Accepted Manuscript

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

review

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

To appear in: Trends in Food Science & Technology

Received Date: 13 September 2013

Revised Date: 15 March 2014
Accepted Date: 22 March 2014

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,*

aDepartment of Plant Sciences, University of Saskatchewan, 51 Campus Dr., Saskatoon, SK S7N

5A8, Canada, bBotanical Alternatives Inc., 176, 8B–3110 8th Street E. Saskatoon, SK S7H 0W2,
Canada and cGuangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and
Engineering, Jinan University, Guangzhou 510632, China

*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

associate biological activity with specific compounds.

KEYWORDS: orbitide; cyclolinopeptide; lignan; linolenic acid; cadmium; linatine; cyanogenic

glycoside; dietary fiber; flaxseed; Linum usitatissimum L.; peptide nomenclature

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

(Turner, et al., 2014; Heimbach, 2009).

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

composition to those in soybean.

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

(Ray, Paynel, Morvan, Lerouge, Driouich, & Ray, 2013).

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

ALA, an essential polyunsaturated fatty acid in flaxseed. It is an intermediate in the biosynthesis of

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.

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 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

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

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

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 (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

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 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.

Biological activity of flaxseed lignin 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 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,

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, 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,

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 (Lane, Derbyshire, Li, & Brennan, 2014; Katare, Saxena,

Agrawal, Prasad, & Bisen, 2012; Rabetafika, van Remoortel, Danthine, Paquot, & Blecker, 2011;

Landete, 2012; Carayol, Grosclaude, & Delpierre, 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 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

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, Garrett, Sens, & Sens, 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; 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

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- 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

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 analogues 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 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-

acid residues with N- and C- terminals linked through a peptide bond.

Proposed nomenclature for CLs

CLs were classified as members of the “Caryophyllaceae Type VI homomonocyclopeptides” that

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

according to notes 1 and 2 (IUBMB/IUPAC, 1993; Giles, 1999).

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).

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 ( 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

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 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)

and Genome Canada.

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,

methionine S-oxide; MetO2, methionine S,S-dioxide.

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Figure captions

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. 2. Structures of four cyanogenic glycosides (mono-glycosides: linamarin, lotaustralin; di-

glycosides: linustatin and neolinustatin) and SDG in flaxseed.

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. 5. Proposed nomenclature for CLs.

Fig. 6. Transformation of CL by the chemical oxidation of Met to MetO and MetO2 modified from

Morita and Takeya (2010).

 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,*

aDepartment of Plant Sciences, University of Saskatchewan, 51 Campus Dr., Saskatoon, SK S7N

5A8, Canada, bBotanical Alternatives Inc., 176, 8B–3110 8th Street E. Saskatoon, SK S7H 0W2,
Canada and cGuangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and
Engineering, Jinan University, Guangzhou 510632, China

*Corresponding authors. Tel.: +1 (306) 966 5027; fax: +1 (306) 966 5015; e-mail:
[email protected]
 ACCEPTED MANUSCRIPT

Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4.
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Fig. 5.
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Fig. 6.
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Table 1. Flaxseed composition.

Cotyledons and embryo Seed coat1

Constituent (%) Whole seed
With fat Without fat With fat Without fat

Moisture 7.13 4.31 NR2 7.89 NR2

Nitrogen 4.01 4.64 10.92 3.18 3.52

Oil 38.71 53.20 NR2 1.84 NR2

Fiber (soluble) 10.22 NR2 NR2 NR2 NR2

Fiber (insoluble) 30.41 NR2 NR2 NR2 NR2

Ash NR2 3.38 7.95 2.99 3.31

Weight fraction NR2 58.60 40.40 41.40 59.96

% of total oil 96.70 3.30

1
Hull
2
Not reported.
Data taken from Hadley et al. (1992), Smith (1958) and source papers cited therein.
 ACCEPTED MANUSCRIPT

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

Brown flaxseed (NorLin)1 Yellow flaxseed (Omega)1 Soybean2

Amino acid
g/100 g protein

Alanine (Ala) 4.4 4.5 4.1

Arginine (Arg) 9.2 9.4 7.3

Aspartic acid (Asp) 9.3 9.7 11.7

Cystine (Cys) 1.1 1.1 1.1

Glutamic acid (Glu) 19.6 19.7 18.6

Glycine (Gly) 5.8 5.8 4.0

Histidine (His)* 2.2 2.3 2.5

Isoleucine (Ile)* 4.0 4.0 4.7

Leucine (Leu)* 5.8 5.9 7.7

Lysine (Lys)* 4.0 3.9 5.8

Methionine (Met)* 1.5 1.4 1.2

Phenylalanine (Phe)* 4.6 4.7 5.1

Proline (Pro) 3.5 3.5 5.2

Serine (Ser) 4.5 4.6 4.9

Threonine (Thr)* 3.6 3.7 3.6

Tryptophan (Trp)* 1.83 NR4 NR4

Tyrosine (Tyr) 2.3 2.3 3.4

Valine (Val)* 4.6 4.7 5.2

*Essential amino acids for humans.
Data taken from 1Oomah and Mazza (1993b), 2Friedman and Levin (1989), and 3Bhatty and
Cherdkiatgumchai (1990) source papers cited therein.
4
Not reported.
 ACCEPTED MANUSCRIPT

Table 3. CLs in L. usitatissimum L.

