Purslane (Portulaca oleracea L.) seed oil as a valuable source of important phytonutrients: effect of the extraction method on the lipid composition

Purslane (Portulaca oleracea L.) seed oil as a valuable source of important phytonutrients: effect of the extraction method on the lipid composition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Purslane (Portulaca oleracea L.) seed oil as a valuable source of important phytonutrients: effect of the extraction method on the lipid composition Sabina Taneva, Svetlana Momchilova This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4743339/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Purslane ( Portulaca oleracea L.) is a widespread weed plant used since ancient times as remedy and as food. Its seed oil possesses significant bioactive potential due to the high content of important phytonutrients, mainly essential fatty acids and phytosterols. Although the total fatty acids composition of oil is well documented, till now there is no data published about individual lipid classes. The information on sterols is scarce as well. Therefore, the aim of this work was to characterize in details for the first time the individual lipid classes and their fatty acids composition, sterols, as well as the oil oxidative stability, depending on the extraction method by either hexane, chloroform-methanol mixtures or super-critical CO 2 . The results revealed no significant effect of extraction method on the individual lipid classes (monoacylglycerols, diacylglycerols, free fatty acids, free sterols, triacylglycerols, sterol esters, wax esters and hydrocarbons), including the fatty acids composition of saponifiables, and quite weak effect on the oil oxidative stability regarding super-critical CO 2 vs. organic solvents. Detailed analyses of lipid composition confirmed the potential of purslane seed oil as a cheap and highly valuable source of phytonutrients as essential fatty acids and phytosterols, for application in food, pharmaceutical and cosmetic industries. phytonutrients purslane Portulaca oleracea lipid classes fatty acids phytosterols extraction oxidative stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Purslane ( Portulaca oleracea L.) is a succulent annual plant widespread all over the world, mostly in tropical and subtropical zones [ 1 ]. Although considered a weed it has been used in traditional medicine since ancient times as a remedy for skin inflammations, liver, kidney, stomach, respiratory and other problems [ 1 – 3 ]. Nowadays there are scientific evidences also for its beneficial effects on people with diabetes and high blood cholesterol levels, even as an anti-cancer remedy [ 4 – 7 ]. Besides as a medicinal plant, purslane is very popular and consumed as fresh salad or cooking vegetable [ 1 , 8 ]. Along with the leaves and stems, its seeds attract more and more attention as well because of the specific fatty acids composition of seed oil, namely quite high (> 30%) content of essential omega-3 18:3 (alpha-linolenic) fatty acid [ 9 ]. Thus, the purslane seed oil reveals its potential for use as a food supplement rich in valuable phytonutrients. In addition to essential fatty acids, phy-tosterols are highly desired components of the healthy diet, too [ 5 ]. However, while the fatty acids composition of purslane seed oil has been studied by many researchers, infor-mation in literature on sterols is scarce. In addition, there is no data about individual lipid classes of seed oil in order to assess comprehensively its value for food, pharmaceutical and cosmetic industries. So, the aim of this work was to characterize purslane seed oil regarding its individual lipid classes and their fatty acids composition, as well as to estimate the oil oxidative stability, depending on the extraction method. For the purpose, four of the most used methods of extraction were applied: three classical procedures with organic solvents ( n -hexane and two chloroform-methanol mixtures) and extraction with super-critical carbon dioxide (SC-CO 2 ) which is increasingly preferred in recent years as non-toxic, non-corrosive, non-flammable and cheap solvent. Yields of oil from purslane seeds extracted by these methods were compared as well in addition to estimation of their effect on the purslane oil lipid composition. 2 Materials and Methods 2.1 Samples and reagents The purslane ( Portulaca oleracea L.) was collected in June-August 2018 in full flowering stage in Novo Zhelezare village (42°25'09.2"N/24°39'53.8"E), Plovdiv region, Bulgaria. The plant material was identified by Dr. Ina Aneva (Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences). Voucher specimen CO 1437 has been deposited at the Herbarium of the Institute of Biodiversity and Ecosystem Research (SOM), Bulgarian Academy of Sciences, Bulgaria. The purslane seeds were dried at room temperature in shadow and then portions of them were subjected to respective extraction method. Solvents of HPLC grade were used for extraction and preparative thin layer chromatography (TLC), whereas the reagents for hydrolysis and methylation were of analytical grade (Merck, Sigma-Aldrich). Carbon dioxide for super-critical extraction was delivered by Messer, Sofia, Bulgaria. Reference fatty acids methyl esters (FAME) mixture, individual lipid classes (monoacylglycerols, diacylglycerols, triacylglycerols, sterols, free fatty acid, sterol ester) and a reference mixture of hydrocarbons (C7-C40) were from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.2 Extraction of oil and determination of fat content 2.2.1 Hexane extraction Portions of about 30 g (precisely weighed) milled purslane seeds were extracted with n-hexane in Soxhlet apparatus for 8 h [ 25 ] and then the solvent was distilled in rotary evaporator. The residue was weighted to calculate the fat content by the Eq. (1): Fat % = (m oil / m seeds ) x 100 (Eq. 1) where m was the mass [g], respectively of the residue (oil) and the initial sample (purslane seeds). Five extractions were performed to collect oil for further investigations. The oil samples were kept at -20 o C until analyses. 2.2.2 Chloroform-methanol (2:1) extraction The procedure of Folch et al. [ 26 ] was used as follows: 10 g (precisely weighed) purslane seeds were milled and homogenized with 50 ml methanol in an Erlenmeyer flask with magnetic stirrer for 30 min. Then 100 ml chloroform were added for subsequent one hour homogenization, followed by addition of 50 ml chloroform-methanol mixture (2:1, v/v) for another 90 minutes homogenization. Finally, the sample was filtered through a Buechner funnel and the precipitate was washed with another portion of 50 ml chloroform-methanol mixture (2:1, v/v). The filtrates were combined and transferred to a separatory funnel after measuring of their volume by a graduated glass cylinder. One fourth of the total volume of 0.88% aqueous KCl solution was added, the mixture was vigorously shaken and allowed to settle. The upper (aqueous) layer was discarded and the bottom (lipid) layer was washed again with one fourth of its volume of mixture of 0.88% aqueous KCl solution-methanol (1:1, v/v). After separation in a funnel the bottom layer was filtered and the solvent was evaporated on a rotary vacuum evaporator. The yield of the extract was determined gravimetrically and calculated by Eq. (1) as explained in Section 2.2.1 . Five extractions were performed to collect oil for further investigations. The oil samples were kept at -20 o C until analyses. 2.2.3 Chloroform-methanol (1:2) extraction The procedure of Bligh & Dyer [ 27 ] was used as follows: portion of 10 g (precisely weighed) milled purslane seeds were homogenized with 200 ml chloroform-methanol mixture (1:2, v/v) in an Erlenmeyer flask with magnetic stirrer for 3 hours. Then the sample was filtered and the precipitate was washed with 50 ml chloroform. The filtrates were combined, their total volume was measured by a graduated glass cylinder and were transferred to a separatory funnel. One fourth of the total volume of 0.88% aqueous KCl solution was added, the mixture was vigorously shaken and allowed to settle. Then the bottom (lipid) layer was collected and the solvent was evaporated on a rotary vacuum evaporator. The yield of the extract was determined gravimetrically and calculated by Eq. (1) as explained in Section 2.2.1 . Five extractions were performed to collect oil for further investigations. The oil samples were kept at -20 o C until analyses. 2.2.4 Super-critical CO2 extraction About 5 g (accurately weighed) milled purslane seeds were subjected to SC-CO 2 extraction using a SFT-110-XW apparatus (Supercritical Fluid Technologies Inc., Newark, DE, USA) with two 50 ml extractors equipped with a temperature controller allowed temperature up to 200℃. The SFT Nex10 SCF piston pump and HYAC50-25 compressor (Hyundai, Seoul, Republic of Korea) provided pressure of maximum 60 MPa. The carbon dioxide flow rate at the extractor outlet was measured with an Alicat Scientific (Tucson, AZ, USA) flow meter, model M-5SLPM-D/5M [ 28 ]. The extractions were carried out with pure CO 2 at 50℃ and pressure of 35 MPa [ 14 ] with an initial 20 min static and then 90 min dynamic time (continuous passage of SC-CO 2 through the extractor). The resulting extract was collected at atmospheric pressure in a vial placed in ice. The oil yield was calculated according to Eq. (1). Five SC-CO 2 extractions were performed to collect oil for further investigations and it was kept at -20 o C until analyses. 2.3 Peroxide value, conjugated Dienes and Trienes, and Oxidative stability index Peroxide value, expressed as meq active О 2 /kg oil, was determined by the modified iodometric method of Yanishlieva et al. [ 29 ] based on titration with Na 2 S 2 O 3 . The contents of conjugated dienes and trienes were measured in 1 cm cuvettes by their absorbances at 232 nm and 270 nm, respectively, of 1% oil solutions in iso-octane using Cecil Series 8000 UV/VIS spectrophotometer with reference of pure solvent [ 30 ]. The results were presented as K 232 and K 270 , respectively. Oxidative stability index as a measure of oxidative stability of oils was determined by Metrohm Professional Rancimat apparatus (Model 892, Switzerland) on 3 g oil samples at 80 o C, 100 o C, 110 o C and 120 o C, with air flow at 20 L/h. 2.4 Lipid classes About 100 mg (accurately weighed) purslane seed oil were applied as hexane solution on a silica gel preparative TLC glass plate (20 x 20 cm). After development with a mobile phase of hexane-acetone (100:8, v/v) the separated zones of individual lipid classes were visualized under UV light (366 nm) by spraying with 2,7-dichlorofluoresceine (ethanolic solution). Identification of each zone was according to reference mixture of lipid classes applied alongside on the plate. After this development each separated zone of wax esters + hydrocarbons, sterol esters, triacylglycerols and the band below them (called zone X) was scraped from the plate, transferred to small glass column and eluted with diethyl ether. After its evaporation under gentle stream of nitrogen, the residue was weighed for determination of lipid class yield (equivalently to Eq. (1)). Then sterol esters were subjected to hydrolysis and methylation [ 31 ] for subsequent determination, respectively, of sterols and fatty acids composition. Triacylglycerols, unidentified lipid zone and a part of zone wax esters + hydrocarbons were transmethylated [ 31 ] for analysis of their fatty acids composition. On the other hand, the whole combined zone of free sterols, free fatty acids, diacylglycerols, monoacylglycerols and polar lipids was scraped from the plate, eluted with diethyl ether and subjected to second development with a mobile phase of hexane-diethyl ether-acetic acid (70:30:1, v/v/v) for better separation of these lipid classes. Except for triacylglycerols the other classes were in minor quantities (below 5%) therefore 10–12 preparative TLC plates were used to collect sufficient amounts for their further analysis including yield evaluation. Free fatty acids, mono- and di-acylglycerols were methylated for further analysis of their fatty acids composition whereas free sterols were analyzed for determination of sterol profile. 