Flaxseed ( Linum usitatissimum L.) bioactive compounds and peptide nomenclature: A review

Flaxseed ( Linum usitatissimum L.) is an oilseed used in industrial andnaturalhealthproducts. Flaxseedaccumulatesmanybiolog-ically active compounds and elements including linolenic acid, linoleic acid, lignans, cyclic peptides, polysaccharides, alkaloids, cyanogenic glycosides, and cadmium. Most biological andclinical studies of ﬂaxseed have focused on extracts containing a -linolenic acid or lignan. Other ﬂaxseed compounds have received less attention and their activity is not well described. The beneﬁts of consumption of whole ﬂaxseed fractions such as oil, mucilage and protein indicate that consideration of the entire portfolio of bioactives present is required to associate biological activity with speciﬁc compounds.


Introduction
Flaxseed (Linum usitatissimum L.), one of the oldest cultivated crops, continues to be widely grown for oil, fiber, and food (Oomah, 2001).The average worldwide flaxseed production between 2007 and 2011 was 1,862,449 tonnes (FAO, 2011).Flaxseed oil is an excellent source of the omega-3 fatty acid linolenic acid with typical levels of 55% in the oil (Oomah, 2001) making it ideal for paints, varnishes, and inks due to its fast polymerization properties.Increasing demand for edible oil sources with significant percentages of omega-3 fatty acids is resulting in consumption of flaxseed as a functional food.Flaxseed is also added to animal feed to improve animal reproductive performance and health (Heimbach, 2009;Turner et al., 2014).

Biological effects of flaxseed and flaxseed fractions
There are many flaxseed products that are consumed including: whole seed, ground whole seed, flaxseed oil, partially defatted flaxseed meal (usually from expeller pressing), fully defatted flaxseed meal (from solvent extraction), flaxseed mucilage extracts, flaxseed hulls, flaxseed oleosomes and flaxseed alcohol extracts.Each of these products is associated with specific beneficial health effects.Although each fraction contains more than one bioactive component reports commonly ignore the presence of a plurality of bioactive compounds in flaxseed fractions or attribute the effect of a component of the flaxseed on the observed effect.
Whole flaxseed is widely accepted as a healthy food that has anticancer activity.Controlled experimental diets have demonstrated numerous beneficial effects of flaxseed consumption (Clark et al., 1995;Cunnane et al., 1993;Jenkins et al., 1999).Dietary flaxseed flour (see description below) reduces epithelial cell proliferation and nuclear aberrations in female rat mammary glands.This finding indicates that flaxseed may reduce the growth rate of mammary cancer (Serraino & Thompson, 1991).Additionally, it has been found that flaxseed lignan reduces mammary tumor growth in the later stages of carcinogenesis (Thompson, Seidl, Rickard, Orcheson, & Fong, 1996).Supplements of 14% flaxseed oil and 20% flaxseed meal reduce the incidence of azoxymethane-induced aberrant crypt foci formation in Fisher 344 male rats (Williams, Verghese, Boateng, et al., 2007;Williams, Verghese, Walker, et al., 2007).Similarly, it has been shown that the substitution of corn meal with flaxseed meal (15%) or corn oil with flaxseed oil (15%) in a basal diet, significantly decreased tumor multiplicity and size in the small intestine and colon of Fisher 344 male rats.The authors concluded that flaxseed meal and oil are effective chemo-preventive agents (Bommareddy et al., 2009).
Inclusion of flaxseed products has also been associated with improvement in blood lipids.Inclusion of 20% flaxseed in diets of rats decreased total plasma cholesterol, triglyceride (TG), and low-density lipoprotein (LDL) cholesterol by 21, 34, and 23%, respectively.Supplementation with 30% flaxseed had a more pronounced effect, reducing the same factors by 33, 67, and 23% (Ratnayake et al., 1992).In human studies, 15 g/d of flaxseed administered for three months was associated with reduction in serum TG and LDL cholesterol without any alteration of high-density lipoprotein (HDL) cholesterol (Bierenbaum, Reichstein, & Watkins, 1993).It has also been reported that consumption of 50 g flaxseed/day for four weeks lowered the plasma LDL cholesterol by 8% in young healthy adults (Cunnane et al., 1995).Indeed, a meta-analysis of studies published from January 1990 to October 2008 revealed a consensus regarding the effect of flaxseed products on blood lipids.All flaxseed interventions, included in the analysis, reduced both total cholesterol [0.10 mmol/L (95% CI: À0.20, 0.00 mmol/L)] and LDL cholesterol [0.08 mmol/L (95% CI: À0.16, 0.00 mmol/L)].While flaxseed oil had no significant effect on either cholesterol or LDL cholesterol.Interventions with whole flaxseed and lignan enriched supplements (flaxseed alcoholic extracts) lowered total cholesterol (À0.21 and À0.16 mmol/L) and LDL cholesterol (À0.28 and À0.16 mmol/L).Female subjects, and individuals with elevated initial cholesterol concentrations, showed a greater response.These experimental findings support the hypothesis that flaxseed consumption has a positive effect on  Oomah and Mazza (1993b), 2  Friedman and Levin (1989), and 3 Bhatty and Cherdkiatgumchai (1990) source papers cited therein.
suppressing the development of atherosclerosis.Recently it was demonstrated that consumption of one whole flaxseed product in the form of a bagel, muffin, bar or bun that containing 30 g of flaxseed by a group of patients displaying pulmonary artery disease and elevated blood pressure significantly reduced both systolic and diastolic blood pressure (Rodriguez- Leyva et al., 2013).In this study the authors reported significant linear correlations between blood pressure lowering effects and markers of flax consumption.Unfortunately, none of the correlations were demonstrated to be causal.Based on the weight of evidence from human clinical trials available prior to 2011 Health Canada's Food Directorate concluded that a claim linking consumption of ground whole flaxseed and blood cholesterol lowering was warranted (Health Canada, 2014).
While there are many known flaxseed varieties and flaxseed composition is known to be affected by genotype and environmental conditions virtually none of the studies of flaxseed effects on physiology have shown definitively that the active ingredient is associated with the observed biological effect.Differential studies that vary just one metabolic constituent in the diet are more useful but such studies are rare.In one such study researchers showed that flaxseed with both high and negligible ALA content showed that flaxseed with very low ALA content lowered hypercholesterolemic atherosclerosis in rabbits (Prasad, Mantha, Muir, & Westcott, 1998).A second approach to differential analysis has been presented by Marambe, Shand, and Wanasundara (2011).In their study the digestion of flaxseed protein was compared for whole seed and for flaxseed meal after removal of mucilage.In vitro digestibility of proteins was slowed by the presence of mucilage.No biological activity was observed in this study but the approach could conclusively demonstrate the role of flaxseed mucilage in flaxseed biological activity.In addition, researchers commonly associate observed effects with metabolic differences that may be spurious and unintentionally misleading.Would the low ALA flax studied by Prasad, Strauss, and Reichart (1999) have evoked the same profound response of blood pressure on individual subjects with peripheral artery disease observed by Rodriguez- Leyva et al. (2013)?Was the flaxseed used in the Rodriguez- Leyva et al. (2013) study a representative variety or was it anomalous?Is one component of the flaxseed or multiple components contributing to the biological effects?While these questions are easily asked where whole flaxseed is studied it is important to note that similar questions may be posed when the biological activity of enriched flaxseed fractions is considered.