Code New name1 Lit. name2 Amino acid sequence (NαC-)3 Chemical formula MW (Da) References
1 [1–9-NC]-CLA CLA Ile-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile C57H85N9O9 1040.34 Kaufmann & Tobschirbel, 1959
2 [1–9-NC]-CLB CLB Met-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile C56H83N9O9S 1058.38 Weygand, 1968; Morita et al., 1997
3 [1–9-NC],[1-MetO]-CLB CLC MetO-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile C56H83N9O10S 1074.38 Morita et al., 1999
4 [1–9-NC],[1-MetO2]-CLB CLK MetO2-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile C56H83N9O11S 1090.38 Matsumoto et al., 2001b
Stefanowicz, 2001, Stefanowicz, 2004,
5 [1–8-NC]-CLD CLD', CLK4, CLO5 Met-Leu-Leu-Pro-Phe-Phe-Trp-Ile C57H77N9O8S 1048.34 Okinyo-Owiti et al., 2014
6 [1–8-NC],[1-MetO]-CLD CLD MetO-Leu-Leu-Pro-Phe-Phe-Trp-Ile C57H77N9O9S 1064.34 Morita et al., 1999
7 [1–8-NC],[1-MetO2]-CLD 1-Msn-CLD MetO2-Leu-Leu-Pro-Phe-Phe-Trp-Ile C57H77N9O10S 1080.34 Olivia, 2013
Stefanowicz, 2001, Stefanowicz, 2004,
8 [1–8-NC]-CLE CLE', CLJ4, CLP5 Met-Leu-Val-Phe-Pro-Leu-Phe-Ile C51H76N8O8S 961.26 Okinyo-Owiti et al., 2014
9 [1–8-NC],[1-MetO]-CLE CLE MetO-Leu-Val-Phe-Pro-Leu-Phe-Ile C51H76N8O9S 977.26 Morita et al., 1999
10 [1–8-NC],[1-MetO2]-CLE CLJ MetO2-Leu-Val-Phe-Pro-Leu-Phe-Ile C51H76N8O10S 993.26 Matsumoto et al., 2001b
11 [1–8-NC]-CLF CLF, CLL Met-Leu-Met-Pro-Phe-Phe-Trp-Val C55H73N9O8S2 1051.50 Stefanowicz, 2001, Stefanowicz, 2004
12 [1–8-NC],[3-MetO]-CLF CLI Met-Leu-MetO-Pro-Phe-Phe-Trp-Val C55H73N9O9S2 1068.35 Matsumoto et al., 2001a
13 [1–8-NC],[1-MetO]-CLF (Mso)-LCP-3 MetO-Leu-Met-Pro-Phe-Phe-Trp-Val C55H73N9O9S2 1068.35 Reaney et al., 2013,
14 [1–8-NC],[1-MetO,3-MetO]-CLF CLF MetO-Leu-MetO-Pro-Phe-Phe-Trp-Val C55H73N9O10S2 1084.35 Matsumoto et al., 2001a
15 [1–8-NC]-CLG CLG, CLM Met-Leu-Met-Pro-Phe-Phe-Trp-Ile C56H75N9O8S2 1066.38 Stefanowicz, 2001, Stefanowicz, 2004
16 [1–8-NC],[3-MetO]-CLG CLN Met-Leu-MetO-Pro-Phe-Phe-Trp-Ile C56H75N9O9S2 1082.38 Stefanowicz, 2004
17 [1–8-NC],[1-MetO]-CLG CLH MetO-Leu-Met-Pro-Phe-Phe-Trp-Ile C56H75N9O9S2 1082.38 Matsumoto et al., 2001a
18 [1–8-NC],[1-MetO,3-MetO]-CLG CLG MetO-Leu-MetO-Pro-Phe-Phe-Trp-Ile C56H75N9O10S2 1098.38 Matsumoto et al., 2001a
19 [1–9-NC]-CLQ [1–9-NC]-CLQ Met-Leu-Lys-Pro-Phe-Phe-Phe-Trp-Ile C66H87N11O9S 1210.53 Okinyo-Owiti et al., 2014
[1–9-NαC],[1-
20 [1–9-NαC],[1-MetO]-CLQ MetO-Leu-Lys-Pro-Phe-Phe-Phe-Trp-Ile C66H87N11O10S 1226.52 Okinyo-Owiti et al., 2014
MetO]-CLQ
21 [1–9-NaC]-CLR [1–9-NaC]-CLS Gly-Ile-Pro-Pro-Phe-Trp-Leu-Thr-Leu C54H76N10O10 1025.24 Okinyo-Owiti et al., 2014
1Post-translational modifications of all ribosomally synthesized and post-translationally modified peptides (RiPPs) are noted according to Arnison 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.
2Name used in first literature description (name used in subsequent literature description).
3The methionine residues of amino acid sequences are highlighted in Fig. 4. Abbreviations are MetO for methionine S-oxide and MetO2 for methionine
S,S-dioxide.
4Stephanowitz (2004) reused CLJ and CLK for 8 and 5 after these names were used originally for 10 and 4 respectively by Matsumoto et al. (2001b).
5Okinyo-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.

10
 ACCEPTED MANUSCRIPT

Highlights

 Flax compounds; linatine, cyanogenic glycosides, Cd and mucilage are discussed.

 The difficulty in attributing bioactivity to flaxseed compounds is discussed.
 A systematic nomenclature of 21 flaxseed orbitides (cyclolinopeptides) is proposed.
 The biological activity and ribosomal origin of flaxseed orbitides are reviewed.