2.5 Fatty acids composition Gas chromatography (GC) on methyl esters was used for determination of fatty acids composition. Prior to analysis, the sample (oil or respective individual lipid class as explained in Section 2.4 .) was (trans)methylated by 1% H 2 SO 4 in methanol to fatty acids methyl esters [ 31 ]. After their purification by preparative TLC on silica gel plate with a mobile phase of hexane-acetone (100:6 v/v), FAME were injected as hexane solution in Shimadzu Nexis GC 2030 chromatograph equipped with a flame ionization detector and Simplicity Wax capillary column (30 m × 0.32 mm × 0.25 µm, Supelco). A temperature program from 170°C to 260°C at 2°C/min and 5 min hold at final temperature was applied. The injector and detector temperatures were 260°C and 280°C, respectively. Nitrogen was used as a carrier gas at a flow rate of 1.0 mL/min; split 1:50. The peaks identification was according to retention times compared to that of FAME in a reference mixture. Amounts of fatty acids were calculated by corresponding peak areas and presented as rel. %. Measurements were done in triplicate. 2.6 Sterols composition Composition of sterols in both classes Free sterols and Sterol esters (after hydrolysis as explained in section 2.4 ) was determined by Shimadzu GC Nexis 2030 chromatograph equipped with a flame-ionization detector and TRB-5 capillary column (30 m × 0.25 mm × 0.25 µm, Teknokroma). Identification of sterols was carried out using GC-MS equipment Shimadzu QP 2020 with Sapiens-5MS column (30 m x 0.25 mm x 0.25 µm) operated from 85°C to 150°C at 15°C/min, then to 320°C at 4°C/min and hold at this temperature for 20 min; split 1:10; injector and detector temperature 280°C and 320°C, respectively; carrier gas helium with a flow rate of 0.8 ml/min. Mass spectral library NIST 2017 was used to identify individual peaks. 2.7 Hydrocarbons and wax esters Hydrocarbons and wax esters were isolated by preparative TLC as one zone as described in Section 2.4 . A part of this zone was analyzed by GC for determination of hydrocarbons and wax esters, and the other part was used for analysis of fatty acids composition of wax esters as described in Section 2.5 . Identification of hydrocarbons and wax esters was done by GC-MS equipment Shimadzu QP 2020 with Sapiens-5MS column (30 m x 0.25 mm x 0.25 µm) operated from 65°C to 320°C at 5°C/min and hold at this temperature for 20 min; split 1:10; injector and detector temperature 280°C and 320°C, respectively; carrier gas helium with a flow rate of 0.8 ml/min. NIST 2017 was used as Mass spectral library, additionally to a standard mixture of hydrocarbons (C7-C40). Quantification was carried out on Shimadzu GC Nexis 2030 chromatograph equipped with a flame-ionization detector and TRB-5 capillary column (30 m × 0.25 mm× 0.25 µm, Teknokroma) under the same chromatographic conditions. 2.8 Statistical analysis Measurements were performed in triplicate. Results are presented as a mean value of 3 parallel determinations ± standard deviation and have been compared by t-test (MS Excel 2010 software). 3 Results and Discussion 3.1 Fat content and oxidative stability of oil Extraction of purslane seeds with different solvents produced dark brownish green oil without obvious difference in appearance (Fig. 1 S). Data about oil yield depending on the extraction method are presented in Table 1 . As could be seen the lowest value for fat content was obtained by SC-CO 2 extraction (15%) while both chloroform-methanol mixtures ensured about 22% and the procedure with hexane in Soxhlet apparatus provided 17% oil. Predominant literature data about fat content of purslane seeds concern Soxhlet extraction by hexane resulting in oil yield from 8–17% [ 10 – 15 ]. A single publication reports oil yield after SC-CO 2 extraction [ 14 ] but much lower (7.5%) than the present result (14.8%, Table 1 ). No information about yield by chloroform-methanol mixtures was found to compare with results in Table 1 . Table 1 Fat content and initial oxidation quality (peroxide value, conjugated dienes and trienes) of purslane seed oil obtained by different extraction methods Purslane seeds Hexane SC-CO 2 Chloroform-Methanol (2:1) Chloroform-Methanol (1:2) Fat content (w/w %) 17.1 ± 1.2 a 14.8 ± 0.4 b 21.0 ± 1.0 c 23.0 ± 1.1 c Peroxide value (meqO 2 /kg) 0.8 ± 0.2 a 0.9 ± 0.2 a 0.7 ± 0.2 a 0.7 ± 0.1 a conj. Dienes (K 232 ) 2.2 ± 0.1 a 2.7 ± 0.2 b 2.1 ± 0.1 a 2.3 ± 0.1 a conj. Trienes (K 270 ) 0.60 ± 0.01 a 0.64 ± 0.01 b 0.59 ± 0.01 a 0.61 ± 0.01 a Values are mean of three determinations ± standard deviation. Different letters within each row indicate statistically significant difference (p < 0.05). Freshly extracted oil samples were subjected to evaluation of their quality concerning the oxidative stability expressed by the peroxide value (PV) and amount of conjugated dienes and trienes (presented by K 232 and K 270 ) as measures of initial products of lipid oxidation (Table 1 ). The results revealed that peroxide value was not affected by the extraction method and was within the limits of freshly produced seed oil. Very similar values (PV of 0.91–0.98 meqO 2 /kg) were published by other authors for hexane- and SC-CO 2 purslane seed extractions [ 14 , 16 ]. As for conjugated dienes and trienes, the results in Table 1 showed slightly higher values for K 232 and K 270 of oil extracted by SC-CO 2 compared to the procedures with organic solvents. Data in literature about that is controversial: K 232 and K 270 of hexane- and SC-CO 2 extracted oils has got practically the same but at levels of 0.2 [ 14 ] or 2.1–2.5 [ 16 ] for K 232 and of 0.03–0.04 [ 14 ] vs. 0.6–0.7 [ 16 ] for K 270 , the latter values [ 16 ] being very similar to present. In addition to PV and conjugated dienes and trienes, oxida-tive stability index (OSI) was estimated as a measure of secondary and subsequent oxida-tion products, and OSI was determined at four temperatures for hexane extracted oil (Table 2 ). As could be seen, the oxidative stability gradually decreased with increasing of tem-perature, and the OSI values were common for highly unsaturated oil. Only two papers were found in literature with OSI data for purslane oil [ 15 , 16 ]. In the first work [ 15 ] two extraction methods were applied: by hexane at room temperature for 36 h, and mechanical pressing with and without preliminary microwave irradiation of the seeds, resulting in OSI values (110 o C, 20 L/h air flow), respectively, of 5.12h, 9.7h and 4.6h. The second work [ 16 ] used SC-CO 2 extraction (at 50℃, pressure of 35 MPa, 210 min dynamic time), and hexane extraction at room temperature for 36 h. The OSI determined at 110 o C and 20 L/h air flow were, respectively, 12.5h and 11.5h. The differences in oxidative stability could be ex-plained by the effect of antioxidants which amount depended on the extraction method. On the other hand, fatty acids composition of oil had a major effect on oxidative stability as well. For that reasons comparison of OSI values of oils produced at different conditions with initially different fatty acids composition and antioxidants is only relative and if could be concluded, the results in Table 2 are generally similar to data presented by Delfan-Hosseini et al. [ 15 ]. Table 2 Oxidative stability index at different temperatures of purslane seed oil obtained by hexane extraction Purslane seed oil 80℃ 100℃ 110℃ 120℃ Oxidative Stability Index (h) 42.7 ± 0.3 a 8.3 ± 0.3 b 4.1 ± 0.2 c 2.3 ± 0.1 d Values are mean of three determinations ± standard deviation. Different letters within each row indicate statistically significant difference (p < 0.05). 3.2 Lipid classes Only one publication has been found in literature to demonstrate some separation of lipid groups during fractionation of oil from purslane leaves and seeds to neutral lipids, glycolipids and phospholipids [ 17 ]. To the best of our knowledge the present work reports for the first time detailed results about the individual neutral lipid classes and their amounts, namely: monoacylglycerols (MAG), diacylglycerols (DAG), free fatty acids (FFA), free sterols (St), some unidentified compounds with slightly lower polarity than that of sterols, triacylglycerols (TAG), sterol esters (StE), hydrocarbons and wax esters (Fig. 1 ). As in typical seed oil TAGs were the main component (> 90%) of the oil whereas the other classes were far less (Table 3 ). Traces of polar lipids were observed faintly on plate start but impossible for quantitation. No effect of extraction procedure on lipid classes proportions was observed. Table 3 Lipid classes in purslane seed oil obtained by different extraction methods Lipid class Hexane SC-CO 2 Chloroform-methanol (2:1) Chloroform-methanol (1:2) MAG 0.2 ± 0.05 0.2 ± 0.05 0.3 ± 0.05 0.3 ± 0.05 FFA 0.3 ± 0.05 0.3 ± 0.05 0.2 ± 0.05 0.2 ± 0.05 DAG 3.1 ± 0.3 2.8 ± 0.3 3.4 ± 0.4 3.0 ± 0.2 St 0.8 ± 0.2 1.2 ± 0.3 1.2 ± 0.2 0.9 ± 0.2 X 1.0 ± 0.2 1.1 ± 0.1 0.8 ± 0.2 1.1 ± 0.2 TAG 90.6 ± 0.8 90.3 ± 0.7 90.1 ± 0.8 90.2 ± 0.5 StE 2.4 ± 0.3 2.5 ± 0.3 2.8 ± 0.2 2.9 ± 0.3 Waxes 1.6 ± 0.3 1.6 ± 0.3 1.2 ± 0.2 1.4 ± 0.2 Values are mean of three determinations ± standard deviation. No statistically significant difference (p < 0.05) was found within each row. Abbreviations of lipid classes: MAG – monoacylglycerols, FFA – free fatty acids, DAG – diacylglycerols, St – free sterols, X – zone with unidentified compounds, TAG – triacylglycerols, StE – sterol esters, Waxes - hydrocarbons and wax esters. 3.3 Fatty acids composition of lipid classes Initially, the total fatty acids composition of purslane oil was determined and revealed quite high amount of the essential alpha-linolenic (18:3 ɷ-3) and linoleic (18:2 ɷ-6) fatty acids, respectively almost 40% and 33% (Table 4 ). Except for palmitic (16:0), oleic (9–18:1) and stearic (18:0) fatty acids about 12%, 10% and 4% respectively, the other fatty acids were near to or below 1%. Having in mind the similar amounts of linolenic and lin-oleic acids in purslane oil it could be expected that in some cases their proportion would be opposite, i.e. linoleic acid to be more than linolenic acid. Indeed half of the publications report predomination of linolenic acid [ 16 – 22 ] in contrast to the others [ 11 – 15 , 23 , 24 ]. In fact purslane oil with predominant alpha-linolenic acid possesses better bioactive potential and is preferable as healthier phytonutrient. No effect of extraction method on the total fatty acids composition was detected. Table 4 Total fatty acids composition of purslane seed oil obtained by different extraction methods Fatty acid, rel.