Flaxseed oil
Authors of a number of studies have suggested that the primary benefit of flaxseed oil consumption is due to its ALA.Flaxseed oil consumption exerts several effects on inflammatory mediators and markers depending on dose.Flaxseed oil given at 14 g/d to human subjects over 4 weeks decreased the levels of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and cytokines.A lower dose did not have this effect (Caughey, Mantzioris, Gibson, Cleland, & James, 1996;Thies et al., 2001;Wallace, Miles, & Calder, 2003).Supplementation of the diet with 6% ALA depressed the levels of IL-6 and IL-10 and increased the production of TNF-a in mice (Chavali, Zhong, & Forse, 1998).A lower omega-6 to omega-3 fatty acid ratio decreased atherosclerosis in comparison to a higher ratio in apolipoprotein E, in LDL receptor double knockout mice.Feeding Golden Syrian hamsters 20 g/d ALA for six weeks reduced serum cholesterol by 17e21% (Yang et al., 2005).However, no changes were found in serum total cholesterol, LDL cholesterol or HDL cholesterol in healthy subjects or hyperlipidemic patients (Freese & Mutanen, 1997;Kestin, Clifton, Belling, & Nestel, 1990;Sanders & Roshanai, 1983;Singer, Wirth, & Berger, 1990).David (1983) suggested that ALA might lower the growth rate of breast and colon cancers.It is noteworthy that almost all the literature extolling the beneficial functions of flaxseed oil fails to confirm that ALA itself, rather than other bioactive compounds found in flaxseed oil produced the observed health benefits.
The quality of flaxseed oil is poorly described in most of the previously mentioned studies.Flaxseed oil contains bioactive peptides that oxidize over the first few weeks of storage under nitrogen (Br€ uhl et al., 2007).In addition, flaxseed oil products were not examined during most studies to determine if markers of oxidation accumulate in the oil.Oxidation of vegetable oil produces potentially toxic biologically active compounds (Esterbauer, 1993;Han & Csallany, 2009).It is interesting that Collins, Shand, and Drew (2011) observed large differences in flaxseed oil metabolism of fish fed flaxseed oil that was protected either by addition of an antioxidant or by a combination of antioxidant and encapsulation when compared with an unprotected oil indicating that flaxseed oil quality may determine ALA metabolism in fish.It is not known if the effect of oil quality on ALA metabolism extends to other species but few studies verify the presence of oxidation products in oil during the research.More recently, Randall, Reaney, and Drew (2013) observed that linolenic acid conversion to eicosapentaenoic acid was enhanced in fish simultaneously fed flaxseed and petroselenic acid (18:1 D6), in the form of coriander oil, a fatty acid that potentially decreases linoleic acid conversion to arachidonic acid.Specific fatty acids included in dietary studies of flaxseed oil may modify ALA metabolism.

