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Accepted Manuscript: 10.1016/j.tifs.2014.03.011
Accepted Manuscript: 10.1016/j.tifs.2014.03.011
Youn Young Shim, Bo Gui, Paul G. Arnison, Yong Wang, Martin J.T. Reaney
PII: S0924-2244(14)00069-7
DOI: 10.1016/j.tifs.2014.03.011
Reference: TIFS 1537
Please cite this article as: Shim, Y.Y., Gui, B., Arnison, P.G., Wang, Y., Reaney, M.J.T., Flaxseed
(Linum usitatissimum L.) bioactive compounds and peptide nomenclature: A review, Trends in Food
Science & Technology (2014), doi: 10.1016/j.tifs.2014.03.011.
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ACCEPTED MANUSCRIPT
Flaxseed (Linum usitatissimum L.) bioactive compounds and
peptide nomenclature: A review
Youn Young Shima, Bo Guia, Paul G. Arnisonb, Yong Wangc, and Martin J.T. Reaneya,c,*
*Corresponding authors. Tel.: +1 (306) 966 5027; fax: +1 (306) 966 5015; e-mail:
[email protected]
1
Flaxseed (Linum usitatissimum L.) is an oilseed used in industrial and natural health products.
Flaxseed accumulates many biologically active compounds and elements including linolenic acid,
linoleic acid, lignans, cyclic peptides, polysaccharides, alkaloids, cyanogenic glycosides, and
cadmium. Most biological and clinical studies of flaxseed have focused on extracts containing α-
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
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
food. Flaxseed is also added to animal feed to improve animal reproductive performance and health
Mature flaxseed is oblong and flattened, comprising an embryo with two cotyledons surrounded by a
thin endosperm and a smooth, often shiny yellow to dark brown seed coat (hull) (Fig. 1). The
composition of flaxseed is presented in Table 1 (Hadley, Lacher, & Mitchel-Fetch, 1992; Smith, 1958).
Analysis of brown Canadian flaxseed conducted by the Canadian Grain Commission showed the
average composition of commercial seed was 41% fat, 20% protein, 28% total dietary fiber, 7.7%
moisture, and 3.4% ash (DeClercq, 2012). Minor components included: cyanogenic glycosides, phytic
acid, phenolics, trypsin inhibitor, linatine, lignans (phytoestrogens), minerals, vitamins, cadmium,
selenium and cyclolinopeptides (CLs) (Bhatty, 1995; Matsumoto, Shishido, Morita, Itokawa, & Takeya,
2002).
The reported protein content of flaxseed varies widely from 10−31% (Bajpai, Pandey, & Vasistha,
1985; Morita et al., 1997; Oomah & Mazza, 1993a; Salunkhe & Desai, 1986). Approximately 56−70%
of the protein is found in cotyledons and about 30% in the seed coat and endosperm (Dev, Quensel, &
Hansen, 1986; Sosulski & Bakal, 1969). Flaxseed protein contains higher amounts of arginine, aspartic
acid, and glutamic acid than other amino acids (Table 2; Bhatty & Cherdkiatgumchai, 1990; Oomah &
Mazza, 1993b). The essential amino acids found in flaxseed meal are similar in concentration and
Flaxseed contains substantial soluble and insoluble fiber. Cui (2001) reported the content of insoluble
and soluble fiber to be 20% and 9%, respectively, whereas Hadley et al. (1992) reported 30% and 10%,
respectively. The differences likely arise from seed and/or extraction protocols used. Soluble fiber, also
known as mucilage, occurs in the seed coat and is readily extracted with hot (Cui, Mazza, Oomah, &
Biliaderis, 1994) and less readily with cold water (Paynel et al., 2013). This soluble fiber includes acidic
[composed of L-rhamnose (25.3%), L-galactose (11.7%), L-fructose (8.4%), and D-xylose (29.1%)] and
neutral polysaccharides [L-arabinose (20%) and D-xylose/D-galactose (76%)] (Anderson & Lowe, 1947).
Insoluble fiber is composed of cellulose (7–11%), lignin (2–7%), and acid detergent fiber (10– 14%) (Cui,
Mazza, Oomah, & Biliaderis, 1994). Flaxseed mucilage arabinogalactans are associated with protein
Flaxseed oil content of ranges from 38–44% due to genotype and environmental parameters (Oomah
& Mazza, 1997; van Uden, Pras, & Woerdenbag, 1994). Microscopic membrane bound oil bodies, known
as oleosomes, are the main storage form of oil in the endosperm and cotyledons. Oleosomes may be
extracted from other seed components and they contain CLs (Gui, Shim, & Reaney, 2012). Fatty acid
composition varies among different flaxseed types and cultivars. The majority of the flaxseed oil (75%) is
found in cotyledons, with much of the remaining oil (22%) present in the seed coat and endosperm
(Dorrell, 1970). The oil is primarily in the form of triacylglycerides with a fatty acid profile typically
including linolenic (52%), linoleic (17%), oleic (20%), palmitic (6%), and stearic (4%) acids (Green,
1990). Minor lipids and lipid soluble compounds include monoacylglycerides, diacylglycerides,
tocopherols, sterols sterol-esters, phospholipids, waxes, CLs, free fatty acids (FFAs), carotenoids,
chlorophyll, and other compounds. The oxidative instability of renders flax less desirable for use as
cooking oil although flaxseed oil is used in northern China for cooking (Choo, Birch, & Dufour, 2007).
