processes Article Lipid Isolation Process and Study on Some Molecular Species of Polar Lipid Isolated from Seed of Madhuca ellitica Doan Lan Phuong 1,2 , Tran Quoc Toan 1,2 , Ly P. T. Dang 1 , Andrey B. Imbs 3 , Pham Quoc Long 1,2, *, Tran Dinh Thang 4 , Bertrand Matthaeus 5 , Long Giang Bach 6,7 and Le Minh Bui 6,7, * 1 2 3 4 5 6 7 * Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet road, Caugiay district, Hanoi 10000, Vietnam; doanlanphuong75@gmail.com (D.L.P.); tranquoctoan2010@gmail.com (T.Q.T.); phuongly1412@gmail.com (L.P.T.D.) Graduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet road, Caugiay district, Hanoi 10000, Vietnam A.V. Zhirmunsky Institute of Marine Biology, Far-Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia; andrey_imbs@hotmail.com School of Chemistry, Biology and Environment, Vinh University, Vinh City, Vietnam 46000; thangtd@vinhuni.edu.vn Working Group of Lipid Research, Department of Safety and Quality of Cereals Max, Federal Research Institute for Nutrition and Food, Rubner-Institut, Schutzenberg 12, DE-32756 Detmold, Germany; bertrand.matthaeus@mri.bund.de NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City 70000, Vietnam; blgiang@ntt.edu.vn Center of Excellence for Biochemistry and Natural Products, Nguyen Tat Thanh University, Ho Chi Minh City 70000, Vietnam Correspondence: mar.biochem@fpt.vn (P.Q.L.); blminh@ntt.edu.vn (L.M.B.) Received: 18 May 2019; Accepted: 10 June 2019; Published: 17 June 2019



 Abstract: This study attempted the lipid extraction process from the seeds of Madhuca ellitica, a lipid-rich plant, and conducted a lipidomic analysis on molecular species of the obtained product. Total lipids of the crude seeds were found to contain 11.2% of polar lipids. The major fatty acids (FAs) of the polar lipids were palmitic (16:0), stearic (18:0), oleic (18:1n-9), and linoleic (18:2n-6) acids, which amounted to 28.5, 12.5, 44.8, and 13.2% of total FAs, respectively. The content and chemical structures of individual molecular species of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA), and sulfoquinovosyldiacylglycerol (SQDG) were determined by HPLC with a tandem high-resolution mass spectrometry (HRMS). The major molecular species were 18:1/18:2 PE, 16:0/18:1 PC, 18:1/18:2 PC, 16:0/18:2 PG, 16:0/18:1 PG, 16:1/18:1 PI, 16:0/18:1 PI, 18:0/18:2 PI, 16:0/18:1 PA, 18:1/18:2 PA, 16:0/18:1 SQDG, and 18:0/18:1 SQDG. The application of a tandem HRMS allows us to determine the content of each isomer in pairs of the monoisotopic molecular species, for example, 18:0/18:2 and 18:1/18:1. The evaluation of the seed polar lipid profile will be helpful for developing the potential of this tree for nutritive and industrial uses. Keywords: Madhuca ellitica; lipid molecular species; phospholipids; sulfoquinovosyldiacylglycerol; seeds; tandem high-resolution mass spectrometry 1. Introduction Buttercup tree or mahua, Madhuca elliptica (Pierre ex Dubard) H.J.Lam, Madhuca longifolia (Koenig) J.F. Macb (Synonyms, Madhuca indica Gmelin, Madhuca latifolia Macb., Bassia latifolia Roxb.; Processes 2019, 7, 375; doi:10.3390/pr7060375 www.mdpi.com/journal/processes Processes 2019, 7, 375 2 of 20 Family, Sapotaceae) is a commercially important tree cultivated throughout the subtropical region of the Indo-Pak subcontinent and regarded highly for its flowers, fruits, timber, and seeds [1–3]. Previous reports have pointed out a myriad of valuable compounds, especially compounds of low molecular-weight including saponins, carbohydrates, triterpenoids, steroids, flavonoids, and glycosides, have been isolated from Madhuca [4–7]. Among these compounds, carotinoids, existing dominantly in the seeds of Madhuca, figure in certain medicinal applications for treatments of skin disease, rheumatism, headache, piles, and use as a laxative [8–10]. The Madhuca seed is also rich in fat (up to 58%) [11,12], suggesting potential use of the seed in the manufacture of laundry soaps, lubricants and biodiesel [13,14]. The base of the oil seeds of the plant is constituted of triacylglycerols (TG), which are neutral lipids. Also, natural seed fats contain a number of polar lipids, which are usually separated and lost during oil manufacturing. Among polar lipids, phospholipids hold great importance due to their nutritional value and bioactivities [15]. Each phospholipid class is a complex mixture of individual compounds, termed as “molecular species”. Structures of phospholipids consists of the same polar group but different acyl chains of their molecules, which defines their nutritional value and bioactivities of phospholipid molecular species. Therefore, unambiguous identification of molecular species profile of the oilseed, through a lipidomic approach, may introduce possible application in the cosmetic, nutraceutical, and pharmaceutical industries [16,17]. Even though plant lipids and TG molecular species have been studied extensively in terms of compositional structure [18–20], information on the molecular species composition of phospholipids is scanty. For M. elliptica, previous studies have revealed its TG molecular species profile in seed fat and its solid and liquid fractions [21]. In addition, fatty acid (FA) composition of polar lipids and individual phospholipid class content of mahua butter from M. elliptica have been described [10]. The present work aimed to identify the chemical structure and quantitative composition of phospholipid molecular species from the M. elliptica seeds. The molecular species of sulfoquinovosyldiacylglycerols (SQDG), which contain a charged chemical group like phospholipid molecules, were also investigated. High performance liquid chromatography (HPLC), in tandem with high -resolution mass spectrometry (HRMS), was used as a lipidomic method [22]. Some important papers concerning molecular species of polar lipids in marine species, such as corals and red (hot) algae, have been recently published. For example, a previous study that investigated the distribution of tetracosapolyenoic acids (TPA) in molecular species of different phospholipid (PL) classes in the soft corals Sinularia macropodia and Capnella sp. from shallow waters of Vietnam showed some interesting results [23]. To be specific, Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI) were found to be major PL classes of S. macropodia and Capnella