonging to three groups of the red seaweed, the brown seaweed and the green seaweed Determination of polar lipid molecular forms of two selected seaweed species collected from Con Dao and Truong Sa Archipelago, Vietnam In vitro bioactivity survey of lipid fractions from two selected seaweed species CHAPTER OVERVIEW 1.1 Overview of the seaweed The overview shows the introduction about the role of seaweeds in the marine ecosystems, the classification and distribution of seaweed in Vietnam In addition, it also mention to the introduction about two genera Lobophora and Halimeda, and studies on chemical composition 1.2 Seaweed lipids General introduction about lipids and seaweed lipids, lipid classes, international and national studies on seaweed lipids 1.3 High-resolution mass spectrometry in the polar lipid studies Introduction about the LCMS-IT-TOF system and its application in the polar lipid research CHAPTER RESEARCH OBJECT AND METHODOLOGY 2.1 Research objects The research objects are 60 samples of 50 seaweed species, 14 families belonging to groups of Red seaweed, Brown seaweed and Green seaweed collected in the sea from Quang Ninh to Khanh Hoa and 02 offshore islands of Con Dao and Truong Sa - Vietnam 2.2 Research methods 2.2.1 Sample collection and preservation method According to “Marine Environment and Natural Resources Investigation Process - Part of Environmental Biology and Chemistry” 2.2.2 Isolation and extraction of lipids and fatty acids 2.2.2.1 Extraction and determination of total lipid content Extraction of total lipids according to the method of E.G Bligh and W.J Dyer 2.2.2.2 Analysis of composition and content of lipid classes in total lipids According to the method of image analysis on Sorbfil TLC Videodensitometer, Krasnodar, Russia 2.2.2.3 Analysis of composition and content of fatty acids Formatted: Left, Space After: pt, Line spacing: Multiple 1,08 li, Tab stops: 8,54 cm, Left Determination of the composition and the content of fatty acids by gas chromatography (GC, GC-MS) according to the method of Carreau & Dubacq 2.2.3 Principal component analysis method SAS-JMP Statistical discovery Pro 13:2:1 69 software was used for principal component analysis (PCA) 2.2.4 Determination of polar lipid molecular species Determination of the content and the structure of molecular forms was carried out by the HPLC-HRMS method of Imbs et al (2015, 2017, 2019) and Sikorskaya et al (2018) on the LCMS-IT-TOF system (Shimadzu, Japan) 2.2.5 Evaluation of biological activity 2.2.5.1 Free radical scavenging activity: according to the method of Kumar, 2013 2.2.5.2 Tested antimicrobial activity: according to the method of Vlietinck A.J., 1998 and Vanden Berghe, 1991 2.2.5.3 Cytotoxic activity on cancer cells: carried out on cancer cell lines by SRB according to the method of Monks A, 2011 2.2.5.4 Anti-inflammatory activity: determined through the ability to inhibit NO production of RAW 264.7 cells stimulated by LPS, according to the method of Liao H, 2014 CHAPTER EXPERIMENT The samples were studied according to the general diagram as follows: Figure 3.1 General study diagram 3.1 Extraction and determination of total lipid content 3.2 Determination of composition and content of fatty acids 3.3 Determination of composition and content of classes 3.4 Determination of polar lipid molecular species The analysis was carried out in an electrospray ionization (ESI) mode that simultaneously recognizes positive and negative ion signals Scanning was performed with m/z range of 100-1200 The individual molecular forms of polar lipids were detected by comparison with validation standards on the Shimadzu LC-MS processing software The percentage of molecules in each molecular group and in the lipid subclass was calculated by the peak area of