% Hexane SC-CO 2 Chloroform-methanol (2:1) Chloroform-methanol (1:2) 14:0 0.1 ± 0.05 0.1 ± 0.05 0.1 ± 0.05 0.1 ± 0.05 16:0 11.7 ± 0.3 12.3 ± 0.6 12.1 ± 0.2 11.8 ± 0.3 16:1 0.1 ± 0.05 0.1 ± 0.05 0.1 ± 0.05 0.1 ± 0.05 18:0 3.6 ± 0.2 3.7 ± 0.2 3.8 ± 0.2 3.7 ± 0.1 9–18:1 9.9 ± 0.3 9.7 ± 0.2 9.5 ± 0.2 9.5 ± 0.1 11–18:1 1.1 ± 0.1 1.0 ± 0.1 1.1 ± 0.2 1.1 ± 0.1 18:2 33.2 ± 0.1 32.7 ± 0.4 32.8 ± 0.4 32.9 ± 0.3 18:3 38.2 ± 0.2 38.5 ± 0.3 38.1 ± 0.3 38.6 ± 0.4 20:0 0.9 ± 0.1 0.9 ± 0.2 0.9 ± 0.1 0.9 ± 0.1 20:1 0.1 ± 0.05 0.1 ± 0.05 0.2 ± 0.05 0.2 ± 0.05 22:0 0.6 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 0.6 ± 0.1 22:1 0.1 ± 0.05 0.1 ± 0.05 0.2 ± 0.05 0.1 ± 0.05 24:0 0.4 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 Values are mean of three determinations ± standard deviation. No statistically significant difference (p < 0.05) was found within each row. After estimation of total fatty acids composition, the fatty acids composition of each lipid class was determined. Results for the main four acids (alpha-linolenic, linoleic, palmitic and oleic acids) are presented on Fig. 2 , and for the others twelve (12:0, 14:0, 16:1, 18:0, 11–18:1, 20:0, 20:1, 22:0, 22:1, 24:0, 24:1 and 26:0) on Figure S2. It can be seen that dis-tribution of fatty acids among classes is different and specific but does not depend on the extraction method. Thus, alpha-linolenic acid (Fig. 2 a) predominates in triacylglycerols and the next X zone with amounts above 36%. The other lipid classes are relatively rich in it, too (> 23%), excepting wax esters (6.5%). Linoleic acid (Fig. 2 b) is at similar levels (> 24%), excluding wax esters (9.3%), reaching almost 42% and 48% in diacylglycerols and sterol esters, respectively. Palmitic acid (Fig. 2 c) has the opposite distribution with the highest amount in wax esters (42%) and levels in the range 11–22% in the other lipid clas-ses. Oleic acid (Fig. 2 d) gradually increases from 6–12% with increasing of lipid class polarity. It should be noted that seven of the eight saturated fatty acids measured in purslane oil, namely 12:0, 14:0, 16:0, 18:0, 22:0, 24:0 and 26:0 are concentrated in wax esters in amounts quite higher than in the other lipid classes, at levels of 42% for 16:0 (Fig. 2 c), 15% for 18:0 (Fig. 2 S a), in the range of 2–7% for 22:0 (Fig. 2 S b), 24:0 (Fig. 2 S c), 26:0 (Fig. 2 S d) and 14:0 (Fig. 2 S f), and 0.7% 12:0 (Fig. 2 S e). Similar predomination in wax esters can be seen also of 20:1 (Fig. 2 S j). On the other hand, 20:0 (Fig. 2 S i) and 11–18:1 (Fig. 2 S h) do not predominate in a single lipid class. The other fatty acids 16:1 (Fig. 2 S g), 22:1 (Fig. 2 S k) and 24:1 (Fig. 2 S l) have specific distribution as well despite their minor to traces amounts. Figure S3 contains diagrams with the same results for fatty acids composition of individual lipid classes but in different presentation to facilitate the comparison between lipid classes regarding their fatty acids composition. Thus, it is obvious that total fatty acids composition of the oil (Fig. 3 S a) is almost equal to that of triacylglycerols (Fig. 3 S b). This result is expected and confirms the content above 90% of triacylglycerols (Table 3 ). Monoacylglycerols (Fig. 3 S c) and free fatty acids (Fig. 3 S d) have similar fatty acids composition but different from that of diacylglycerols (Fig. 3 S e), unidentified lipid zone (Fig. 3 S f) and sterol esters (Fig. 3 S g). Wax esters (Fig. 3 S h) differ the most from the other lipid classes in the high content of saturated fatty acids as discussed above. To the best of our knowledge such detailed fatty acids composition of individual lipid classes in purslane oil is published for the first time. This information could be necessary and useful as base for further biochemical and physiological investigations and conclusions, and, on the other hand, for practical purposes in production of food supplements, pharmaceutical and cosmetic goods. 3.4 Sterols composition Sterols in both fractions of free and esterified sterols in purslane oil were determined. In the first fraction nine sterols were detected (Fig. 3 ) and sitosterol was much more (about 77%) than the others (below 10%). In the fraction of sterol esters thirteen sterols were measured (Fig. 4 ). However, in contrast to the free sterols, sterol esters contained far less sitosterol (about 48%) but much more campesterol and delta-5 avenasterol. In the single publication yet in literature with results about sterol composition of purslane oil (15 identified sterols but without data about sterol esters) beta-sitosterol predominated (53%) followed by campesterol (17%) and delta-5-avenasterol (11%). The other sterols were below 3% [ 12 ]. Similarly to the fatty acids composition, the present results revealed no effect of extraction method on sterol composition of purslane oil concerning both free sterols and sterol esters. 3.5 Waxes The most unpolar compounds in purslane seed oil were separated and isolated as lipid zone Waxes (Fig. 1 , Table 3 ) containing mainly hydrocarbons and minor amounts of wax esters. Concerning the former, twenty-five hydrocarbons with chain length from 14 to 37, as well as squalene, were identified and quantified (Fig. 5 ). Among them only two (C33 and C31) and squalene were more than 10%. As for wax esters, traces peaks were de-tected but with ambiguous identification. For that reason they are not discussed here and require additional investigation. No data from other authors have been found in literature for comparison. As with the other lipid classes no effect of extraction method on hydrocarbons was observed. 4 Conclusions Detailed analysis of purslane seed oil regarding its lipid composition including separation and quantitation of seven individual lipid classes (monoacylglycerols, diacylglycerols, free fatty acids, free sterols, triacylglycerols, sterol esters, wax esters + hydrocarbons) and determination of fatty acids composition of saponifiables was done for the first time, in order to study the effect of extraction method on lipids and oxidative stability of purslane seed oil extracted by three procedures with organic solvents (hexane, chloroform-methanol mixtures) vs. extraction with super-critical CO 2 . The results revealed no significant effect of extraction method on the individual lipid classes and their fatty acids composition, and quite weak effect on the oil oxidative stability regarding super-critical CO 2 vs. organic solvents. Analyses of lipid composition confirmed the potential of purslane seed oil as a cheap and highly valuable source of phytonutrients as essential ɷ-3 fatty acids and phytosterols, for application in food, pharmaceutical and cosmetic industries. Declarations Acknowledgments The support by the Operational Program “Science and Education for Smart Growth”, co-financed by the European Union through the European Structural and Investment Funds, grant BG05M2OP001-1.002-0012 Center of Competence “Sustainable utilization of bio-resources and waste of medicinal and aromatic plants for innovative bioactive products”, is gratefully acknowledged. Funding This research was funded by the Operational Program “Science and Education for Smart Growth”, co-financed by the European Union through the European Structural and Investment Funds, grant BG05M2OP001-1.002-0012 Center of Competence “Sustainable utilization of bio-resources and waste of medicinal and aromatic plants for innovative bioactive products”. Conflicts of interest The authors declare no conflicts of interest. Data availability All data supporting the findings of this study are available within the paper. Any raw data files are available from the corresponding author upon reasonable request. Code availability Not applicable. Authors' contributions Sabina Taneva: Investigation, Chemical Analyses, Acquisition of Data, Data Analysis and Interpretation; Svetlana Momchilova: Methodology, Data Analysis and Interpretation, Drafting of article and Editing. Ethics approval Experiments with the plant material adhere to international/national guidelines. Consent for publication Not applicable. References Srivastava R, Srivastava, V. Multipurpose Benefits of an Underexplored Species Purslane (Portulaca oleracea L.): A Critical Review. Environm Manag 2021; https://doi.org/10.1007/s00267-021-01456-z Montoya-García CO, García-Mateos R. Bioactive compounds of purslane (Portulaca oleracea L.) according to the production system: A review. Sci Hortic 2023; https://doi.org/10.1016/j.scienta.2022.111584 Uddin K, Juraimi A. Purslane Weed (Portulaca oleracea): A Prospective Plant Source of Nutrition, Omega-3 Fatty Acid, and Antioxidant Attributes. Sci World J 2014; https://doi.org/10.1155/2014/951019 Arshad Z, Rezapour-Firouzi S. The Sources of Essential Fatty Acids for Allergic and Cancer Patients; a Connection with Insight into Mammalian Target of Rapamycin: A Narrative Review. Asian Pac J Cancer Prev 2018; https://doi.org/10.22034/APJCP.2018.19.9.2391 Li K, Xia T. A review on ethnopharmacology, phytochemistry, pharmacology and potential uses of Portulaca oleracea L. J Ethnopharm 2024; https://doi.org/10.1016/j.jep.2023.117211 Saad B, Kmail A, Haq SZH. Anti-Diabesity Middle Eastern Medicinal Plants and Their Action Mechanisms. Evid-Based Complem Altern Med 2022; https://doi.org/10.1155/2022/2276094 Naeem F, Khan S. Purslane (Portulaca oleracea L.) as Phytogenic Substance - A Review. J Herbs Spices Med Plants 2013; https://doi.org/10.1080/10496475.2013.782381 Petropoulos S, Karkanis A. Phytochemical composition and bioactive compounds of common purslane (Portulaca oleracea L.) as affected by crop management practices. Trends Food Sci Technol 2016; https://doi.org/10.1016/j.tifs.2016.06.010 Zhou Y-X, Xin H-L, Rahman K, Wang S-J, Peng C, Zhang H. Portulaca oleracea L.: A Review of Phytochemistry and Pharmacological Effects. BioMed Res Intern 2015; https://doi.org/10.1155/2015/925631 Desta M, Molla A, Yusuf Z. Characterization of physico-chemical properties and antioxidant activity of oil from seed, leaf and stem of purslane (Portulaca oleracea L.). Biotech Rep 2020; https://doi.org/10.1016/j.btre.2020.e00512 Gunenc A, Rowland O, Xu H, Marangoni A, Hosseinian F. Portulaca oleracea seeds as a novel source of alkylresorcinols and its phenolic profiles during germination. LWT-Food Sci Technol 2019; https://doi.org/10.1016/j.lwt.2018.10.075 Matthäus B, Babiker EE, Özcan MM, Al-Juhaimi FY, Ahmed IAM, Ghafoor K. Changes in Fatty Acid, Tocopherol and Sterol Contents of Oils Extracted from Several Vegetable Seeds. J Oleo Sci 2021; http://doi.org/10.5650/jos.ess21225 Srivastava M, Banerji