Introduction

Plant leaves generally contain up to 7 wt% total lipids (TL) per dry weight [1]. We have analyzed TL levels of 13 types of commercial green leafy vegetables harvested in Hokkaido, Japan, and found that the variation in the TL ranged from 6.1 to 13.0 wt% per dry weight [2]. This TL level was relatively higher than those of fruits, flowers, and stem vegetables (2–4 wt% per dry weight) (unpublished data). The higher TL level of green leafy vegetables is due to the large amounts of thylakoid membrane lipids in the tissues. The major constituents of leaf lipids are monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) with moderate amounts of sulfoquinovosyl diacylglycerol (SQDG) and phospholipids (PL), while only small amounts of neutral lipids are present [3]. The high levels of MGDG and DGDG in the leaf lipids originate from the unusually high composition of both galactolipids (GL) in the thylakoid membranes of chloroplasts [4]. Usually, MGDG and DGDG from plant leaf lipids have very high amounts of α-linolenic acid (18:3n-3, ALA) [1]. The ALA content of acylated fatty acids has been reported to be approximately 95 % in MGDG from leaf chloroplasts of cucurbits [5], alfalfa [6], wheat [7], and green holly [8], while that in DGDG has been reported in the range from 79 to 88 %. Thus, green leafy vegetable lipids are potential dietary sources of ALA.

ALA is an essential fatty acid that must be consumed through the diet. There have been many epidemiological and clinical studies on the cardiovascular-protective effects of ALA [9]. ALA is a precursor of eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids. Both n-3 EPA and DHA have sometimes been regarded as active forms of ALA in biological systems. EPA and DHA have been shown to cause significant biochemical and physiological changes in the body that often result in a positive influence on human nutrition and health. EPA and DHA consumption have benefits of reducing the risk of cardiovascular disease, probably due to regulation of membrane structure, lipid metabolism, blood clotting, blood pressure, and inflammation [10–14]. Thus, the bioconversion of ALA to EPA and DHA is important for understanding the biological importance of ALA.

Humans have been generally considered to have a poor ability to form DHA from ALA. Tracer studies have shown that the proportion of ALA conversion to DHA in infants is very low, less than 1 % [15]. Another study has demonstrated that in adult men, the conversion of ALA to EPA is limited (approximately 8 %), and conversion to DHA is extremely low (<0.1 %) [16]. However, studies in normal healthy adults consuming Western diets showed that supplemental ALA raised EPA and DPA statuses in the blood and breast milk. Addition of ALA to the diets of formula-fed infants has been shown to raise DHA levels [17]. Another study showed that there was no difference in brain DHA accretion between rats fed ALA and DHA [18]. This was due to decreased DHA metabolism and an increased rate of DHA synthesis in rats fed ALA. The conversion of ALA to DHA by the liver and other specific DHA-requiring tissues, such as the brain, provides ample DHA when sufficient ALA has been consumed [19]. Thus, the need for ALA is extremely apparent because ALA is by far the predominant form of n-3 PUFA consumed in the typical Western and vegetarian diets [20].

ALA is present in notable amounts in plant sources, including green leafy vegetables and commonly consumed oils, such as rapeseed and soybean oils. Additionally, ALA-rich oils, such as flaxseed oil, are commercially available. These ALA-containing products are a major source of n-3 PUFAs, especially in Western and vegetarian diets. ALA in seed oils exists as triacylglycerol (TAG), while in green leafy vegetables most ALA is bound to GLs, such as MGDG and DGDG. MGDG and DGDG digestion is known to be based on lipase hydrolysis of pancreatic juice, similar to TAG digestion [21, 22]; however, the ALA absorption as these GL may be different from that of TAG because several studies have reported different absorption rates of EPA and DHA in different ester forms, such as TAG, PL, and ethyl esters (EE) [23–28]. Among these, EPA and DHA of PL have been reported to show the highest bioavailability, followed by those of TAG and EE [25–27]. Higher absorption rates of EPA and DHA derived from PL have been reported in a human study using krill oil as a dietary lipid [24, 28].

Although the intake of each PUFA from the different dietary form such as EE, TAG, and PL have been investigated, no study has been performed on those lipids bound to GL. Thus, in the present study, the intake of ALA from GL was compared with that from TAG using spinach leaf as a source of ALA-rich GL.