negative ions [MH]-, excepted that molecular forms PC, MGDG, DGDG, etc., were determined by the peak area of the positive ions [M+H]+ or [M+Na]+ Determination of each molecular group in polar lipids according to the retention time and high-resolution mass spectrometry (HRMS) data of 158 polar lipid molecules from Lobophora sp and 64 polar lipid molecules from the species Halimeda incrassata Lamx have shown on table 3.1, figure 3.2 Table 3.1 Retention time of the groups Group Classes Retention time (minute) Phospholipid PA PG PC 3.5-5.6 5.4-6.8 7.5-11.5 Glycolipid Betaine lipid PI MGDG DGDG SQDG DGTS DGTA 16.5-20 2.5-3.5 8.5-14 3.5-5.5 4.5-7 10-16 Figure 3.2 Retention time of classes when using Diol column CHAPTER RESULTS AND DISCUSSION 4.1 Study and examination of lipids and fatty acids of seaweed 4.1.1 Total lipid content and lipid classes - The total lipid content of 60 seaweed samples mostly fluctuates in the range of 0.35 - 0.58% (based on fresh weight) Some samples with considerably high lipid content are 29KT (1.7%), 18KT (1.6%), 10A (1.49%), 9A (1.34%), 33KT (1.1%), and 1KT (1.06%) The total lipid content and composition in seaweed samples depends on the biological nature of the species, growth conditions and sampling time - The results of lipid analysis by Sorbfil TLC Videodensitometer showed that most of seaweed lipids consist of lipid classes: polar lipids (Pol), sterols (ST), free fatty acids (FFA), triacylglycerol (TG), hydrocarbons and waxes (HW), in which Pol and TG have the highest content 4.1.2 Composition and content of fatty acids 4.1.2.1 Composition and content of fatty acids of red seaweeds Analysis of 20 red seaweed samples identified 30 fatty acids with carbon chains from C12 to C22 Table 4.4 Values of the parameters that measure the tendency to concentrate fatty acids Parameter SFA MUFA PUFA Mean Min Q1 Median Q3 Max 50.17 20.66 39.50 54.92 61.52 64.32 23.39 9.58 19.48 21.97 27.66 45.51 19.93 4.73 8.36 12.97 26.56 63.89 n-3 (ω3) 11.19 2.17 4.56 6.94 10.76 44.86 n-6 (ω6) 7.76 0.83 2.99 3.72 14.21 24.42 n-9 (ω9) 15.51 6.01 11.67 13.29 20.05 29.48 PUFA /SFA 0.54 0.09 0.15 0.22 0.70 3.09 ω3/ ω6 3.88 0.48 0.89 1.32 1.78 44.41 According to WHO, PUFA/SFA value > 0.4: healthy food and ω3/ω6 value > 0.1: good for health Some samples in the red seaweed group should be noted: + Palisada parvipapillata species (sample 23KT) has the highest PUFA and ω6 content and PUFA/SFA ratio, and the second highest ω3 content + Hypnea panosa species (sample 12KT) has the highest ω3 and DHA content (43.69%) and ω3/ω6 ratio, and the second highest PUFA content and PUFA/SFA ratio 4.1.2.2 Composition and content of fatty acids of brown seaweeds Analysis of 31 samples of brown seaweed identified 30 fatty acids with carbon chains from C12 to C22 Table 4.5 Values of parameters that measure the tendency to concentrate fatty acids Parameter SFA Mean Min Q1 Median Q3 Max 50.26 19.62 38.59 45.63 54.97 87.69 MUFA PUFA 24.52 4.87 19.87 24.64 28.27 45.21 21.54 0.00 11.86 20.82 28.31 59.98 n-3 (ω3) 9.56 0.00 3.16 9.42 12.52 34.15 n-6 (ω6) 11.00 0.00 4.21 9.36 16.51 28.48 n-9 (ω9) 17.17 3.80 13.75 17.73 19.87 29.28 PUFA /SFA 0.59 0.00 0.20 0.52 0.66 2.71 ω3/ ω6 2.04 0.00 0.48 0.63 1.45 26.43 + Lobophora sp (sample 1KT) has the highest ω3 and PUFA content, and the second highest PUFA/SFA ratio + Sargassum polycystum species (sample 20KT) has the highest ω6 content and PUFA/SFA ratio, and the second highest PUFA content (AA reaching 27.12%) 4.1.2.3 Composition and content of fatty acids of green seaweeds Analysis of samples of green seaweed identified 28 fatty acids Table 4.6 Values of parameters that measure the tendency to concentrate fatty acids Parameter Mean Min Q1 Median Q3 Max SFA MUFA PUFA n-3 (ω3) n-6 (ω6) n-9 (ω9) PUFA /SFA ω3/ ω6 41.10 