Biological activity of flaxseed lignan and lignan complex
Secoisolariciresinol diglucoside (SDG, MW 686.7), is a phytochemical and antioxidant that acts as a precursor of mammalian lignans and a phytoestrogen (Adolphe, Whiting, Juurlink, Thorpe, & Alcorn, 2010;Prasad, 2004).Extraction of flaxseed with aqueous alcohol yields a solution containing SDG, a form of lignan, in a polymer with phenylpropanoids and hydroxymethylglutaric acid referred to as flaxseed lignan complex (FLC).The oil-insoluble complex is reported to exert multiple physiological effects in animals and humans.Westcott and Paton (2001) reported a FLC is composed of 34e38% SDG, 15e21% cinnamic acid glucoside and 9.6e11% hydroxymethylglutaric acid.Consumption of the lignans or the FLC is reported to slow the progression of atherosclerosis in humans and other mammals (Prasad, 2005(Prasad, , 2009a(Prasad, , 2009b;;Zhang et al., 2008).Treatment of subjects with FLC [40 mg/kg body weight (bw)/d] for eight weeks suppressed the development of hypercholesterolemic atherosclerosis by 34% in rabbits (Prasad, 2005).Hypercholesterolemic humans were treated with 300 mg or 600 mg of FLC for eight weeks.The 300 mg dose reduced total cholesterol and LDL cholesterol by 15 and 17%, respectively, without any change in the ratio of total cholesterol/ HDL cholesterol.A higher dose of 600 mg reduced the serum total cholesterol and LDL cholesterol by 24 and 22%, respectively, with a decrease in the total cholesterol/HDL cholesterol ratio (Zhang et al., 2008).Prasad (2009b) also found that FLC was effective in slowing the progression of atherosclerosis by 31% in hyperlipidemic rabbits, as well as reducing oxidative stress.
SDG produced by chemical hydrolysis and isolated from flax alcohol extracts can present biological activity that is similar to the complex.SDG can prevent the development of atherosclerosis and diabetes (Fukumitsu et al., 2008;Prasad, 1999Prasad, , 2009b)), and additional benefits include modification of blood lipids and cholesterol levels (Fukumitsu, Aida, Shimizu, & Toyoda, 2010).As substantial recent reviews of SDG activity have been presented it is not reviewed further here.In some studies alcoholic extracts that contain SDG have been used without further separation.These fractions may contain a range of ethanol soluble compounds including linatine, cyanogenic glycosides, CLs etc. Interpretation of these studies requires caution.

Biological activity of selected flaxseed components
Based on the complexity of the flaxseed fractions used for much of the research discussed above it is not possible to attribute the health benefits of flaxseed consumption to a sole bioactive component present in flaxseed.The exploration of the biological roles of flaxseed polyunsaturated fatty acids and lignan has been substantial in contrast to the modest efforts made on CLs and other components.Others have recently reviewed much of this research (Carayol, Grosclaude, & Delpierre, 2010;Landete, 2012;Lane, Derbyshire, Li, & Brennan, 2014;Katare, Saxena, Agrawal, Prasad, & Bisen, 2012;Rabetafika, van Remoortel, Danthine, Paquot, & Blecker, 2011;Rodriguez-Leyva, Bassett, McCullough, & Pierce, 2010).