R, Rawat AKS, Mehrotra S. Fatty Acid Composition of Some Medicinally Useful Seeds. J Herb Pharmacother 2006; http://doi.org/10.1300/J157v06n01_04 Zadeh JH, Özdikicierler O, Pazı F. A Comparative Study on Response Surface Optimization of Supercritical Fluid Extraction Parameters to Obtain Portulaca Oleracea Seed Oil with Higher Bioactive Content and Antioxidant Activity Than Solvent Extraction. Eur J Lipid Sci Technol 2022; http://doi.org/10.1002/ejlt.202200136 Delfan-Hosseini S, Nayebzadeh K, Mirmoghtadaie L, Kavosi M, Hosseini SM. Effect of extraction process on composition, oxidative stability and rheological properties of purslane seed oil. Food Chem 2017; http://dx.doi.org/10.1016/j.foodchem.2016.11.150 Sodeifian G, Ardestani NS, Sajadian SA, Moghadamian K. Properties of Portulaca oleracea seed oil via supercritical fluid extraction: Experimental and optimization. J Supercr Fl 2018; https://doi.org/10.1016/j.supflu.2017.12.026 Venkateshwari V, Vijayakumar A, Vijayakumar AK, Prasanna Anjaneya Reddy L, Srinivasan M, Rajasekharan R. Leaf lipidome and transcriptome profiling of Portulaca oleracea: characterization of lysophosphatidylcholine acyltransferase. Planta 2018; https://doi.org/10.1007/s00425-018-2908-8 Abozed SS, Elaraby GM, Zahran HA. Application of Spray-dried Microcapsules of Purslane (Portulaca oleracea L.) Seed Oil Enhances Quality of Mango Juice. Open Agric J 2021; https://doi.org/10.2174/1874331502115010001 El-Sayed M-I K. Effects of Portulaca oleracea L. seeds in treatment of type-2 diabetes mellitus patients as adjunctive and alternative therapy. J Ethnopharmacol 2011; https://doi.org/10.1016/j.jep.2011.06.020 Liu L, Howe P, Zhou Y-F, Xu Z-Q, Hocart C, Zhang R. Fatty acids and beta-carotene in Australian purslane (Portulaca oleracea) varieties. J Chromatogr A 2000; https://doi.org/10.1016/S0021-9673(00)00747-0 Petropoulos SA, Fernandes A, Arampatzis DA, Tsiropoulos NG, Petrović J, Soković M, Barros L, Ferreira ICFR. Seed oil and seed oil byproducts of common purslane (Portulaca oleracea L.): A new insight to plant-based sources rich in omega-3 fatty acids. LWT-Food Sci Technol 2020; https://doi.org/10.1016/j.lwt.2020.109099 Petropoulos SA, Fernandes A, Calhelha RC, Rouphael Y, Petrović J, Soković M, Ferreira ICFR, Barros L. Antimicrobial Properties, Cytotoxic Effects, and Fatty Acids Composition of Vegetable Oils from Purslane, Linseed, Luffa, and Pumpkin Seeds. Appl Sci 2021; https://doi.org/10.3390/app11125738 Mousavi SRJ, Niazmand R. Fatty Acids Composition and Oxidation Kinetic Parameters of Purslane (Portulaca oleracea) Seed Oil. Agric Res 2017; https://doi.org/10.1007/s40003-017-0271-9 Stroescu M, Stoica-Guzun A, Ghergu S, Chira N, Jipa I. Optimization of fatty acids extraction from Portulaca oleracea seed using response surface methodology. Ind Crops Prod 2013; https://doi.org/10.1016/j.indcrop.2012.07.051 ISO 659:2009, Oilseeds – Determination of oil content (Reference method), p. 12 (2009). Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; https://doi.org/10.1016/S0021-9258(18)64849-5 Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; https://doi.org/10.1139/o59-099 Boyadzhieva S, Coelho JAP. Errico M, Reynel-Avilla HE, Yankov DS, Bonilla-Petriciolet A, Stateva RP. Assessment of Gnaphalium viscosum (Kunth) valorization prospects: Sustainable recovery of antioxidants by different tech-niques. Antioxidants 2022; https://doi.org/10.3390/antiox11122495 Yanishlieva N, Popov A, Marinova E. Eine Modifizierte Jodometrische Methode zur Bestimmung der Peroxidzahl in kleinen Lipidproben. Copt Rend Acad Bulg Sci 1978;31:869-871. AOCS Official method Ch 5-91. Specific Extinction of Oils and Fats, Ultraviolet Absorption. In: Official methods and rec-ommended practices of the AOCS. Urbana, IL: AOCS Press. Christie WW. Lipid analysis: isolation, separation, identification, and structural analysis of lipids. 3rd ed. The Oily Press: Bridgwater, England, 2003; pp. 205-224. Additional Declarations No competing interests reported. Supplementary Files SupplementaryPurslaneoil.pdf Supplementary Materials: FigS1 Purslane seed oil extracted by different methods: 1 – Soxhlet extraction with hexane; 2 – Super critical CO 2 extraction; 3 – extraction with chloroform-methanol (2:1, v/v); 4 - extraction with chloroform-methanol (1:2, v/v); Fig S2 Content of fatty acids in individual lipid classes in purslane seed oil obtained by different extraction methods: (a) 18:0; (b) 22:0; (c) 24:0; (d) 26:0; (e) 12:0; (f) 14:0; (g) 16:1; (h) 11-18:1; (i) 20:0; (j) 20:1; (k) 22:1; (l) 24:1. Abbreviations: MAG – monoacylglycerols, FFA – free fatty acids, DAG – diacylglycerols, X – minor zone with unidentified compounds, TAG – triacylglycerols, StE – sterol esters, WE – wax esters; Fig S3 Content of fatty acids in individual lipid classes in purslane seed oil obtained by different extraction methods: (a) total lipids; (b) triacylglycerols; (c) monoacylglycerols; (d) free fatty acids; (e) diacylglycerols; (f) zone X with unidentified compounds; (g) sterol esters; (h) wax esters. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Aug, 2024 Reviews received at journal 07 Aug, 2024 Reviews received at journal 04 Aug, 2024 Reviewers agreed at journal 03 Aug, 2024 Reviewers agreed at journal 02 Aug, 2024 Reviewers invited by journal 31 Jul, 2024 Editor assigned by journal 25 Jul, 2024 Submission checks completed at journal 24 Jul, 2024 First submitted to journal 15 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4743339","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":337328952,"identity":"18badac2-d61d-47b0-86da-98f3e37cd507","order_by":0,"name":"Sabina Taneva","email":"","orcid":"","institution":"Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sabina","middleName":"","lastName":"Taneva","suffix":""},{"id":337328953,"identity":"7257b351-ec43-421b-94f8-2c0246e65793","order_by":1,"name":"Svetlana Momchilova","email":"data:image/png;base64,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","orcid":"","institution":"Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Svetlana","middleName":"","lastName":"Momchilova","suffix":""}],"badges":[],"createdAt":"2024-07-15 13:50:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4743339/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4743339/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62868247,"identity":"2e0a1e1a-b987-4027-958f-37c235b6cde2","added_by":"auto","created_at":"2024-08-20 12:15:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150098,"visible":true,"origin":"","legend":"\u003cp\u003eLipid classes in purslane seed oil separated on preparative silica gel TLC plate by mobile phase of hexane-acetone (100:8, v/v); UV visualization at 366 nm after spraying with 2,7-dichlorofluoresceine ethanolic solution. Abbreviations: MAG – monoacylglycerols, FFA – free fatty acids, DAG – diacylglycerols, St – free sterols, X – zone with unidentified compounds, TAG – triacylglycerols, StE – sterol esters, Waxes - hydrocarbons and wax esters.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4743339/v1/a742a4f9f46f48b7ed31224a.png"},{"id":62867865,"identity":"a09cc3d0-1560-4a01-8b51-5175c13344c5","added_by":"auto","created_at":"2024-08-20 12:07:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103456,"visible":true,"origin":"","legend":"\u003cp\u003eContent of the main fatty acids in individual lipid classes in purslane seed oil obtained by different extraction methods: (a) Linolenic acid (9,12,15-18:3); (b) Linoleic acid (9,12-18:2); (c) Palmitic acid (16:0); (d) Oleic acid (9-18:1). Content of the other fatty acids is given in Figure S2 (a-l). Abbreviations: MAG – monoacylglycerols, FFA – free fatty acids, DAG – diacylglycerols, X – minor zone with unidentified compounds, TAG – triacylglycerols, StE – sterol esters, WE - wax esters.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4743339/v1/ad5096d4f25a423e4a9c497d.png"},{"id":62867867,"identity":"3098d09d-8b60-4a9b-b35a-c5bacd94ed9a","added_by":"auto","created_at":"2024-08-20 12:07:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74610,"visible":true,"origin":"","legend":"\u003cp\u003eFree sterols in purslane seed oil obtained by different extraction methods\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4743339/v1/522ade065232d6cce1729d53.png"},{"id":62867862,"identity":"7c3d3100-e043-4aca-870d-e9fdc92b2272","added_by":"auto","created_at":"2024-08-20 12:07:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":182594,"visible":true,"origin":"","legend":"\u003cp\u003eSterols of sterol esters in purslane seed oil obtained by different extraction methods\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4743339/v1/2f24e1e08c291ba2ee345d42.png"},{"id":62868248,"identity":"e0307d6e-9d64-4ddb-9632-b1d5b849f036","added_by":"auto","created_at":"2024-08-20 12:15:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":48613,"visible":true,"origin":"","legend":"\u003cp\u003eHydrocarbons in purslane seed oil obtained by different extraction methods\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4743339/v1/f694b73226cb072ae866662d.png"},{"id":62868269,"identity":"b478cfae-9bb7-44d0-b3f8-f7befab87169","added_by":"auto","created_at":"2024-08-20 12:16:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1304404,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4743339/v1/19e74c5d-abad-4cc4-aa6a-c8f9c3255f3d.pdf"},{"id":62868249,"identity":"46358782-1974-4cbb-96ca-1eacbaf53a7a","added_by":"auto","created_at":"2024-08-20 12:15:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":493814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u003c/strong\u003e \u003cstrong\u003eFigS1\u003c/strong\u003e Purslane seed oil extracted by different methods: 1 – Soxhlet extraction with hexane; 2 – Super critical CO\u003csub\u003e2\u003c/sub\u003e extraction; 3 – extraction with chloroform-methanol (2:1, v/v); 4 - extraction with chloroform-methanol (1:2, v/v); \u003cstrong\u003eFig S2\u003c/strong\u003e Content of fatty acids in individual lipid classes in purslane seed oil obtained by different extraction methods: (a) 18:0; (b) 22:0; (c) 24:0; (d) 26:0; (e) 12:0; (f) 14:0; (g) 16:1; (h) 11-18:1; (i) 20:0; (j) 20:1; (k) 22:1; (l) 24:1. Abbreviations: MAG – monoacylglycerols, FFA – free fatty acids, DAG – diacylglycerols, X – minor zone with unidentified compounds, TAG – triacylglycerols, StE – sterol esters, WE – wax esters; \u003cstrong\u003eFig S3\u003c/strong\u003e Content of fatty acids in individual lipid classes in purslane seed oil obtained by different extraction methods: (a) total lipids; (b) triacylglycerols; (c) monoacylglycerols; (d) free fatty acids; (e) diacylglycerols; (f) zone X with unidentified compounds; (g) sterol esters; (h) wax esters.\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaryPurslaneoil.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4743339/v1/def9ac5bdbeccb10ef1a9bb9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Purslane (Portulaca oleracea L.) seed oil as a valuable source of important phytonutrients: effect of the extraction method on the lipid composition","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePurslane (\u003cem\u003ePortulaca oleracea\u003c/em\u003e L.) is a succulent annual plant widespread all over the world, mostly in tropical and subtropical zones [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although considered a weed it has been used in traditional medicine since ancient times as a remedy for skin inflammations, liver, kidney, stomach, respiratory and other problems [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nowadays there are scientific evidences also for its beneficial effects on people with diabetes and high blood cholesterol levels, even as an anti-cancer remedy [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Besides as a medicinal plant, purslane is very popular and consumed as fresh salad or cooking vegetable [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Along with the leaves and stems, its seeds attract more and more attention as well because of the specific fatty acids composition of seed oil, namely quite high (\u0026gt;\u0026thinsp;30%) content of essential omega-3 18:3 (alpha-linolenic) fatty acid [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, the purslane seed oil reveals its potential for use as a food supplement rich in valuable phytonutrients. In addition to essential fatty acids, phy-tosterols are highly desired components of the healthy diet, too [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, while the fatty acids composition of purslane seed oil has been studied by many researchers, infor-mation in literature on sterols is scarce. In addition, there is no data about individual lipid classes of seed oil in order to assess comprehensively its value for food, pharmaceutical and cosmetic industries.\u003c/p\u003e \u003cp\u003eSo, the aim of this work was to characterize purslane seed oil regarding its individual lipid classes and their fatty acids composition, as well as to estimate the oil oxidative stability, depending on the extraction method. For the purpose, four of the most used methods of extraction were applied: three classical procedures with organic solvents (\u003cem\u003en\u003c/em\u003e-hexane and two chloroform-methanol mixtures) and extraction with super-critical carbon dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e) which is increasingly preferred in recent years as non-toxic, non-corrosive, non-flammable and cheap solvent. Yields of oil from purslane seeds extracted by these methods were compared as well in addition to estimation of their effect on the purslane oil lipid composition.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Samples and reagents\u003c/h2\u003e \u003cp\u003eThe purslane (\u003cem\u003ePortulaca oleracea\u003c/em\u003e L.) was collected in June-August 2018 in full flowering stage in Novo Zhelezare village (42\u0026deg;25'09.2\"N/24\u0026deg;39'53.8\"E), Plovdiv region, Bulgaria. The plant material was identified by Dr. Ina Aneva (Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences). Voucher specimen CO 1437 has been deposited at the Herbarium of the Institute of Biodiversity and Ecosystem Research (SOM), Bulgarian Academy of Sciences, Bulgaria. The purslane seeds were dried at room temperature in shadow and then portions of them were subjected to respective extraction method. Solvents of HPLC grade were used for extraction and preparative thin layer chromatography (TLC), whereas the reagents for hydrolysis and methylation were of analytical grade (Merck, Sigma-Aldrich). Carbon dioxide for super-critical extraction was delivered by Messer, Sofia, Bulgaria. Reference fatty acids methyl esters (FAME) mixture, individual lipid classes (monoacylglycerols, diacylglycerols, triacylglycerols, sterols, free fatty acid, sterol ester) and a reference mixture of hydrocarbons (C7-C40) were from Sigma-Aldrich Co. (St. Louis, MO, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Extraction of oil and determination of fat content\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Hexane extraction\u003c/h2\u003e \u003cp\u003ePortions of about 30 g (precisely weighed) milled purslane seeds were extracted with n-hexane in Soxhlet apparatus for 8 h [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and then the solvent was distilled in rotary evaporator. The residue was weighted to calculate the fat content by the Eq.\u0026nbsp;(1):\u003c/p\u003e \u003cp\u003eFat % = (m\u003csub\u003eoil\u003c/sub\u003e / m\u003csub\u003eseeds\u003c/sub\u003e) x 100 (Eq.\u0026nbsp;1)\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003em\u003c/em\u003e was the mass [g], respectively of the residue (oil) and the initial sample (purslane seeds). Five extractions were performed to collect oil for further investigations. The oil samples were kept at -20\u003csup\u003eo\u003c/sup\u003eC until analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Chloroform-methanol (2:1) extraction\u003c/h2\u003e \u003cp\u003eThe procedure of Folch et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] was used as follows: 10 g (precisely weighed) purslane seeds were milled and homogenized with 50 ml methanol in an Erlenmeyer flask with magnetic stirrer for 30 min. Then 100 ml chloroform were added for subsequent one hour homogenization, followed by addition of 50 ml chloroform-methanol mixture (2:1, v/v) for another 90 minutes homogenization. Finally, the sample was filtered through a Buechner funnel and the precipitate was washed with another portion of 50 ml chloroform-methanol mixture (2:1, v/v). The filtrates were combined and transferred to a separatory funnel after measuring of their volume by a graduated glass cylinder. One fourth of the total volume of 0.88% aqueous KCl solution was added, the mixture was vigorously shaken and allowed to settle. The upper (aqueous) layer was discarded and the bottom (lipid) layer was washed again with one fourth of its volume of mixture of 0.88% aqueous KCl solution-methanol (1:1, v/v). After separation in a funnel the bottom layer was filtered and the solvent was evaporated on a rotary vacuum evaporator. The yield of the extract was determined gravimetrically and calculated by Eq.\u0026nbsp;(1) as explained in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.2.1\u003c/span\u003e. Five extractions were performed to collect oil for further investigations. The oil samples were kept at -20\u003csup\u003eo\u003c/sup\u003eC until analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Chloroform-methanol (1:2) extraction\u003c/h2\u003e \u003cp\u003eThe procedure of Bligh \u0026amp; Dyer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] was used as follows: portion of 10 g (precisely weighed) milled purslane seeds were homogenized with 200 ml chloroform-methanol mixture (1:2, v/v) in an Erlenmeyer flask with magnetic stirrer for 3 hours. Then the sample was filtered and the precipitate was washed with 50 ml chloroform. The filtrates were combined, their total volume was measured by a graduated glass cylinder and were transferred to a separatory funnel. One fourth of the total volume of 0.88% aqueous KCl solution was added, the mixture was vigorously shaken and allowed to settle. Then the bottom (lipid) layer was collected and the solvent was evaporated on a rotary vacuum evaporator. The yield of the extract was determined gravimetrically and calculated by Eq.\u0026nbsp;(1) as explained in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.2.1\u003c/span\u003e. Five extractions were performed to collect oil for further investigations. The oil samples were kept at -20\u003csup\u003eo\u003c/sup\u003eC until analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Super-critical CO2 extraction\u003c/h2\u003e \u003cp\u003eAbout 5 g (accurately weighed) milled purslane seeds were subjected to SC-CO\u003csub\u003e2\u003c/sub\u003e extraction using a SFT-110-XW apparatus (Supercritical Fluid Technologies Inc., Newark, DE, USA) with two 50 ml extractors equipped with a temperature controller allowed temperature up to 200℃. The SFT Nex10 SCF piston pump and HYAC50-25 compressor (Hyundai, Seoul, Republic of Korea) provided pressure of maximum 60 MPa. The carbon dioxide flow rate at the extractor outlet was measured with an Alicat Scientific (Tucson, AZ, USA) flow meter, model M-5SLPM-D/5M [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The extractions were carried out with pure CO\u003csub\u003e2\u003c/sub\u003e at 50℃ and pressure of 35 MPa [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] with an initial 20 min static and then 90 min dynamic time (continuous passage of SC-CO\u003csub\u003e2\u003c/sub\u003e through the extractor). The resulting extract was collected at atmospheric pressure in a vial placed in ice. The oil yield was calculated according to Eq.\u0026nbsp;(1). Five SC-CO\u003csub\u003e2\u003c/sub\u003e extractions were performed to collect oil for further investigations and it was kept at -20\u003csup\u003eo\u003c/sup\u003eC until analyses.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Peroxide value, conjugated Dienes and Trienes, and Oxidative stability index\u003c/h2\u003e \u003cp\u003ePeroxide value, expressed as meq active О\u003csub\u003e2\u003c/sub\u003e/kg oil, was determined by the modified iodometric method of Yanishlieva et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] based on titration with Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e. The contents of conjugated dienes and trienes were measured in 1 cm cuvettes by their absorbances at 232 nm and 270 nm, respectively, of 1% oil solutions in iso-octane using Cecil Series 8000 UV/VIS spectrophotometer with reference of pure solvent [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The results were presented as K\u003csub\u003e232\u003c/sub\u003e and K\u003csub\u003e270\u003c/sub\u003e, respectively. Oxidative stability index as a measure of oxidative stability of oils was determined by Metrohm Professional Rancimat apparatus (Model 892, Switzerland) on 3 g oil samples at 80\u003csup\u003eo\u003c/sup\u003eC, 100\u003csup\u003eo\u003c/sup\u003eC, 110\u003csup\u003eo\u003c/sup\u003eC and 120\u003csup\u003eo\u003c/sup\u003eC, with air flow at 20 L/h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Lipid classes\u003c/h2\u003e \u003cp\u003eAbout 100 mg (accurately weighed) purslane seed oil were applied as hexane solution on a silica gel preparative TLC glass plate (20 x 20 cm). After development with a mobile phase of hexane-acetone (100:8, v/v) the separated zones of individual lipid classes were visualized under UV light (366 nm) by spraying with 2,7-dichlorofluoresceine (ethanolic solution). Identification of each zone was according to reference mixture of lipid classes applied alongside on the plate. After this development each separated zone of wax esters\u0026thinsp;+\u0026thinsp;hydrocarbons, sterol esters, triacylglycerols and the band below them (called zone X) was scraped from the plate, transferred to small glass column and eluted with diethyl ether. After its evaporation under gentle stream of nitrogen, the residue was weighed for determination of lipid class yield (equivalently to Eq.