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

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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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,*

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

RI PT

aDepartment

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

AC C

EP

TE D

M AN U

SC

Engineering, Jinan University, Guangzhou 510632, China

*Corresponding authors.

Tel.: +1

(306)

966

5027;

fax:

+1

(306) 966

5015;

e-mail:

[email protected]

1

ACCEPTED MANUSCRIPT 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-

RI PT

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

SC

associate biological activity with specific compounds.

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

AC C

EP

TE D

M AN U

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

2

ACCEPTED MANUSCRIPT

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

RI PT

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

SC

food. Flaxseed is also added to animal feed to improve animal reproductive performance and health

M AN U

(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

TE D

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,

AC C

2002).

EP

selenium and cyclolinopeptides (CLs) (Bhatty, 1995; Matsumoto, Shishido, Morita, Itokawa, & Takeya,

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 &

3

ACCEPTED MANUSCRIPT 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%,

RI PT

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

SC

[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,

M AN U

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

TE D

& 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

EP

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

AC C

(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; LinolaTM with linoleic acid above 65% and α4

ACCEPTED MANUSCRIPT 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

oil.

Biological effects of flaxseed and flaxseed fractions

RI PT

(Mantzioris, James, Gibson, & Cleland, 1994, 1995) and may contribute to effects of dietary flaxseed

SC

There are many flaxseed products that are consumed including: whole seed, ground whole seed,

M AN U

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

TE D

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.,

EP

1995; Cunnane et al., 1993; Jenkins et al., 1999). Dietary flaxseed flour (see description below) reduces

AC C

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

5

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

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

M AN U

(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

TE D

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

EP

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

AC C

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, 6

ACCEPTED MANUSCRIPT 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).

RI PT

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.

SC

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

M AN U

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

TE D

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

EP

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

AC C

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.

7

ACCEPTED MANUSCRIPT 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

RI PT

tumour necrosis factor-α (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

SC

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

M AN U

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,

TE D

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

EP

produced the observed health benefits.

The quality of flaxseed oil is poorly described in most of the previously mentioned studies. Flaxseed

AC C

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 8

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

Secoisolariciresinol diglucoside (SDG, MW 686.7), is a phytochemical and antioxidant that acts as a

M AN U

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%

TE D

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

EP

(bw)/d] for eight weeks suppressed the development of hypercholesterolemic atherosclerosis by 34% in

AC C

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.

9

ACCEPTED MANUSCRIPT 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, &

RI PT

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,

SC

cyanogenic glycosides, CLs etc. Interpretation of these studies requires caution.

M AN U

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

TE D

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;

EP

Landete, 2012; Carayol, Grosclaude, & Delpierre, 2010).

Protein and hydrolysates

AC C

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 10

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

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;

M AN U

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.

TE D

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

just 12 %.

AC C

Flaxseed mucilage

EP

mucilage or both increased total digestion. The amount of undigested protein in whole ground seed was

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 11

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

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)

M AN U

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.

TE D

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,

EP

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

Linatine

AC C

SDG (Fig. 3). Paradoxcally, these compounds may also have contributed to the observed health benefits.

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 12

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

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

M AN U

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

TE D

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.

EP

Cyanogenic glycoside consumption may not always produce undesirable effects. Consumption of

AC C

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).

13

ACCEPTED MANUSCRIPT 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

RI PT

(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

SC

Many plants, including flaxseed, absorb the toxic heavy metal cadmium from the soil which then

M AN U

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

TE D

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)

2009).

EP

though vegetarians consumed higher levels of cadmium (up to 5.4 µg/kg bw/week) (Alexander et al.,

AC C

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 14

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

burden on susceptible individuals.

Cadmium is toxic to specific cancer cell lines and has been considered for use in chemotherapy

(Satarug, Garrett, Sens, & Sens, 2010).

Cyclolinopeptides

M AN U

(Waalkes & Diwan, 1999). However, chronic exposure to cadmium increases the risks of cancer

Although CLs have been known for more than half a century research has largely been restricted to

TE D

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

EP

vitro studies of CL biological activity have been described in various publications (Gaymes, Cebrat,

AC C

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 15

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

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

M AN U

immunosuppressant. Both 1 and CsA function by inhibiting the action of Interleukin-1-α and Interleukin-2. This finding strongly indicates that 1 shares the same mechanism as CsA in the plaqueforming 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

TE D

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

EP

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

AC C

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 16

ACCEPTED MANUSCRIPT (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

RI PT

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

SC

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

M AN U

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

TE D

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.

EP

Flaxseed CLs are orbitides

Plant cyclopeptides are cyclic compounds found in higher plants. They typically comprise 2 to 37

AC C

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. 17

ACCEPTED MANUSCRIPT 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

RI PT

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.

SC

Proposed nomenclature for CLs

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

M AN U

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

TE D

(Matsumoto, Shishido, Morita, Itokawa, & Takeya, 2001a, 2002; Matsumoto, Shishido, & Takeya, 2001b; Morita, Shishido, Matsumoto, Itokawa, & Takeya, 1999; Stefanowicz, 2001, 2004; OkinyoOwiti, Young, Burnett, & Reaney, 2014). In addition, another cyclic peptide with a non-proteinaceous

EP

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

AC C

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 18

ACCEPTED MANUSCRIPT 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).

Methionine S-oxide (methionine oxide)

[1] [2]

RI PT

Methionine S,S-dioxide (methionine dioxide)

O MetO or Met O2 MetO2 or Met

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

SC

known that mRNA directs orbitide synthesis it is appropriate that the linear sequence presented for

M AN U

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).

