The effects of seed from Linum usitatissimum cultivar with increased phenylpropanoid compounds and hydrolysable tannin in a high cholesterol-fed rabbit

According to the World Health Organization, cardiovascular disease (CVD) is the leading cause of death worldwide, which represents 30% of all global deaths [1]. Dyslipidemia, including higher serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) and lower high-density lipoprotein cholesterol (HDL-C), is a major risk factor for atherosclerosis. When atherosclerotic processes take hold in the arteries that supply blood to the heart, the condition becomes coronary artery disease (CAD). Moreover, inflammations and elevated platelet counts are associated with poor cardiovascular outcomes [2]. Dietary fat is considered one of the most important factors associated with blood lipid metabolism and plays a significant role in the cause and prevention of atherosclerosis [3]. In addition to hypercholesterolemia, hypertriglyceridemia may be another factor for diseases. Because cholesterol lowering is a major target for reducing CVD risk, dietary interventions to reduce TC and TG levels in individuals with borderline dyslipidemia and obesity without overall cardiovascular risk are becoming mandatory [3]. Large-scale clinical trials using statins have shown a significant reduction in cardiovascular events, mainly due to the lowering of plasma concentrations of LDL-C; however, the capacity of statins to prevent cardiovascular events are still limited to 30% to 40% of the patients treated even when intensive statin therapy is used [3, 4]. Nutraceuticals and functional food ingredients that are beneficial to vascular health may represent useful compounds that are able to reduce the overall cardiovascular risk induced by dyslipidaemia by acting parallel to statins or as an adjuvants [5].

Flax (Linum usitatissimum L.) is among the oldest plants with great significance in food production, healthcare, pharmaceutics, and industry as a source of fibre and oil. Moreover, flaxseed is one of the key sources of phytochemicals in the functional food area [6]. In addition to being one of the richest sources of ω-3 fatty acids and lignans, flaxseed is an excellent source of high-quality protein and soluble fibre and has considerable potential as a source of phenolic compounds [6]. It is established that polyunsaturated fatty acids (PUFA) like ω-3 (α-linolenic acid, ALA) and ω-6 (linoleic acid, LA) play important roles in human health and disease. Both are considered essential because they are not endogenously synthesized and must be obtained from the diet [7].

Due to the opposing effects of ω-3 and ω-6 fatty acids, a healthy diet should contain a balanced ω-6: ω-3 ratio. A balanced ω-6/ω-3 ratio 1–2/1 is one of the most important dietary. Human beings evolved eating a diet with a ω-6:ω-3 ratio of about 1:1 but modern diets exhibit LA: ALA ratios ranging between 15:1 to 30:1 [8, 9].

Excessive amounts of ω-6 and a very high ω-6/ω-3 ratio, promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased level of ω-3 (a low ω-6/ω-3 ratio) exert suppressive effects [8, 10, 11]. L. usitatissimum cv. Linola seed exhibits an extremely high ω-6 to a ω-3 ratio (~ 31:1), so its health beneficial effect is rather limited [12]. However, new genetically modified L. usitatissimum L. namely W86 show the ~ 30-fold increase in α-linolenic acid level compared to the Linola cultivar [12]. Thus, the ratio of ω-6 to ω-3 in seed from W86 is about 1.3:1 [12], which is quite close to that recommended for the human diet [9]. Obviously, the bioavailability of ALA is dependent on the type of flax ingested [13].

Although, there are no data that suggest the LDL-C increases associated with some ω-3 fatty acid formulations lead to adverse outcomes, these increases in LDL-C may compromise the achievement of lipid targets; thus, there is a need for agents that can lower TG levels without increasing LDL-C levels [7] especially in individuals with high cholesterol levels.

Perhaps most importantly, there are some human conditions that cannot be adequately modelled by invertebrate or rodent species we chose rabbits. These animals are phylogenetically closer to primates than rodents and further offer a more diverse genetic background than inbred and outbred rodent strains [14]. The special characteristics of rabbit anatomy and physiology make it uniquely suitable for the study of hypercholesterolemia and atherosclerosis through diet which makes the model a better overall approximation to humans [15]. The rabbit model is highly reproducible with minimal variation between animals in a single laboratory and between laboratories.