Protein and hydrolysates
Flaxseed protein products are rich in arginine, an amino acid that, when present in the vascular endothelia, can lower blood pressure (Udenigwe et al., 2012).Flaxseed protein isolates (FPI) fed at 200 mg/kg bw to spontaneously hypertensive rats (SHR) effectively lowered blood pressure four hours after administration.Hydrolysis of the FPI followed by isolation of a peptide cation rich fraction produced peptides with highly elevated arginine.This fraction produced a marked decrease in blood pressure in the same SHR system in just 2 h.Flaxseed protein, like protein from other sources, produces bioactive linear peptide fractions when hydrolyzed by proteases.These fractions have been tested for a number of modes of action.For example, flaxseed proteins digested with Flavourzyme Ò inhibited angiotensin converting enzyme (Marambe, Shand, & Wanasundara, 2008).The same protein fraction is also effective at scavenging hydroxyl radicals.Flaxseed peptides produced by FPI hydrolysis by thermolysin and pronase (Udenigwe & Aluko, 2010) or alcalase (Omoni & Aluko, 2006) were biologically active in a number of assays.A recent review of flaxseed proteins concluded that the association of flaxseed proteins with mucilage was an advantage in their applications in food formulations.However, mucilage increases the viscosity of aqueous solutions making the separation of protein difficult.It was noted that preparing protein isolates that were free from mucilage was technically difficult due to viscosity (Omoni & Aluko, 2006;Udenigwe & Aluko, 2010;Udenigwe, Lin, Hou, & Aluko, 2009).
Flaxseed protein hydrolysis products may inhibit angiotensin I-converting enzyme but only a few studies have focused on the hydrolysis of flaxseed protein as would occur under gut conditions (Marambe et al., 2008(Marambe et al., , 2011) ) investigated the in vitro digestion of flaxseed protein and found that whole ground flaxseed resisted digestion in both the modeled stomach and intestinal digestion conditions but that treatments that would inactivated flaxseed protease inhibitors or removed mucilage or both increased total digestion.The amount of undigested protein in whole ground seed was just 12%.

Flaxseed mucilage
Effects of flaxseed mucilage on the digestive tract have been investigated recently in human and animal trials.The products were supplied in solution or incorporated into food.As would be expected for consumption of fiber, feelings of satiety and fullness were expressed by subjects consuming 2.5 g of soluble fiber in contrast to control subjects.A significant decrease in subsequent energy intake was observed after the flaxseed drink compared to the control (2937 vs. 3214 kJ).There was no difference in either appetite ratings or energy intake by subjects that consumed soluble flaxseed fiber in a drink or as a tablet.Consuming flaxseed mucilage liquid three times a day was compared to consuming the same amount in the form of bread (Kristensen et al., 2012).Fiber consumed by both means lowered total fasting cholesterol and LDL cholesterol after seven days.Fecal fat excretion increased with both treatments while the liquid form of the mucilage, and not the bread product, increased energy excretion in feces.The authors concluded that soluble flaxseed fiber might be used to reduce cholesterol and may be useful in controlling energy balance.Similar observations were made for rats provided soluble flaxseed fiber as part of their diet.Weight gain was also limited in rats fed soluble fiber and dietary linseed in contrast to controls (Kristensen, Knudsen, et al., 2013).The effects of soluble flaxseed fiber on appetite, blood triglycerides and appetite regulating hormones were observed for seven hours after a test meal (Kristensen, Savorani, et al., 2013).Higher levels of dietary fiber served with the meal (3.4 g/MJ from flaxseed) were associated with decreased blood triglyceride (18%) after the meal.Additionally, satiety and fullness ratings increased while insulin decreased with the higher dietary fiber treatment.The concentrations of hormones ghrelin, cholecystokinin and glucagon-like peptide 1 were not affected by the treatment.
Polyunsaturated fatty acids, the lignan complex, polysaccharides, flaxseed proteins and CLs are major functional classes of compounds that might impart some or all of the observed experimental results of flaxseed products.While compounds associated with health benefits were consumed, compounds that are also bioactive but potentially toxic were also present in many of the aforementioned studies.These compounds include: linatine, cyanogenic glycosides, cadmium, phytate, selenium, and SDG (Fig. 3).Paradoxically, these compounds may also have contributed to the observed health benefits.

Linatine
When fed flaxseed meal as a major portion of the diet poultry suffer symptoms similar to vitamin B deficiency.Addition of vitamin B in the form of pyridoxine overcame this deficiency.Linatine, a component of flaxseed meal, may cause the decreased growth (Klosterman, Lamoureux, & Parsons, 1967).Most studies have not demonstrated direct effects of linatine on health but rather have shown weight gain responses to vitamin B supplementation.For example it was concluded that linatine did not affect the performance of swine fed meal from low linolenic acid flaxseed as their growth rate was unaffected by the inclusion of pyridoxine (Batterham, Andersen, & Green, 1994).Another route to showing the potential impact of linatine is to determine the vitamin B status.Bishara and Walker (1977) fed pigs a diet containing 30% flaxseed meal by dry weight and challenged the test animals with tryptophan to determine vitamin B status.An observable change in tryptophan metabolites was reported suggesting that the animals were experiencing marginal vitamin B6 deficiency (Bishara & Walker, 1977).While there are no reports of vitamin B deficiency in humans that have consumed flaxseed this is potentially related to the relatively lower consumption of flaxseed products as a portion of diet.The risk of flaxseed causing a deficiency could be related to the level of consumption and the vitamin B status of the individual consuming the seed.The elderly, vegetarians, and vegans have been noted as groups that are more likely to have a deficiency in vitamin B (Allen, 2009;Gilsing et al., 2010).