Materials and Methods

Standards and Chemicals

Standard MGDG, DGDG, and SQDG were purchased from Lipid Products (Redhill, UK), while standard phosphatidyl choline (PC) was from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All of the other chemicals and solvents used in this study were of analytical grade.

Separation and Analysis of Spinach Leaf Lipids

Dried spinach leaf powder (GABAN Co. Ltd., Tokyo, Japan) was obtained from a local food market. The spinach powder (2 kg) was extracted with six volumes (v/w) of methanol. The methanol extracts were dissolved into a separatory funnel using a chloroform/methanol/water (10:5:3, v/v/v) solution. After being shaken, the funnel was allowed to stand overnight, and the lower layer was concentrated under vacuum using a rotary evaporator. The last traces of organic solvent and water were removed in a desiccator under high vacuum to obtain spinach lipids (SPL). The lipid class profile of SPL was analyzed by thin layer chromatography (TLC). The lipid fraction was dissolved in a chloroform/methanol/water (65:25:4, v/v/v) solution and spotted onto 0.25 mm silica gel plates (Merck, Darmstadt, Germany). The plates were developed with a chloroform–methanol–water (65:25:4, v/v/v) solution, and the spots were visualized by spraying the plates with orcinol sulfuric acid or Dittmer reagent, followed by charring. The lipid samples were also analyzed by silica gel TLC using n-hexane/diethyl ether/acetic acid (80:20:1, v/v/v) as the developing solvent. The spot was detected using 50 % aqueous sulfuric acid charring. The chromatogram was taken with a charge coupled device camera, and then the digital image of the plate was acquired and transferred to the computer. The image was properly cropped and saved in bitmap format using Vistascan software on a Windows-controlled system (Hewlett–Packard, Tokyo, Japan). The ratio of each lipid fraction in the sample was expressed as the bitmap percentage of the total bitmap intensities.

The fatty acid compositions of SPL and linseed oil (LO) used as dietary lipids were determined by gas chromatography (GC) after converting fatty acyl groups in the lipids to their corresponding methyl esters by transesterification using sodium methoxide (CH3ONa) as the catalyst [29]. Briefly, 1 mL of n-hexane and 0.2 mL of 2 N NaOH in methanol were added to an aliquot of sample lipids, vortexed and incubated at 50 °C for 30 s. Next, 0.2 mL of 2 N HCl in methanol solution was added to the solution and mixed. The upper hexane layer was recovered and subjected to GC analysis. GC analysis was performed on a Shimadzu GC-14B instrument (Shimadzu Seisakusho, Kyoto, Japan) equipped with a flame-ionization detector and a capillary column [Omegawax 320 (30 m × 0.32 mm i.d.); Supelco, Bellefonte, PA, USA]. The injection port and flame ionization detector were set at 250 and 260 °C, respectively; the column temperature was maintained at 200 °C. The carrier gas was helium at a flow rate of 50 kPa. The fatty acid contents in lipid samples were expressed as the weight percentages of total fatty acids. For the reference, fresh leafy vegetables obtained in the local market in Hakodate, Japan, were extracted with chloroform/methanol/water (10:5:3, v/v/v) after freezed-drying, treatment and the fatty acid composition of the lipids was analyzed as described above.

Animals and Diets

The aim of this study was to compare the SPL and LO as dietary ALA source under normal conditions. Thus, normal and healthy C57BL/6J mice were used the rodent model. A total of 27 normal and healthy C57BL/6J mice (4 weeks old, male) were purchased from Charles River Laboratories (Japan, Inc., Yokohama, Kanagawa, Japan). All mice were housed in stainless cages (seven mice per a cage, four cages in total) and acclimatized for 2 weeks on a normal rodent diet MF (Oriental Yeast Co., Ltd., Tokyo, Japan). Mice had free access to food and tap water. Room temperature and humidity were controlled at 23 ± 1 °C and 50 ± 10 % with a 12 h light/12 h dark cycle. In most animal experiments using C57BL/6 J mice, more than six mice were used. The mice were, therefore, randomly divided into four groups of seven mice in one cage and then fed experimental diets for 4 weeks. The body weight, diet, and water intake of each mouse was recorded every day. The compositions of the diets are shown in Table 1. All dietary components except for lipids were obtained from CLEA (Japan, Inc. Tokyo, Japan). Lard was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Corn oil and LO were obtained from Wako Pure Chemical Ind. Ltd. (Osaka, Japan). For the preparation of SPL containing diet, SPL and lard were dissolved in chloroform. After mixing well, the solvent was completely removed. The mixed lipid was heated at 50 °C, and then the liquid was mixing with other dietary components. The two experimental diets, SPL (3 %) and LO, were designed to contain equal amounts of ALA, equating to approximately 0.7 g/g of the diet (Table 2). However, the resulting manufactured diets had slightly different of total fatty acid intake because of the difference in the non-acyl component levels of SPL and LO. By GC analysis using 17:0 as an internal standard, fatty acid content of the SPL (3 %) diet was 5.2 g/7 g lipids, while that of the LO diet was 6.8 g/7 g lipids. All procedures for the use and care of animals for this research were approved by the ethical committee of Experimental Animal Care at Hokkaido University.

