Lipidomics and Anti-Inflammation Activity of Brown Algae, Lobophora sp., in Vietnam

Graduate Univesity of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam Vietnam Naval Academy, Nha Trang, Vietnam Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam Research Institute for Marine Fisheries, Hai Phong, Vietnam Institute of Marine Environment and Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam Nhatrang Institute of Technology Research and Application, Vietnam Academy of Science and Technology, Hanoi, Vietnam


Introduction
Plants produce secondary metabolites as signals to interact with the environment and stresses [1,2]. A number of secondary metabolites from seaweeds have been detected to have such valuable bioactivities as antibacterial, anti-viral, anticancer, and antioxidant. In Vietnam, the brown seaweed genus Lobophora belongs to the family Dictyotaceae, found worldwide in tropical to temperate waters and discovered in the coral reef. From the early 1980s to 2017, 49 scientific works have been reported on chemicals and bioactivities of Lobophora genus, in which 40 have been reported with bioactivities. Particularly, most of the studies have been centered on Lobophora variegata, while other species have been still poorly studied and reported [3]. Until now, there have been few studies on lipidomic profile and their bioactivities.
Certain lipid classes have been identified in several algae including polar (Pol), sterol (ST), diacylglycerol (DG), free fatty acids (FFA), triacylglycerol (TG), monoalkyldiacylglycerol (MADG), and hydrocarbons and wax (HW) [4], while neutral lipid classes play various roles such as storing energy or pre-hormones for the body; the polar lipid class has been especially considered for its various bioactivities such as acting as antioxidant and helping in curing cardiovascular diseases and cancer caused by long chain polyunsaturated fatty acids (PUFAs) [5,6]. Some high level PUFAs containing C20 fatty acids such as 20:4n-6 (AA), 20:5n-3 (EPA), and 20:3n-6 have been identified and evaluated bioactivities in 7 brown seaweed species belonging to the genera Sargassum, Cystoseira, Padina, and Turbinaria. Other PUFAs have been identified as 22:6n-3 (DHA), which is a highly valuable bioactive fatty acid for application in medicine and food [7]. In addition to being sources of PUFA, the polar lipids have an important role in structural function as components of cell membranes [8].
In algae, fatty acids are found in the esterified structure such as glycoglycerolipids (GLs), glycerophospholipids (PLs), and betaine lipids [9]. e research on the lipidomics is essential to identify new bioactive compounds and properties based on the polar lipid composition [10]. Moreover, it is also a critical step to the discovery of fostering bio-prospection of lipidic extracts.
Nowadays, mass spectrometry (MS) coupled with liquid chromatography (LC) technique is usually used to determine the detailed structural characterization of lipids and to fully explore lipidomic signature of distinct matrices [11,12] and identify lipidome signature of cultivated seaweeds Ulva lactuca Linnaeus [13], Chondrus crispus Stackhouse [14], and Codium tomentosum Stackhouse [15]. However, there has been no report on Lobophora genus lipidome.
is study aims to analyse and identify the lipid characterisation of species Lobophora sp. first collected at Con Dao, Ba Ria-Vung Tau, Vietnam, by using high-performance liquid chromatography in combination with high-resolution mass spectrometry (HPLC-HRMS). e lipid extracts have also been pre-tested for anti-inflammatory effects by inhibiting the production of NO.

Materials and Methods
e Lobophora sp. samples were collected in Con Dao, Ba Ria-Vung Tau, Vietnam. Chemicals were obtained from Sigma, Merck, that reached the purification standards for analysis and HPLC grade.

Total Lipid Extraction.
Total lipid was extracted according to method of Bligh and Dyer [16] and Nguyen et al. [17]. Polar lipids were obtained by the silica column. e 200 mg of total lipid was dissolved in chloroform and loaded on the silica column (Phillipsburg, NJ), washed by 44 ml chloroform to remove the pigments and neutral lipid, and then eluted by 120 ml MeOH 95% to obtain polar lipids. e obtained fractions were stored in chloroform at -5°C for analysis.

Analyses of Lipid Classes.
Total lipids were dissolved in CHCl 3 (10 mg/ml) and spotted on the Sorbfil thin plate (6 × 6 cm). e n-hexane/diethyl ether/acetic acid (85/15/1, v/v/v) solvent was applied to identify neutral lipid layers; then, the CHCl 3 /CH 3 OH (2/1, v/v) solvent was applied to identify the polar lipid layer. e TLC was displayed by 10% H 2 SO 4 /CH 3 OH reagent at 240°C for 10 minutes. e image of thin layer was obtained using Epson Perfection 2400 scanner, Japan, with grayscale mode. e lipid layers on the thin plate and the percentage of the layers were identified by light sensitivity on the Sorbfil TLC Analysis software Video densitometer, Krasnodar, Russia [17,18].

