Study of phospholipid molecular species of the green seaweed Halimeda incrassata Lamx. from Truong Sa islands, Vietnam

Nội dung text: Study of phospholipid molecular species of the green seaweed Halimeda incrassata Lamx. from Truong Sa islands, Vietnam

1. Vietnam Journal of Marine Science and Technology; Vol. 21, No. 1; 2021: 77–84 DOI: Study of phospholipid molecular species of the green seaweed Halimeda incrassata Lamx. from Truong Sa islands, Vietnam Pham Thu Hue1,2, Nguyen Van Tuyen Anh3, Pham Quoc Long3, Le Tat Thanh2,3,* 1Graduate University of Science and Technology, VAST, Vietnam 2Vietnam Naval Academy, Nha Trang, Vietnam 3Institute of Nature Products Chemistry, VAST, Vietnam *E-mail: thanh.biotech@gmail.com Received: 2 January 2021; Accepted: 8 March 2021 ©2021 Vietnam Academy of Science and Technology (VAST) Abstract In this report, by a high performance liquid chromatography (HPLC) - high resolution mass spectrometry (HRMS) method, 7 molecular species of phospholipid in the polar lipid class were identified including phosphatidylinositol (PI) and phosphatidylglycerol (PG). The PI 32:0 (16:0/16:0) and PG 34:3 (16:1/18:2) molecular species have the highest content. PI molecular species are mainly formed by saturated fatty acids (16:0, 18:0) and only one polyunsaturated fatty acids C20:4, while PG species are formed by unsaturated fatty acids (16:1, 18:2 and 18:3) and only one saturated fatty acid C16:0. Keywords: Phospholipid, molecular species, calcified seaweeds, Halimeda incrassata, Truong Sa islands. Citation: Pham Thu Hue, Nguyen Van Tuyen Anh, Pham Quoc Long, Le Tat Thanh, 2021. Study of phospholipid molecular species of the green seaweed Halimeda incrassata Lamx. from Truong Sa islands, Vietnam. Vietnam Journal of Marine Science and Technology, 21(1), 77–84. 77
2. Pham Thu Hue et al. INTRODUCTION Vietnam in 1980 [9]. H. incrassata sample was The seaweed genus Halimeda, belonging to then treated and soaked in formaldehyde 5% the Halimedaceae family, the Chlorophyta before identifying the Latin name. The phylum, widely spread over the world specimen of sample was stored at the Institute especially near the outlying islands in the of Marine Environment and Resources and the tropical sea. This species in combination with Institute of Natural Products Chemistry corals is regarded as a structural component of (VAST). reefs and also protects seabed from disasters Total lipid extraction and coastal currents [1]. In the marine Total lipid was extracted with a solvent environment, the spread of seaweed across system of chloroform and methanol (1:2) [10] seabed is not only an aesthetic scene but also a in the combination with sonication. 100 g of place for marine animals to spawn and prevent seaweed was extracted two times by the solvent their offspring from danger. In Vietnam, system of 300 ml CHCl :CH OH and 100 ml Halimeda incrassate Lamx. is mainly 3 3 CHCl under ultrasound condition for 180 distributed in Truong Sa archipelago (Khanh 3 minutes. The extracted mixture was obtained Hoa) [2]. This is a rare marine plant that by filtering, and shaken after adding 80 ml CaCO is synthesized in the tissues (around 35– 3 H O. The lower layer was collected using 45%) at a speed equivalent to those of the reefs, 2 funnel. The total lipid was obtained using thus it is named a calcified seaweed [3]. vacuum rotary evaporator Eyela N1300, then Halimeda incrassata is a potential resource of stored at (-)20oC prior to analysis. compounds with various bioactivities such as antibacterial, anti-inflammation, [4, 5]. Along Analysis of molecular species of with natural products from marine organism, phospholipid lipid is increasingly recognized because of its The total lipid was dissolved in MeOH and positive effects on bones, cardiovascular and processed in HPLC-HRMS Shimadzu to nervous systems. The effects of lipid are recognize molecular species of the phospholipid mainly related to the biological and subclass [11]. The chromatography system pharmacological roles of polyunsaturated fatty contains 2 high-pressure pumps, CTO-20A acids (PUFAs) [6–8]. oven, Shim-Pack diol column (50 mm × 4.6 mm In this report, we aim to report the ID, 5 μm, Shimadzu, Kyoto, Japan), using two composition and the content of fatty acids, lipid solvent systems of A hexane:2- classes and