Therefore, the purpose of presenting research was to identify the effect of flaxseed, a bioactive food compound on rabbits with high TC and TG levels. Particularly, this study aims to verify the clinical outcome of the change in the lipid profile due to the use of seeds from W86, a genetically modified Linum usitatissimum cv. Linola.

Moreover, the second objective was to determine if use of flax seeds with increase in α-linolenic acid level, and increased phenylpropanoid compounds, and hydrolysable tannin [12] has deleterious effects of red blood cells (RBCs), white blood cells (WBCs) and platelets and if these effects are different in normal and hypercholesterolemic rabbits.

Naturally derived dietary supplements capable of modifying dyslipidemias are becoming more popular and attractive to patients. Among them are Linum usitatissimum L., the richest known source of PUFA, and lignans [6]. Most commonly used forms include flaxseed, flaxseed flour and flaxseed oil [37–39]. Moreover, nutraceuticals are an interesting option for patients with dyslipidemia owing to their safety, tolerability, and ability to lower plasma lipid levels and play a peculiar role in ameliorating human dyslipidemia [5, 40]. These roles of nutraceuticals were detailed reviewed by Scicchitano, et al. [5]. In their review, the authors focused on the mechanism of action of nutraceuticals and functional food ingredients on lipids and their role in the management of lipid disorders.

The aim of this study was to assess the impact of W86 flaxseed with increased phenylpropanoid compounds and hydrolysable tannin on hematological parameters and atherogenic indexes in a hypercholesterolemic diet based on the results in alterations in the lipid profile. Moreover, we investigated the effect of short-term use of flaxseed, with special emphasis on variety W86, in rabbits on the hemopoietic and lymphopoietic system.

Our results show that exposure of rabbits to W86 flaxseed during the full fattening period did not have a major impact on the body weight and feed intake. However, the feed intake in the experimental groups was lower at the end of this period than in 6 weeks and significantly greater (P < 0.05) decline was observed in the LIN group than in W86 group during the experiment.

In spite of the well-known variability of hematological parameters in rabbits with regard to breed-related and individual differences, the measured values of RBC, HGB, and PCV count in this research occur within the range of reference physiologic values for rabbits [29]. Although, the values of the RBC in blood in three groups (CHOL, LIN, and W86), were near the bottom level of the range stipulated for adult New Zealand white rabbits (4–7.2 × 10¹² L^− 1), in our opinion the RBC, HGB and PCV values just like MCV, MCH, MCHC, and RDW were physiological for the age of the tested rabbits [28, 29]. Moreover, demonstrated results suggest that use of W86 flaxseed does not have deleterious effects on the hemopoietic system.

Studies on HLR, PLR, and MPV have grown recently following the discovery of their immense values in prediction and prognosis of many medical conditions and are potent markers of inflammation which underlies the basic pathologies of various diseases [41–43]. Furthermore, the chronic inflammatory response is a critical element in the pathogenesis of atherosclerosis and is associated with the production of lymphocytes and PLT. Elevated activated platelets count promotes atherothrombosis, atherosclerosis formation, progression, and destabilization of atherosclerotic plaques, and are associated with an incidence of CAD [44, 45]. The present results demonstrated that there was an increase in platelet counts in both CHOL and LIN groups. However, PLT values in rabbits supplemented with W86 flaxseed at week 10 were the same as in the CTRL group.

Cardiovascular events are primarily associated with a high circulating platelet count and low blood lymphocytes. Moreover, the PLR has come into use recently as an indicator of the balance between inflammation and thrombosis and act as an effective biomarker to predict CVD risk [44]. Furthermore, PLR gives an idea about both the aggregation and inflammation pathways and it may be more valuable than either platelet or lymphocyte count alone in the prediction of coronary atherosclerotic burden [46]. High PLR values are associated with cardiovascular diseases and adverse outcomes [47]. It has been reported that NLR (analogue of HLR) is a marker for progression of atherosclerosis in coronary artery disease and a marker for possible cardiac events and mortality in patients with stable coronary artery disease [34, 48]. The present results demonstrated that W86 flaxseed significantly decreased PLR and HRL ratios in rabbits fed high cholesterol diet. Moreover, W86 flaxseed significantly decreased MPVLR in contrast to parental Linola flaxseed (group LIN). Several studies reviewed by Nording, et al. [32] support the roles of platelets in chronic low-grade inflammation driving atherosclerosis. In our study, we found that W86 flaxseed positively influenced on the PLT count. The PLT of the high cholesterol-W86 fed groups was statistically significantly lower than that of the CHOL, and LIN groups and similar to CTRL. This result suggests that W86 flaxseed with increased phenylpropanoid compounds, hydrolysable tannin, and the well balanced ω-6/ω-3 ratio can prevent thrombocytosis and eventually atherosclerotic lesions.