28.92 36.34 38.89 47.27 56.80 22.15 20.05 20.93 22.04 22.60 25.40 22.78 9.66 16.27 25.88 27.12 33.25 15.90 3.38 10.25 16.93 20.87 25.84 6.03 0.20 1.76 5.14 7.57 15.00 11.02 6.99 7.91 9.49 12.42 21.54 0.60 0.17 0.34 0.67 0.75 1.05 29.52 0.61 1.13 2.76 5.61 126.98 Further study on Halimeda incrassata Lamx is needed (TSL sample) has the highest PUFA and ω6 content, and the second ω3 and PUFA/SFA ratio in the green seaweed group 14 6.38 36:6 5.27 35:1 6.02 36:5 16:0/20:6 16:0/19:1 17:0/18:1 0.88 5.69 40:7 0.47 5.39 40:6 16:0/20:5 1.83 5.21 40:5 20:3/20:4 20:2/20:4 20:3/20:3 20:1/20:4 20:2/20:3 DGTS is an isomer structure and precursor for DGTA biosynthesis in the brown seaweed Therefore, the co-existence of 27 molecular forms of DGTS and 37 molecular forms of DGTA in the new brown seaweed Lobophora sp can be explained by a partial conversion from DGTS to DGTA; these betaine lipids are as amphoteric as phospholipids because they have a positively charged quaternary ammonium group and are thought to have a similar structural function to phospholipids in various types of seaweed 4.3 Determination of polar lipid molecular forms of green seaweed Halimeda incrasata Lamx 4.3.1 Determination of phospholipid molecular forms The table below shows 07 molecular forms of phospholipids of groups of PG (4), PI (3) and the way to determine the molecular forms with high content in each group of PG and PI Rt (minute) PG Diacyl % PG Rt (minute) PI Diacyl % PI 6.99 32:2 16:1/16:1 7.81 18.09 32:0 16:0/16:0 52.03 7.08 34:4 16:1/18:3 6.76 17.39 34:0 16:0/18:0 17.66 6.72 34:3 16:1/18:2 61.02 16.68 36:4 16:0/20:4 30.31 6.54 34:2 16:0/18:2 24.41 Discussion: In the phospholipid subclass of the green seaweed Halimeda incrassata Lamx., there are molecular forms in groups of PI and PG, and there is no appearance of PC, PE and PS like other previously announced green seaweed species, possibly because this 1.72 0.98 0.44 15 seaweed species has a very high CaCO3 synthesis capacity (35-45%) equally with corals 4.3.2 Determination of glycolipid molecular forms The table below shows 40 types of glycolipid molecular forms of groups of MGDG (12), DGDG (11), SQDG (17) and the way to determine the molecular forms with high content in each group Rt MGDG (minute) 3.11 3.02 2.92 30:2 30:1 30:0 3.14 32:4 2.96 32:2 Rt MGDG (minute) Diacyl % MGDG 14:1/16:1 14:0/16:1 14:0/16:0 16:1/16:3 14:1/18:3 16:1/16:1 14:0/18:2 1.34 3.70 20.26 Diacyl % MGDG Rt (minute) DGDG Diacyl % DGDG 10.34 10.58 30:0 32:2 23.26 4.37 10.04 32:1 9.51 32:0 9.71 34:2 14:0/16:0 14:0/18:2 16:0/16:1 14:0/18:1 16:0/16:0 14:0/18:0 16:0/18:2 Rt (minute) DGDG Diacyl % DGDG 9.23 8.78 34:1 34:0 11.42 9.15 % SQDG 2.50 2.19 3.52 2.51 2.10 4.69 0.46 1.26 3.68 4.31 9.12 2.78 3.21 3.08 2.91 2.81 32:0 34:6 34:5 34:3 36:4 16:0/16:0 16:3/18:3 16:3/18:2 16:1/18:2 16:0/20:4 4.22 9.86 18.83 2.31 7.69 Rt (minute) SQDG 28:0 30:1 30:0 32:3 32:1 32:0 33:0 34:4 Diacyl 14:0/14:0 14:0/16:1 14:0/16:0 16:0/16:3 16:0/16:1 16:0/16:0 16:0/17:0 16:0/18:4 % SQDG 14.99 14.69 14.09 14.78 13.77 13.35 12.63 14.43 2.54 1.05 29.47 1.89 4.26 36.58 0.48 0.82 8.57 36:1 Rt (minute) SQDG 16:0/18:1 16:0/18:0 16:0/20:1 18:0/18:1 Diacyl 13.83 13.59 12.72 12.27 13.81 13.38 12.03 14.31 13.68 34:3 34:2 34:1 34:0 36:5 36:4 36:1 42:11 42:10 16:0/18:3 16:0/18:2 16:0/18:1 16:0/18:0 16:0/20:5 16:0/20:4 16:0/20:1 20:5/22:6 20:4/22:6 6.19 38.84 5.91 0.87 Discussion: Some other studies showed antiviral and antibacterial activities of SQDG 32:0 and 30:0 (diacyl 16:0/16:0 and 14:0/16:0, respectively) The study of Wang et al also showed that SQDG 32:0 (16:0/16:0) has antiviral activity against HSV (antiviral) Because these are also two types of molecules with high content in the 16 subclass SQDG of the green seaweed Halimeda incrassata Lamx with content of 36.58% and 