Analyses of Fatty Acids.
Fatty acid methyl esters (FAMEs) were obtained by incubating total lipid with 2% H 2 SO 4 in CH 3 OH at 80°C for 2 hrs and then cleaned by TLC in the hexane:diethyl ether, 95/5 (v/v) solvent. FAMEs were analysed on Shimadzu GC-2010 gas chromatograph (Kyoto, Japan) using flame ionization detector (FID) on Capillary Equity 5 (Merck, L × ID 30 m × 0.25 mm, df 0.25 µm). Carrier gas was He at the speed of 20 ml/min. Temperature program in the oven operating was at 160°C, raised 2°C/min to 240°C, and then kept for 20 minutes. Fatty acids were identified by the equivalent retention time value, Equivalent Chain Length, with the standard system of fatty acids C16 : 0 and C18 : 0. e fatty acid structures were identified by GC-MS. e spectra were compared with the NIST library and fatty acid mass spectra archive [17,18].

Analysis of Molecular Species of Polar Lipids.
e molecules of polar lipids were analysed by high-performance liquid chromatography combined high resolution mass spectrometry (HPLC-HRMS) with Shim-Pack diol column (ID 50 mm × 4.6 mm, carrier size 5 μm, Shimadzu, Kyoto, Japan). e polar lipid classes were separated by HPLC in a solvent A: n-hexane/2-propanol/formic acid/(C 2 H 5 ) 3 N and the solvent B: 2-propanol/H 2 O/formic acid/(C 2 H 5 ) 3 N [19] and then detected by the high-resolution ion trap over time of mass spectrometry using the LC/MS-IT-TOF (Shimadzu) device, an electronic atomizing ionization source (ESI) device. Flow rate was 0.2 ml/min [17,20,21]. Assay. In vitro antiinflammatory activity of lipid samples was elucidated by using nitric oxide (NO) assay as described previously by Mosmann [22] using the Griess reagents (Promega, USA). e RAW 264.7 cells at the concentration of 2 × 10 5 cells/mL were dropped on the 96-well plate and incubated in 37°C with 5% CO 2 in 24 h. e culture medium was exchanged to Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, USA) with no fetal bovine serum (FBS) for 3 hours. e cells were incubated with the lipid fractions at different concentrations before stimulating the NO production by LPS (1 µg/mL) for 24 hours. en, the amount of nitrite in the culture medium was measured by Griess reaction at room temperature for 10 minutes. e fresh culture medium was used as a blank, while L-NMMA (Sigma) was tested as the positive sample. e mixtures were quantified spectrophotometrically at 540 nm using a micro-plate reader (ELx800 Biotekm, USA). e sodium nitrite was used as a standard compound to establish the standard curve. e NO inhibition was calculated following the formula: %NO inhibition � 100% − [concentration of NO sample /concentration of NO LPS ] * 100. IC50s were calculated by TableCurve 2Dv4 software. e cell viability test was performed parallel with the NO inhibition assay. e 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was used to evaluate cell viability because active cells transform the water-soluble MTT to an insoluble purple formazan. 20 μl lipid sample and 180 µL of RAW 264.7 cells were put into 96-well plates and then incubated with 5% CO 2 for 72 hours at 37°C. After adding MTT (5 µg/mL), the mixtures were incubated for 4 hours and supernatant was removed. e formazan crystals were dissolved in DMSO and measured at 540 nm. e percentage of cell viability was determined by comparing to the control samples. 3.1. Total Lipid. Total lipid content from brown algae Lobophora sp. is 1.06 ± 0.2% weight of fresh algae, similar to the brown samples previously studied [4,23]. Compositions and contents of total lipid classes of Lobophora sp. are shown in Figure 1 and Table 1