sp. and more than 32 molecular species of these four PL classes were determined by high-resolution tandem mass spectrometry. The major molecular species of PL in both coral species were 18:1e/20:4 PE, 18:0e/20:4 PC, 18:0e/24:5 PS, and 18:0/24:5 PI. In two model red algae, Polysiphonia sp. and Porphyridium sp., forms of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), the two most commonly found galactolipids in chloroplast membranes, were determined via positive-ion electrospray ionization/mass spectrometry (ESI/MS) and ESI/MS/MS [24]. In the study of Honda et al. (2019) the characteristics of glycerolipids, which are the substrates of eicosanoids production of A. chilensis, were investigated and compared to the reported values of A. vermiculophyllum. The results showed that monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylcholine (PC) were the major lipid classes in A. chilensis and accounted for 44.4% of the total lipid extract [25]. 2. Materials and Methods 2.1. Materials Ripe fruits of M. ellitica (1000 g) were collected from a plantation in Binh Duong province of Vietnam in February 2016 and were botanically identified by Dr. Nguyen Quoc Binh, Vietnam National Processes 2019, 7, 375 3 of 20 Museum of Nature—Vietnam Academy of Science and Technology. Seeds were cleaned, dried in cabinet dryer at 50 ◦ C for 12 h, and stored at 4 ◦ C after collection. Water content (%) in the M. ellitica seed was 10.27%. To prepare the sample for analysis, 100 g of sample seeds were finely ground using an electric grinder (KIKA Labortechnik M20), after which they were immediately subjected to lipid extraction. Neutral lipid standards, phospholipid standards (phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA)), and sulfoquinovosyldiacylglycerol (SQDG) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.2. Extraction of Total Lipids (TL) M. ellitica powder was then extracted for TL following a previously described method of Folch et al. (1957) [26] in which a Soxhlet extractor in combination with a solvent mixture of CHCl3 /MeOH (2:1, v/v) and aqueous solution of sodium chloride (0.75%) were used. After being separated overnight at 4 ◦ C, the CHCl3 layer was recovered and dehydrated with sodium sulfate. The extract was filtered and subjected to a rotary evaporator at 40 ◦ C, and TL obtained were dissolved in CHCl3 and stored at −20 ◦ C. The content of TL was determined gravimetrically. 2.3. Lipid Fractionation A column incorporated with 100 g of silica gel 60 (70–230 mesh, Sigma-Aldrich Co., St. Louis, MO, USA) was loaded with TL (3.4 g). Neutral lipids, glycolipids, and phospholipids were eluted with CHCl3 (450 mL), acetone (230 mL), MeOH (60 mL), and MeOH/water (200 mL, 95:5, v/v). The MeOH-contained fractions were combined, taken to dryness, and polar lipids obtained were dissolved in CHCl3 and stored at −20 ◦ C. 2.4. Analysis of Lipid Class Compositions One-dimensional thin layer chromatography (TLC) using precoated silica gel plates (6 cm × 6 cm) Sorbfil PTLC-AF-V (Sorbfil, Krasnodar, Russia) was employed to analyze TL composition. The development of plates commenced with n-hexan/Et2 O/AcOH (70:20:1, v/v/v) to their full length, followed with CHCl3 /MeOH/C6 H6 /NH4 OH (65:30:5:10, v/v/v) to 25% length. Following that, plates were subjected to a stream of air for drying, sprayed with 10% H2 SO4 in MeOH and heated at 240 ◦ C for 10 min. An image scanner (Epson Perfection 2400 PHOTO) operating in grayscale mode was used to record the chromatograms. An image analysis software (Sorbfil TLC Videodensitometer, Krasnodar, Russia) was used to determine percentages of lipid contents based on band intensity. Peak areas and lipid class percentages were calculated according to Hamoutene (2008) [27]. In the first and second direction of separation of polar lipids using two-dimensional silica gel TLC, CHCl3 /MeOH/C6 H6 /28% NH4 OH (65:30:10:6, v/v/v) and CHCl3 /MeOH/AcOH/CH3 COCH3 /C6 H6 /H2 O (70:30:4:5:10:1, v/v/v) were used, respectively. Identification of phospholipids on TLC plates was made using aforementioned authentic standards and the specific spray reagents [28]. The phospholipid content was evaluated with spectrophotometry following the digestion with perchloric acid [29]. 2.5. Analysis of Lipid Class Compositions To obtain fatty acid methyl esters (FAMEs), the lipids, contained in a screw cap vial, were treated with 2% H2 SO4 in MeOH for 2 h at 80 ◦ C under Ar. The purification was performed by TLC development in benzene. The preparation of 4,4-Dimethyloxazoline (DMOX) derivatives of FAs from FAMEs follows the study of Svetashev (2011) [30]. FAMEs were analyzed by gas chromatography (GC) employed on a Shimadzu GC-2010 chromatograph (Kyoto, Japan) at 210 ◦ C. GC was performed in tandem with a flame ionization detector on a SUPELCOWAX 10 (Supelco, Bellefonte, PA, USA) capillary column (30 m × 0.25 mm× 0.25 µm). Temperatures of both injector and detector were maintained at 240 ◦ C. Carrier gas was He at Processes 2019, 7, 375 4 of 20 30 cm/s. The identification of FAMEs was made by comparison with authentic standards (Supelco 37 Component FAME Mix; Supelco, Bellefonte, PA, USA) and a table of equivalent chain-lengths [31]. To identify structural composition of FAs, corresponding methyl esters and DMOX derivatives were analyzed by gas chromatography mass spectrometry (GC–MS) on a Shimadzu GCMS-2010 instrument (Kyoto, Japan) (electron impact at 70 eV) with a MDN-5s (Supelco, Bellefonte, PA, USA) capillary column (30 m × 0.25 mm ID). Carrier gas was He at 30 cm/s. The temperature parameter for analysis of FAMEs started at 160 ◦ C, followed by an increase of 2 ◦ C/min to 240 ◦ C that was held for 20 min. Temperatures for both injector and detector temperatures were maintained at 250 ◦ C. For analysis of GC–MS of DMOX derivatives, the temperature started at 210 ◦ C, increased by 3 ◦ C/min to 270 ◦ C that was held for 40 min. The injector and detector temperatures were 250 ◦ C for this analysis. The mass spectra of FAMEs were compared with the Mass Spectral Library: WILEY275.L and NIST 98 [32]. 