29.47%, respectively, this glycolipid subclass can also have good antiviral activity potential 4.3.3 Determination of betaine lipid molecular forms From HPLC-HRMS, in the polar lipids of the green seaweed sample H incrassata Lamx., we have identified 17 types of betaine lipid molecules, all of which are belonging to the diacylglycerylN,N,N-trimethylhomoserine (DGTS) group Rt (minute) 5.16 4.83 5.07 4.68 4.44 4.92 4.71 DGTS 28:0 30:0 32:3 32:1 32:0 34:4 34:3 Diacyl % DGTS 14:0/14:0 14:0/16:0 14:0/18:3 16:0/16:3 14:0/18:1 16:0/16:1 16:0/16:0 6.57 18.26 5.71 4.15 15.22 6.60 12.56 Rt (minute) DGTS Diacyl 4.32 4.26 4.47 4.14 4.47 3.99 34:1 34:0 36:4 36:1 38:6 38:1 16:0/18:1 16:0/18:0 16:0/20:4 16:0/20:1 16:0/22:6 16:0/22:1 % DGTS 10.38 1.29 9.18 7.02 1.95 1.11 Below are examples of the way to determine specific molecular forms for groups of subclasses of phospholipids, glycolipids and betaine lipids in the polar lipids class of seaweed • Determine the molecular form of PI 34:1 with Rt = 19.04 minutes and the highest content of 44.09% in the phospholipid class of brown seaweed Lobophora sp On MS- spectrum, ion [M-H]- has the strongest signal at m/z 835.5283 (Figure 4.6-b) Ion [M-H]- at m/z 835.5283 with formula [C43H79O13P]-, calculated 835.5221, different 0.00620 is selected to analyse on MS2- On MS2- spectrum (Figure 4.6-c), the signal at m/z 297.0334 is a semimolecular ion with inositol [C9H14O9P]- due to the loss of acyl groups This fragmentation is important in determining the molecular 17 forms of PI Signals at m/z 255.2338 corresponding to the fatty acid anion 16:0 [C16H31O2]-, at m/z 281.2471 corresponding to the fatty acid anion 18:1 [C18H33O2]-, and at m/z 391.2226 are signals corresponding to [M-H]- ion which loses simultaneously the fatty acid molecule 18:1 [C18H34O2] and inositol [C6H10O5]; signal at m/z 409.2304 is fragment of ion [C19H38O7P]- when [M-H]- ion loses simultaneously keten molecule of fatty acid 18:1 [C18H32O] and inositol [C6H10O5]; signal at m/z 417.2373 is the signal of [C21H38O6P]- ion when [M-H]- ion loses simultaneously fatty acid molecule 16:0 [C16H32O2] and inositol [C6H10O5]; at m/z 553.2744 [C25H46O11P]- ion is the signal of [MH]ion which loses fatty acid molecule 18:1 [C18H34O2]; at m/z 571.2832 [C25H48O12P]- is the signal of [M-H]- ion which loses keten molecule of fatty acid 18:1 [C18H32O]; at m/z 579.2906 the signal is [C27H48O11P]- ion when [MH]- ion loses the fatty acid molecule 16:0 [C16H32O2] Thus, mass spectrometry data proves that the considered molecular form is diacyl glycerophosphoinositol PI 16:0/18:1 By GC and GC-MS analysis, the fatty acid in total lipids and polar lipid fractions of brown seaweed Lobophora sp is PI 16:0/18:1n-9 By the same method, we identified the remaining phospholipid molecular forms of seaweed 18 Figure 4.6 HPLC-HRMS and fragmentation of PI 34:1 [C43H79O13P]- (a) HPLC chromatogram of [C43H79O13P]-, (b) Mass spectrum (MS-) of [C43H79O13P]-, (c) Mass spectrum (MS2-) at m/z 835.5283 • Determine the molecular form of SQDG 32:0 with Rt=13.35 minutes and the highest content of 36.58% in the glycolipid subclass of the green seaweed Halimeda incrassata Lamx On the negative ion spectrum MS-, there is a signal of the strongest negatively charged ion [M-H]- at m/z 793.4977 [C41H77O12 S]- The molecular formula determined is C41H78O12S This negative ion is selected to bombard MS2- (Figure 4.18) 19 Figure 4.18 HPLC-HRMS and fragmentation of SQDG 32:0 [C41H78O12S]; (a) HPLC chromatogram of [C41H77O12S]-; (b) Mass spectrum (MS-) of [C41H77O12S]-; (c) Mass spectrum (MS2-) at m/z 793,4977 On spectrum MS2- (Figure 4.18-c), the signals at m/z 537.2656 appear simultaneously corresponding to [C25H45O10S]- ion because the [M-H]- ion loses a neutral molecule at m/z 256.2321 of