TE D

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

EP

acids substituents are numbered and grouped as shown. Modifications of amino acids use conventions of

AC C

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 MetO2 (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

19

ACCEPTED MANUSCRIPT 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 &

RI PT

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,

SC

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

M AN U

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).

TE D

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

EP

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)

AC C

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, 20

ACCEPTED MANUSCRIPT 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

RI PT

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

SC

activity to ALA that is contributed by cyclic peptides. Additioally the oxidative state of the oil in many

M AN U

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.

TE D

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

EP

appears to be negligible for most individuals when flaxseed products are consumed in moderation.

AC C

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

21

ACCEPTED MANUSCRIPT 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

RI PT

varieties of flax.

Acknowledgements

This work was supported by the Saskatchewan Agriculture Development Fund (Project 20080205)

SC

and Genome Canada.

M AN U

Abbreviations used

CLs, cyclolinopeptides; ALA, α-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,

AC C

EP

TE D

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

22

ACCEPTED MANUSCRIPT

References Adolphe, J. L., Whiting, S. J., Juurlink, B. H. J., Thorpe, L. U., & Alcorn, J. (2010). Health effects with consumption of the flax lignan secoisolariciresinol diglucoside. British Journal of Nutrition, 103, 929-938.

RI PT

Aladedunye, F., Sosinska, E., & Przybylski, R. (2013). Flaxseed cyclolinopeptides: analysis and storage stability. Journal of the American Oil Chemists' Society, 90, 419-428.

Alexander, J., Benford, D., Cockburn, A., Cravedi, J.-P., Dogliotti, E., Domenico, A. D., &

SC

Férnandez-Cruz, M. L. (2009). Cadmium in food - Scientific opinion of the Panel on Contaminants in the Food Chain. The European Food Safety Authority Journal, 980, 1-139.

M AN U

Allen, L. H. (2009). How common is vitamin B-12 deficiency? American Journal of Clinical Nutrition, 89, 693S-696S.

Anderson, E., & Lowe, H. J. (1947). The composition of flaxseed mucilage. Journal of Biochemistry, 168, 289-297.

TE D

Arnison, P. G., Bibb, M. J., Bierbaum, G., Bowers, A. A., Bugni, T. S., Bulaj, G., et al. (2013). Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Natural Product Reports, 30, 108-160.

EP

Bajpai, M., Pandey, S., & Vasistha, A. K. (1985). Spectrum of variability of characteristics and composition of the oils from different genetic varieties of linseed. Journal of the American Oil Chemists'

AC C

Society, 62, 628.

Batterham, E. S., Andersen, L. M., & Green, A. G. (1994). Pyridoxine supplementation of LinolaTM meal for growing pigs. Animal Feed Science and Technology, 50, 167-174. Benedetti, E., & Pedone, C. (2005). Cyclolinopeptide A: inhibitor, immunosuppressor or other? Journal of Peptide Science, 11, 268-272. Bhatty, R. S. (1995). Nutrient composition of whole flaxseed and flaxseed meal. In S. C. Cunnane & L. U. Thompson (Eds.), Flaxseed in Human Nutrition (pp. 22-42). Champaign, IL: AOCS Press. 23

ACCEPTED MANUSCRIPT Bhatty, R. S., & Cherdkiatgumchai, P. (1990). Compositional analysis of laboratory-prepared and commercial samples of linseed meal and of hull isolated from flax. Journal of the American Oil Chemists' Society, 67, 79-84. Bierenbaum, M. L., Reichstein, R., & Watkins, T. R. (1993). Reducing atherogenic risk in

College of Nutrition, 12, 501-504.

RI PT

hyperlipemic humans with flax seed supplementation: a preliminary report. Journal of the American

Bishara, H. N., & Walker, H. F. (1977). The vitamin B6 status of pigs given a diet containing linseed

SC

meal. British Journal of Nutrition, 37, 321-331.

Bommareddy, A., Zhang, X., Schrader, D., Kaushik, R. S., Zeman, D., Matthees, D. P., et al. (2009).

276-283.

M AN U

Effects of dietary flaxseed on intestinal tumorigenesis in Apc Min mouse. Nutrition and cancer, 61,

Brühl, L., Matthäus, B., Fehling, E., Wiege, B., Lehmann, B., Luftmann, H., et al. (2007). Identification of bitter off-taste compounds in the stored cold pressed linseed oil. Journal of Agricultural

TE D

and Food Chemistry, 55, 7864-7868.

Carayol, M., Grosclaude, P., & Delpierre, C. (2010). Prospective studies of dietary alpha-linolenic acid intake and prostate cancer risk: a meta-analysis. Cancer Causes & Control, 21, 347-355.

EP

Caughey, G. E., Mantzioris, E., Gibson, R. A., Cleland, L. G., & James, M. J. (1996). The effect on human tumor necrosis factor α and interleukin 1β production of diets enriched in n-3 fatty acids from

AC C

vegetable oil or fish oil. American Journal of Clinical Nutrition, 63, 116-122. Chavali, S. R., Zhong, W. W., & Forse, R. A. (1998). Dietary alpha-linolenic acid increases TNFalpha, and decreases IL-6, IL-10 in response to LPS: effects of sesamin on the delta-5 desaturation of omega-6 and omega-3 fatty acids in mice. Prostaglandins, Leukotrienes and Essential Fatty Acids, 58, 185-191. Choo, W. S., Birch, E. J., & Dufour, J. P. (2007). Physicochemical and stability characteristics of flaxseed oils during pan-heating. Journal of the American Oil Chemists' Society, 84, 735-740. 24

ACCEPTED MANUSCRIPT Clark, W. F., Parbtani, A., Huff, M. W., Spanner, E., de Salis, H., Chin-Yee, I., et al. (1995). Flaxseed: a potential treatment for lupus nephritis. Kidney International, 48, 475-480. Collins, S. A., Shand, P. J., & Drew, M. D. (2011). Stabilization of linseed oil with vitamin E, butylated hydroxytoluene and lipid encapsulation affects fillet lipid composition and sensory

RI PT

characteristics when fed to rainbow trout. Animal Feed Science and Technology, 170, 53-62. Covello, P. S., Datla, R., S. S., Stone, S. L., Balsevich, J. J., Reaney, M. J. T., Arnison, P. G., et al. (2012). DNA sequences encoding Caryophyllaceae and Caryophyllaceae-like cyclopeptide percursors

SC

and methods of use. US Patent Number 58905 A1.