2.6. Analysis of Molecular Species of Polar Lipids Determination of chemical structures and molecular species of PL was performed by high performance liquid chromatography, in conjunction with high-resolution mass spectrometry (HPLC−HRMS). In the HPLC separation of the polar lipids, content of Et3 N/AcOH (0.08:1, v/v) was fixed in the solvent system [33]. This permits an efficient electrospray ionization (ESI) and stabilizes ion signal by the simultaneous registration of positive and negative ions. A Shimadzu Prominence liquid chromatograph (Kyoto, Japan) was used to perform the HPLC−HRMS analysis of polar lipids. The instrument was equipped with two LC-20AD pump units, a high pressure gradient forming module, CTO-20A column oven, SIL-20A auto sampler, CBM-20A communications bus module, DGU-20A3 degasser, and a Shim-Pack diol column (50 mm × 4.6 mm ID, 5 µm particle size) (Shimadzu, Kyoto, Japan). Two solvent mixtures, A and B, formed the binary solvent gradient for HPLC separation. The mixture A consisted of n-hexane/2-propanol/AcOH/Et3 N (82:17:1:0.08, v/v/v/v) and mixture B consisted of 2-propanol/H2 O/AcOH Et3 N (85:14:1:0.08, v/v/v/v). The acceleration of gradient started at 5% of mixture B and reached 80% throughout the course of 25 min. After 1 min, the composition changed to 5% of mixture B over 10 min and maintained at 5% for another 4 min (the total run time was 40 min). The flow rate was 0.2 mL/min. Determination of lipids was performed by a high resolution tandem ion trap time of flight mass spectrometry with a Shimadzu LCMS-IT-TOF instrument (Kyoto, Japan). The instrument operates at both positive and negative ion mode during each analysis under ESI conditions. Temperature for ion source was set to 200 ◦ C. The range of detection was m/z 100–1200. Negative and positive modes had their potential set at-3.5 and 4.5 kV, respectively. The drying gas (N2 ) pressure was 200 kPa. The nebulizer gas (N2 ) flow was 1.5 L/min. For identification of lipids, authentic standards were compared with the samples, which was performed using Shimadzu LCMS Solution control and processing software (v.3.60.361). Molecular species of each phospholipid class were determined by HRMS fragmentation pathways in comparison with standards [30]. Individual molecular species within each polar lipid class were quantified with respect to the peak areas for the individual extracted ion chromatograms [34]. 3. Results 3.1. Total Lipid Composition Total lipids (TL) constituted 34.0 ± 0.2% of the seed. The composition of TL is presented in Table 1 and Figure 1. The level of triacylglycerols (TG) was highest (63.2% of TL), followed by waxes (WX, 9.28% of TL), free fatty acids (FFA, 6.14% of TL), and diacylglycerols (DG, 5.17% of TL). Polar lipids constituted 11.2% of TL and contained glycolipids (GL, 6.78% of TL) and phospholipids (PL, 4.45% of TL). Five major classes were found in the total PL including Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). These five classes amounted to 13.2, 13.1, 7.7, 4.9, and 2.9% of total PL, respectively. − Processes 2019, 7, 375 5 of 20 Table 1. Lipid class composition (% of total lipid) and Phospholipid (PL) class (% of total Phospholipids) obtained from Madhuca ellitica seeds. Lipid Class Content (%) Phospholipid (PL) Class Content (%) Waxes Triacylglycerols Diacylglycerols Free fatty acids Glycolipids Phospholipids 5.28 ± 0.1 63.2 ± 1.5 5.17 ± 0.9 6.14 ± 0.6 5.78 ± 0.2 14.43 ± 1.3 PC PE PI PG PA LPE 30.7 ± 0.4 29.6 ± 0.3 17.2 ± 0.1 10.9 ± 0.2 6.8 ± 0.1 4.8± 0.1 Results are given as the average of triplicate determinations ± standard deviation. PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PG: phosphatidylglycerol; PA: phosphatidic acid; LPE: Lyso-PE. Figure 1. Lipid classes of the M. ellitica on Sorbfil thin layer chromatography (TLC). 3.2. Fatty Acid Composition of Total Lipids and Lipid Fractions The FA composition of TL and their fractions, obtained from of M. elliptica seeds, is presented in Table 2. Oleic (18:1n-9), palmitic (16:0), stearic (18:0), and linoleic (18:2n-6) acids were the major FAs, collectively composing more than 98% of the total identified FAs. In general, the prevalence of unsaturated FAs is the major characteristic of the FA composition of TL. Oleic acid was the main FA (49.5%) followed by palmitic acid. The FA profiles of TL, neutral, and polar lipid fractions were similar. In addition, low levels of myristic (14:0), α-linolenic (18:3n-3), and arachidic (20:0) acids were detected. Table 2. Fatty acid composition (% of total fatty acids) of total, neutral, and polar lipids obtained from Madhuca ellitica seeds. Fatty Acids Total Lipids Neutral Lipids Polar Lipids 14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6 18:3n-3 20:0 Σ SFA Σ UFA SFA/UFA 0.1 ± 0.1 24.4 ± 1.2 0.1 ± 0.1 11.5 ± 0.5 49.5 ± 3.5 13.6 ± 1.1 0.1 ± 0.1 0.4 ± 0.2 36.4 ± 0.4 63.3 ± 1.7 0.58 0.2 ± 0.1 25.7 ± 1.4 0.1 ± 0.1 13.5 ± 2.3 46.1 ± 2.8 13.6 ± 0.9 0.2 ± 0.1 0.4 ± 0.2 39.8 ± 0.6 60.0 ± 1.1 0.66 0.1 ± 0.1 28.5 ± 1.7 nd 12.5 ± 1.8 44.8 ± 2.9 13.2 ± 1.3 0.3 ± 0.1 0.3 ± 0.1 41.4 ± 0.5 58.3 ± 1.3 0.71 nd: not detected or under 0.1% of fatty acid composition in each type phospholipid; SFA: saturated fatty acids. UFA: unsaturated fatty acids. 3.3. Chemical Structure of Molecular Species of Polar Lipids The phospholipid classes of M. ellitica seeds were separated by HPLC−HRMS. The retention times of different molecular species of PE, PG, PA, PC, and PI were 5.90–6.87 min, 8.54–8.89 min, Processes 2019, 7, 375 − 6 of 20 9.18–9.37 min, 9.84–10.35 min, and 12.31–13.28 min, respectively. Among molecular species, four species including PE, PG, PA, and PI were revealed to be detectable at the MS2 stage, whereas PC species were identified at the MS3 stage [35]. HRMS spectra of PE molecular species are presented in detail as the examples of MS fragmentation (Figure 2A–D). Figure 2. The high-resolution mass spectrometry (HRMS) spectra of (A) negative quasi-molecular − − and (B) positive cluster ions [M + H + (C2 H5 )3 N]+ of phosphatidylethanolamine ions [M − H] − − [M (PE) molecular species, MS2 spectra of negative quasi-molecular ions − H]− at m/z 714.4988 (A), 716.5163 (B), 738.4975 (C), and 740.5140 (D) corresponded to 16:0/18:2 PE, 16:0/18:1 PE, 18:2/18:2 PE, and 18:1/18:2 PE molecular species of phosphatidylethanolamine MS2 spectra of negative quasi-molecular ions [M −−H]−−at m/z 742.5296 (A) and 744.5436 (C) corresponded to a mixture of 18:1/18:1 PE + 18:0/18:2 PE and 18:0/18:1 PE molecular species of phosphatidylethanolamine from Madhuca longifolia seeds. The sub-pictures (B) and (D) show the spectra in a large