the fatty acid 16:0 [C16H32O2], and the signal at m/z 255.2286 corresponds to the anion fragment value of the fatty acid 16:0 [C16H31O2]- Thus, with the mass spectrometry data on MS2-, the molecular form SQDG 32:0 is 16:0/16:0 By the same method, we identified the remaining glycolipid molecular forms of seaweed Comments: Some other studies showed antiviral and antibacterial activities of SQDG 32:0 and 30:0 (diacyl 16:0/16:0 and 14:0/16:0, respectively) The study of Wang et al also showed that SQDG 32:0 (16:0/16:0) has antiviral activity against HSV (antiviral) Because these are also two types of molecules with high content in the class SQDG of the green seaweed Halimeda incrassata Lamx with 20 content of 36.58% and 29.47% respectively, this glycolipid subclass can also have good antiviral activity potential • Determine the molecular form of DGTS 34:3 with Rt= 4.71 minutes and high content of 12.56% in betaine lipid subclass of green seaweed Halimeda incrassata Lamx On the positive ion spectrum MS+, the strongest signal [M+H]+ at m/z 734.5857 corresponds to the ion formula [C44H80NO7]+ (Figure 4.20b) On the spectrum MS2+ (Figure 4.20c), the signal at m/z 506.3562 corresponds to the neutral molecular fragments lost as m/z 228.2295 [C14H28O2] (the fatty acid 14:0) Figure 4.20 HPLC-HRMS and fragmentation of DGTS 34:3 [C44H80NO7]+; (a) HPLC chromatogram of [C44H80NO7]+, (b) Mass spectrum MS+ of [C44H80NO7]+; (c) Mass spectrum MS2+ at m/z 734,5857 In addition, the signal at m/z 428.3346 corresponds to the missing fatty acid molecule m/z 306.2511 [C20H34O2] (the fatty acid molecule 21 20:3) The fragmentation at m/z 236.1668 [C10H22NO5]+ is an important signal in determining the molecular forms of DGTS Therefore, DGTS 34:3 is determined to be specific for diacylglycerylN,N,N-trimethylhomoserine DGTS 14:0/20:3 Additionally, the signals at m/z 496.3571 and m/z 478.3535 are obtained because the [M+H]+ ion loses the neutral ketenite fragments of the fatty acid 16:0 with m/z 238.2286 [C16H30O] (the fatty acid 16:0 losing water molecule) and m/z 256.2322 [C16H32O2] (the fatty acid16:0) Simultaneously, on MS2+, two signals appeared at m/z 474,3801 and m/z 456.3654, corresponding to the neutral ketenite fragments at m/z 260.2056 [C18H28O] (the fatty acid 18:3 losing water molecule) and m/z 278.2203 [C18H30O2] (the fatty acid 18:3) Thus, at the same mass value of [M+H]+ ion at m/z 734.5857, we can identify two molecular forms that are isomers of each other, including DGTS 14:0/20:3 and DGTS 16 :0/18:3, in which DGTS 16:0/18:3 occupies the main content By GC and GC-MS analysis, fatty acid composition in total lipids and polar lipid fractions in the green seaweed Halimeda incrassata Lamx are DGTS 14:0/20:3n-3 and 16:0/18:3n-3 By the same method, we determined the remaining molecular forms of the lipid betaine of seaweed Comments: Several studies have shown that the DGTS group in the betaine lipid subclass is capable of replacing the PC group in the outer membrane of chloroplasts Thus, the absence of PC subclass in the green seaweed Halimeda incrassata Lamx is reasonable because in the betaine lipid subclass only DGTS group with 17 molecular forms was detected 22 4.4 Results of bioactivity evaluation 4.4.1 Free radical scavenging activity At a concentration of 200 g/mL, all lipid fractions show only low DPPH radical “scavenging” activity with levels of 16.27-26.36% This shows that the lipid fractions of two species of seaweed Lobophora sp and Halimeda incrassata Lamx are quite resistant to oxidation Previous publications on the oxidative stability of lipid fractions in seaweed showed that glycolipid omega-3 fatty acids strongly exhibit this property 4.4.2 Antimicrobial activity All fractions are resistant to several test strains of P aeruginosa, F