especially phospholipid molecular propanol:CH3COOH:N(CH3)3 82:17:1:0.08 species of the green algae Halimeda incrassata (v/v) and B (propanol-2): Lamx. collected from Truong Sa islands. As H2O:CH3COOH:N(CH3)3 85:14:1:0.08 (v/v). well known, Truong Sa is a place with the Phospholipid molecular species are identified by harsh weather conditions, difficulties in the combination IT and TOF techniques in the commuting and collecting samples. Thus, this Shimadzu LCMS-IT-TOF device (Kyoto, publication would greatly contribute practical Japan). The machine could be operated in both effects in the novel knowledge of seaweed with negative and positive ion modes in each analysis large yield in the far islands. under electrospray ionisation (ESI) conditions. The quantification of the phospholipid METHODS molecular species was calculated according to Sample collection the area per substance peak obtained on the The Halimeda incrassata Lamx. sample positive or negative ion spectrums. was collected in 7/2017 by DSc. Dam Duc Tien from Truong Sa archipelago, Khanh Hoa, Viet RESULT AND DISCUSSION Nam. The sample collecting process has been Identifying molecular species of phospholipid performed following the Provisional In the total lipid of the green algae H. Regulations for Field Survey issued by the incrassata sample, polar lipid class (Pol) has State Committee for Science and Technology accounted for 32.3% of the total lipid. Using 78
3. Study of phospholipid molecular species HRMS technique, molecular species in phosphatidylglycerol (PG) (figure 1). phospholipid (PL) subclass have been Molecular and ion forms of each PL have been identified and classified into 2 groups of analyzed and determined on the mass phosphatidylinositol (PI) and spectrum [11]. PI PG Figure 1. Formula of PI and PG molecular species Identifying molecular species of identified, in which alkenyl acyl phosphatidylinositol (PI) glycerophosphoinositol has not appeared, all of In the PI group of the green algae H. molecular species are incrassta, 3 molecular species have been diacylglycerophosphoinositol (table 1). Table 1. Composition of PI’s molecular species Phospholipid (C:N)* Diacyl [M-H]- m/z Molecular formula Area % in PI PI 32:0 16:0/16:0 809.5076 C41H79O13P 24745446 52.03 PI 34:0 16:0/18:0 837.5406 C43H83O13P 8401740 17.66 PI 36:4 16:0/20:4 857.4986 C45H79O13P 27318082 30.31 ∑ 47563676 100.00 Note: *: The number of carbon atoms (C) and number of double bonds (N) in the acyl chain. - For example, PI 32:0 has the highest is the mass of anion [C16H31O2] (fatty acid percentage of 52.03% (table 1, figure 2). 16:0). In conclusion, the spectrum data have On the MS- spectrum, the ion peak [M-H]- shown that the considered molecular species is has the strongest signal at m/z 809.5076 diacylglycerophosphoinositol PI 16:0/16:0. - [C41H78O13P] corresponding to molecular According to table 1, the PI’s molecular formula C41H79O13P (m/z 810.5104). This species with the content ranked second is PI negative ion was chosen to analyze on the MS2- 36:4 (30.31%). . MS2- spectrum of PI 32:0 also has the m/z On the MS- spectrum of PI, the signal [M- - - signals corresponding to a half-molecular ion H] at m/z 857.4986 [C45H78O13P] has been containing inositol, acyl group and anion identified with formula of C45H79O13P (m/z carboxylate of the fatty acid disappearing 858.5161). On the MS2- spectrum of ion [M-H]- simultaneously. The signal at m/z 553.2717 (figure 3c), the ion fragment at the signal at m/z corresponds to the lost neutral fragment at m/z 601.2548 is similar to the lost fragment of fatty 256.2359 (C16H32O2) (fatty acid 16:0). The acid 16:0 (C16H32O2) at m/z 256.2438. signal at m/z 391.224 corresponds to ion Additionally, the signal at m/z 601.2548 - [C41H78O13P] at m/z 809.5076 losing both the corresponds to ions of glycerophosphate - fragment m/z 256.2307 (C16H32O2) (fatty acid [C3H6O5P] at m/z 152.9980, inositol (C6H10O5) 16:0) and inositol m/z 162.0528 (C6H10O5). at m/z 162.0528 and ketene of fatty acid 20:4 2- Besides, the signal at m/z 255.2285 on the MS (C20H30O) at m/z 286.2040 (a fatty acid lost 1 79