Cui, S. W. (2001). Polysaccharide gums from agricultural products: processing, structures and

M AN U

functionality. Lancaster PA: Technomic Publishing Co., Inc.

Cui, W., Mazza, G., Oomah, B. D., & Biliaderis, C. G. (1994). Optimization of an aqueous extraction process for flaxseed gum by response surface methodology. LWT - Food Science and Technology, 27, 363-369.

TE D

Cunnane, S. C., Ganguli, S., Menard, C., Liede, A. C., Hamadeh, M. J., Chen, Z.-Y., et al. (1993). High α-linolenic acid flaxseed (Linum usitatissimum): some nutritional properties in humans. British Journal of Nutrition, 69, 443-453.

EP

Cunnane, S. C., Hamadeh, M. J., Liede, A. C., Thompson, L. U., Wolever, T. M., & Jenkins, D. J. (1995). Nutritional attributes of traditional flaxseed in healthy young adults. American Journal of

AC C

Clinical Nutrition, 61, 62-68.

David, P. (1983). Cancer chemotherapy: new recruits. Nature, 304, 675. DeClercq,

D.

R.

(2012).

Flaxseed

quality

in

Canada.

Available

at:

http://www.grainscanada.gc.ca/flax- lin/trend-tendance/qfc-qlc-eng.htm Accessed June 20, 2013. Dev, D. K., Quensel, E., & Hansen, R. (1986). Nitrogen extractability and buffer capacity of defatted linseed (Linum usitatissimum L.) flour. Journal of the Science of Food and Agriculture, 37, 199-205.

25

ACCEPTED MANUSCRIPT Di Blasio, B., Benedetti, E., Pavone, V., Pedone, C., & Goodman, M. S. (1987). Conformations of bioactive peptides: cyclolinopeptide A. Biopolymers - Peptide Science Section, 26, 2099-2101. Di Blasio, B., Rossi, F., Benedetti, E., Pavone, V., Pedone, C., Temussi, P. A., et al. (1989). Bioactive peptides: solid-state and solution conformation of cyclolinopeptide A. Journal of the

RI PT

American Chemical Society, 111, 9089-9098. Dorrell, D. G. (1970). Distribution of fatty acids within seed of flax. Canadian Journal of Plant Science, 50, 71-75.

SC

Esterbauer, H. (1993). Cytotoxicity and genotoxicity of lipid-oxidation products. American Journal of Clinical Nutrition, 57, 779S-785S.

M AN U

FAO (Food and Agriculture Organization of the United Nations). (2011). Flaxseed production in Canada and the world. Available at: http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567 ancor Accessed May 20, 2013.

Freese, R., & Mutanen, M. (1997). Alpha-linolenic acid and marine long-chain n-3 fatty acids differ

Nutrition, 66, 591-598.

TE D

only slightly in their effects on hemostatic factors in healthy subjects. American Journal of Clinical

Friedman, M., & Levin, C. E. (1989). Composition of Jimson weed (Datura stramonium) seeds.

EP

Journal of Agricultural and Food Chemistry, 37, 998-1005. Fukumitsu, S., Aida, K., Ueno, N., Ozawa, S., Takahashi, Y., & Kobori, M. (2008). Flaxseed lignan

AC C

attenuates high-fat diet-induced fat accumulation and induces adiponectin expression in mice. British Journal of Nutrition, 100, 669-676. Fukumitsu, S., Aida, K., Shimizu, H., & Toyoda, K. (2010). Flaxseed lignan lowers blood cholesterol and decreases liver disease risk factors in moderately hypercholesterolemic men. Nutrition Research, 30, 441-446.

26

ACCEPTED MANUSCRIPT Gaymes, T. J., Cebrat, M., Siemion, I. Z., & Kay, J. E. (1997). Cyclolinopeptide A (CLA) mediates its immunosuppressive activity through cyclophilin-dependent calcineurin inactivation. FEBS Letters, 418, 224-227. Giles, P. M. Jr. (1999). Revised section F: Natural products and related compounds (IUPAC

RI PT

Recommendations 1999). Pure and Applied Chemistry, 71, 587-643. Gilsing, A. M., Crowe, F. L., Lloyd-Wright, Z., Sanders, T. A., Appleby, P. N., Allen, N. E., et al. (2010). Serum concentrations of vitamin B12 and folate in British male omnivores, vegetarians and

SC

vegans: results from a cross-sectional analysis of the EPIC-Oxford cohort study. European Journal of Clinical Nutrition, 64, 933-939.

M AN U

Górski, A., Kasprzycka, M., Nowaczyk, M., Wieczoreck, Z., Siemion, I. Z., Szelejewski, W., et al. (2001). Cyclolinopeptide: a novel immunosuppressive agent with potential anti-lipemic activity. Transplantation Proceedings, 33, 553.

Green, A. G. (1990). Mutation breeding for fatty acid composition in flax (Linum usitatissimum). In

TE D

FAO/IAEA Co-ordinated Research Programme (pp. 2-4). Bombay.