oxysporum, S aureus, E coli, A niger at MIC of 150 and 200 g/mL In the green seaweed sample Halimeda incrassata (TSL), the polar lipid (Pol) fraction exhibits antibacterial activity against P.aeruginosa, the non-polar lipid (NPL) fraction exhibits activity against the mold F Oxysporum, and the total lipid (TL) fraction exhibits activity against Gram (+) S aureus In the brown seaweed sample Lobophora sp (1KT), the total lipid (TL) fraction is well resistant to both strains of E coli and A niger, the non-polar lipid (NPL) fraction shows activity against A niger, and the polar lipid (Pol) fraction is resistant to E coli 4.4.3 Cytotoxic activity on cancer cells At a concentration of 100 g/mL, only the polar lipid fraction of Lobophora sp has cytotoxic effects on SK-LU-1, MCF7 and Hep G2 cell lines with growth inhibition levels of 44.94, 49.97 and 48.41, respectively Polar lipid fraction of Halimeda incrassata Lamx 23 inhibits the growth of the MCF7 cell line by 42.5% The lipid fractions of these two seaweeds may have antitumor effects, but by a different mechanism, such as a sulfolipid (R-12) of the SQDG group (SQDG 16:0/20:5) isolated by Ohta et al., which is also present in the polar lipids of two species of Lobophora sp and Halimeda incrassata Lamx with concentrations of 2.3 and 2.1% respectively, inhibiting the activity of polymerase enzymes and TdT with IC50 1-2 µg/ml 7.5 µg/ml 4.4.4 Anti-inflammatory activity The anti-inflammatory activity was evaluated in vitro for inhibition of NO production in LPS-stimulated RAW 264.7 cell line Test results showed that lipid fractions of seaweed species Lobophora sp and Halimeda incrassata Lamx well inhibited NO production with IC50 of 52.1- 66.21 µg/mL and 32.57- 41.66 µg/mL (positive control L-NMMA with IC50 of 8.90 µg/mL) Table 4.26 Determination results of anti-inflammatory activity Concentration (µg/mL) 100 20 0.8 IC50 100 20 0.8 IC50 TL (1KT) % NO inhibition % living cells 80.93 96.06 15.25 99.47 5.86 -2.82 61.09±6.06 TL (TSL) 74.68 94.96 35.06 102.75 12.66 1.61 41.66±1.95 - PoL (1KT) % NO inhibition % living cells 69.07 97.29 18.47 98.00 4.66 -1.29 66.21±6.24 PoL (TSL) 79.22 98.99 37.99 101.55 23.05 5.04 32.57±3.75 - NPL (1KT) % NO inhibition % living cells 84.75 98.24 20.76 102.00 7.20 -3.61 52.10±4.43 NPL (TSL) 76.30 97.93 39.61 100.90 24.03 10.75 33.59±3.86 Most clinical studies use omega-3 and omega-6 fatty acids that are in the class of triglycerides or ethyl esters Recently, however, 24 products derived from marine organisms rich in omega and omega fatty acids are in polar lipid subclasses with more oxidation stability, so they are better than free fatty acids SQDG 28:0 and MGDG 28:0 (14:0/14:0) inhibiting inflammatory proteins are combined with omega-3 to treat and restore joint chondrocytes, so polar lipids are increasingly attracting the attention of researchers due to their outstanding health benefits CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS This is the first work that comprehensively examined lipids and fatty acids of 60 samples of 50 seaweed species in Vietnam 1.1 Total lipid content mostly fluctuated in the range of 0.35 0.58% (based on fresh weight) Some samples with significantly high content such as Sargassum paniculatum J Ag (1.7%), Gracilaria tenuistipitata Chang & Xia (1.6%), Turbinaria ornata (Turn.) J Ag (1.49%), Chnoospora implexa J Ag (1.34%), Hypnea fragelliformis Grev (1.1%) and Lobophora sp (1.06%) 1.2 The results of lipid analysis by Sorbfil TLC Videodensitometer showed that the majority of seaweed lipids consisted of classes: polar lipids (Pol), sterols (ST), free fatty acids (FFA), triacylglycerols (TG), hydrocarbons and waxes (HW), in which Pol and TG had the highest content 1.3 The content and composition of total lipids and fatty