4. Pham Thu Hue et al. H2O). Hence, PI 36:4 is characteristic of Similarly, the other molecular species of PI 34:0 diacylglycerophosphoinositol PI 16:0/20:4. (PI 16:0/18:0) has been identified in table 1. - Figure 2. HPLC-HRMS and fragmentation of PI 32:0 [C41H78O13P] : a- HPLC chromatogram - - - 2- of [C41H78O13P] ; b- Mass spectrometry (MS ) of [C41H78O13P] ; c- Mass spectrometry (MS ) of signal at m/z 809.5076 Figure 3. HPLC-HRMS and the fragmentation of PI 36:4 [C45H78O13P]: a- HPLC chromatogram - - - 2- of [C45H78O13P] ; b- Mass spectrometry (MS ) of [C45H78O13P] ; c- Mass spectrometry (MS ) of signal at m/z 857.4986 80
5. Study of phospholipid molecular species Identifying molecular species of spectrum of [M-H]- (figure 4c), the observed phosphatidylglycerol (PG) ion fragment at m/z 507.2671 corresponded - In the PG group of green algae H. to the ion [C40H72O10P] losing a ketene of the incrassate, 4 molecular species with exact fatty acid 16:1 at m/z 236.2101 (C16H28O) (a formula have been identified (table 2). fatty acid removed 1 H2O). The signal at m/z Data from table 2 presented that PG 34:3 415.2241 corresponds to the disappearance of has the highest content of 61.02% (figure 4). both ketene of the 16:1 fatty acid m/z - On the negative ion MS spectrum of PG, the 236.2101 and glycerol (C3H8O3) m/z 92.0430 - - strongest signal of the negative ion [M-H] at from considered ion [C40H72O10P] . The ion - m/z 743.4772 [C40H72O10P] has been recorded. fragment at m/z 279.2294 is the segment of - Thus, the formula has been considered anion [C18H31O2] (fatty acid 18:2). Hence, C40H73O10P (m/z 744.4758). PG 34:3 has been assumed as the character of This anion has been chosen to analyze on diacylglycerolphosphoglyerol, PG 16:1/18:2. the MS2- spectrum. On the negative ion MS2- Table 2. Composition of PG molecular species Phospholipid (C:N)* Diacyl [M-H]- m/z Molecular formula Area % in PG PG 32:2 16:1/16:1 717.4629 C38H71O10P 10909490 7.81 PG 34:4 16:1/18:3 741.4642 C40H71O10P 9443406 6.76 PG 34:3 16:1/18:2 743.4772 C40H73O10P 85255153 61.02 PG 34:2 16:0/18:2 745.4902 C40H75O10P 34097828 24.41 ∑ 139705877 100.00 Note: *: The number of carbon atoms (C) and number of double bonds (N) in the acyl chain. - Figure 4. HPLC-HRMS and the fragmentation of PG 34:3 [C40H72O10P] : a- HPLC chromatogram - - - 2- of [C40H72O10P] ; b- Mass spectrometry (MS ) of [C40H72O10P] ; c- Mass spectrometry (MS ) of signal at m/z 743.4772 Moreover, table 2 also shows that the PG shows the signal at m/z 741.4642 - lowest content of PG (6.76%) is PG 34:4 [C40H70O10P] . Thus, the formula is C40H71O10P (figure 5). The negative ion MS- spectrum of with m/z 742.4578. 81
6. Pham Thu Hue et al. - Figure 5. HPLC-HRMS and the fragmentation of PG 34:4 [C40H70O10P] : a- HPLC chromatogram - - - 2- of [C40H70O10P] ; b- Mass spectrometry (MS ) of [C40H70O10P] ; c- Mass spectrometry (MS ) of signal at m/z 741.4642 On the MS2-spectrum of [M-H]- (figure 5c), group belonging to the betaine lipid subclass the ion fragment at m/z 505.2500 corresponds can substitute for PC at the chloroplast to the lost ketene of fatty acid 16:1 at m/z membrane. Thus, the absence of PC in this 236.2142 (C16H28O). On the other hand, the seaweed agrees with our recent publication signal at m/z 505.2500 is created from the about the betaine lipid subclass of the H. glycerophosphate segment at m/z 152.998 incrassata species which has detected only one - [C3H6O5P] , glycerol molecule at m/z 92.0430 DGTS group with 17 molecular species. (C3H8O3) and a ketene of the fatty acid 18:3 In 7 molecular species identified, fatty (C18H28O) (a fatty acid 18:3 lost 1 H2O) m/z acids containing 16 carbons (C16) are the main 260.2090. Hence, PG 34:4 is regarded as the composition of both PG and PI groups (9/14 character of diacylglycerolphosphoglyerol, PG acyl radicals). By the GC analysis of the 16:1/18:3. Similarly, 2 other molecular species composition and content of fatty acids, C16 of PG in table 2 have been determined. fatty acids were detected with various contents Discussion in green algae H. incrassata, which is similar to By using HPLC-HRMS technique to the report of Victor et al., on the fatty acid analyze green algae H. incrassata, 2 groups composition of 26 seaweed species including 8 including PI (3 molecular species) and PG (4 green algae [13]. Regarding the composition of molecular species) in the phospholipid subclass fatty acids in different phospholipid subclasses, have been identified, without the appearance of the PI group (table 2) is formed mainly from phosphatidylcholine (PC), two saturated fatty acids C16:0, C18:0 and phosphatidylethanolamine (PE) and unsaturated fatty acids C20:4. These C20:4 phosphatidylserine (PS), which is similar to fatty acids are important in the composition of other reports of green algae [12]. It may be some prostaglandins in order to control the related to the characteristic of the algae about inflammation [16, 17]. In contrast, the PG CaCO3 synthesis. Previous studies [12–15] group (table 3) is formed by only one saturated have demonstrated that the fatty acid C16:0 and unsaturated fatty acids diacylglyceryltrimethylhomoserine (DGTS) C16:1, C18:2, C18:3. Notably, C18:2 and 82
7. Study of phospholipid molecular species C18:3 acids appear on the fatty acid Technology, 16(3), 297–305. composition of this species by the GC analysis 10.15625/1859-3097/16/3/8124. in the forms of C18:2n-6 and C18:3n-3, which [3] Carneiro, P. B. D. M., Pereira, J. U., and are the important fatty acids for the synthesis Matthews-Cascon, H., 2018. Standing process in omega-3 and omega-6 disorder to stock variations, growth and CaCO3 endothelial cell and nervous system balance production by the calcareous green alga [18, 19]. It is remarkably related to bioactivities Halimeda opuntia. Journal of the Marine of these compounds and the function of Biological Association of the United phospholipid subclass, suggesting further Kingdom, 98(2), 401–409. studies about effects on the structure and /10.1017/S0025315416001247. activities of phospholipid species. [4] Silva, A. M. O., Novoa, A. V., Gutierrez, D. D., and Mancini-Filho, J., 2017. CONCLUSION Seaweeds from Halimeda genus as By using HPLC-HRMS to analyze green sources of natural antioxidants. J. Anal. algae H. incrassate, this study has identified 7 Pharm. Res., 5(6), 1–5. molecular species of phospholipid belonging to [5] Novoa, A. V., Andrade-Wartha, E. R., the PI group (3 species) and the PG group (4 Linares, A. F., Genovese, M. I., González, species). Amongst those, PI 32:0 (16:0/16:0) A. E. B., Vuorela, P., Costa, A., and has the highest content of 52.03% and PG 34:3 Mancini-Filho, J., 2011. Antioxidant (16:1/18:2) has the highest content of 61.02%. activity and possible bioactive The compositions of fatty acids in the components in hydrophilic and lipophilic phospholipid subclass are mainly C16, which is fractions from the seaweed Halimeda rich in the green algae total lipid, but there is a incrassata. Revista Brasileira de Farmacognosia, 21(1), 53–57. difference between two groups of phospholipid molecular species. While the PI group mainly 05000010. contains two saturated fatty acids (C16:0, [6] El Gamal, A. A., 2010. Biological C18:0) and 1 unsaturated fatty acid C20:4, PG importance of marine algae. Saudi has only 1 saturated fatty acid C16:0 and three Pharmaceutical Journal, 18(1), 1–25. unsaturated fatty acids (C16:1, C18:2, C18:3). [7] La Guardia, M., Giammanco, S., Di Majo, Acknowledgements: We would like to thank D., Tabacchi, G., Tripoli, E., and the Laboratory of Comparative Biochemistry Giammanco, M., 2005. Omega 3 fatty A.V. Zhirmunsky Institute of Marine Biology, acids: biological activity and effects on Far-Eastern Branch of the Russian Academy of human health. Panminerva Medica, 47(4), Sciences, 17 Palchevskogo str., Vladivostok 245–257. 690041, Russian Federation. [8] Calder, P. C., and Yaqoob, P., 2009. Omega‐3 polyunsaturated fatty acids and REFERENCES human health outcomes. Biofactors, 35(3), [1] Dam, D. T., 2008. Initial research on the 266–272. distribution and coverage of some [9] State Committee for Science and calcified seaweed species in Truong Sa Technology, 1981. Temporary rules of archipelago, Vietnam Sea. Vietnam marine integrated investigation (section Association of Marine Science and Seaweed). Science and Technics Technology, 10, 18–22. Publishing House, Hanoi. 205 p. [2] Tien, D. D., 2016. Species composition [10] Bligh, E. G., and Dyer, W. J., 1959. A and distribution of seaweeds from some rapid method of total lipid extraction and small islands (Nam Yet, Son Ca, Song Tu purification. Canadian Journal of Tay, Sinh Ton) of Truong Sa archipelago. Biochemistry and Physiology, 37(8), 911– Vietnam Journal of Marine Science and 917. 83
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