scale. Phosphatidylethanolamine (PE). Six signals of negative quasi-molecular ions [M − H]−− at m/z 714.4988, 716.5163, 738.4975, 740.5140, 742.5296, and 744.5436, as well as six signals of positive cluster ions [M + H + (C2 H5 )3 N]+ at m/z 817.6365, 819.6499, 841.6331, 843.6484, 845.6627, and 847.6749, were observed in the HRMS spectra of PE of M. ellitica seeds (Figure 1). Other less intensive peaks on Figure 1 corresponded to the signals of isotopic ions. − − Six components were analyzed as described below. The MS2 spectrum of the ions [M − H] of component 1 (m/z 714.4988) (Table 3) contained the signal at m/z 452.2697 formed by the loss of neutral fragment of 262.2291 (C18 H30 O, calculated 262.2297) (Figure 2A). This neutral fragment Processes 2019, 7, 375 7 of 20 corresponded to dehydrated 18:2 acid. The MS2 spectrum also contained the signals of carboxylate anions of 16:0 and 18:2 acids at m/z 255.2294 ([C16 H31 O2 ]− , calculated 255.2324) and m/z 279.2284 ([C18 H31 O2 ]– , calculated 279.2324), respectively. According to the elemental composition calculated and the value of monoisotopic molecular mass, component 1 was identified as palmitoyl linoleyl glycerophosphoethanolamine, 16:0/18:2 PE (Table 3). The MS2 spectrum of the ions [M − H]− of component 2 (m/z 716.5163) contained the signal at m/z 452.2713 formed by the loss of neutral fragment of 264.2450 (C18 H32 O, calculated 264.2453) (Figure 2B). This neutral fragment corresponded to dehydrated 18:1 acid. The MS2 spectrum also contained the signals of carboxylate anions of 16:0 and 18:1 acids at m/z 255.2294 ([C16 H31 O2 ]– , calculated 255.2324) and m/z 281.2443 ([C18 H33 O2 ]– , calculated 281.2480), respectively. Component 2 was identified as palmitoyl oleoyl glycerophosphoethanolamine, 16:0/18:1 PE (Table 1). The MS2 spectrum of the ions [M − H]– of component 3 (m/z 738.4975) contained the signal at m/z 476.2778 formed by the loss of dehydrated 18:2 acid (262.2197, C18 H30 O, calculated 262.2297) (Figure 2C). Only one signal of carboxylate anions at m/z 279.2283 ([C18 H31 O2 ]– ) was found. This result showed that two acyl groups were identical. Component 3 was identified as dilinoleyl glycerophosphoethanolamine, 18:2/18:2 PE (Table 3). The MS2 spectrum of the ions [M − H]– of component 4 (m/z 740.5140) contained the signals at m/z 478.2854 and 476.2705 formed by the loss of neutral fragments of dehydrated 18:2 (262.2286, C18 H30 O, calculated 262.2297) and 18:1 acids (264.2435, C18 H32 O, calculated 264.2453) (Figure 2D). The other two signals at m/z 281.2444 and 279.2284 were attributed to the anions of 18:1 and 18:2 acids, respectively. Component 4 was identified as oleoyl linoleyl glycerophosphoethanolamine, 18:1/18:2 PE (Table 3). The MS2 spectrum of component 5 [M − H]– , m/z 742.5296) contained six signals formed two compact groups (Figure 2A). Three major signals at m/z 283.2545 ([C18 H35 O2 ]− ), 281.2443 ([C18 H33 O2 ]− ), and 279.2285 ([C18 H31 O2 ]− ) (Figure 2B) corresponded to the anions of 18:0, 18:1, and 18:2 acids, respectively. Three weak signals at m/z 478.2866, 476.2707, and 460.2808 were formed by the loss of neutral fragments of 264.2430 (C18 H32 O, calculated 264.2453), 266.2589 (C18 H34 O, calculated 266.261), and 282.2488 (C18 H34 O2 , calculated 282.2559), respectively. These neutral fragments also originated from 18:1 and 18:2 acids. Component 5 was identified as a mixture of isomeric dioleoyl glycerophosphoethanolamine (18:1/18:1 PE) and stearoyl linoleyl glycerophosphoethanolamine, 18:0/18:2 PE (Table 3). The ratio between 18:1/18:1 PE and 18:0/18:2 PE was 4.4/1 according to the intensity of the corresponding ion peaks. The MS2 spectrum of the ions [M − H]– of component 6 (m/z 744.5436) showed the signals at m/z 480.3046, 283.2577, and 281.2441 (Figure 2C,D). Similar to components 1 and 2, component 6 was identified as stearoyl oleoyl glycerophosphoethanolamine, 18:0/18:1 PE (Table 3). Thus, seven PE molecular species, such as 16:0/18:2 PE, 16:0/18:1 PE, 18:2/18:2 PE, 18:1/18:2 PE, 18:1/18:1 PE, 18:0/18:2 PE, and 18:0/18:1 PE, were identified. Phosphatidylcholine (PC). Six signals of negative acetylated molecular ions [M + CH3 COO]– at m/z 816.5760, 818.5889, 820.5943, 840.5712, 842.5888, and 844.6059, as well as six corresponded signals of positive quasi-molecular ions [M+ H]+ , were observed in the HRMS spectra of PC of M. ellitica seeds (Table 4). The ions [M + CH3 COO]– of each component lost methyl acetate (CH3 COOCH3 ) at the MS2 state. The subsequent fragmentation formed the anions characterized acyl groups of most components at the MS3 stage. In MS3 spectra of ions [M − H − CH3 COOCH3 ]– , the signals at m/z 255.2320, 283.2602, 281.2474, and 279.2332 indicated the presence of acyl groups of 16:0, 18:0, 18:1, and 18:2 acids, respectively. According to the MS3 data, the elemental composition calculated, and the value of mono-isotopic molecular mass, we identified components 1, 2, and 5 as 16:0/18:2 PC, 16:0/18:1 PC, and 18:1/18:2 PC, respectively (Table 4). Component 6 were identified as a mixture of 18:1/18:1 PC and 18:0/18:2 PC with the ratio 1.58:1. We could not observe MS3 fragmentation of components 3 and 4 because of their low concentration. We suggest that these components are 16:0/18:0 PC and 18:2/18:2 PC on the base of their elemental composition and mono-isotopic molecular mass (Table 4). Processes 2019, 7, 375 8 of 20 Table 3. Molecular species of phosphatidylethanolamine (PE) from Madhuca ellitica seeds. ESI-MS No. Molecular Species [M + H + Et3 N]+ m/z MS2 Monoisotopic Molecular Mass [M − H]– m/z Measured Calculated Molecular Formula Fragment Ion [M − H − X]– * m/z Composition X 1 16:0/18:2 PE 817.6365 714.4988 715.5061 715.5152 C39 H74 NO8 P 452.2697 279.2284 255.2294 C21 H44 NO7 P C18 H31 O2 C16 H31 O2 C18 H30 O C21 H43 NO6 P C23 H43 NO6 P 2 16:0/18:1 PE 819.6499 716.5163 717.5236 717.5309 C39 H76 NO8 P 452.2713 281.2443 255.2294 C21 H44 NO7 P C18 H33 O2 C16 H31 O2 C18 H32 O C21 H43 NO6 P C23 H45 NO6 P 3 18:2/18:2 PE 841.6331 738.4975 739.5048 739.5152 C41 H74 NO8 P 476.2778 279.2283 C23 H44 NO7 P C18 H31 O2 C18 H30 O C23 H43 NO6 P 4 18:1/18:2 PE 843.6484 740.5140 741.5213 741.5309 C41 H76 NO8 P 478.2854 476.2705 281.2444 279.2284 C23 H46 NO7 P C23 H44 NO7 P C18 H33 O2 C18 H31 O2 C18 H30 O C18 H32 O C23 H43 NO6 P C23 H45 NO6 P C23 H46 NO7 P C23 