Australian scientists selected a new genetic variant; Linola TM with linoleic acid above 65% and α-
linolenic acid (ALA) below 2% that has improved oxidative stability (Green & Dribnenki, 1994;
Haumann, 1990) compared to conventional flaxseed. Human metabolic pathways do not synthesize
hormone-like eicosanoids, which regulate inflammation and immune function in higher animals
(Mantzioris, James, Gibson, & Cleland, 1994, 1995) and may contribute to effects of dietary flaxseed oil.
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
Whole flaxseed is widely accepted as a healthy food that has antcancer 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 et al., 2007ab). 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: −0.20,
0.00 mmol/L)] and LDL cholesterol [0.08 mmol/L (95% CI: −0.16, 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 (−0.21 and −0.16
mmol/L) and LDL cholesterol (−0.28 and −0.16 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 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 et al. (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 et al. (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
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
tumour necrosis factor- (TNF-α), 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-α 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 17–21% (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
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 (Brühl 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 et al. (2011)
observed large differences in flaxseed oil metabolism of fish fed flaxseed oil that was protected either by
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 et al. (2013) observed that linolenic acid conversion to eicosapentaenoic acid was
enhanced in fish simultaneously fed flaxseed and petroselenic acid (18:1 6), 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.
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
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 34–38%
SDG, 15–21% cinnamic acid glucoside and 9.6–11% hydroxymethylglutaric acid. Consumption of the
lignans or the FLC is reported to slow the progression of atherosclerosis in humans and other mammals
(Prasad, 2005, 2009ab; 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,
activity that is similar to the complex. SDG can prevent the development of atherosclerosis and diabetes
(Fukumitsu, Aida, Ueno, Ozawa, Takahashi, & Kobori, 2008; Prasad, 1999, 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,
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 (Lane, Derbyshire, Li, & Brennan, 2014; Katare, Saxena,
Agrawal, Prasad, & Bisen, 2012; Rabetafika, van Remoortel, Danthine, Paquot, & Blecker, 2011;
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 hours. Flaxseed protein, like protein from other sources, produces
bioactive linear peptide fractions when hydrolysed by proteases. These fractions have been tested for a
number of modes of action. For example, flaxseed proteins digested with Flavourzyme ® 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) and 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 (Udenigwe, Lin, Hou, & Aluko, 2009; Udenigwe & Aluko, 2010; Omoni &
Aluko, 2006).
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, Shand, & Wanasundara, 2008, 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 a 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, lowered 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 et al., 2013a). The effects of soluble flaxseed fiber on appetite, blood
triglycerides and appetite regulating hormones were observed for seven hours after a test meal
(Kristensen et al., 2013b). 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). Paradoxcally, 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 observed 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 µM of cyanogenic compound/g of flaxseed. A thirty-gram
dose of flaxseed could release 240 µM 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 µg/kg bw/week (range from 1.9 to 3.0 µg/kg bw/week)
though vegetarians consumed higher levels of cadmium (up to 5.4 µg/kg bw/week) (Alexander et al.,
2009).
A Proposed Tolerable Weekly Intake (PTWI) was established to be 7 µg/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
µg/per week (Walpole, Prieto-Merino, Edwards, Cleland, Stevens, & Roberts, 2012). Thirty grams of
flaxseed consumption (Leyva et al., 2011) containing 500 µg cadmium/kg (Saastamoinen, Pihlava,
Eurola, Klemola, Jauhiainen, & Hietaniemi, 2013) on a daily basis would contribute 105 µg/per week to
the diet or 1.69 µg/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 µg/kg bw/week may
place specific individuals at risk. The panel recommended lowering the TWI to just 2.5 µg/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
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
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; Górski et al., 2001; Kessler et al., 1986a; Kessler, Klein, & Muller, 1986b;
Siemion, Cebrat, & Wieczorek, 1999; Wieczorek, Bengtsson, Trojnar, & Siemion, 1991).