\u0026nbsp;(1)). Then sterol esters were subjected to hydrolysis and methylation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] for subsequent determination, respectively, of sterols and fatty acids composition. Triacylglycerols, unidentified lipid zone and a part of zone wax esters\u0026thinsp;+\u0026thinsp;hydrocarbons were transmethylated [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] for analysis of their fatty acids composition. On the other hand, the whole combined zone of free sterols, free fatty acids, diacylglycerols, monoacylglycerols and polar lipids was scraped from the plate, eluted with diethyl ether and subjected to second development with a mobile phase of hexane-diethyl ether-acetic acid (70:30:1, v/v/v) for better separation of these lipid classes. Except for triacylglycerols the other classes were in minor quantities (below 5%) therefore 10\u0026ndash;12 preparative TLC plates were used to collect sufficient amounts for their further analysis including yield evaluation. Free fatty acids, mono- and di-acylglycerols were methylated for further analysis of their fatty acids composition whereas free sterols were analyzed for determination of sterol profile.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fatty acids composition\u003c/h2\u003e \u003cp\u003eGas chromatography (GC) on methyl esters was used for determination of fatty acids composition. Prior to analysis, the sample (oil or respective individual lipid class as explained in Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e.) was (trans)methylated by 1% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in methanol to fatty acids methyl esters [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. After their purification by preparative TLC on silica gel plate with a mobile phase of hexane-acetone (100:6 v/v), FAME were injected as hexane solution in Shimadzu Nexis GC 2030 chromatograph equipped with a flame ionization detector and Simplicity Wax capillary column (30 m \u0026times; 0.32 mm \u0026times; 0.25 \u0026micro;m, Supelco). A temperature program from 170\u0026deg;C to 260\u0026deg;C at 2\u0026deg;C/min and 5 min hold at final temperature was applied. The injector and detector temperatures were 260\u0026deg;C and 280\u0026deg;C, respectively. Nitrogen was used as a carrier gas at a flow rate of 1.0 mL/min; split 1:50. The peaks identification was according to retention times compared to that of FAME in a reference mixture. Amounts of fatty acids were calculated by corresponding peak areas and presented as rel. %. Measurements were done in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sterols composition\u003c/h2\u003e \u003cp\u003eComposition of sterols in both classes Free sterols and Sterol esters (after hydrolysis as explained in section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e) was determined by Shimadzu GC Nexis 2030 chromatograph equipped with a flame-ionization detector and TRB-5 capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m, Teknokroma). Identification of sterols was carried out using GC-MS equipment Shimadzu QP 2020 with Sapiens-5MS column (30 m x 0.25 mm x 0.25 \u0026micro;m) operated from 85\u0026deg;C to 150\u0026deg;C at 15\u0026deg;C/min, then to 320\u0026deg;C at 4\u0026deg;C/min and hold at this temperature for 20 min; split 1:10; injector and detector temperature 280\u0026deg;C and 320\u0026deg;C, respectively; carrier gas helium with a flow rate of 0.8 ml/min. Mass spectral library NIST 2017 was used to identify individual peaks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Hydrocarbons and wax esters\u003c/h2\u003e \u003cp\u003eHydrocarbons and wax esters were isolated by preparative TLC as one zone as described in Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e. A part of this zone was analyzed by GC for determination of hydrocarbons and wax esters, and the other part was used for analysis of fatty acids composition of wax esters as described in Section \u003cspan refid=\"Sec11\" class=\"InternalRef\"\u003e2.5\u003c/span\u003e. Identification of hydrocarbons and wax esters was done by GC-MS equipment Shimadzu QP 2020 with Sapiens-5MS column (30 m x 0.25 mm x 0.25 \u0026micro;m) operated from 65\u0026deg;C to 320\u0026deg;C at 5\u0026deg;C/min and hold at this temperature for 20 min; split 1:10; injector and detector temperature 280\u0026deg;C and 320\u0026deg;C, respectively; carrier gas helium with a flow rate of 0.8 ml/min. NIST 2017 was used as Mass spectral library, additionally to a standard mixture of hydrocarbons (C7-C40). Quantification was carried out on Shimadzu GC Nexis 2030 chromatograph equipped with a flame-ionization detector and TRB-5 capillary column (30 m \u0026times; 0.25 mm\u0026times; 0.25 \u0026micro;m, Teknokroma) under the same chromatographic conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eMeasurements were performed in triplicate. Results are presented as a mean value of 3 parallel determinations\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and have been compared by t-test (MS Excel 2010 software).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Fat content and oxidative stability of oil\u003c/h2\u003e \u003cp\u003eExtraction of purslane seeds with different solvents produced dark brownish green oil without obvious difference in appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS). Data about oil yield depending on the extraction method are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As could be seen the lowest value for fat content was obtained by SC-CO\u003csub\u003e2\u003c/sub\u003e extraction (15%) while both chloroform-methanol mixtures ensured about 22% and the procedure with hexane in Soxhlet apparatus provided 17% oil. Predominant literature data about fat content of purslane seeds concern Soxhlet extraction by hexane resulting in oil yield from 8\u0026ndash;17% [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A single publication reports oil yield after SC-CO\u003csub\u003e2\u003c/sub\u003e extraction [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] but much lower (7.5%) than the present result (14.8%, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No information about yield by chloroform-methanol mixtures was found to compare with results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFat content and initial oxidation quality (peroxide value, conjugated dienes and trienes) of purslane seed oil obtained by different extraction methods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePurslane seeds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexane\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC-CO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroform-Methanol (2:1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroform-Methanol (1:2)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFat content (w/w %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeroxide value (meqO\u003csub\u003e2\u003c/sub\u003e/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003econj. Dienes (K\u003csub\u003e232\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003econj. Trienes (K\u003csub\u003e270\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean of three determinations\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters within each row indicate statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFreshly extracted oil samples were subjected to evaluation of their quality concerning the oxidative stability expressed by the peroxide value (PV) and amount of conjugated dienes and trienes (presented by K\u003csub\u003e232\u003c/sub\u003e and K\u003csub\u003e270\u003c/sub\u003e) as measures of initial products of lipid oxidation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results revealed that peroxide value was not affected by the extraction method and was within the limits of freshly produced seed oil. Very similar values (PV of 0.91\u0026ndash;0.98 meqO\u003csub\u003e2\u003c/sub\u003e/kg) were published by other authors for hexane- and SC-CO\u003csub\u003e2\u003c/sub\u003e purslane seed extractions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As for conjugated dienes and trienes, the results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed slightly higher values for K\u003csub\u003e232\u003c/sub\u003e and K\u003csub\u003e270\u003c/sub\u003e of oil extracted by SC-CO\u003csub\u003e2\u003c/sub\u003e compared to the procedures with organic solvents. Data in literature about that is controversial: K\u003csub\u003e232\u003c/sub\u003e and K\u003csub\u003e270\u003c/sub\u003e of hexane- and SC-CO\u003csub\u003e2\u003c/sub\u003e extracted oils has got practically the same but at levels of 0.2 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] or 2.1\u0026ndash;2.5 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] for K\u003csub\u003e232\u003c/sub\u003e and of 0.03\u0026ndash;0.04 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] vs. 0.6\u0026ndash;0.7 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] for K\u003csub\u003e270\u003c/sub\u003e, the latter values [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] being very similar to present. In addition to PV and conjugated dienes and trienes, oxida-tive stability index (OSI) was estimated as a measure of secondary and subsequent oxida-tion products, and OSI was determined at four temperatures for hexane extracted oil (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As could be seen, the oxidative stability gradually decreased with increasing of tem-perature, and the OSI values were common for highly unsaturated oil. Only two papers were found in literature with OSI data for purslane oil [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the first work [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] two extraction methods were applied: by hexane at room temperature for 36 h, and mechanical pressing with and without preliminary microwave irradiation of the seeds, resulting in OSI values (110\u003csup\u003eo\u003c/sup\u003eC, 20 L/h air flow), respectively, of 5.12h, 9.7h and 4.6h. The second work [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] used SC-CO\u003csub\u003e2\u003c/sub\u003e extraction (at 50℃, pressure of 35 MPa, 210 min dynamic time), and hexane extraction at room temperature for 36 h. The OSI determined at 110\u003csup\u003eo\u003c/sup\u003eC and 20 L/h air flow were, respectively, 12.5h and 11.5h. The differences in oxidative stability could be ex-plained by the effect of antioxidants which amount depended on the extraction method. On the other hand, fatty acids composition of oil had a major effect on oxidative stability as well. For that reasons comparison of OSI values of oils produced at different conditions with initially different fatty acids composition and antioxidants is only relative and if could be concluded, the results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e are generally similar to data presented by Delfan-Hosseini et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOxidative stability index at different temperatures of purslane seed oil obtained by hexane