Cyanogenic glycosides
Cyanogenic compounds have profound biological effects.Flaxseed contains linamarin, linustatin and neolinustatin with the potential to release 7.8 mM of cyanogenic compound/g of flaxseed.A thirty-gram dose of flaxseed could release 240 mM of cyanide.Researchers have found no effect of prolonged consumption of flaxseed.Cyanogenic glycoside consumption may not always produce undesirable effects.Consumption of 20% flaxseed meal by weight of diet counteracted the toxicity of selenium fed in the form of sodium selenite (Olson & Halverson, 1954).Furthermore, it was discovered that the protective factor could be extracted from flaxseed meal by aqueous methanol (MeOH).Chromatography of these protective extracts revealed that fractions that protected against selenium toxicity were also rich in the cyanogenic glycosides linustatin and neolinustatin.HPLC peaks that contained these compounds and pure linamarin both provided some protective effect against selenium toxicity (Palmer, Olson, Halverson, Miller, & Smith, 1980).
The capacity of individuals to detoxify cyanide is related to the presence of sulfur containing amino acids in the diet.Most research into the toxicity of cyanogenic compounds to humans is related to the consumption of cassava as a staple in the diet.The toxicity of cassava is exacerbated due to the typical high levels of consumption and the low availability of dietary protein to those that rely on this staple (World Health Organization, 2004).The toxicity of flaxseed cyanogenic glycosides is likely to be rare except in cases where flaxseed fractions are consumed in relatively large amounts in low protein diets.

Cadmium
Many plants, including flaxseed, absorb the toxic heavy metal cadmium from the soil which then accumulates in the seed.Plant products contribute about two thirds of dietary cadmium (Satarug, Garrett, Sens, & Sens, 2010).Of this, whole oilseeds and oilseed meal including products of sunflower, peanut and flaxseed represent the richest cadmium sources.Cadmium toxicity is typically observed as the loss of kidney function and bone density although many other symptoms are recognized.These effects are cumulative and may require decades of exposure.For most of the population exposure to cadmium through food occurs at a low level.For example, it has been estimated that cadmium exposure through diet in European countries was 2.3 mg/ kg bw/week (range from 1.9 to 3.0 mg/kg bw/week) though vegetarians consumed higher levels of cadmium (up to 5.4 mg/kg bw/week) (Alexander et al., 2009).
A Proposed Tolerable Weekly Intake (PTWI) was established to be 7 mg/kg bw/week (WHO Food Additives, 1989).The choice of weekly dietary exposure was chosen over the daily exposure due to the high levels of cadmium in specific foods and the slow adsorption and excretion characteristics of cadmium.Cadmium has a very long half-life in the human system.For a 62 kg human this amounts to 434 mg/per week (Walpole et al., 2012).Thirty grams of flaxseed consumption (Leyva et al., 2011) containing 500 mg cadmium/kg (Saastamoinen et al., 2013) on a daily basis would contribute 105 mg/per week to the diet or 1.69 mg/kg bw/week.This level of cadmium consumption is below the PTWI and normal exposure levels for Europeans.However, the Contaminants in the Food Chain (CONTAM) panel (Alexander et al., 2009) advised that the cadmium TWI recommendation be lowered to protect more sensitive individuals in the population.Based on a meta-analysis of kidney function and exposure to cadmium it was established that the formerly established PTWI for cadmium of 7 mg/kg bw/week may place specific individuals at risk.The panel recommended lowering the TWI to just 2.5 mg/kg bw.Consumption of 30 g of flaxseed/day would contribute 70% of the newly proposed TWI.It is possible that consuming larger amounts of flaxseed over longer periods of time may place a significant cadmium burden on susceptible individuals.
Cadmium is toxic to specific cancer cell lines and has been considered for use in chemotherapy (Waalkes & Diwan, 1999).However, chronic exposure to cadmium increases the risks of cancer (Satarug et al., 2010).