H44 NO7 P C23 H44 NO6 P C18 H35 O2 C18 H33 O2 C18 H31 O2 C18 H32 O C18 H34 O C18 H34 O2 C23 H43 NO6 P C23 H45 NO6 P C23 H47 NO6 P C23 H48 NO7 P C18 H35 O2 C18 H33 O2 C18 H32 O C23 H45 NO6 P C23 H47 NO6 P 5 18:1/18:1 PE (4.4) 18:0/18:2 PE (1) 845.6627 742.5296 743.5369 743.5465 C41 H78 NO8 P 478.2866 476.2707 460.2808 283.2545 281.2443 279.2285 6 18:0/18:1 PE 847.6749 744.5436 745.5509 745.5622 C41 H80 NO8 P 480.3046 283.2577 281.2441 ESI-MS: electrospray ionization-mass spectrometry. * Precursor ion [M − H]– . Processes 2019, 7, 375 9 of 20 Table 4. Molecular species of phosphatidylcholine (PC) from Madhuca ellitica seeds. ESI-MS No. Molecular Formula [M + CH3 COO]− m/z 1 16:0/18:2 PC MS2 Monoisotopic Molecular Mass Molecular Species 816.5760 Measured 757.5621 Calculated 757.5622 C42 H80 NO8 P MS3 Fragmentation ** Fragment Ion * [M + CH3 COO − C3 H6 O2 ]− m/z m/z Composition 742.5389 279.2274 C18 H31 O2 C18 H33 O2 C16 H31 O2 - 2 16:0/18:1 PC 818.5889 759.5750 759.5778 C42 H82 NO8 P 744.5532 281.2462 255.2320 3 16:0/18:0 PC 820.5943 761.5804 761.5935 C42 H84 NO8 P 746.5599 ND 4 18:2/18:2 PC 840.5712 781.5573 781.5622 C44 H80 NO8 P 766.5309 ND C18 H33 O2 C18 H31 O2 C18 H35 O2 C18 H33 O2 C18 H31 O2 5 18:1/18:2 PC 842.5888 783.5749 783.5778 C44 H82 NO8 P 768.5511 281.2432 279.2352 6 18:1/18:1 PC (1.58) 18:0/18:2 PC (1) 844.6059 785.5920 785.5935 C44 H84 NO8 P 770.5674 283.2602 281.2474 279.2332 ND: not detected. * Precursor ion [M+CH3 COO]– . ** Precursor ion [M+CH3 COO–C3 H6 O2 ]– . Processes 2019, 7, 375 10 of 20 Phosphatidylinositol (PI). The main molecular species of PI from M. ellitica seeds produced five negative quasi-molecular ions [M − H]– at m/z 833.5188 ([C43 H78 O13 P]– ), 835.5328 ([C43 H80 O13 P]– ), 857.5155 ([C45 H78 O13 P]– ), 861.5463 ([C45 H80 O13 P]– ), 863.5635 ([C45 H82 O13 P]– ) (Table 5). These five components constituted more than 96% of total PI. Positive quasi-molecular ions also formed. MS2 fragmentation of the ions [M − H]– of PI were more complex than that of PE or PC and gave more ions. Generally, the MS2 spectra the ions [M − H]– of PI contained the signals of FA carboxylate anions and several fragments, which arise from the loss of each FA, each dehydrated FA, two FAs, two dehydrated FAs, and different combinations of inositol and acyl fragments. As an example, MS2 fragmentation of the ions [M − H]– of component 1 (m/z 833.5188) is explained in detail (Table 5). The ions at m/z 281.2474 [C18 H33 O2 ]– , 279.2307 [C18 H31 O2 ]– , and 255.2346 [C16 H31 O2 ]– corresponded to carboxylate anions of 18:1, 18:2, and 16:0, respectively. The loss of neutral acids 16:1, 16:0, and 18:2 gave ions at m/z 579.2901 ([M − H − C16 H32 O2 ]– ), 577.2784 ([[M − H − C16 H30 O2 ]– ), and 553.2813 ([M − H − C18 H32 O2 ]– , calculated 553.2783), respectively. The loss of dehydrated 18:2 and 16:0 led to the formation of ions at m/z 571.2904 ([M − H − C18 H30 O]– ) and 595.2769 ([M − H − C18 H30 O]– ), respectively. The appearance of ions at m/z 297.0388 (calculated 297.0381) was caused by the simultaneous loss of 16:0 and 18:2 (or 16:1 and 18:1). The ions at m/z 315.0464 (calculated 315.0487) were formed by the simultaneous loss of dehydrated 16:0 and 18:2 (or 16:1 and 18:1). The loss of inositol and acyl fragments, namely ([C6 H10 O5 + C16 H30 O2 ], ([C6 H10 O5 + C16 H32 O2 ], and ([C6 H10 O5 + C18 H32 O2 ], gave the ions at m/z 417.2418, 415.2268, and 391.2253, respectively, characteristic for PI. Thus, compound 1 was identified as a mixture of 16:1/18:1 PI and 16:0/18:2 PI. Similar to our approach described above, compounds 2, 3, and 5 were identified as individual 16:0/18:1 PI, 18:2/18:2 PI, and 18:0/18:1 PI, respectively, whereas compound 4 contained a mixture of 18:0/18:2 PI and 18:1/18:1 PI (Table 5). Phosphatidylglycerol (PG). Molecular species of PG of M. ellitica seeds were identified according to the monoisotopic molecular mass of negative quasi-molecular ions [M − H]– , their elemental compositions, and MS2 fragmentation indicated acyl groups of PG molecules. Six major signals of the negative quasi-molecular ions [M − H]– at m/z 745.4944, 747.5092, 749.5212, 771.5092, 773.5253, and 775.5380 were observed in the HRMS spectra (Table 6). MS2 spectra of the ions [M − H]– contained the signals of carboxylate anions of 16:0 ([C16 H31 O2 ]– ), 18:0 ([C18 H35 O2 ]– ), 18:1([C18 H33 O2 ]– ), and 18:2 ([C18 H31 O2 ]– ) acids. The signals of the ions, which lost neutral FAs, or dehydrated FAs, or glycerol fragment (C3 H6 O2 ) were also observed in some MS2 spectra of the ions [M − H]– of PI molecular species. According to MS/MS data, the molecular species 16:0/18:0 PG, 16:0/18:1 PG, 16:0/18:2 PG, 18:0/18:1 PG, 18:0/18:2 PG, 18:1/18:1 PG, and 18:1/18:2 PG were identified (Table 6). These seven molecular species constituted about 99% of total PG. Phosphatidic acid (PA). Six signals of the negative quasi-molecular ions [M − H]– at m/z 671.4643, 673.4798, 695.4660, 697.4803, 699.4949, and 701.5098 were detected in the HRMS spectra of molecular species of PA from M. ellitica seeds (Table 7). Similar to other phospholipid classes, MS2 spectra of the ions [M − H]– of PA contained the signals of carboxylate anions of 16:0 ([C16 H31 O2 ]– ), 18:0 ([C18 H35 O2 ]– ), 18:1([C18 H33 O2 ]– ), and 18:2 ([C18 H31 O2 ]– ) acids. The signals of the ions [M − H]– , which lost two neutral FAs or two dehydrated FAs, were also observed in these MS2 spectra. The components, which produced ions related to three FAs, were considered as a mixture of two isotopes. For example, compound 5 (m/z 699.4949, [M − H]– ) was identified as a mixture of 18:1/18:1 PA and 18:0/18:2 PA. On the whole, the molecular species 16:0/18:1 PA, 16:0/18:2 PA, 18:0/18:1 PA, 18:0/18:2 PA, 18:1/18:1 PA, 18:1/18:2 PA, and 18:2/18:2 PA were identified (Table 7). Processes 2019, 7, 375 11 of 20 Table 5. Molecular species of phosphatidylinositol (PI) from Madhuca ellitica seeds. ESI-MS No. 1 Molecular Species 16:1/18:1 PI 16:0/18:2 PI [M − H]– m/z 833.5188 MS2 Monoisotopic Molecular Mass Molecular Formula Measured 834.5256 Calculated Fragment Ion * [M − H − X]− m/z X 834.5258 C43 H79 O13 P 595.2769 579.2901 577.2784 571.2904 553.2813 417.2418 415.2268 409.2352 391.2253 297.0388 281.2474 279.2307 255.2346 C16 H30 O C16 H30 O2 C16 H32 O2 C18 H30 O C18 H30 O2 C22 H40 O7 (C6 H10 O5 + C16 H30 O2 ) C22 H42 O7 (C6 H10 