extraction\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePurslane seed oil\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80℃\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100℃\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e110℃\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e120℃\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidative Stability Index (h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean of three determinations\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters within each row indicate statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Lipid classes\u003c/h2\u003e \u003cp\u003eOnly one publication has been found in literature to demonstrate some separation of lipid groups during fractionation of oil from purslane leaves and seeds to neutral lipids, glycolipids and phospholipids [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To the best of our knowledge the present work reports for the first time detailed results about the individual neutral lipid classes and their amounts, namely: monoacylglycerols (MAG), diacylglycerols (DAG), free fatty acids (FFA), free sterols (St), some unidentified compounds with slightly lower polarity than that of sterols, triacylglycerols (TAG), sterol esters (StE), hydrocarbons and wax esters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As in typical seed oil TAGs were the main component (\u0026gt;\u0026thinsp;90%) of the oil whereas the other classes were far less (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Traces of polar lipids were observed faintly on plate start but impossible for quantitation. No effect of extraction procedure on lipid classes proportions was observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLipid classes in purslane seed oil obtained by different extraction methods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipid class\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexane\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC-CO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroform-methanol (2:1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroform-methanol (1:2)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e90.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e90.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e90.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e90.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWaxes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean of three determinations\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. No statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was found within each row. Abbreviations of lipid classes: MAG \u0026ndash; monoacylglycerols, FFA \u0026ndash; free fatty acids, DAG \u0026ndash; diacylglycerols, St \u0026ndash; free sterols, X \u0026ndash; zone with unidentified compounds, TAG \u0026ndash; triacylglycerols, StE \u0026ndash; sterol esters, Waxes - hydrocarbons and wax esters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Fatty acids composition of lipid classes\u003c/h2\u003e \u003cp\u003eInitially, the total fatty acids composition of purslane oil was determined and revealed quite high amount of the essential alpha-linolenic (18:3 ɷ-3) and linoleic (18:2 ɷ-6) fatty acids, respectively almost 40% and 33% (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Except for palmitic (16:0), oleic (9\u0026ndash;18:1) and stearic (18:0) fatty acids about 12%, 10% and 4% respectively, the other fatty acids were near to or below 1%. Having in mind the similar amounts of linolenic and lin-oleic acids in purslane oil it could be expected that in some cases their proportion would be opposite, i.e. linoleic acid to be more than linolenic acid. Indeed half of the publications report predomination of linolenic acid [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] in contrast to the others [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In fact purslane oil with predominant alpha-linolenic acid possesses better bioactive potential and is preferable as healthier phytonutrient.\u003c/p\u003e \u003cp\u003eNo effect of extraction method on the total fatty acids composition was detected.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTotal fatty acids composition of purslane seed oil obtained by different extraction methods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatty acid, rel.%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexane\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC-CO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroform-methanol (2:1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroform-methanol (1:2)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u0026ndash;18:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u0026ndash;18:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e33.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e32.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e32.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18:3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e38.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean of three determinations\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. No statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was found within each row.\u003c/p\u003e \u003cp\u003eAfter estimation of total fatty acids composition, the fatty acids composition of each lipid class was determined. Results for the main four acids (alpha-linolenic, linoleic, palmitic and oleic acids) are presented on Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and for the others twelve (12:0, 14:0, 16:1, 18:0, 11\u0026ndash;18:1, 20:0, 20:1, 22:0, 22:1, 24:0, 24:1 and 26:0) on Figure S2. It can be seen that dis-tribution of fatty acids among classes is different and specific but does not depend on the extraction method. Thus, alpha-linolenic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) predominates in triacylglycerols and the next X zone with amounts above 36%. The other lipid classes are relatively rich in it, too (\u0026gt;\u0026thinsp;23%), excepting wax esters (6.5%). Linoleic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) is at similar levels (\u0026gt;\u0026thinsp;24%), excluding wax esters (9.3%), reaching almost 42% and 48% in diacylglycerols and sterol esters, respectively. Palmitic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) has the opposite distribution with the highest amount in wax esters (42%) and levels in the range 11\u0026ndash;22% in the other lipid clas-ses. Oleic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) gradually increases from 6\u0026ndash;12% with increasing of lipid class polarity. It should be noted that seven of the eight saturated fatty acids measured in purslane oil, namely 12:0, 14:0, 16:0, 18:0, 22:0, 24:0 and 26:0 are concentrated in wax esters in amounts quite higher than in the other lipid classes, at levels of 42% for 16:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), 15% for 18:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS a), in the range of 2\u0026ndash;7% for 22:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS b), 24:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS c), 26:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS d) and 14:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS f), and 0.7% 12:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS e). Similar predomination in wax esters can be seen also of 20:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS j). On the other hand, 20:0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS i) and 11\u0026ndash;18:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS h) do not predominate in a single lipid class. The other fatty acids 16:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS g), 22:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS k) and 24:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS l) have specific distribution as well despite their minor to traces amounts. Figure S3 contains diagrams with the same results for fatty acids composition of individual lipid classes but in different presentation to facilitate the comparison between lipid classes regarding their fatty acids composition. Thus, it is obvious that total fatty acids composition of the oil (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS a) is almost equal to that of triacylglycerols (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS b). This result is expected and confirms the content above 90% of triacylglycerols (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Monoacylglycerols (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS c) and free fatty acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS d) have similar fatty acids composition but different from that of diacylglycerols (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS e), unidentified lipid zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS f) and sterol esters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS g). Wax esters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS h) differ the most from the other lipid classes in the high content of saturated fatty acids as discussed above.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge such detailed fatty acids composition of individual lipid classes in purslane oil is published for the first time. This information could be necessary and useful as base for further biochemical and physiological investigations and conclusions, and, on the other hand, for practical purposes in production of food supplements, pharmaceutical and cosmetic goods.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Sterols composition\u003c/h2\u003e \u003cp\u003eSterols in both fractions of free and esterified sterols in purslane oil were determined. In the first fraction nine sterols were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and sitosterol was much more (about 77%) than the others (below 10%). In the fraction of sterol esters thirteen sterols were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, in contrast to the free sterols, sterol esters contained far less sitosterol (about 48%) but much more campesterol and delta-5 avenasterol. In the single publication yet in literature with results about sterol composition of purslane oil (15 identified sterols but without data about sterol esters) beta-sitosterol predominated (53%) followed by campesterol (17%) and delta-5-avenasterol (11%). The other sterols were below 3% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilarly to the fatty acids composition, the present results revealed no effect of extraction method on sterol composition of purslane oil concerning both free sterols and sterol esters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Waxes\u003c/h2\u003e \u003cp\u003eThe most unpolar compounds in purslane seed oil were separated and isolated as lipid zone Waxes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) containing mainly hydrocarbons and minor amounts of wax esters. Concerning the former, twenty-five hydrocarbons with chain length from 14 to 37, as well as squalene, were identified and quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Among them only two (C33 and C31) and squalene were more than 10%. As for wax esters, traces peaks were de-tected but with ambiguous identification. For that reason they are not discussed here and require additional investigation.