acids depend on the biological nature of the species, growing conditions and time of sampling Hypnea panosa J Ag had an abnormal phenomenon in samples collected from Con Dao with DHA content up to 43.69% of total lipid 1.4 The content of total saturated fatty acids (∑SFA) mostly fluctuated in the range from 38 - 55%, monounsaturated fatty acids 25 (∑MUFA) 20 - 28%, polyunsaturated fatty acids (∑PUFA) 12 - 25% Some samples had considerably high PUFA content such as Palisada parvipapillata (C K Tseng) K W Nam (63.89%, of which EPA 33.58% and AA 23.4%), Lobophora sp (59.98%, of which DHA 14.26%, EPA 11.56% and AA 12.14%) 90% of seaweed samples (53/60) had lipids exceeding WHO standards for healthy omega3/omega-6 ratio (>0.1) By PCA analysis of fatty acid data, fatty acids were identified as chemical markers in seaweed classification by group The fatty acids C16:1n-7, C18:1n-9 and C20:4n-6 are typical for brown seaweed group; C15:0, C16:0 and C18:0 are typical to the red seaweed group and the fatty acids C18:3n-6; and C18:1n-7 are typical to the green seaweed group Polar lipids of Lobophora sp accounted for about 27.5% of total lipids 158 molecular forms were identified, including 48 phospholipid molecular forms belonging to subclasses PI (11), PC (14), PG (22) and PA (1); 46 glycolipid molecular forms belonging to subclasses MGDG (9), DGDG (1) and SQDG (36); 64 betaine lipid molecular forms belonging to subclasses DGTA (37) and DGTS (27) In the glycolipid subclass, the content of unsaturated fatty acids decreased in the order MGDG > DGDG > SQDG Except for SQDG 14:0/16:0, 16:0/16:0 and 16:0/18:1n-9, the remaining polar lipid molecular forms were first reported in the genus Lobophora Polar lipids of Halimeda incrassata Lamx accounted for about 33.7% of total lipids 64 molecular forms were identified, including 07 phospholipid molecular forms belonging to groups of PI (3) and PG (4); 40 glycolipid molecular forms belonging to groups of MGDG (12), DGDG (11) and SQDG (17) and 17 betaine lipid molecular forms belonging to the DGTS group This is the first study on the polar lipid molecular forms of seaweed of the genus Halimeda 26 The molecular forms SQDG 20:5/22:6 and SQDG 20:4/22:6 were first discovered in seaweed The bioactivity test results showed that all lipid fractions of Lobophora sp and Halimeda incrassata Lamx inhibited NO production on RAW 264.7 cell line stimulated by LPS with IC50 of 52.1- 66.22 g/ml and 32.57 - 41.66 g/ml, respectively With high oxidation stability and antimicrobial activity against a number of tested microorganisms, the lipids of these two seaweed species can be used for nutritional, pharmaceutical and cosmetic products The findings of the thesis are not only a basic survey data on Vietnamese seaweeds, supporting the field of seaweed taxonomy, the correlation between species diversity and chemical diversity, but also a scientific basis to orient conservation, cultivation and effective use of seaweeds RECOMMENDATIONS It is recommended to study on use of lipids of Lobophora sp and Halimeda incrassata Lamx to create nutritional, pharmaceutical and cosmetic products It is necessary to further study some promising species such as Sargassum paniculatum J Ag., Palisada parvipapillata (C K Tseng) K W Nam and find out the cause of the superior DHA content of samples belonging to Hypnea panosa J.Ag collected from Con Dao It is necessary to continue to study polar lipids of species with high polar lipid content and rich in omega-3 and omega-6 27 NEW FINDINGS OF THE DOCTORAL THESIS This is the first work that comprehensively examined lipids and fatty acids of 50 species of red seaweed, brown seaweed and green seaweed in the seas and islands of Vietnam The species Hypnea panosa J.Ag collected from Con Dao had DHA