O5 + C16 H32 O2 ) C24 H40 O6 (C6 H10 O5 + C18 H30 O) C24 H42 O7 (C6 H10 O5 + C18 H32 O2 ) C34 H64 O4 (C16 H32 O2 + C18 H32 O2 ) C25 H45 O11 P C25 H47 O11 P C27 H47 O11 P 597.3036 579.2939 571.2876 553.2805 435.2438 417.2389 409.2351 391.2250 297.0373 281.2484 255.2337 C16 H30 O C16 H32 O2 C18 H32 O C18 H34 O2 C22 H40 O6 (C6 H10 O5 + C16 H30 O) C22 H42 O7 (C6 H10 O5 + C16 H32 O2 ) C24 H42 O6 (C6 H10 O5 + C18 H32 O) C24 H44 O7 (C6 H10 O5 + C18 H34 O2 ) C34 H64 O4 (C16 H32 O2 + C18 H34 O2 ) C25 H47 O11 P C27 H49 O11 P 577.2805 415.2264 279.2337 C18 H32 O2 C24 H42 O7 (C6 H10 O5 + C18 H32 O2 ) C27 H47 O11 P 2 16:0/18:1 PI 835.5328 836.5401 836.5415 C43 H81 O13 P 3 18:2/18:2 PI 857.5155 858.5228 858.5258 C45 H79 O13 P Processes 2019, 7, 375 12 of 20 Table 5. Cont. ESI-MS No. 4 5 Molecular Species 18:0/18:2 PI 18:1/18:1 PI 18:0/18:1 PI [M − H]– m/z 861.5463 863.5635 MS2 Monoisotopic Molecular Mass Molecular Formula Measured 862.5536 864.5708 Calculated Fragment Ion * [M − H − X]− m/z X 862.5571 C45 H81 O13 P 599.3202 597.3037 595.2894 581.3095 579.2945 577.2775 437.2665 435.2511 433.2306 419.2571 417.2394 415.2241 297.0370 283.2622 281.2484 279.2328 C18 H30 O C18 H32 O C18 H34 O C18 H32 O2 C18 H34 O2 C18 H36 O2 C24 H40 O6 (C6 H10 O5 + C18 H30 O) C24 H42 O6 (C6 H10 O5 + C18 H32 O) C24 H44 O6 (C6 H10 O5 + C18 H34 O) C24 H42 O7 (C6 H10 O5 + C18 H32 O2 ) C24 H44 O7 (C6 H10 O5 + C18 H34 O2 ) C24 H46 O7 (C6 H10 O5 + C18 H36 O2 ) C36 H68 O4 C27 H45 O11 P C27 H47 O11 P C27 H49 O11 P C45 H83 O13 P 701.5027 599.3203 597.3032 581.3097 579.2943 419.2573 417.2393 297.0376 283.2627 281.2485 C6 H10 O5 C18 H32 O C18 H34 O C18 H34 O2 C18 H36 O2 C24 H44 O7 (C6 H10 O5 + C18 H34 O2 ) C24 H46 O7 (C6 H10 O5 + C18 H36 O2 ) C36 H70 O4 C27 H47 O11 P C27 H49 O11 P 864.5728 * Precursor ion [M − H]– . Processes 2019, 7, 375 13 of 20 Table 6. Molecular species of phosphatidylglycerol (PG) from Madhuca ellitica seeds. ESI-MS No. Molecular Species MS2 Monoisotopic Molecular Mass Molecular Formula [M − H]– m/z Measured Calculated Fragment Ion * [M − H − X]− m/z X 1 16:0/18:2 PG 745.4944 746.5017 746.5098 C40 H75 O10 P 391.2224 279.2303 255.2336 C21 H38 O4 (C3 H6 O2 + C18 H32 O2 ) C22 H43 O8 P C24 H43 O8 P 2 16:0/18:1 PG 747.5092 748.5164 748.5254 C40 H77 O10 P 281.2482 255.2323 C22 H43 O8 P C24 H45 O8 P 3 16:0/18:0 PG 749.5212 750.5285 750.5411 C40 H79 O10 P 283.2598 255.2321 C22 H43 O8 P C24 H47 O8 P 4 18:1/18:2 PG 771.5092 772.5164 772.5254 C42 H77 O10 P 491.2788 281.2477 279.2325 C18 H32 O2 C24 H43 O8 P C24 H45 O8 P 5 18:1/18:1 PG 18:0/18:2 PG 773.5253 774.5326 774.5411 C42 H79 O10 P 283.2598 281.2478 279.2328 C24 H43 O8 P C24 H45 O8 P C24 H47 O8 P C42 H81 O10 P 493.2940 491.2773 419.2554 417.2406 283.2608 281.2478 C18 H34 O2 C18 H36 O2 C21 H40 O4 (C3 H6 O2 + C18 H34 O2 ) C21 H42 O4 (C3 H6 O2 + C18 H36 O2 ) C24 H45 O8 P C24 H47 O8 P 6 18:0/18:1 PG 775.5380 776.5524 776.5567 * Precursor ion [M − H]– . Processes 2019, 7, 375 14 of 20 Table 7. Molecular species of phosphatidic acid (PA) from Madhuca ellitica seeds. ESI-MS No. 1 Molecular Species 16:0/18:2 PA [M − H]– m/z 671.4643 MS2 Monoisotopic Molecular Mass Molecular Formula Measured 672.4716 Calculated Fragment ion * [M − H − X]− m/z X 672.4730 C37 H69 O8 P 409.2375 391.2271 279.2274 255.2313 C18 H30 O C18 H32 O2 C19 H37 O6 P C21 H37 O6 P C16 H32 O2 C18 H32 O C18 H34 O2 C19 H37 O6 P C21 H39 O6 P 2 16:0/18:1 PA 673.4798 674.4871 674.4887 C37 H71 O8 P 417.2394 409.2332 391.2242 281.2469 255.2318 3 18:2/18:2 PA 695.4660 696.4727 696.4730 C39 H69 O8 P 433.2324 415.2228 279.2319 C18 H30 O C18 H32 O2 C21 H37 O6 P C39 H71 O8 P 435.2511 433.2351 417.2405 415.2241 281.2458 279.2316 C18 H30 O C18 H32 O C18 H32 O2 C18 H34 O2 C21 H37 O6 P C21 H39 O6 P C39 H73 O8 P 437.2641 435.2511 419.256 417.2382 415.2223 283.2608 281.2479 279.2322 C18 H30 O C18 H32 O C18 H32 O2 C18 H34 O2 C18 H36 O2 C21 H37 O6 P C21 H39 O6 P C21 H41 O6 P C39 H75 O8 P 437.2633 435.2503 419.2546 417.2385 283.2621 281.247 C18 H32 O C18 H34 O C18 H34 O2 C18 H36 O2 C21 H39 O6 P C21 H41 O6 P 4 5 6 18:1/18:2 PA 18:0/18:2 PA 18:1/18:1 PA 18:0/18:1 PA 697.4803 699.4949 701.5098 698.4876 700.5022 702.5171 698.4887 700.5043 702.5200 * Precursor ion [M − H]– . Processes 2019, 7, 375 15 of 20 3.4. Composition of the Molecular Species of the Phospholipids The percentages of the individual molecular species described above within each phospholipid class (PE, PC, PI, PG, and PA) are combined in Table 8. The composition of total phospholipids (Table 1) was used to determine the percentages of the individual molecular species within total phospholipids of M. ellitica seeds (Table 8). The weight content of each molecular species was calculated with regard to the content of phospholipids in total seed lipids (44.5 g/kg). Five molecular species (16:0/18:1, 16:0/18:2, 18:1/18:1, 18:1/18:2, and 18:2/18:2) amounted about 91% of total PE. The major molecular species of PC were 16:0/18:1, 18:0/18:2, and 18:1/18:2. It was found that 16:0/18:1 was the main molecular species of both PI and PG. Table 8. Content of molecular species of phospholipids obtained from Madhuca ellitica seeds. Concentration Phospholipid Class Molecular Species PE % of Each Phospholipid Class % of Total Phospholipids mg/kg of Total Lipids 16:0/18:1 PE 16:0/18:2 PE 18:0/18:1 PE 18:0/18:2 PE 18:1/18:1 PE 18:1/18:2 PE 18:2/18:2 PE 19.21 16.13 4.64 3.96 17.43 27.18 11.44 5.77 4.85 1.39 1.19 5.23 8.16 3.44 2567 2158 620 529 2329 3632 1529 PC 16:0/18:0 PC 16:0/18:1 PC 16:0/18:2 PC 18:0/18:2 PC 18:1/18:1 PC 18:1/18:2 PC 18:2/18:2 PC 3.96 30.11 13.27 17.10 10.79 19.78 4.98 1.19 9.05 3.99 5.14 3.24 5.94 1.50 530 4026 1774 2287 1443 2645 666 PI 16:0/18:1 PI 16:0/18:2 PI 16:1/18:1 PI 18:0/18:2 PI 18:1/18:1 PI 18:2/18:2 PI Other PI 44.18 1.01 20.92 23.08 5.25 2.02 3.54 7.70 0.18 3.65 4.02 0.92 0.35 0.62 3427 78 1623 1790 407 157 275 PG 16:0/18:0 PG 16:0/18:1 PG 16:0/18:2 PG 18:0/18:1 PG 18:0/18:2 PG 18:1/18:1 PG 18:1/18:2 PG Other PG 9.42 43.47 22.05 7.93 2.77 8.32 5.14 0.90 1.06 4.87 2.47 0.89 0.31 0.93 0.58 0.10 469 2167 1099 395 138 415 256 45 PA 16:0/18:1 PA 16:0/18:2 PA 18:0/18:1 PA 18:0/18:2 PA 18:1/18:1 PA 18:1/18:2 PA 18:2/18:2 PA 29.01 15.98 7.05 4.74 18.19 19.62 5.41 1.91 1.05 0.47 0.31 1.20 1.29 0.36 852 469 207 139 534 576 159 Results are given as the average of triplicate determinations. PE: phosphatidylethanolamine; PC: phosphatidylcholine; PI: phosphatidylinositol; PG: phosphatidylglycerol; PA: phosphatidic acid. About 82% of PA were comprised of four molecular species (16:0/18:1, 16:0/18:2, 18:1/18:1, and 18:1/18:2). Overall, three molecular species, namely 16:0/18:1 PC (9.05%), 18:1/18:2 PE (8.16%), and 16:0/18:1 PI (7.7%), were mostly abundant in total phospholipids. Total phospholipids contained 32.05, 38.68, 15.97, and 5.65% of the unsaturated molecular species with one, two, three, and four double bonds in their acyl groups, respectively. The content of saturated molecular species was low (2.25% of total phospholipids). Molecular species with 18:3 were not identified, probably, because of their low concentrations. Processes 2019, 7, 375 16 of 20 3.5. Sulfoquinovosyldiacylglycerol (SQDG) A pure SQDG fraction was isolated from polar lipids by preparative TLC and analyzed by HPLC–HRMS. Ten major signals of the negative quasi-molecular ions [M − H]– .at m/z 765.4858, 793.5157, 815.4939, 817.5106, 819.5253, 839.4893, 841.5061, 843.5215, 845.5368, and 847.5527 were detected in the HRMS spectra of the SQDG fraction (Table 9). The elemental composition and the value of monoisotopic molecular mass confirmed the presence of SQDG in total lipids of M. ellitica seeds. The signals of carboxylate anions in MS2 spectra of the ions [M − H]– of SQDG allowed us to identify fourteen molecular species which constituted about 99% of total SQDG. There were 14:0/16:0, 16:0/16:0, 16:0/18:1, 16:0/18:2, 16:0/18:3, 16:1/18:1, 18:0/18:1, 18:0/18:2, 18:0/18:3, 18:1/18:1, 18:1/18:2, 18:1/18:3, 18:2/18:2, and 18:2/18:3 SQDG (Table 9). The main molecular species was 16:0/18:1 SQDG (28.09%), followed by 18:0/18:1 SQDG (17.97%), and 16:0/18:2 SQDG (12.65%). Unsaturated components predominated in the SQDG fraction, whereas the content of saturated molecular species was low (7.51% of total SQDG). Several molecular species of SQDG contained one 18:3 acyl group, but a component with two 18:3 acyl groups was not found. Table 9. Molecular species of sulfoquinovosyldiacylglycerol (SQDG) from Madhuca ellitica seeds. No. Molecular Species Content, mol. % ESI–MS Monoisotopic Molecular Mass Measured Calculated Molecular Formula 1 14:0/16:0 0.36 765.4858 766.4901 672.4730 C39 H74 O12 S 2 3 4 16:0/16:0 16:0/18:3 16:0/18:2 + 16:1/18:1 7.15 2.04 12.65 4.72 793.5157 815.4939 817.5106 794.5198 816.5011 818.5179 794.5214 816.5057 818.5214 C41 H78 O12 S C43 H76 O12 S C43 H78 O12 S 5 16:0/18:1 28.09 819.5253 820.5325 820.537 C43H80O12S 6 18:2/18:3 0.77 839.4893 840.4965 840.5057 C45 H76 O12 S 7 18:2/18:2 + 18:1/18:3 2.44 0.75 841.5061 842.5134 842.5214 C45 H78 O12 S 8 18:0/18:3 + 18:1/18:2 0.99 5.76 843.5215 844.5287 844.537 C45 H80 O12 S 9 18:0/18:2 + 18:1/18:1 7.12 8.75 845.5368 846.5441 846.5527 C45 H82 O12 S 10 18:0/18:1 17.97 847.5527 848.5599 848.5683 C45 H84 O12 S a MS2 Fragment Ion a [M − H − X]− m/z X 537.2731 509.2461 537.2723 559.2593 563.2875 561.2734 537.2730 563.2892 537.2747 561.2694 559.2581 563.2841 561.2733 559.2638 565.3002 563.2883 561.2728 559.2573 565.3005 563.2894 561.2738 565.3009 563.2903 C14 H28 O2 C16 H32 O2 C16 H32 O2 C16 H32 O2 C16 H30 O2 C16 H32 O2 C18 H32 O2 C16 H32 O2 C18 H34 O2 C18 H30 O2 C18 H32 O2 C18 H30 O2 C18 H32 O2 C18 H34 O2 C18 H30 O2 C18 H32 O2 C18 H34 O2 C18 H36 O2 C18 H32 O2 C18 H34 O2 C18 H36 O2 C18 H34 O2 C18 H36 O2 Precursor ion [M − H]– . 4. Discussion The seeds of Madhuca are the base of numerous food products including a variety of lipid substances. Some of which are commonly referred to as “fat”, “oil” or “butter” [13]. Technologies of their manufacturing are adopted to obtain the substances with target properties. Different methods are used for lipid analysis of these seed products and fresh seeds. Therefore, the comparable information on the lipid composition of M. ellitica seeds is limited. The seeds of M. ellitica were found to be rich in lipids. The seed contains a considerable amount of crude non-polar compounds (up to 61% of dw), which can be extracted by organic solvents. However, the lipid percentage does not seem to exceed 50% and depends on the extraction method [10,11,13]. Classic Folch’s method yielded 34% of total lipids from fresh seeds and indicated that the lipid contents in M. ellitica seeds and other oilseed crops are similar [23]. Quality and utility of seed oils are mainly determined by their FA composition. The previous studies of the FA composition of total lipids Processes 2019, 7, 375 17 of 20 have shown that 16:0 (11.7–25.9%), 18:0 (19.1–32.2%), 18:1n-9 (32.9–48.6%), and 18:2n -6 (9.4–15.4%) were the major FAs found in Indian M. longifolia seeds [8,13,21]. The FAs of total lipids of our samples (Table 2) contained 18:0 at low content (11.5%) and 18:1n-9 at high content (49.5%). The oils with the high content of 18:1n-9 and the very low level of 18:3n-3 (0.1%) are suitable for some cosmetic and pharmaceutical preparations. Some polyunsaturated FAs (PUFAs), such as 20:5n-3 and 22:6n-3, were earlier detected in mahua butter from the Indian buttercup [10] but were not found in the present study (Table 2). To our knowledge, these n-3 PUFAs are common for marine plants and animals. The enrichment of the polar lipid fraction with PUFAs was reported for animal lipids, but not for plant lipids (Table 2). The FA profiles of total, neutral, and polar lipids from M. ellitica seeds were quite similar (Table 2). Unusual FAs were not found in neutral lipids from M. ellitica seeds, in spite of the fact that seed TG often contain rare FAs [32]. The elevated content of 16:0 in the polar lipids (Table 2) has been previously observed in M. ellitica seeds [10]. In total lipids (TL) of oil seeds, the level of neutral lipids is highest, followed by glycolipids (GL) and phospholipids (PL). Neutral lipid classes, first of all, triacylglycerols (TG), prevailed in TL from M. ellitica seeds (Table 1). Extraction with hexane produced TL containing 91.2 and 0.2% of TG and PL, respectively [10]. The use of Folch’s method [23] showed that M. ellitica seed TL contained 4.5% of PL (Table 1). According to Ramadan and others (2006), the predominant PL subclasses were PE (57.7%) followed by PC (30.6%), while PI and PS were isolated in smaller quantities [10]. Our results confirmed that PE, PC and PI were the major PL subclasses, while PG was detected instead of PS. Additionally, PA was observed in