Green, A. G., & Dribnenki, J. C. P. (1994). Linola-a new premium polyunsaturated oil. Lipid Technology, 6, 29-33.

EP

Gui, B., Shim, Y. Y., & Reaney, M. J. T. (2012). Distribution of cyclolinopeptides in flaxseed fractions and products. Journal of Agricultural and Food Chemistry, 60, 8580-8589.

AC C

Hadley, M., Lacher, C., & Mitchel-Fetch, J. (1992). Fibre in flaxseed. In Proceedings of the 54th Flax Institute of United States (pp. 79-83). Fargo, ND: Flax Institute of United States. Han, I., & Csallany, A. (2009). Formation of toxic alpha, beta-unsaturated 4-hydroxy-aldehydes in thermally oxidized fatty acid methyl esters. Journal of the American Oil Chemists' Society, 86, 253-260. Haumann, B. F. (1990). Low-linolenic flax: variation on familiar oilseed. American Oil Chemists' Society, 1, 934-941.

27

ACCEPTED MANUSCRIPT Health Canada (2014). Summary of Health Canada's assessment of a health claim about ground whole flaxseed and blood cholesterol lowering. Available at: http://www.hc-sc.gc.ca/fn-an/labeletiquet/claims-reclam/assess-evalu/flaxseed-graines-de-lin-eng.php Accessed March 4, 2014. Heimbach, J. T. (2009). Determination of the generally recognized as safe status of the addition of

RI PT

whole and milled flaxseed to conventional foods and meat and poultry products. In Flax Cnada 2015 (pp. 1-178). Port Royal, VA: JHeimbach LLC.

IUBMB/IUPAC (International Union of Biochemistry and Molecular Biology/International Union of

SC

Pure and Applied Chemistry). (1993). Nomenclature and symbolism for amino acids and peptides. Available at: http://www.chem.qmul.ac.uk/iupac/AminoAcid/AA172.html Accessed August 20, 2013.

M AN U

Jadhav, P. D., Okinyo-Owiti, D. P., Ahiahonu, P. W. K., & Reaney, M. J. T. (2013). Detection, isolation and characterisation of cyclolinopeptides J and K in ageing flax. Food Chemistry, 138, 17571763.

Jenkins, D. J., Kendall, C. W., Vidgen, E., Agarwal, S., Rao, A. V., Rosenberg, R. S., et al. (1999).

TE D

Health aspects of partially defatted flaxseed, including effects on serum lipids, oxidative measures, and ex vivo androgen and progestin activity: a controlled crossover trial. American journal of clinical nutrition, 69, 395-402.

EP

Katare, C., Saxena, S., Agrawal, S., Prasad, G., & Bisen, P. S. (2012). Flax seed: a potential medicinal food. Journal of Nutrition & Food Sciences, 2, 120-128.

AC C

Kaufmann, H. P., & Tobschirbel, A. (1959). Über ein Oligopeptid aus Leinsamen. Chemische Berichte-Recueil, 92, 2805-2809. Kessler, H., Gehrke, M., Haupt, A., Klein, M., Muller, A., & Wagner, K. (1986a). Common structural features for cytoprotection activities of somatostatin, antamanide and related peptides. Wien Klin Wochenschr, 64, 74-78.

28

ACCEPTED MANUSCRIPT Kessler, H., Klein, M., & Muller, A. (1986b). Conformational prerequisites for the in vitro inhibition of cholate uptake in hepatocytes by cyclic analogues of antamanide and somatostatin. Angewandte Chemie - International Edition in English, 25, 997-999. Kestin, M., Clifton, P., Belling, G. B., & Nestel, P. J. (1990). n-3 Fatty acids of marine origin lower

RI PT

systolic blood pressure and triglycerides but raise LDL cholesterol compared with n-3 and n-6 fatty acids from plants. American Journal of Clinical Nutrition, 51, 1028-1034.

Klosterman, H. J., Lamoureux, G. L., & Parsons, J. L. (1967). Isolation, characterization, and

SC

synthesis of linatine. A vitamin B6 antagonist from flaxseed (Linum usitatissimum). Biochemistry, 6, 170-177.

M AN U

Kristensen, M., Jensen, M., Aarestrup, J., Petersen, K., Søndergaard, L., Mikkelsen, M. S., & Astrup, A. (2012). Flaxseed dietary fibers lower cholesterol and increase fecal fat excretion, but magnitude of effect depend on food type. Nutrition & Metabolism, 9, 1-8.

Kristensen, M., Knudsen, K. E. B., Jørgensen, H., Oomah, D., Bügel, S., Toubro, S., Tetens, I., &

TE D

Astrup, A. (2013a). Linseed dietary fibers reduce apparent digestibility of energy and fat and weight gain in growing rats. Nutrients, 5, 3287-3298.

Kristensen, M., Savorani, F., Christensen, S., Engelsen, S. B., Bügel, S., Toubro, S., Tetens, I., &

EP

Astrup, A. (2013b). Flaxseed dietary fibers suppress postprandial lipemia and appetite sensation in young men. Nutrition, Metabolism & Cardiovascular Diseases, 23, 136-143.