Molecular Species of Polar Lipids.
ree subclasses including phospholipid, glycolipid, and betaine lipid were identified in 26.80% polar lipid (Pol).
First, the phospholipid subclass contains 4 groups of phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidic acid (PA). In PI group, 13 molecular forms were found, of which 11 forms had complete formula with one PI isomer form and no alkenyl acyl glycerophosphoinositol (Table 3).
In particular, PI 34 : 1 is recognized at the highest rate of 44.09% PI Figure  us, the mass spectrometry data prove that the considered molecule is diacyl glycerophosphoinositol PI 16 : 0/18 : 1 or PI 34 : 1. In the PC group, 18 molecular forms have been identified, in which 14 molecular forms are completely identified with one isomer (Table 3) In the PG group, 15 molecular forms have been determined with complete formula, in which 6 PGs are detected with isomers (Table 3) With the PA molecular forms, only one PA 40 : 8 is identified (Table 3) Second, in the glycolipid subclass, 3 groups have been identified including monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). In the MGDG group, 21 molecular forms are identified, in which 8 have been completely identified (Table 4).
For example, with the highest ratio of 13.92%, MGDG 38 : 9 has formed molecular ions (Figure 5S      In the DGDG group, only one DGDG form has been found as DGDG 34 : 1 which is described in Table 4 and Figure 6S. On  In the SQDG group, 26 complete molecular forms have been identified with 10 isomers (Table 4) In addition to phospholipid and glycolipid classes, betaine lipid subclass includes 2 groups of DGTA and DGTS. In the DGTA group, 37 molecular forms have been identified, in which 29 forms are completely identified with 8 isomers (Table 5). Among those, DGTA 34 : 1 is the highest content of 10 3783. is ion eliminates water molecule and shows signal at m/z 456. 3806. In addition, the signal at m/z 236.1485 is semimolecule ion of DGTA that lost diacyl groups, which is a very important signal in determining the

Anti-Inflammatory Activity.
ree lipid fractions yield the inhibitory effects on NO production in RAW 264.7 cells.
e IC 50 values have been evaluated from 52.10 to 66.21 µg/ mL (Table 6). e unpolar lipid fraction (UPol) has a strongest activity IC 50 of 52.10 ± 4.43, following by total lipid and polar lipid samples with IC 50 of 61.09 ± 6.06 and 66.21 ± 6.24 µg/mL, respectively. All fractions do not exhibit the cytotoxicity with the cell viability from 96.06 to 100% at the concentration of 100 µg/mL, while the control sample shows the cell viability percentage of 89.90% with the same concentration (Table 6).