Cyclolinopeptides
Although CLs have been known for more than half a century research has largely been restricted to structural and physical characterization.Most of what is known about biological activities is anecdotal or based on small animal studies with modest numbers of specimens or in vitro analysis.CLs occur as a significant seed component in flaxseed, but the role of these compounds in planta is largely unknown.In vitro studies of CL biological activity have been described in various publications (Gaymes, Cebrat, Siemion, & Kay, 1997;G orski et al., 2001;Kessler, Gehrke, et al., 1986;Kessler, Klein, & Muller, 1986;Siemion, Cebrat, & Wieczorek, 1999;Wieczorek, Bengtsson, Trojnar, & Siemion, 1991).Kessler, Klein, et al. (1986) reported that CLA (1) inhibits cholate uptake into hepatocytes.Later, the tripeptide -Phe-Phe-Pro-observed in isolated form from 1, which is similar to structures in antamanide and somatostatin, was proved to suppress the hepatocyte cell transport system.It is possible that this peptide sequence imparts the observed cytoprotective effects of 1 on hepatocytes (Rossi & Bianchini, 1996).Immunomodulatory activity of 1 was studied using: Jerne's plaque-forming cell number determination test for the primary and secondary humoral immune response, delayed type hypersensitivity reaction, the skin-allograft rejection, the graft-versus-host reaction for the cellular immune response in mice, human lymphocyte proliferation test in vitro and the post-adjuvant polyarthritis test in rats and hemolytic anemia test in New Zealand black mice (Wieczorek et al., 1991).The results show 1 affected both the humoral and cellular immune responses.An increased skin-allograft rejection time and reduced graft-versus-host reaction index was also observed.Human lymphocyte proliferation was inhibited in vitro by 1 via phytohemagglutinin (Wieczorek et al., 1991).The symptoms associated with two immune diseases, postadjuvant polyarthritis in rats and hemolytic anemia of New Zealand black mice, were alleviated.In the research of G orski et al. (2001), the immunosuppressive effects of 1 were compared with cyclosporine A (CsA), a known immunosuppressant.Both 1 and CsA function by inhibiting the action of Interleukin-1-a and Interleukin-2.This finding strongly indicates that 1 shares the same mechanism as CsA in the plaque-forming cells test and the autologous rosette-forming cells test.This study also compared the effects of both compounds on human lymphocytes in vitro.It was found that at very low concentrations, 1 induced the same effects as CsA on T-and B-cell proliferation, acquisition of activation antigens and immunoglobulin synthesis (G orski et al., 2001).Overall, these studies demonstrated that 1 had similar biological effects to CsA.The toxicity of 1 was evaluated by intravenous and oral administration in rats and mice (Wieczorek et al., 1991).Oral administration of 4 g/kg CL 1 in olive oil, 2% gelatin solution did not harm mice while a concentration of 3 g/kg in rats was also well tolerated.Intravenous administration of 1 at 230 mg/kg is non-toxic to mice.The combined strong immunosuppressive activity and low toxicity at relatively large doses of 1 indicates a potential as an immunosuppressive drug.
Other CLs and their analogs were also investigated for immunosuppressive activities.According to the research of Morita et al. (1997), CL 2 inhibits concanavalin-A induced proliferation of human peripheral blood lymphocytes at treatment levels comparable to that of CsA.CLs 2 and 9 also manifested a moderate inhibitory effect on concanavalin-A induced mouse lymphocyte proliferation (Morita et al., 1997).Many chemical analogs of 1 were tested for their effects on the immune response (Benedetti & Pedone, 2005;Picur, Cebrat, Zabrocki, & Siemion, 2006;Siemion et al., 1999).Many of these compounds including a -Pro-Xxx-Phe-sequence (where Xxx means a hydrophobic, aliphatic, or aromatic residue) were found to exert immunosuppressive activity although none of them were greater than CL 1 (Picur et al., 2006).The immunosuppressive activity of CLs and analogs make them potential value-added natural products of flaxseed and should lead to further investigation of the biological activities of CLs.
Flaxseed CLs belong to the peptide class of orbitides that are plant cyclic peptides produced from ribosomal precursors (Arnison et al., 2013).These natural products occur in significant quantities in some plant species.Databases containing expressed sequence tags and genome sequences were investigated for information about orbitide biosynthesis.This revealed genes that appeared to encode precursors, which are subsequently cyclized to mature orbitides (Covello et al., 2012).Moreover, the presence of new orbitides, which were predicted by sequence analysis, has been confirmed by mass spectrometry (MS) (Okinyo-Owiti, Young, Burnett, & Reaney, 2014).Sequence analysis also predicts the presence of many similar precursor genes in Euphorbiaceae, Caryophyllaceae, Linnaceae, and Rutaceae (Arnison et al., 2013).These peptides are made of proteinogenic amino acids and are produced by ribosomal synthesis.A more thorough discussion of CLs is provided below.