AC C

Landete, J. M. (2012). Plant and mammalian lignans: a review of source, intake, metabolism, intestinal bacteria and health. Food Research International, 46, 410-424. Lane, K., Derbyshire, E., Li, W., & Brennan, C. (2014). Bioavailability and potential uses of vegetarian sources of omega 3 fatty acids: a review of the literature. Critical Reviews in Food Science and Nutrition, 54, 572-579. Leyva, D. R., Zahradka, P., Ramjiawan, B., Guzman, R., Aliani, M., & Pierce, G. N. (2011). The effect of dietary flaxseed on improving symptoms of cardiovascular disease in patients with peripheral 29

ACCEPTED MANUSCRIPT artery disease: rationale and design of the FLAX-PAD randomized controlled trial. Contemporary Clinical Trials, 32, 724-730. Mantzioris, E., James, M. J., Gibson, R. A., & Cleland, L. G. (1994). Dietary substitution with an αlinolenic acid-rich vegetable oil increases eicosapentaenoic acid concentrations in tissues. American

RI PT

Journal of Clinical Nutrition, 59, 1304-1309. Mantzioris, E., James, M. J., Gibson, R. A., & Cleland, L. G. (1995). Differences exist in the relationships between dietary linoleic and alpha-linolenic acids and their respective long-chain

SC

metabolites. American Journal of Clinical Nutrition, 61, 320-324.

Marambe, H. K., Shand, P. J., & Wanasundara, J. P. D. (2011). Release of angiotensin I-converting

M AN U

enzyme inhibitory peptides from flaxseed (Linum usitatissimum L.) protein under simulated gastrointestinal digestion. Journal of Agricultural and Food Chemistry, 59, 9596-9604. Marambe, H. K., Shand, P. J., & Wanasundara, J. P. D. (2008). An in-vitro investigation of selected biological activities of hydrolysed flaxseed (Linum usitatissimum L.) Proteins. JAOCS, Journal of the

TE D

American Oil Chemists' Society, 85, 1155-1164.

Matsumoto, T., Shishido, A., Morita, H., Itokawa, H., & Takeya, K. (2001a). Cyclolinopeptides F–I, cyclic peptides from linseed. Phytochemistry, 57, 251-260.

EP

Matsumoto, T., Shishido, A., & Takeya, K. (2001b). New cyclic peptides from higher plants. Tennen Yuki Kagobutsu Toronkal Koen Yoshishu, 43, 407-412.

AC C

Matsumoto, T., Shishido, A., Morita, H., Itokawa, H., & Takeya, K. (2002). Conformational analysis of cyclolinopeptides A and B. Tetrahedron, 58, 5135-5140. Morita, H., Shishido, A., Matsumoto, T., Takeya, K., Itokawa, H., Hirano, T., et al. (1997). A new immunosuppressive cyclic nonapeptide, cycloinopeptide B from Linum usitatissimum. Bioorganic and Medicinal Chemistry Letters, 7, 1269-1272. Morita, H., Shishido, A., Matsumoto, T., Itokawa, H., & Takeya, K. (1999). Cyclolinopeptides B–E, new cyclic peptides from Linum usitatissimum. Tetrahedron, 55, 967-976. 30

ACCEPTED MANUSCRIPT Morita, H., & Takeya, K. (2010). Bioactive cyclic peptides from higher plants. Heterocycles, 80, 739-764. Okinyo-Owiti, D. P., Young, L. W., Burnett, P.-G. G., & Reaney, M. J. T. (2014). Flaxseed orbitides: Detection, sequencing and 15N incorporation. Peptide Science, 102, 168-175.

RI PT

Olson, O. E., & Halverson, A. W. (1954). Effect of linseed oil meal and arsenicals on selenium poisoning in the rat. Proceedings of the South Dakota Academy of Science, 33, 90-94.

Omoni, A. O., & Aluko, R. E. (2006). Mechanism of the inhibition of calmodulin-dependent

SC

neuronal nitric oxide synthase by flaxseed protein hydrolysates. JAOCS, Journal of the American Oil Chemists' Society, 83, 335-340.

M AN U

Oomah, B. D. (2001). Flaxseed as a functional food source. Journal of the Science of Food and Agriculture, 81, 889-894.

Oomah, B. D., & Mazza, G. (1993a). Processing of flaxseed meal: effect of solvent extraction on physicochemical characteristics. LWT - Food Science and Technology, 26, 312-317.

TE D

Oomah, B. D., & Mazza, G. (1993b). Flaxseed proteins - a review. Food Chemistry, 48, 109-114. Oomah, B. D., & Mazza, G. (1997). Effect of dehulling on chemical composition and physical properties of flaxseed. LWT - Food Science and Technology, 30, 135-140.

EP

Palmer, I. S., Olson, O. E., Halverson, A. W., Miller, R., & Smith, C. (1980). Isolation of factors in linseed oil meal protective against chronic selenosis in rats. Journal of Nutrition, 110, 145-150.

AC C

Paynel, F., Pavlov, A., Ancelin, G., Rihouey, C., Picton, L., Lebrun, L., et al. (2013). Polysaccharide hydrolases are released with mucilages after water hydration of flax seeds. Plant Physiology and Biochemistry, 62, 54-62.

Picur, B., Cebrat, M., Zabrocki, J., & Siemion, I. Z. (2006). Cyclopeptides of Linum usitatissimum. Journal of Peptide Science, 12, 569-574. Picur, B., Lisowski, M., & Siemion, I. Z. (1998). A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline. Letters in Peptide Science, 5, 183-187. 31

ACCEPTED MANUSCRIPT Prasad, K. (2004). Antihypertensive activity of secoisolariciresinol diglucoside (SDG) isolated from flaxseed: Role of guanylate cyclase. International Journal of Angiology, 13, 7-14. Prasad, K. (2005). Hypocholesterolemic and antiatherosclerotic effect of flax lignan complex isolated from flaxseed. Atherosclerosis, 179, 269-275.

RI PT

Prasad, K. (2009a). Flax lignan complex slows down the progression of atherosclerosis in hyperlipidemic rabbits. Journal of Cardiovascular Pharmacology and Therapeutics, 14, 38-48. Prasad, K. (2009b). Flaxseed and cardiovascular health. Journal of cardiovascular pharmacology,

SC

54, 369-377.