Table 1 Composition (g/kg) of experimental diets
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Table 2 Fatty acid content of total fatty acids and total diet
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Fatty Acid Compositions of Dietary Lipids and ALA Levels of Each Diet

Dietary lipids were extracted from each diet with a chloroform/methanol (2:1, v/v) solution after being prepared as described previously by Folch et al. [30]. The chloroform/methanol solution contained a known amount of 17:0 as an internal standard. Fatty acid compositions of the lipids were analyzed by GC after converting fatty acyl groups in the lipids to their methyl esters, as described above. The ALA levels of the diets were calculated by comparing the peak ratios of ALA to that of the internal standard (17:0) and the lipid content.

Sample Collections

Blood samples were taken from the caudal vena cava of the mice. Mice were euthanized, and each tissue was immediately excised and weighed. The livers were immediately stored in RNA later™ (Sigma Chemical Co., St. Louis, MO, USA) for quantitative real-time PCR analysis. Blood serum analyses were conducted by the Analytical Center of Hakodate Medical Association (Hakodate, Japan). The analyses included measuring the following parameters: neutral lipids, free fatty acids, phospholipids, total cholesterol, HDL cholesterol, LDL cholesterol, and free fatty acids.

Tissue and Blood Lipid Analysis

Livers, small intestines, and brains were extracted with a chloroform/methanol (2:1, v/v) solution containing a known amount of internal standard (17:0), as described previously by Folch et al. [30]. The tissue samples from each mouse were analyzed separately. The major fatty acid contents of each tissue were analyzed by GC after converting fatty acyl groups in the lipid to their methyl esters, as described above. The contents were reported as milligrams per gram tissue. However, only small amounts of serum samples remained after the lipid parameter analyses. Therefore, all serum samples were combined in each group, and then the lipids were extracted with a chloroform/methanol/water (1:2:0.8, v/v/v) solution, as described previously by Bligh and Dyer [31]. Fatty acid compositions (wt% of total fatty acids) were analyzed by GC after converting fatty acyl groups in the lipid to their methyl esters, as described above.

Quantitative Real-Time PCR

Total RNA was extracted from the livers of mice using RNeasy Lipid Tissue Mini Kits (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. The cDNA was then synthesized from total RNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems Japan Ltd., Tokyo, Japan). Quantitative real-time PCR analyses of individual cDNA were performed with ABI Prism 7500 (Applied Biosystems Japan Ltd., Tokyo, Japan) using TaqMan Gene Expression Assays (Applied Biosystems Japan Ltd., Tokyo, Japan). The mRNA analyses were performed on genes associated with the bioconversion of ALA to DHA, which included Δ6-desaturase (Fads2) (from ALA to 18:4n-3 and 24:5n-3 to 24:6n-3), elongase-5 (Elov5) (from 18:4n-3 to 20:4n-3), Δ5-desaturase (Fads1) (from 20:4n-3 to EPA), elongase-2 (Elov2) (from EPA to DPA and DPA to 24:5n-3), and peroxisomal acyl CoA oxidase (Acox1) (from 24:6n-3 to DHA). The gene-specific primers Mm00507605_m1 (Fads1 mRNA), Mm00517221_m1 (Fads2 mRNA), Mm00517086_m1 (Elov2 mRNA), Mm00506717_m1 (Elov5 mRNA), Mm01246834_m1 (Acox1 mRNA), and Mm99999915_g1 (GAPDH mRNA; internal control), respectively.

Statistical Analysis

Data are presented as the mean ± SEM (n = 6 or 7). Analysis of variance (ANOVA) was used to test for significant differences between different groups. Statistical comparisons were performed using Scheffe’s F test. Differences with P < 0.05 were considered significant.