Kessler and co-workers (1986b) 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 humoural 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 humoural 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, post-adjuvant polyarthritis in rats and hemolytic
anemia of New Zealand black mice, were alleviated. In the research of Górski et al. (2001), the
immunosuppressant. Both 1 and CsA function by inhibiting the action of Interleukin-1- 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
(Górski 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
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 analogues make them
potential value-added natural products of flaxseed and should lead to further investigation of the
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
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
et al. (2013) suggested calling homodetic plant cyclic peptides, comprising those peptides arising from
ribosomal synthesis that do not contain cysteine knots and having 5–12 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-
contain eight or nine amino acid residues with molecular masses of approximately 1 kDa. The first CL,
(first orbitide CL 1), was identified after it was isolated from sediments recovered from crude flaxseed
oil (Kaufmann & Tobschirbel, 1959). In 1968, Weygand discovered a similar cyclic nonapeptide, CL 2.
Between 1999 and 2014, 17 CLs (3–6, 8–12, and 14–21) were identified from the seed and root of flax
(Matsumoto, Shishido, Morita, Itokawa, & Takeya, 2001a, 2002; Matsumoto, Shishido, & Takeya,
2001b; Morita, Shishido, Matsumoto, Itokawa, & Takeya, 1999; Stefanowicz, 2001, 2004; Okinyo-
Owiti, Young, Burnett, & Reaney, 2014). In addition, another cyclic peptide with a non-proteinaceous
amino acid residue (N-methyl-4-aminoproline) was isolated from L. album in 1998 (Picur, Lisowski, &
Siemion, 1998). Subsequently, cyclic peptides from flaxseed were named according to the date of their
discovery with each newly discovered peptide being ascribed the next letter in the alphabet (old name,
Table 3). Type VI nomenclature and flaxseed orbitide nomenclature are highly irregular with publications
defining novel designations for oxidation products and authors using a single name for two distinct
compounds. International Union of Pure and Applied Chemists (IUPAC) recommendation relating to
methionine sulfur oxidation products is clear. “Indication of the modification of this sulfur should not
suggest the addition of further sulfur. Hence calling… methionine S-oxide by the name
methionine sulfoxide, and methionine S,S-dioxide by the name methionine sulfone may be confusing
and is not recommended.” Therefore methionine S-oxide and methionine S,S-dioxide are designated
O
Methionine S-oxide (methionine oxide) MetO or Met [1]
O2
Methionine S,S-dioxide (methionine dioxide) MetO2 or Met [2]
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 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 (1−21) are shown in Table 3 and Fig. 5. Linkage [1–#-
NαC] occurs between amino acid 1 and amino acid “#” through the alpha amino group that forms a N-C
cyclization of the core peptide. The en dash (–) 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 post-translational
modification, for example, oxidation of the Met residue to MetO and MetO 2 (omitted Met). CL name
(CLX) is recognized by 3 capital letters. 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 (Weygand, 1968; Morita et al., 1997), 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 (MetO2) 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/MetO2 residue in a given CL could be a good indicator of flaxseed oil storage
duration since these processes occur over a period of time (Brühl 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 (Brühl et al., 2007). Met amino acids present in the CLs present
in partially defatted flaxseed products were resistant to oxidation (Aladedunye, Sosinska, & Przybylski,
2013).
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 ( 1–3, 6, 9, 12, 14, 17, and 18)
have been elucidated by 2D FT-NMR spectroscopy (Matsumoto, Shishido, Morita, Itokawa, & Takeya,
2001a, 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, Shishido, Morita, Itokawa, & Takeya, 2002; Quail, Shen,
Reaney, & Sammynaiken, 2009). Recently, Reaney and co-workers (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,
Okinyo-Owiti, Ahiahonu, & Reaney, 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. Additioally 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
Flaxseed cyclic peptide nomenclature has not been applied with rigor. It is recommended that
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 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.
Acknowledgements
This work was supported by the Saskatchewan Agriculture Development Fund (Project 20080205)
Abbreviations used
CLs, cyclolinopeptides; ALA, -linolenic acid; TG, triglyceride; LDL, low-density lipoprotein;
HDL, high-density lipoprotein; FFAs, free fatty acids; CsA, cyclosporine A; MeOH, methanol; HPLC,
high-performance liquid chromatography, NMR, nuclear magnetic resonance; Met, methionine; MetO,
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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 (× 1,000 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. 3. Classification of plant cyclopeptides modified from Tan and Zhou (2006).
Fig. 4. Structures of CLs. Abbreviations are Ile for Isoleucine, Gly for Glycine, Met for methionine,
MetO for methionine S-oxide, and MetO2 for methionine S,S-dioxide. Predicted products of NCBI
genes AFSQ01016651.1 (1, 2, 8, and 19), AFSQ01025165.1 (5, 11, and 15), AFSQ01011783.1
(21), and AFSQ01011783.1 (21) are highlighted in green, blue, yellow, and yellow, respectively.
Fig. 6. Transformation of CL by the chemical oxidation of Met to MetO and MetO2 modified from
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