Prasad, K., Mantha, S. V., Muir, A. D., & Westcott, N. D. (1998). Reduction of hypercholesterolemic

M AN U

atherosclerosis by CDC-flaxseed with very low alpha-linolenic acid. Atherosclerosis, 136, 367-375. Prasad, N., Strauss, D., & Reichart, G. (1999). Cyclodextrins inclusion for food, cosmetics and pharmaceuticals. European Patent Number 1084625.

Quail, J. W., Shen, J., Reaney, M. J. T., & Sammynaiken, R. (2009). Cyclolinopeptide A methanol

TE D

solvate. Acta Crystallographica Section E, 65, 1913-1914.

Rabetafika, H. N., van Remoortel, V., Danthine, S., Paquot, M., & Blecker, C. (2011). Flaxseed proteins: Food uses and health benefits. International Journal of Food Science and Technology, 46, 221-

EP

228.

Randall, K. M., Reaney, M. J. T., & Drew, M. D. (2013). Effect of dietary coriander oil and

AC C

vegetable oil sources on fillet fatty acid composition of rainbow trout. Canadian Journal of Animal Science, 1-8.

Ratnayake, W. M. N., Behrens, W. A., Fischer, P. W. F., L'Abbé, M. R., Mongeau, R., & BeareRogers, J. L. (1992). Chemical and nutritional studies of flaxseed (variety Linott) in rats. The Journal of Nutritional Biochemistry, 3, 232-240.

32

ACCEPTED MANUSCRIPT Ray, S., Paynel, F., Morvan, C., Lerouge, P., Driouich, A., & Ray, B. (2013). Characterization of mucilage polysaccharides, arabinogalactanproteins and cell-wall hemicellulosic polysaccharides isolated from flax seed meal: a wealth of structural moieties. Carbohydrate polymers, 93, 651-660. Reaney, M. J. T., Jia, Y., Shen, J., Schock, C., Tyler, N., Elder, J., et al. (2013). Recovery of

RI PT

hydrophobic peptides from oils. US Patent Number 8383172. Rodriguez-Leyva, D., Bassett, C. M. C., McCullough, R., & Pierce, G. N. (2010). The cardiovascular effects of flaxseed and its omega-3 fatty acid, alpha-linolenic acid. Canadian Journal of Cardiology, 26,

SC

489-496.

Rodriguez-Leyva, D., Weighell, W., Edel, A. L., LaVallee, R., Dibrov, E., Pinneker, R., Maddaford,

M AN U

T. G., Ramjiawan, B., Aliani, M., Guzman, R., & Pierce, G. N. (2013). Potent antihypertensive action of dietary flaxseed in hypertensive patients. Hypertension, 62, 1081-1089.

Rossi, F., & Bianchini, E. (1996). Synergistic induction of nitric oxide by beta-amyloid and cytokines in astrocytes. Biochemical and Biophysical Research Communications, 225, 474-478.

TE D

Saastamoinen, M., Pihlava, J.-M., Eurola, M., Klemola, A., Jauhiainen, L., & Hietaniemi, V. (2013). Yield, SDG lignan, cadmium, lead, oil and protein contents of linseed (Linum usitatissimum L.) cultivated in trials and at different farm conditions in the south-western part of Finland. Agricultural and

EP

Food Science, 22, 296-306.

Salunkhe, D. K., & Desai, B. B. (1986). Linseed, niger and cottonseed. In Postharvest Biotechnology

AC C

of Oilseeds (pp. 171-186). Boca Raton, FL: CRC Press. Sanders, T. A. B., & Roshanai, F. (1983). The influence of different types of ω3 polyunsaturated fatty acids on blood lipids and platelet function in healthy volunteers. Clinical Science, 64, 91-99. Satarug, S., Garrett, S. H., Sens, M. A., & Sens, D. A. (2010). Cadmium, environmental exposure, and health outcomes. Environ Health Perspect, 118, 182-190. Serraino, M., & Thompson, L. U. (1991). The effect of flaxseed supplementation on early risk markers for mammary carcinogenesis. Cancer Letters, 60, 135-142. 33

ACCEPTED MANUSCRIPT Shim, Y. Y., Gui, B., Arnison, P. G., Wang, Y., & Reaney, M. J. T. (2014) Flaxseed (Linum usitatissimum L.) compositions and processing: A review. Trends in Food Science & Technology. Submitted, Manuscript ID: TIFS-D-14-00009. Siemion, I. Z., Cebrat, M., & Wieczorek, Z. (1999). Cyclolinopeptides and their analogs - A new

RI PT

family of peptide immunosuppressants affecting the calcineurin system. Archivum Immunologiae et Therapiae Experimentalis, 47, 143-153.

Singer, P., Wirth, M., & Berger, I. (1990). A possible contribution of decrease in free fatty acids to

SC

low serum triglyceride levels after diets supplemented with n-6 and n-3 polyunsaturated fatty acids. Atherosclerosis, 83, 167-175.

M AN U

Smith, A. K. (1958). Vegetable protein isolates. In I. A. M. Altschul (Ed.), In processed plant protien foodstuffs (pp. 249-276). New York: Academic Press Inc.

Sosulski, F. W., & Bakal, A. (1969). Isolated proteins from rapeseed, flax and sunflower meals. Canadian Institute of Food Science and Technology Journal, 2, 28-32.

TE D

Stefanowicz, P. (2001). Detection and sequencing of new cyclic peptides from linseed by electrospray ionization mass spectrometry. Acta Biochimica Polonica, 48, 1125-1129. Stefanowicz, P. (2004). Electrospray mass spectrometry and tandem mass spectrometry of the natural

10, 665-671.