Flaxseed CLs are orbitides
Plant cyclopeptides are cyclic compounds found in higher plants.They typically comprise 2 to 37 amino acids.The cyclic peptides of flaxseed specifically contain proteinogenic amino acids and their oxidized products.Tan and Zhou (2006) reviewed the chemistry of plant cyclopeptides and reported structures of 455 cyclopeptides in 24 plant families.In their review they divided plant cyclopeptides into two classes, five subclasses, and eight types according to their bonding structures (Fig. 3).Individual classes were named according to taxonomic distribution.Cyclopeptide alkaloids (Type I), Caryophyllaceae-type cyclopeptides (Type VI), and Cyclotides (Type VIII) are the three largest groups numbering 185, 168, and 51 members, respectively.Flaxseed cyclic peptides belong to type VI peptides.Although Tan and Zhou (2006) classified all cyclopeptides found in plants calling Type VI cyclopeptides Caryophyllaceae-type homomonocyclopeptides.This name was cumbersome and Arnison et al. (2013) suggested calling homodetic plant cyclic peptides, comprising those peptides arising from ribosomal synthesis that do not contain cysteine knots and having 5e12 amino acids, orbitides (Arnison et al., 2013).This distinguishes orbitides from the cyclotides that contain cysteine knots.CLs are orbitides.Both orbitides and cyclotides are homodetic cyclic peptides as the ring consists solely of amino-acid residues with Nand C-terminals linked through a peptide bond.
Designation of the first amino acid in a homodetic cyclic peptide is problematic and not described by IUPAC.Mass spectrometry fragmentation has allowed analysts to choose the preferred first amino acid in each sequence as the N-terminal of the first linear cleavage product in MS/MS analysis.Now that it is known that mRNA directs orbitide synthesis it is appropriate that the linear sequence presented for flaxseed cyclic peptides matches the linear sequence of the DNA and RNA that gave rise to the cyclic peptide preprotein and peptide.This designation is recommended in a recent review of all cyclic peptides synthesized from ribosomal precursors (Arnison et al., 2013).The DNA and amino acid sequences of the CL precursor protein are provided by Shim, Gui, Arnison, Wang, and Reaney (2014) and Okinyo-Owiti et al., 2014.Sequences of DNA bases and amino acids that are processed to CLs are highlighted (Fig. 4).
The structures proposed nomenclature for CLs (1e21) are shown in Table 3 and Fig. 5. Linkage [1e#-NaC] occurs between amino acid 1 and amino acid "#" through the alpha amino group that forms a NeC cyclization of the core peptide.The en dash (e) is used and placed in square brackets.Identical amino acids substituents are numbered and grouped as shown.Modifications of amino acids use conventions of UniProt and IUPAC in square brackets.Authors only indicate when there is posttranslational modification, for example, oxidation of the Met residue to MetO and MetO 2 (omitted Met).CL name (CLX) is recognized by 3 capital letters.There is no provision for a nomenclature that involves more than 26 CLs.
Fresh aqueous methanolic (70%) extracts of flaxseed comprise as major CL constituents, 1 and methionine (Met) containing 2 (Morita et al., 1997;Weygand, 1968), 5, 8, 11, and 15 (Stefanowicz, 2001) (curiously, Stefanowicz (2004) renamed these same peptides three years later citing the structures as being "new".The latter publication did not cite the earlier report).On the other hand, aged flaxseed oil contains mainly 1 and methionine S-oxide (MetO) possessing CLs such as 3, 6, 9, 14, and 18 (Reaney et al., 2013).The MetO residues derive from Met oxidation, with further oxidation of these residues leading to methionine S,S-dioxide (MetO 2 ) containing CLs such as 4 and 10 (Fig. 6) (Morita & Takeya, 2010).CL 1 does not contain Met and is, therefore, not prone to this form of oxidation.Met oxidation to MetO/MetO 2 residue in a given CL could be a good indicator of flaxseed oil storage duration since these processes occur over a period of time (Br€ uhl et al., 2007;Jadhav, Okinyo-Owiti, Ahiahonu, & Reaney, 2013).For instance 9 confers a bitter flavor to linseed oil and is a product of oxidation of 8.The oxidative transformation of 8 to 9 in freshly prepared oil begins after 2 days at room temperature and the intensity of bitterness increases over a period of 20 weeks, corresponding with increasing accumulation of 9 in the oil (Br€ uhl et al., 2007).Met amino acids present in the CLs present in partially defatted flaxseed products were resistant to oxidation (Aladedunye, Sosinska, & Przybylski, 2013).