the lipids of M. ellitica seeds. It is undoubted that the lipids of M. elliptica contained PG because its’ chemical structures were confirmed by mass-spectrometry (Table 6). Generally, both PG and PS are known to be in seed lipids [19]. The discrepancy in PL class composition of M. ellitica may be explained by different extraction methods applied for TL preparation. Phospholipids (PL) are recognized to play multiple roles in cell processes. PL form a bilayer of cell membranes and, therefore, are involved in important functions of the cell, such as energy transduction, signal transduction, trans-membrane transport and cell-cell recognition. The wide range of these biochemical processes explains the need for high diversity in phospholipid structure [36]. At the same time, only a few molecular species within each PL class demonstrate high biological activities. A potential pharmaceutical importance of an individual molecular species mainly depends on the chemical structure of its acyl groups [37–39]. Thus, common chemical characteristics, such as lipid class composition and total FA composition of each lipid class, are not enough for the detailed description of polar components of seed lipids. A lipidomic study of lipid molecular species is necessary for the use of these seeds effectively [40]. Fast determination of the profile of lipid molecular species became available as a result of the development of chromatography–mass-spectrometry [41–43]. The amounts of neutral lipids in seed oils are known to be the highest, followed by glycolipids and phospholipids [44]. Correspondingly, analysis of molecular species of neutral lipids and glycolipids are performed on a regular basis [19,45], but data on PL molecular species are scanty [46]. For M. ellitica seeds, molecular species profiles of TG of different fat products were earlier described [21], but data on PL molecular species were absent. In the present study, the chemical structure and the content of PL molecular species of M. ellitica seeds were determined for the first time by a high-resolution tandem mass-spectrometry. This MS method allows both the detection of lipid molecular species in a presence of other low-molecular weight compounds and the determination of acyl group chemical structures of each lipid molecular species [22]. Thirty-four molecular species belonging to five PL classes were identified (Table 3). It is likely that using the molecular species profile as a description of the PL class may be preferable to using the FA profile. Indeed, total FAs of PL contained 58.3% of unsaturated FAs (Table 2), while unsaturated PL molecular species amounted to 97.7% of total PL (Table 3). In animal PL, polyunsaturated components are concentrated in position sn-2 with saturated FAs most abundant in position sn-1. In plants, the differences between the two positions are relatively minor. We did not determine the positional distributions of FAs in PL from the seeds of M. longifolia, but we showed Processes 2019, 7, 375 18 of 20 that half of the molecular species contain two unsaturated acyl groups (Table 8). This distribution is explained by some differences between lipid metabolism in plants and other organisms [43]. The results will be important to determine nutraceutical and economical utility of M. ellitica seeds. Similar to PL, a charged group presents in molecules of sulfoquinovosyldiacylglycerol (SQDG), which is one of the important glycolipid (GL) classes in plants [47]. Membranes of chloroplasts and other plastids are enriched in GL. Thylakoid membranes of the chloroplasts are the site of the light reactions of photosynthesis. According to the important role of GL, these classes have been found in all seed oils. However, Kadri recently observed a lack of SQDG in polar lipids of Pinus halepensis seeds [19]. SQDG has been previously detected in the seeds of M. longifolia by Ramadan and others [10]. To confirm the presence and the structure of SQDG, this lipid class was obtained during the isolation of the polar lipid fraction from the seeds of M. ellitica. A profile of molecular species of SQDG was investigated similarly to that of PL. Among fourteen molecular species, three major species (16:0/18:1, 18:0/18:1, and 16:0/18:2) amounted to 58.7% of total SQDG (Table 9). The molecular species of SQDG containing two unsaturated acyl groups (for example, 18:1/18:1, 18:1/18:2, 18:2/18:2, 18:2/18:3) were also found. The compositions of the SQDG molecular species have been described in different phyla, for example, a soil bacterium [48], cyanobacteria [49,50], microalgae, seaweeds [51], Arabidopsis leaves [45], and sea urchin [52]. These molecular species were characterized by the high degree of saturation of FAs and contained one or two saturated acyl groups. Antibacterial, antitumor, and antiviral activities were reported for SQDG. Since the biological activities depend on a FA saturation degree, we suppose that unsaturated molecular species of SQDG from the seeds of M. ellitica enhance the pharmacological potential of these compounds. 5. Conclusions The seed oil from the buttercup tree M. ellitica is widely used in India and Indochina. However, a detailed analysis of its polar lipid fractions has not been performed. A lipidomic approach showed that the seeds of M. ellitica contain a variety of polar lipid compounds with both biotechnological potential and pharmaceutical interest. Our research enhances the industrial potential of M. ellitica and shows that their seeds may be a good source of polar lipids, which contain about 95% of unsaturated components. Further studies are needed to extend knowledge concerning the distribution of SQDG molecular species between oil seeds. Author Contributions: Investigation, D.L.P., T.Q.T., L.P.T.D., A.B.I., T.D.T., B.M. and L.G.B.; Supervision, P.Q.L. and L.M.B.; Writing—original draft, D.L.P. Funding: This research received no external funding Conflicts of Interest: The authors declare no conflict of interest References 1. 2. 3. 4. 5. 6. Kundu, M. Madhuca longifolia (Koenig) J. F. Macb. Seed Leaflets 2012, 156. Chopra, R.N.; Nayar, S.L.; Chopra, I.C. Glossary of Indian Medicinal Plant; National Institute of Science communication and Information resource (CSIR): New Delhi, India, 1985. Benerji, D.S.N.; Rajini, K.; Rao, B.S. 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