Results

Fatty Acid Contents of Dietary Lipids

The major fatty acids of SPL were ALA (53.2 %), 16:0 (13.9 %), and linoleic acid (18:2n-6, LA) (12.3 %). Analysis of other leafy vegetable lipids showed the highest content of ALA in the fatty acids: Komatsuna (Brassica rapa var. perviridis), 52.5 %; Mizuna (Brassica rapa var. laciniifolia), 50.8 %, Perilla (Perilla frutescens var. acuta), 52.3 %; Sweet basil (Ocimum basilicum), 56.8 %; Mistuba (Cryptotaenia japonica), 35.0 %; Parsley (Petroselium crispum), 30.5 %; Garland chrysanthemum (Chrysanthemum coronarium), 68.3 %; Garlic chives (Allium tuberosum), 48.6 %; Welsh onion (Allium fistulosum), 47.2 %. These leafy vegetables contained 6.1–13.0 wt% lipids per dry weight and most of lipids composed of GL rich in ALA.

LO also contained a high level of ALA (45.3 %), followed by 18:1n-9 (25.3 %), LA (16.3 %), and 16:0 (5.9 %). However, the lipid class compositions of LO and SPL were different. TLC analysis showed that LO was mainly composed of TAG; however, only a small amount of TAG was detected in SPL. When each lipid composition of SPL was roughly analyzed based on the spot intensities of TLC, the main lipid class of SPL was found to be DGDG (22.0 %), followed by MGDG (17.3 %), SQDG (13.5 %), chlorophylls (11.6 %), PC (3.5 %), and lutein (1.3 %). Chlorophylls and lutein are non-acyl lipids. MGDG, DGDG, SQDG, and PC are diacyl glycerols with non-acyl components bound to the remaining position of glycerol, resulting in the relatively lower percentage of fatty acids in SPL than in LO. Therefore, for the comparison of the dietary SPL with that from LO, LO (15 g/1 kg diet) was added to the diet so that the ALA level of the diet was almost the same as that in the diet containing SPL (3 %) (Table 2).

Fatty Acid Levels of Livers (Table 3), Small Intestines (Table 4), Brains (Table 5), and Sera (Table 6)

All animals remained healthy throughout the experimental period. There were no significant differences in the body weights, food and water intake, liver, small intestine, brain, muscle, heart, or kidney weights of mice fed four types of diets. Food intake (g) per day of each group was 24.16 ± 2.14, 24.13 ± 2.63, 23.45 ± 2.53, 23.80 ± 3.99 for control, LO, SPL (1 %), and SPL (2 %) group, respectively. There was also no significant difference in dairy food intake among different four groups. Supplementation of ALA-rich diets, including LO, SPL (1 %), and SPL (3 %), significantly increased hepatic n-3 PUFA levels, including ALA, EPA, and docosapentaenoic acid (DPA, 22:5n-3), compared with the control, while a significant decrease in n-6 arachidonic acid (ARA, 20:4n-6) was found in the ALA-rich diet feedings (Table 3). The LA level of mice fed ALA-rich diets also decreased, although the LA content in the ALA diets was higher than that in the control (Table 2). Mice fed LO and SPL (3 %) showed significantly higher DHA levels relative to the control. The DHA level in mice fed SPL (1 %) also increased, but the difference compared with the control was not significant.

Table 3 TL level and fatty acid content of liver
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Table 4 TL level and fatty acid content of small intestine
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Table 5 Fatty acid composition of serum (wt% of total fatty acids)
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Table 6 TL level and fatty acid content of brain
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Although the ALA content of the diet was the same for LO and SPL (3 %) (Table 2), significantly higher contents of hepatic EPA and DPA were found in mice fed SPL (3 %) than in the LO group (Table 3). Additionally, the ALA level of the mice fed SPL (3 %) was higher than those fed LO, but the difference was not significant. The higher ALA level in the mice fed SPL (3 %) than those fed LO was also found in the small intestinal lipids (Table 4) and in the serum lipids (Table 5). In serum lipids, EPA and DPA were also much higher in mice fed SPL (3 %) than in the LO group. However, the difference in the fatty acid contents in brain lipids was small (Table 6).