The rabbit fed with typical laboratory chow diets contain typical plasma cholesterol concentrations in the range of ~ 0.78–0.91 mmol L^− 1 as we showed in CTRL group [29, 49]. The rabbit rapidly develops severe hypercholesterolemia leading to premature atherosclerosis in response to dietary manipulation. Diets containing high cholesterol amounts (0.3–0.5%) have demonstrated that the hypercholesterolemic rabbit can develop complex, advanced lesions that more closely resemble those found in humans [50, 51]. In this study, a high-cholesterol diet resulted in hypercholesterolemia in rabbits, and blood TG and TC levels were increased compared to that measured in CTRL counterparts group.

The increased use of ALA is a powerful example of one such nutritional strategy that may produce significant cardiovascular benefits. Based on the results of clinical trials, epidemiological investigations and experimental studies, ingestion of ALA has been suggested to have a positive impact on CVD. Increased linoleic intake has been shown to promote platelet aggregation and oxidation of LDL. Renaud, et al. [52] demonstrated that α-linolenic acid has antithrombotic properties and may also be antiarrhythmic. Moreover, α-linolenic acid is a precursor of other omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which may have independent beneficial effects [53]. Harper, et al. [54] showed that patients which received linseed capsules demonstrated a 25% increase in the plasma values of docosapentaenoic acid (DPA), a cardioprotective fatty acid in the linseed group while the olive oil group showed no changes.

The HDL-C is recently gaining sample scientific attention due to its antiatherogenic properties providing cardioprotection. The relationship between HDL-C and cardiovascular diseases is primarily due to cholesterol efflux facilitated by apolipoprotein A1 during the process called the reverse cholesterol transport and HDL-C acceptors or by diffusion leads to HDL-C mobilization from cell membrane to hepatic storage. In addition to this, HDL-C exerts antithrombotic activity by preventing platelet aggregation [55]. Furthermore, HDL-C prevents oxidative modification of LDL and blocks the pro-inflammatory effects of oxidized LDL [56]. However, Prasad, et al. [57] showed that HDL-C can also have proinflammatory effects. In the research of Bierenbaum, et al. [58] hyperglycaemic patients who received linseed together with vitamin E daily for 3 months exhibited a significant reduction in serum TC, LDL-C and serum lipid oxidation products while HDL-C did not change at all. In this trial, statistically insignificant changes (P = 0.09) between group CTRL, LIN, and W86 were observed in HDL-C levels in rabbits at week 10 in contrast to group CHOL. However, this statistically significant increase should be rather considered together with generally high TC and lipoprotein content in rabbit fed cholesterol without flaxseed.

Peng, et al. [59] stated in their review, that a large number of studies have shown that hypertriglyceridemia contributes to the development and progression of atherosclerosis. However, the proatherogenic mechanism of hypertriglyceridemia seems rather complicated and needs to be further investigated. Based on current knowledge and the evidence of clinical studies, controlling and lowering plasma TG levels is one of the important measures to further reduce the residual risk of CVD events in atherosclerotic CAD patients. Stuglin and Prasad [60] suggested that the linseed increased the TG level while the others demonstrated the reduction in TG concentration induced by the linseed [61]. In our study, TG concentration was lowered through both linseed supplementation in contrast to CHOL group. However, herein we show that W86 flaxseed showed more beneficial effect than flaxseed from the parental variety. What is more important is that W86 flaxseed in contrast to parental variety reduced not only TG but also TC level in hypercholesterolemic rabbits (P = 0.034). This result is opposed to the previous observation of Dupasquier, et al. [62]. These authors showed that flaxseed in the diet reduced serum TG but had no effect on serum TC in rabbits. Moreover, Prasad [63] reported an increase in serum TC and no change in serum TG in rabbits on a flaxseed diet as we have shown for Linola flaxseed (group LIN).