content up to 43.69% of total lipids while samples collected from Nha Trang had almost no this active ingredient Eight fatty acids were identified that can be used as chemical markers in seaweed taxonomy by group This is the first work that studied the polar lipid molecular forms of seaweed species of the genus Lobophora 158 polar lipid molecular forms of Lobophora sp were identified, of which 155 molecular forms were reported for the first time in seaweed species of the genus Lobophora This is the first work that studied the polar lipid molecular forms of seaweed species of the genus Halimeda The molecular forms SQDG 20:5/22:6 and SQDG 20:4/22:6 were first discovered in seaweed Lipid fractions of two species Lobophora sp and Halimeda incrassata Lamx were first evaluated for inhibitory effect of NO production on RAW 264.7 cell line stimulated by LPS 25 THESIS-RELATED PUBLICATIONS Pham Thu Hue, Nguyen Van Tuyen Anh, Nguyen Thi Cam Binh, Dam Duc Tien, Le Tat Thanh, Pham Quoc Long, Research on the content of lipid classes and fatty acids from Sargassum seaweed, the 6th Asian Symposium on Advanced Materials: Chemistry, Physics and Biomedicine of Functional and Novel Materials, Hanoi-Vietnam, September 27-30th, 2017, pp 577-582 Thu Hue Pham, Van Tuyen Anh Nguyen, Thi Thanh Trung Do, Anh Duy Do, Duc Tien Dam, Thi Thanh Van Tran, Quoc Long Pham and Tat Thanh Le, Lipidomics and anti-inflammation activity of the brown algae Lobophora sp in Vietnam, Journal of Chemistry, 2020, 10 pages, ISSN: 2090-9063 (SCI-E,Q2), DOI:https://doi.org/10.1155/2020/8829054 Pham Thu Hue, Nguyen Van Tuyen Anh, Do Thi Thanh Trung, Pham Quoc Long, Dam Duc Tien, Tran Thi Thanh Van, Le Tat Thanh, Lipid, fatty acid composition and molecular species of betaine lipid in the green algae Halimeda incrassata Lamx collected from Truong Sa archipelago, Vietnam J Chem., 2021, 59(3), 369-375 (Scopus), DOI: 10.1002/vjch.202000201 Le Tat Thanh, Nguyen Van Tuyen Anh, Pham Thu Hue, Molecular species of glycolipid and anti- inflamation activity of lipid fractions in the green algae Halimeda incrassata Lamx collected from Truong Sa, Viet Nam, Vietnam J Chem., 2021, 59(5), 640-648 (Scopus), DOI: 10.1002/vjch.202100027 Thu Hue Pham, Van Tuyen Anh Nguyen, Thi Hoang Yen Kieu, Le My Anh Nguyen, Hai Nam Hoang, Quoc Long Pham, Duc Tien Dam, Thi Thanh Van Tran, Tat Thanh Le, Content and composition of lipid classes, fatty acid from Sargassum seaweed collected at Con Dao and Van Phong bay, Vietnam Journal of Marine Science and Technology, 2021, 21(2), 1–8, ISSN 1859-3097, DOI: https://doi.org/10.15625/1859-3097/15942 Pham Thu Hue, Nguyen Van Tuyen Anh, Pham Quoc Long, Le Tat Thanh, Studying on phospholipid molecular species of the green seaweed Halimeda incrassata Lamx from Truong Sa in Vietnam, Vietnam Journal of Marine Science and Technology, 2021, Vol 21(1),1–8, ISSN 1859-3097, DOI: https://doi.org/10.15625/1859-3097/15939 Le Tat Thanh, Pham Thu Hue, Nguyen Van Tuyen Anh, Dam Duc Tien, Study on using fatty acid data in the botanical chemotaxonomy for Vietnamese seaweed species, Viet Nam Journal of Biotechnology, 2021, 19(2), 371-379 ... Hanoi-Vietnam, September 27-30th, 2017, pp 577-582 Thu Hue Pham, Van Tuyen Anh Nguyen, Thi Thanh Trung Do, Anh Duy Do, Duc Tien Dam, Thi Thanh Van Tran, Quoc Long Pham and Tat Thanh Le, Lipidomics... Thi Thanh Van, Le Tat Thanh, Lipid, fatty acid composition and molecular species of betaine lipid in the green algae Halimeda incrassata Lamx collected from Truong Sa archipelago, Vietnam J Chem.,... phospholipids, glycolipids and betaine lipids in the polar lipids class of seaweed • Determine the molecular form of PI 34:1 with Rt = 19.04 minutes and the highest content of 44.09% in the phospholipid
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