Crystal structure of CLs
The biological activity of orbitides is a product of their 3D shape and primary amino acid composition.The genome of flax is published but the primary sequence will provide only partial information regarding the mechanism of protein function.Detailed 3D structure of each gene product is vital to understand its full function.The structures of nine different CLs (1e3,6,9,12,14,17,and 18) have been elucidated by 2D FT-NMR spectroscopy (Matsumoto, Shishido, Morita, et al., 2001;Matsumoto et al., 2002;Morita et al., 1999).Structures of 1 with different co-crystallized solvent molecules have been determined by single-crystal X-ray diffraction (Di Blasio, Benedetti, Pavone, Pedone, & Goodman, 1987;Di Blasio et al., 1989;Matsumoto et al., 2002;Quail, Shen, Reaney, & Sammynaiken, 2009).Recently, Reaney et al. (2013) have reported the solid state and solution structures of four CLs using X-ray diffraction and 2D-NMR.1, 2, and 4 were readily crystallized; two adjacent prolines form a cis bond that contributes rigidity to the molecule (Jadhav et al., 2013).In contrast, 9 and 10, that contain only one proline residue, do not crystallize readily.Difficulty in crystallization of compounds containing MetO may be caused, in part, by the optical activity of the MetO moiety.It is typically more difficult or impossible to form crystals of diastereomers.

Conclusions
Flaxseed oil is a rich source of ALA but this oil also contains cyclic peptides.Most studies of ALA acid are truly studies of cold pressed flaxseed oil.These studies may erroneously attribute biological activity to ALA that is contributed by cyclic peptides.Additionally the oxidative state of the oil in many studies is not known.Flaxseed coat materials are a rich source of lignans and the polysaccharide mucilage.The latter has profound effects on digestive health.Studies that attribute biological activity of flaxseed hull and flaxseed hull extracts to either lignans or polysaccharides alone may also be in error.Alcohol extracts of flaxseed meal contain lignans, cyanogenic glycosides and cyclic peptides.These materials may all contribute biological activity.
Flaxseed cyclic peptide nomenclature has not been applied with rigor.It is recommended that peptides be named as described in Table 3 for future publications.
The risk of toxicity from flaxseed consumption due to linatine, cyanogenic glycosides and cadmium appears to be negligible for most individuals when flaxseed products are consumed in moderation.Regular consumption of flaxseed or flaxseed meal products could place a significant portion of the "cadmium burden" on individuals.However, current recommendations for maximum weekly cadmium consumption are not likely to be exceeded with reasonable flaxseed product consumption levels.The cyanogenic glycoside levels in flaxseed do not appear to be sufficiently concentrated to contribute any biological effect.There is a reported interaction of cyanogenic glycosides with selenium toxicity.This interaction has not been studied in sufficient detail to use flaxseed as a treatment for selenium poisoning.The level of the vitamin B antagonist, linatine, in flaxseed has never been associated with toxicity in humans but the consumption of large amounts of flaxseed can lead to evidence of limited vitamin B availability in swine.It is not known if individuals with compromised vitamin B might become deficient when consuming flaxseed.The level of linatine is not known for most current flaxseed varieties and thus it is not possible to suggest that the dose of this compound is acceptable in untested varieties of flax.

Fig. 1 .
Fig. 1.Hand-cut sections of flaxseed (L.usitatissimum L., var.CDC Bethune) mounted in distilled water showing anatomical structures.(A) The side of flaxseed (B) Hand-cut section of flaxseed.Images were obtained (Â1000 magnification) with a Canon Eos 300D digital camera mounted on a Zeiss Stemi SV 11 light microscope.The images were subsequently processed in Photoshop 7.

Fig. 6 .
Fig. 6.Transformation of CL by the chemical oxidation of Met to MetO and MetO 2 modified from Morita and Takeya (2010).
a Hull.b Not reported.Data taken from

Table 2 .
Amino acid compositions of flaxseed in comparison to soybean.

Table 3 .
Stefanowicz (2004)13)um L. Post-translational modifications of all ribosomally synthesized and post-translationally modified peptides (RiPPs) are noted according toArnison et al. (2013).All CLs listed are either a fundamental parent structure or oxidized products of a fundamental parent structure.The first recognized compound of each fundamental parent structure is used for all structure names.Fundamental parent structures are provided for CLA, CLB, CLD, CLE, CLF, CLG, CLQ, and CLR.b Name used in first literature description (name used in subsequent literature description).cThemethionineresidues of amino acid sequences are highlighted in Fig.4.Abbreviations are MetO for methionine S-oxide and MetO 2 for methionine S,S-dioxide.dStefanowicz(2004)reusedCLJandCLK for 8 and 5 after these names were used originally for 10 and 4 respectively byMatsumoto, Shishido, and Takeya (2001).e Okinyo-Owiti et al. (2014) proposed the use of new naming convention of CLO and CLP for Met-containing CLPs to distinguish them from MetO containing forms CLD and CLE, respectively. a