Although there were no significant differences in serum total cholesterol (Fig. 1a), LDL cholesterol (Fig. 1c), neutral lipids (Fig. 1d), PL (Fig. 1e), and free fatty acid (FFA) (Fig. 1f) levels among the four dietary groups, HDL cholesterol (Fig. 1b) significantly increased in mice fed SPL (1 %) and SPL (3 %).

Fig. 1
figure 1

Effects of dietary lipids on serum lipid parameters of C57BL/6 J mice. a Total cholesterol; b HDL cholesterol; c LDL cholesterol; d neutral lipids; e phospholipids (PL); f free fatty acids (FFA). Values represent mean ± SE of seven mice per group. Different letters show significant differences at P < 0.05

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Gene Expression of Elongase, Desaturase, and Peroxisomal Enzymes Involved in ALA Bioconversion to DHA

ALA is converted to DHA through a series of desaturation and chain elongation processes. Figure 2 shows the effect of the experimental diets on the relative mRNA expression levels of these fatty acids desaturase, elongase, and acyl CoA oxidase in the liver. SPL (3 %) supplementation significantly decreased Fads1 and Fads2, while a significant increase in Acox1 was found in the SPL (3 %) group. However, LO had no significant effect on these gene expressions compared with the control group.

Fig. 2
figure 2

Expression of mRNA of genes associated with the bioconversion of α-linolenic acid (ALA) to docosahexaenoic acid (DHA) in C57BL/6J mice fed different dietary lipids. Values represent mean ± SE of seven mice per group. Different letters show significant differences at P < 0.05

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Discussion

Supplementation of ALA-rich lipids, LO, and SPL resulted in increased ALA and its n-3 metabolites (EPA, DPA, and DHA) and decreased n-6 PUFA (LA and ARA) in mice livers, small intestines, and sera (Tables 3, 4, 5). Increased n-3 PUFA and decreased n-6 PUFA in rat organs, including the brain, as a result of ALA feeding have been reported in other studies [32–34]; however, in the present study, little effect on the fatty acid composition of brain lipids was observed (Table 6). Lipid and fatty acid compositions of the brain are usually less affected by dietary lipids in normal conditions and strictly regulated through DHA uptake from the plasma and brain DHA metabolism [18]. Therefore, the result in Table 6 may be due to homeostasis found in normal C57BL/6J mice administered the experimental diet for only 4 weeks in the present study. The same result has also been obtained in female ddy mice fed DHA-rich lipids [26]. They reported that the DHA levels of the serum and the liver lipids were significantly increased by feeding DHA lipids, but the fatty acid composition of the brain did not change drastically.

Although the ALA contents in the LO and SPL (3 %) diets were the same (Table 2), the EPA and DPA levels in the liver lipids from the mice fed the SPL (3 %) diet were significantly higher than those fed the LO diet (Tables 3, 5). Additionally, the ALA level of lipids from the livers, small intestines, brains, and plasma of mice fed the SPL (3 %) diet were also higher than those fed the LO diet (Tables 3, 4, 5). These results suggest that ALA originated from SPL may be absorbed more efficiently than that from LO. However, a significant increase in EPA and DPA levels of hepatic lipids from mice fed SPL (3 %), shown in Table 2, may be derived from up-regulation of ALA bioconversion to EPA and DPA. This reaction is regulated by the activities of different enzymes, including Δ6-desaturase (Fads2), elongase-5 (Elov5), Δ5-desaturase (Fads1), and elongase-2 (Elov2). However, the gene expressions of Fads2 and Fads1 significantly decreased in the SPL fed group (Fig. 2). Additionally, SPL feeding had no significant effect on both elongases, showing little effect of SPL feeding on up-regulating bioconversion of ALA to EPA and DPA.

In LO, greater than 99 % of ALA was present as TAG, whereas most ALA in SPL was incorporated into GLs, such as MGDG or DGDG [3, 5–8]. We have reported 75.2 and 77.2 % ALA in MGDG and DGDG from spinach powder lipids, respectively [35]. Both GL were the major lipid components of SPL, and a high ALA level (53.2 %) was found in the SPL used in the present study. The higher levels of ALA and of its metabolites, EPA and DPA, in the mice fed the SPL (3 %) diet was probably due to the higher absorption rate of ALA in the MGDG and/or DGDG forms from an intestinal part.