Discussion
In the total lipid of Lobophora sp., 5 classes including Pol, ST, FFA, TG, and HW have been found, while other brown algae  ey have been considered to be beneficial for the human brain and overall health, including cardiovascular effects, improving the function of the heart and the liver, reducing blood pressure, anti-thrombosis, and helping with arthritis, cancer, and lung diseases [24][25][26][27][28]. Among those, α-and c-linolenic acids (C18 : 3n-3-ALA and C18 : 3n-6-GLA) have the total content of 11.93%, which are very important fatty acids in the formation and protection of the skin to potentially apply in the cosmetic industry [29].
Previous research has shown that the fatty acid composition in seaweed contains saturated fatty acids (SFAs) with high content and polyunsaturated fatty acids (MUFAs, PUFAs) with very low content. For example, the green algae Ulva rigida has 64.5% SFAs, 23.78% PUFAs, and 11.71% MUFAs [9]. e brown algae Padina pavonica has those with content of 43.45%, 1.7%, and 23.67%, respectively [30]; some red algae species belonging to Hypnea genus in Vietnam had the content of SFAs >60%, PUFAs from 3-12%, and MUFAs is 8.55-27.88% [31]; other red algae have the similar fatty acid profile [32,33]. Interestingly, the fatty acid profile of this Lobophora sp. revealed the high level of polyunsaturated fatty acids (MUFAs, PUFAs) with the content of 75.10%, which is extremely noteworthy to further study for potential application.
Particularly, the fatty acid C22 : 6n-3 (DHA) has been identified in the total lipid of this species with the content of 14.26% which is a valuable fatty acid in foods that helps increase the brain development of children and young animals [36]. DHA content of this species is much higher than that of other brown seaweeds collected in Vietnam and of oysters, fish, and corals [4,18,35]. is can be a sign to distinguish seaweeds living in coral reefs from those living in coastal areas.
Recent researches on lipid usually have applied GC-MS, GC-FID techniques to detect the composition and content of fatty acids; however, fatty acids exist in both free and linked types. Nowadays, the application of HR-MS technique helps identify the hold of lipid molecules and even molecular species based on ion values of MS [17,18,21]. Some novel techniques such as LC-MS/MS have been applied to study the lipidome of seaweed [9,10,14,15,37]. With HPLC-HRMS technique, 157 polar lipids molecular forms have been, for the first time, identified belonging to phospholipids, glycolipids, and betaine lipids of Lobophora sp.
Betaine lipids are an amphoteric subclass like phospholipids because they have a positively charged ammonium group. ey are determined to have a function similar to phospholipids in many algae, fungi, and seedless plants [38][39][40][41][42]. DGTA is a glycerol-lipid that contains two fatty acids esterified with glycerol and an ether-linked polar group derived from an amino acid, while DGTS is an isomer structure and biosynthetic precursor of DGTA in the algae [43,44]. e coexistence of DGTS and DTGA in many types of algae can be explained by a partial conversion of DGTS to DGTA. In addition to molecular species analysis, the total lipid from Lobophora sp. has been separated into polar lipid and unpolar lipid fractions, in which polar lipid contained phospholipid, betaine lipid, and glycolipid classes. In recent studies, the extracts from Lobophora species have been reported on such bioactivities as anti-oxidant, anti-inflammation, anti-microbial, and cytotoxic activities [3]. In this study, we have preliminarily screened the anti-inflammation activity of three lipid fractions through the NO production inhibition assay because NO is a key signaling molecule in the inflammation process [45]. e results show that the lipid fractions exhibited the strong NO production inhibition activity.
Particularly, the unpolar lipid fraction displays a higher NO inhibitory activity than polar lipid. is result contrasts with what has been reported on lipid fraction from a mud crab Scylla paramamosain, which shows the inhibition of the polar lipid higher than the unpolar lipid fraction [19]. is may be caused by the polar lipid content Journal of Chemistry 7 of Lobophora sp. which is lower than that of the mud crab S. paramamosain, 26.8% and 40.02%, respectively. Other authors have demonstrated that an abundance of PUFAs in seaweed composition related to the display of antiinflammatory activity [9,46]. It has also been reported that anti-inflammatory activities (IC 50 ) of seafood range from 64.6 to 306.4 µg/mL. In this report, the NO inhibition activity of lipid fractions is comparable to those of Octopus lipids [47] and sulfated polysaccharide of the marine brown algae Lobophora variegata [48]. Particularly, it is the first time that the Lobophora sp. lipid fractions have been demonstrated strong NO inhibition activity. e recent reports indicate that the production of NO is increased by neuronal nitric oxide synthase (nNOS) mediated by the NF-κB factor [49]. In addition, the transcription of Nf-κB has been regulated by n-3 PUFAs [50]. Also, the report of Echeverria shows that n-3 PUFAs (especially EPA and DHA) partially reduce the proinflammatory indexes such as NF-κB and Nrf2 [51]. us, it suggests that lipid fractions containing n-3 PUFAs may inhibit NO inhibition through regulation of factors NF-κB and Nrf2.
In several expert studies, total extracts, their fractions, and molecular species have demonstrated bioactivities [52][53][54][55][56]. Particularly, glycolipids from different algae species have anti-viral, antibacterial, and anti-tumoral activity [53,54]. SQDG (32 : 0) and SQMG (16 : 0) are both described to have anti-microbial activity [55,56]. Some reports suggest that DGTS has the same function as PC due to their similar zwitterionic structure, and they are interchangeable in the cell [44]. It is significant that the MGDG displays anti-inflammatory activity and is combined with omega-3 to treat the regeneration of articular cartilage in the osteoarthritis adult.
us, further study will be focusing on evaluating the bioactivities of the composition of Lobophora sp. lipid such as fatty acids, lipid classes, and molecular species.

Conclusions
In summary, lipid classes and fatty acid profile of seaweed Lobophora sp. have been well defined. e simultaneous detection of high-value long-chain polyunsaturated fatty acids such as AA, EPA, and DHA in this seaweed has inspired researches to study the origin of these fatty acids in the reef ecosystem. HPLC-HRMS technique has allowed identifying 157 molecular forms in polar lipid that are classified into betaine lipid, glycolipid, and phospholipid groups with 64, 45, and 48 molecular forms, respectively. e NO inhibition effects of lipid fractions including total lipid, polar lipid, and unpolar lipid have been reported for the first time with IC 50 from 52.10 to 66.21 µg/mL. ese all suggest that Lobophora sp. lipids need to be further studied for potential application in food, medicine, and cosmetics.

Data Availability
No data were used to support this study.