EP

mixture of cyclic peptides from linseed. European Journal of Mass Spectrometry (Chichester, England),

AC C

Tan, N. H., & Zhou, J. (2006). Plant cyclopeptides. Chemical Reviews, 106, 840-895. Thies, F., Miles, E. A., Nebe-von-Caron, G., Powell, J. R., Hurst, T. L., Newsholme, E. A., et al. (2001). Influence of dietary supplementation with long-chain n-3 or n-6 polyunsaturated fatty acids on blood inflammatory cell populations and functions and on plasma soluble adhesion molecules in healthy adults. Lipids, 36, 1183-1193.

34

ACCEPTED MANUSCRIPT Thompson, L. U., Seidl, M. M., Rickard, S. E., Orcheson, L. J., & Fong, H. H. (1996). Antitumorigenic effect of a mammalian lignan precursor from flaxseed. Nutrition and Cancer, 26, 159165. Turner, T. D., Mapiye, C., Aalhus, J. L., Beaulieu, A. D., Patience, J. F., Zijlstra, R. T., & Dugan, M.

RI PT

E. R. (2014). Flaxseed fed pork: n-3 fatty acid enrichment and contribution to dietary recommendations. Meat Science, 96, 541-547.

Udenigwe, C. C., Adebiyi, A. P., Doyen, A., Li, H., Bazinet, L., & Aluko, R. E. (2012). Low

SC

molecular weight flaxseed protein-derived arginine-containing peptides reduced blood pressure of spontaneously hypertensive rats faster than amino acid form of arginine and native flaxseed protein.

M AN U

Food Chemistry, 132, 468-475.

Udenigwe, C. C., & Aluko, R. E. (2010). Antioxidant and angiotensin converting enzyme-inhibitory properties of a flaxseed protein-derived high fischer ratio peptide mixture. Journal of Agricultural and Food Chemistry, 58, 4762-4768.

TE D

van Uden, W., Pras, N., & Woerdenbag, H. J. (1994). Linum species (Flax): In vivo and in vitro accumulation of lignans and other metabolites. In Y. P. S. Bajaj (Ed.), Biotechnology in agriculture and forestry Vol. 26. Medicinal and aromatic plants VI (pp. 219-244). Berlin, Germany: Springer-Verlag.

EP

Waalkes, M. P., & Diwan, B. A. (1999). Cadmium-induced inhibition of the growth and metastasis of human lung carcinoma xenografts: role of apoptosis. Carcinogenesis, 20, 65-70.

AC C

Wallace, F. A., Miles, E. A., & Calder, P. C. (2003). Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects. British Journal of Nutrition, 89, 679-689.

Walpole, S. C., Prieto-Merino, D., Edwards, P., Cleland, J., Stevens, G., & Roberts, I. (2012). The weight of nations: an estimation of adult human biomass. BMC Public Health, 12(439), 1-6. Westcott, N. D., & Paton, D. (2001). Complex containing lignan, phenolic and aliphatic substances from flax and process for preparing. US Patent Number 6264853. 35

ACCEPTED MANUSCRIPT Weygand, F. (1968). Entwicklunglinien biochemischer analytik. Fresenius' Zeitschrift Für Analytische Chemie, 243, 2-17. WHO (World Health Organization) Food Addictives. (1989). Toxicological evaluation of certain food additives and contaminants. The 33rd meeting of the Joint FAO/WHO Expert Committee on Food

20, 2013.

RI PT

Additives. Available at: http://www.inchem.org/documents/jecfa/jecmono/v024je01.htm Accessed May

Wieczorek, Z., Bengtsson, B., Trojnar, J., & Siemion, I. Z. (1991). Immunosuppressive activity of

SC

cyclolinopeptide A. Peptide Research, 4, 275-283.

Williams, D. S., Verghese, M., Boateng, J., Walker, L. T., Shackelford, L., Khatiwada, J., et al.

M AN U

(2007a). Dietary flax products suppress the formation of azoxymethane-induced colon cancer in Fisher 344 male rats. Journal of Nutrition, 137, 2785.

Williams, D. S., Verghese, M., Walker, L. T., Boateng, J., Shackelford, L., & Chawan, C. B. (2007b). Flax seed oil and flax seed meal reduce the formation of aberrant crypt foci (ACF) in

TE D

azoxymethane-induced colon cancer in Fisher 344 male rats. Food and Chemical Toxicology, 45, 153159.

World Health Organization. (2004). Hydrogen cyanide and cyanides: human health aspects. Geneva,

EP

Switzerland: Concise International Chemical Assessment Document 61. Yang, L., Leung, K. Y., Cao, Y., Huang, Y., Ratnayake, W. M. N., & Chen, Z. Y. (2005). α-

AC C

Linolenic acid but not conjugated linolenic acid is hypocholesterolaemic in hamsters. British Journal of Nutrition, 93, 433-438.

Zhang, W., Wang, X., Liu, Y., Tian, H., Flickinger, B., Empie, M. W., et al. (2008). Dietary flaxseed lignan extract lowers plasma cholesterol and glucose concentrations in hypercholesterolaemic subjects. British Journal of Nutrition, 99, 1301-1309.

36

ACCEPTED MANUSCRIPT

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

RI PT

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; diglycosides: linustatin and neolinustatin) and SDG in flaxseed.

SC

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,

M AN U

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.

AC C

EP

Morita and Takeya (2010).

TE D

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

37

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,*

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

RI PT

aDepartment

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

AC C

EP

TE D

M AN U

SC

Engineering, Jinan University, Guangzhou 510632, China

*Corresponding authors. [email protected]

Tel.: +1

(306)

966

5027;

fax:

+1

(306) 966

5015;

e-mail:

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 1.