Several studies have demonstrated the absorption efficacy of n-3 PUFA from different chemical forms [25, 36]. Generally, PL has been considered to be better absorbed than the TAG form, especially in infants [25]. A comparative study using free fatty acids (FFA), EE, TAG, and PL forms of DHA showed that DHA-PL was more effective at increasing DHA in the liver and the brain of male Balb/c mice than other DHA forms [27]. A human study on the uptake of n-3 PUFA as PL form has been reported using krill oil containing 30–65 % of the fatty acids as the PL form. In a double-blinded crossover trial, Schuchardt et al. [24] compared the uptake of EPA + DHA from three different oral administrations of fish oil TAG, EE, and krill oil (mainly PL). Although the intake levels of EPA and DHA were the same among the three groups, the krill oil group showed the highest incorporation of EPA + DHA into plasma, followed by TAG and EE.

The amphiphilic character of PL has been proposed as the most likely reason for the higher intake of EPA + DHA from PL compared with that from TAG [24]. ALA bound to GL is also due to the amphiphilic character of this lipid, similarly to PL. GL possesses emulsification properties due to the presence of a galactosyl group. As a result, GL has been shown to influence the surface composition of fat droplets and increase the binding rate of hydrolyzing enzymes [37]. This surfactant ability of GL has also been shown to promote the formation of mixed micelles and, therefore, the digestion. The first step in GL digestion is hydrolysis of the sn-1 position on the glycerol backbone by pancreatic lipase, yielding monoacylgalactolipids [22]. This lyso compound shows higher emulsification properties than the corresponding diacylglycerols from TAG. Thus, it may be possible that the high GL level in SPL leads to an enhanced absorption of lipids rich in ALA. However, the present study only analyzed fatty acid distribution of several tissues and sera. This study has a small sample size and was short in duration (only 4 weeks). In addition, there were no measurements of n-3 PUFA excretion and no direct measurement of n-3 PUFA intake. While we did measure fatty acids in serum, liver, brain, and intestine, several tissues were not examined, including the heart and skeletal muscle, that can accumulate and serve as deposits of n-3 PUFA [38]. Longer experiments in different types of animals to ensure fatty acid levels reach equilibrium and a comprehensive fatty acid analysis of blood lipids (plasma, erythrocytes, or leukocytes) and other tissues are needed. For further insight on absorption and metabolism of n-3 PUFA from glycolipids, intake, and excretion measurements as well as tracer studies using isotope-labeled ALA are needed [39–41].

Numerous epidemiological studies, clinical trials, genetic, and nutrigenetic approaches have demonstrated the health benefits of n-3 PUFA such as ALA, EPA, and DHA. The most apparent benefit of these n-3 PUFA is reduction of cardiovascular risk, probably due to regulation of membrane structure, lipid metabolism, blood clotting, blood pressure, and inflammation [42]. Dietary ALA has been known to improve blood lipid levels [9, 19]. However, there was no significant effect of dietary ALA on plasma total cholesterol, LDL cholesterol, neutral lipids, PL, and FFA levels found in the present study using normal mice fed normal diets, although a decreasing trend in neutral lipids, FFA, and LDL cholesterol was observed in ALA-containing diets (Fig. 1). However, significant increases in HDL cholesterol were found in the mice fed SPL (1 %) and (3 %) diets (Fig. 1). Although the reason for the higher HDL levels is unknown, the effect of SPL should be attributed to the biological activities of the characteristic lipid constituents of SPL containing MGDG, DGDG, and SQDG as major components. Studies on both synthetic and natural MGDG and DGDG have revealed their activities, including anti-tumor, anti-inflammatory, and cell cycle regulation [43–45]; however, no studies regarding the effect of GL on lipid metabolism have been conducted. SPL also contained lutein as a major carotenoid. Lutein has attracted great attention for preventing and reversing certain serious eye diseases [46, 47]; however, little is known about the effect of lutein on lipid metabolism. More studies may need to be conducted on the biological effects of these leafy lipid components.

In conclusion, the present study demonstrated the important role of green leafy vegetables as n-3 sources. Leafy vegetable lipids, such as SPL, are mainly composed of DGDG and MGDG rich in ALA. A wide range of plant products contain ALA, including seeds, nuts, vegetables, legumes, grains, and fruits. Among them, several types of seed oils, such as LO, flaxseed oil, and walnut oil, are known to be ALA sources. In addition to these seed oils, green leafy vegetable lipids should also be considered because they are rich in ALA as GL forms and in phytochemicals, such as carotenoids.