The non-HDL-C incorporates the harmful elements of the lipoprotein profile to include TG rich remnant particles from VLDL and IDL, as well as LDL-cholesterol. It is also better for monitoring response to treatments such as lifestyle changes and medication. The non-HDL-C has been recommended as the target for lipid modification in guidelines by the National Institute for Health and Care Excellence. Moreover, the non-HDL-C includes all ApoB containing lipoproteins and for this reason, the non-HDL-C/HDL-C ratio provides cardiovascular risk stratification similar to the ApoB/ApoA1 ratio in diabetics as a comparison with reference lipid marker [64]. Herein, we provided evidence for the non-HDL-C, VLDL-C and LDL-C lowering properties of W86 flaxseed in contrast to Linola seeds.

Atherogenic indexes are considered as a better indicator of coronary heart disease risk than individual lipoprotein concentration. The results of the present study showed that W86 flaxseed also caused a significant reduction in all calculated atherogenic indexes. Furthermore, this beneficial effect is more valuable than was observed for Linola.

The high-fat diet also causes oxidative stress, thus, increases oxidation of LDL which plays a key role in the genesis of atherosclerosis but antioxidants such as flavonoids are known to effectively prevent this kind of damage [65]. The presence of the strong antioxidant in the flaxseed such as anthocyanins, flavones, and proanthocyanins may also offer additional benefit against oxidative stress caused by high cholesterol. Flaxseed from W86 cultivar contains more lignans and tannins than its parental Linola (Additional file 1: Table S1). The most remarkable lignan is secoisolariciresinol diglucoside (SDG), a very potent antioxidant [66]. The beneficial effect of flax lignan complex was reviewed by Prasad [67]. Moreover, Gato, et al. [68] indicate that tannin is a useful food material for treating hypercholesterolemia while Si, et al. [69] reported antioxidant activity of tannins. In the present study, we used SOD radical scavenging properties to examine the antioxidant blood activity. Nevertheless, another recently developed method measuring the total antioxidant status (TAS) provides the evaluation efficiency of the biological interactions between individual antioxidant species such as antioxidant enzymes and low-molecular-weight antioxidants and also a measure of the capacity of biological systems to withstand oxidative attack [70]. Statistical analysis showed significant differences among study groups in the levels of TAS and SOD between 6 and 10 weeks and in all groups both parameters decreased after 10 weeks. No statistically significant differences were found in serum TAS values among groups after 6 weeks. In group LIN we demonstrated a drastic decrease of TAS at 10 weeks in contrast to a W86 group. The present findings are in agreement with previous observations showed that flaxseed oil had no effect on the activity of SOD or flaxseed oil increased the levels of lipid peroxidation and that this effect was associated with the reduction in SOD activity [71, 72]. The research data suggest that antioxidant activity of flaxseed seems to be due to the SDG present in these components [73]. The findings of the present investigation confirm those reports and expand previous observations by demonstrating that W86 flaxseed richer in phenylpropanoid compounds, flavonoids and hydrolysable tannin than Linola can better slow the progression of atherosclerosis by reducing oxidative stress. Additionally, the amount of ALA that is present in the diet is very small, and dietary supplementation is needed whenever increased oxidative stress in the body leads to clinically significant consumption of endogenous antioxidants.

Moreover, McCullough, et al. [74] demonstrated that ALA content of flaxseed is associated with an induction of adipose leptin expression encoded by the “obese” gene. Leptin protein levels were elevated in animals taking diet supplemented with 10% flaxseed. Changes in leptin expression were strongly and positively correlated with adipose ALA levels and inversely correlated with risk of atherosclerosis. W86 flaxseed contains ~ 30-fold excess of ALA than parental Linola and may be a good supplement to obesity prevention but this suggestion should be further investigated.

The present study suggested that W86 flaxseed from genetically modified Linola affect lipid metabolism and lower atherogenic indexes in rabbits. Accurate documentation of the therapeutic effects of W86 flaxseed and its components that contribute uniquely to disease prevention health protection and as a deterrent to degenerative diseases will increase its potential for use as a functional food and food ingredient. Hence, we can conclude that W86 flaxseed provides a safe and relatively inexpensive alternative for reducing cholesterol and thereby the risk of CVD and be a good supplement in atherosclerosis and obesity prevention. Moreover, we believe that this result supports the hypothesis of about the use of nutraceuticals in primary cardiovascular prevention, protocols to reduce the overall burden of cardiovascular disease morbidity and mortality as was suggested by Scicchitano, et al. [5]. Nevertheless, further studies are needed to implement the actual findings associated with this hypothesis.