\u003c/p\u003e \u003cp\u003eNo data from other authors have been found in literature for comparison. As with the other lipid classes no effect of extraction method on hydrocarbons was observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eDetailed analysis of purslane seed oil regarding its lipid composition including separation and quantitation of seven individual lipid classes (monoacylglycerols, diacylglycerols, free fatty acids, free sterols, triacylglycerols, sterol esters, wax esters\u0026thinsp;+\u0026thinsp;hydrocarbons) and determination of fatty acids composition of saponifiables was done for the first time, in order to study the effect of extraction method on lipids and oxidative stability of purslane seed oil extracted by three procedures with organic solvents (hexane, chloroform-methanol mixtures) vs. extraction with super-critical CO\u003csub\u003e2\u003c/sub\u003e. The results revealed no significant effect of extraction method on the individual lipid classes and their fatty acids composition, and quite weak effect on the oil oxidative stability regarding super-critical CO\u003csub\u003e2\u003c/sub\u003e vs. organic solvents. Analyses of lipid composition confirmed the potential of purslane seed oil as a cheap and highly valuable source of phytonutrients as essential ɷ-3 fatty acids and phytosterols, for application in food, pharmaceutical and cosmetic industries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e The support by the Operational Program \u0026ldquo;Science and Education for Smart Growth\u0026rdquo;, co-financed by the European Union through the European Structural and Investment Funds, grant BG05M2OP001-1.002-0012 Center of Competence \u0026ldquo;Sustainable utilization of bio-resources and waste of medicinal and aromatic plants for innovative bioactive products\u0026rdquo;, is gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis research was funded by the Operational Program \u0026ldquo;Science and Education for Smart Growth\u0026rdquo;, co-financed by the European Union through the European Structural and Investment Funds, grant BG05M2OP001-1.002-0012 Center of Competence \u0026ldquo;Sustainable utilization of bio-resources and waste of medicinal and aromatic plants for innovative bioactive products\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflicts of interest\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e All data supporting the findings of this study are available within the paper. Any raw data files are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCode availability\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e Sabina Taneva: Investigation, Chemical Analyses, Acquisition of Data, Data Analysis and Interpretation; Svetlana Momchilova: Methodology, Data Analysis and Interpretation, Drafting of article and Editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval\u003c/strong\u003e Experiments with the plant material adhere to international/national guidelines.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSrivastava R, Srivastava, V. 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J Herb Pharmacother 2006; http://doi.org/10.1300/J157v06n01_04 \u003c/li\u003e\n\u003cli\u003eZadeh JH, \u0026Ouml;zdikicierler O, Pazı F. A Comparative Study on Response Surface Optimization of Supercritical Fluid Extraction Parameters to Obtain Portulaca Oleracea Seed Oil with Higher Bioactive Content and Antioxidant Activity Than Solvent Extraction. Eur J Lipid Sci Technol 2022; http://doi.org/10.1002/ejlt.202200136 \u003c/li\u003e\n\u003cli\u003eDelfan-Hosseini S, Nayebzadeh K, Mirmoghtadaie L, Kavosi M, Hosseini SM. Effect of extraction process on composition, oxidative stability and rheological properties of purslane seed oil. Food Chem 2017; http://dx.doi.org/10.1016/j.foodchem.2016.11.150 \u003c/li\u003e\n\u003cli\u003eSodeifian G, Ardestani NS, Sajadian SA, Moghadamian K. Properties of Portulaca oleracea seed oil via supercritical fluid extraction: Experimental and optimization. J Supercr Fl 2018; https://doi.org/10.1016/j.supflu.2017.12.026 \u003c/li\u003e\n\u003cli\u003eVenkateshwari V, Vijayakumar A, Vijayakumar AK, Prasanna Anjaneya Reddy L, Srinivasan M, Rajasekharan R. Leaf lipidome and transcriptome profiling of Portulaca oleracea: characterization of lysophosphatidylcholine acyltransferase. Planta 2018; https://doi.org/10.1007/s00425-018-2908-8 \u003c/li\u003e\n\u003cli\u003eAbozed SS, Elaraby GM, Zahran HA. Application of Spray-dried Microcapsules of Purslane (Portulaca oleracea L.) Seed Oil Enhances Quality of Mango Juice. Open Agric J 2021; https://doi.org/10.2174/1874331502115010001 \u003c/li\u003e\n\u003cli\u003eEl-Sayed M-I K. Effects of Portulaca oleracea L. seeds in treatment of type-2 diabetes mellitus patients as adjunctive and alternative therapy. J Ethnopharmacol 2011; https://doi.org/10.1016/j.jep.2011.06.020 \u003c/li\u003e\n\u003cli\u003eLiu L, Howe P, Zhou Y-F, Xu Z-Q, Hocart C, Zhang R. Fatty acids and beta-carotene in Australian purslane (Portulaca oleracea) varieties. J Chromatogr A 2000; https://doi.org/10.1016/S0021-9673(00)00747-0 \u003c/li\u003e\n\u003cli\u003ePetropoulos SA, Fernandes A, Arampatzis DA, Tsiropoulos NG, Petrović J, Soković M, Barros L, Ferreira ICFR. Seed oil and seed oil byproducts of common purslane (Portulaca oleracea L.): A new insight to plant-based sources rich in omega-3 fatty acids. LWT-Food Sci Technol 2020; https://doi.org/10.1016/j.lwt.2020.109099 \u003c/li\u003e\n\u003cli\u003ePetropoulos SA, Fernandes A, Calhelha RC, Rouphael Y, Petrović J, Soković M, Ferreira ICFR, Barros L. Antimicrobial Properties, Cytotoxic Effects, and Fatty Acids Composition of Vegetable Oils from Purslane, Linseed, Luffa, and Pumpkin Seeds. Appl Sci 2021; https://doi.org/10.3390/app11125738 \u003c/li\u003e\n\u003cli\u003eMousavi SRJ, Niazmand R. Fatty Acids Composition and Oxidation Kinetic Parameters of Purslane (Portulaca oleracea) Seed Oil. Agric Res 2017; https://doi.org/10.1007/s40003-017-0271-9 \u003c/li\u003e\n\u003cli\u003eStroescu M, Stoica-Guzun A, Ghergu S, Chira N, Jipa I. Optimization of fatty acids extraction from Portulaca oleracea seed using response surface methodology. Ind Crops Prod 2013; https://doi.org/10.1016/j.indcrop.2012.07.051 \u003c/li\u003e\n\u003cli\u003eISO 659:2009, Oilseeds \u0026ndash; Determination of oil content (Reference method), p. 12 (2009).\u003c/li\u003e\n\u003cli\u003eFolch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; https://doi.org/10.1016/S0021-9258(18)64849-5 \u003c/li\u003e\n\u003cli\u003eBligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; https://doi.org/10.1139/o59-099 \u003c/li\u003e\n\u003cli\u003eBoyadzhieva S, Coelho JAP. Errico M, Reynel-Avilla HE, Yankov DS, Bonilla-Petriciolet A, Stateva RP. Assessment of Gnaphalium viscosum (Kunth) valorization prospects: Sustainable recovery of antioxidants by different tech-niques. Antioxidants 2022; https://doi.org/10.3390/antiox11122495 \u003c/li\u003e\n\u003cli\u003eYanishlieva N, Popov A, Marinova E. Eine Modifizierte Jodometrische Methode zur Bestimmung der Peroxidzahl in kleinen Lipidproben. Copt Rend Acad Bulg Sci 1978;31:869-871.\u003c/li\u003e\n\u003cli\u003eAOCS Official method Ch 5-91. Specific Extinction of Oils and Fats, Ultraviolet Absorption. In: Official methods and rec-ommended practices of the AOCS. Urbana, IL: AOCS Press.\u003c/li\u003e\n\u003cli\u003eChristie WW. Lipid analysis: isolation, separation, identification, and structural analysis of lipids. 3rd ed. The Oily Press: Bridgwater, England, 2003; pp. 205-224.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"phytonutrients, purslane Portulaca oleracea, lipid classes, fatty acids, phytosterols, extraction, oxidative stability","lastPublishedDoi":"10.21203/rs.3.rs-4743339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4743339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurslane (\u003cem\u003ePortulaca oleracea\u003c/em\u003e L.) is a widespread weed plant used since ancient times as remedy and as food. Its seed oil possesses significant bioactive potential due to the high content of important phytonutrients, mainly essential fatty acids and phytosterols. Although the total fatty acids composition of oil is well documented, till now there is no data published about individual lipid classes. The information on sterols is scarce as well. Therefore, the aim of this work was to characterize in details for the first time the individual lipid classes and their fatty acids composition, sterols, as well as the oil oxidative stability, depending on the extraction method by either hexane, chloroform-methanol mixtures or super-critical CO\u003csub\u003e2\u003c/sub\u003e. The results revealed no significant effect of extraction method on the individual lipid classes (monoacylglycerols, diacylglycerols, free fatty acids, free sterols, triacylglycerols, sterol esters, wax esters and hydrocarbons), including the fatty acids composition of saponifiables, and quite weak effect on the oil oxidative stability regarding super-critical CO\u003csub\u003e2\u003c/sub\u003e vs. organic solvents. Detailed analyses of lipid composition confirmed the potential of purslane seed oil as a cheap and highly valuable source of phytonutrients as essential fatty acids and phytosterols, for application in food, pharmaceutical and cosmetic industries.\u003c/p\u003e","manuscriptTitle":"Purslane (Portulaca oleracea L.) seed oil as a valuable source of important phytonutrients: effect of the extraction method on the lipid composition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-20 12:07:50","doi":"10.21203/rs.3.rs-4743339/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-07T16:55:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-07T15:12:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-04T15:13:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293834260772390786409654499581149818112","date":"2024-08-03T11:51:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145042607375832394832269404343516539950","date":"2024-08-02T20:12:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-31T15:20:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-25T06:30:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-24T05:48:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Food","date":"2024-07-15T13:48:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"103bdbf5-4769-4b94-a3d4-4fe36887d635","owner":[],"postedDate":"August 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-09-26T05:08:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-20 12:07:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4743339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4743339","identity":"rs-4743339","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}