Fig. 2.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 3.

Fig. 4.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

Fig. 5.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 6.

ACCEPTED MANUSCRIPT Table 1. Flaxseed composition. Seed coat1

Cotyledons and embryo Constituent (%)

Whole seed

With fat

Without fat

With fat

Without fat

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

Ash

NR2

3.38

7.95

Weight fraction

NR2

58.60

SC

M AN U

% of total oil

RI PT

Moisture

40.40

NR2

NR2

2.99

3.31

41.40

59.96

96.70

1

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

AC C

EP

TE D

2

3.30

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 4.4

4.5

4.1

Arginine (Arg)

9.2

9.4

7.3

Aspartic acid (Asp)

9.3

9.7

Cystine (Cys)

1.1

1.1

19.6

19.7

Glycine (Gly)

5.8

5.8

Histidine (His)*

2.2

Isoleucine (Ile)*

4.0

Leucine (Leu)*

5.8

Lysine (Lys)*

4.0

Methionine (Met)*

SC

M AN U

Glutamic acid (Glu)

RI PT

Alanine (Ala)

11.7 1.1

18.6 4.0 2.5

4.0

4.7

5.9

7.7

3.9

5.8

1.5

1.4

1.2

4.6

4.7

5.1

3.5

3.5

5.2

4.5

4.6

4.9

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

Proline (Pro) Serine (Ser)

AC C

Threonine (Thr)*

EP

Phenylalanine (Phe)*

TE D

2.3

*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 1 [1–9-NαC]-CLA

Lit. name2 CLA

Amino acid sequence (NαC-)3 Ile-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile

Chemical formula MW (Da) References C57H85N9O9 1040.34 Kaufmann & Tobschirbel, 1959

2 3 4

[1–9-NαC]-CLB [1–9-NαC],[1-MetO]-CLB [1–9-NαC],[1-MetO2]-CLB

CLB CLC CLK

Met-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile MetO-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile MetO2-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile

C56H83N9O9S C56H83N9O10S C56H83N9O11S

5

[1–8-NαC]-CLD

CLD', CLK4, CLO5 Met-Leu-Leu-Pro-Phe-Phe-Trp-Ile

C57H77N9O8S

6

[1–8-NαC],[1-MetO]-CLD

CLD

MetO-Leu-Leu-Pro-Phe-Phe-Trp-Ile

C57H77N9O9S

7

[1–8-NαC],[1-MetO2]-CLD

1-Msn-CLD

MetO2-Leu-Leu-Pro-Phe-Phe-Trp-Ile

C57H77N9O10S

8

[1–8-NαC]-CLE

CLE', CLJ4, CLP5

Met-Leu-Val-Phe-Pro-Leu-Phe-Ile

9 10 11

[1–8-NαC],[1-MetO]-CLE [1–8-NαC],[1-MetO2]-CLE [1–8-NαC]-CLF

CLE CLJ CLF, CLL

MetO-Leu-Val-Phe-Pro-Leu-Phe-Ile MetO2-Leu-Val-Phe-Pro-Leu-Phe-Ile Met-Leu-Met-Pro-Phe-Phe-Trp-Val

12

[1–8-NαC],[3-MetO]-CLF

CLI

13

[1–8-NαC],[1-MetO]-CLF

(Mso)-LCP-3

14

RI PT

1058.38 Weygand, 1968; Morita et al., 1997 1074.38 Morita et al., 1999 1090.38 Matsumoto et al., 2001b 2001, Stefanowicz, 2004, 1048.34 Stefanowicz, Okinyo-Owiti et al., 2014 1064.34 Morita et al., 1999

Met-Leu-MetO-Pro-Phe-Phe-Trp-Val

C55H73N9O9S2

1068.35 Matsumoto et al., 2001a

MetO-Leu-Met-Pro-Phe-Phe-Trp-Val

C55H73N9O9S2

1068.35 Reaney et al., 2013,

[1–8-NαC],[1-MetO,3-MetO]-CLF CLF

MetO-Leu-MetO-Pro-Phe-Phe-Trp-Val

C55H73N9O10S2

1084.35 Matsumoto et al., 2001a

15 16 17 18

[1–8-NαC]-CLG [1–8-NαC],[3-MetO]-CLG [1–8-NαC],[1-MetO]-CLG [1–8-NαC],[1-MetO,3-MetO]-CLG

Met-Leu-Met-Pro-Phe-Phe-Trp-Ile Met-Leu-MetO-Pro-Phe-Phe-Trp-Ile MetO-Leu-Met-Pro-Phe-Phe-Trp-Ile MetO-Leu-MetO-Pro-Phe-Phe-Trp-Ile

C56H75N9O8S2 C56H75N9O9S2 C56H75N9O9S2 C56H75N9O10S2

1066.38 1082.38 1082.38 1098.38

19

[1–9-NC]-CLQ

C51H76N8O8S

M AN U

TE D

CLG, CLM CLN CLH CLG

SC

C51H76N8O9S C51H76N8O10S C55H73N9O8S2

1080.34 Olivia, 2013 2001, Stefanowicz, 2004, 961.26 Stefanowicz, Okinyo-Owiti et al., 2014 977.26 Morita et al., 1999 993.26 Matsumoto et al., 2001b 1051.50 Stefanowicz, 2001, Stefanowicz, 2004

Stefanowicz, 2001, Stefanowicz, 2004 Stefanowicz, 2004 Matsumoto et al., 2001a Matsumoto et al., 2001a

AC C

EP

[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],[120 [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.

AC C

EP

TE D

M AN U

SC

RI PT

• The biological activity and ribosomal origin of flaxseed orbitides are reviewed.