Hypertension afflicts over 7.3 million Canadians [1] and is the number-one modifiable risk factor for death and disability globally [2], accounting for 13.5% of all global premature deaths [3]. Over the last 25 years, treatments to control hypertension have increased markedly in Canada [4]; however, one third of the diagnosed hypertensive population remains uncontrolled and many people remain undiagnosed. Uncontrolled hypertension can lead to a spectrum of diseases including stroke, coronary artery disease, as well as heart and kidney failure [5].

The exact etiology of the development and progression of primary hypertension remains uncertain; however, both genetic and lifestyle factors, including diet in particular, have been implicated. Many interconnected contributing factors have been proposed to explain the increase in blood pressure (BP) leading to hypertension. Factors include over-activation of the renin-angiotensin-aldosterone system (RAAS), endothelial dysfunction, elevated sympathetic activation, and other genetic predispositions [6–8]. Current therapeutic agents for BP control have proven to be reasonably effective; however, the incidence of mortality due to high BP is still on the rise [9]. Consequently, an important need exists for alternative therapeutic strategies to prevent or manage hypertension.

Lifestyle interventions that have been shown to prevent hypertension include maintaining a healthy body weight, taking moderate physical activity, eating a low-sodium diet, reducing alcohol consumption, and adherence to a diet rich in fruit and vegetables [10]. Another emerging potential therapeutic lifestyle strategy to prevent or manage hypertension is increasing dietary protein intake [11, 12]. Both increases in dietary protein intake as a macronutrient [12–16], and as smaller doses as bioactive peptides [17–19], have shown the potential to reduce BP. Indeed, evidence exists that plant-based protein sources may be more effective than animal sources; however, these data stem mainly from observational trials and are inconsistent [11, 20]. Randomized controlled trials comparing plant-based protein sources to animal-based protein sources are limited [12, 21–23]. An essential plant-based protein source that may have anti-hypertensive properties is hemp seed.

Hemp seed (Cannabis sativa L.) utilization into foods has been limited due to legislation restricting the cultivation of all Cannabis species on account of the presence of varieties high in the psychoactive compound tetrahydrocannabinol (THC). However, as of 1998 in Canada, cultivation of hemp seed containing < 0.3% THC has been allowed. Despite heightened consumer demand for hemp food products, due to historical restrictions on the growing of hemp, the nutritional science literature on hemp-seed protein (HSP) remains limited [24]. HSP has a unique amino-acid profile, with high levels of arginine, which makes HSP an ideal protein to consume for BP-reducing effect, or from which to make BP-reducing bioactive peptides [25].

Both observational and intervention trials have identified associations between dietary protein intake and BP [11, 12, 22]. A meta-analysis of 40 randomized controlled trials demonstrated that replacing a median of 40 g/day of dietary carbohydrate with dietary protein is associated with small reductions in both systolic − 1.76 mmHg (95% confidence interval (CI) − 2.33, − 1.20) and diastolic − 1.15 mmHg (95% CI − 1.59, − 0.71) BP (SBP and DBP, respectively) [22]. The meta-analysis did not find a difference between animal- and plant-based-protein sources in terms of BP-lowering effect. However, trials specifically comparing animal proteins to vegetable proteins are limited and have inconsistent results.

The anti-hypertensive effects of protein may not only be related to the amount of protein in the diet but may also be related to the type of protein. Whether a protein is animal or plant based is likely not as important as the type of amino acids contained in the protein source in terms of BP-lowering ability. Animal sources of protein are considered complete proteins, in that they contain an adequate proportion of all nine essential amino acids necessary for human dietary needs. Most vegetable sources individually are not considered complete because they are limited in one or more of the essential amino acids. This is important because the specific amino-acid content of a protein is likely critical to its anti-hypertensive potential. Additionally, different protein sources may affect BP through alternate mechanisms depending on their amino-acid content. Specific amino acids which have been identified as having potential BP-reducing properties include arginine, cysteine, tryptophan and glutamic acid [26].

Casein is the main protein found in cow’s milk [27], with whey protein making up most of the remaining protein. Pal and Ellis [28] investigated the effects of consuming casein or whey protein, compared to glucose, for 12 weeks in 70 men and women using a randomized, parallel-designed trial. Whey- and casein-protein consumption lowered SBP and DBP compared to baseline after 12 weeks and lowered DBP compared to the glucose control. No difference in BP was seen between the two milk-protein fractions. Soybean protein, which has been well-studied for its potential BP-reducing effects [15, 16, 29], is a representative vegetable protein with a high arginine content. This high arginine content may be responsible for some of soybean protein’s anti-hypertensive properties [26]. Unlike soybean, HSP consumption has not been well-characterized in terms of its effects on BP. HSP has even higher concentrations of arginine than soy, casein or whey protein, which may make it an ideal candidate to evaluate for BP control [30]. Consumer demand for hemp food products is growing and health food retailers are looking to add hemp foods to their product offerings. However, hemp foods, unlike casein, whey and soy, have a very limited database of peer-reviewed publications [31–34] outlining the health benefits of consumption due to restrictions in hemp cultivation, which have only recently been lifted in many countries.

Arginine, conditionally an essential amino acid depending on age and health [35], has been shown to reduce BP when supplemented in the diet [35, 36]. The main source of endogenous nitric oxide (NO) is L-arginine, which is oxidized to NO and citrulline by the action of endothelial NO synthase (eNOS) [37]. However, some proteins with incorporated arginine can undergo methylation and, with subsequent proteolysis, can yield methyl-arginine compounds such as asymmetric dimethylarginine (ADMA). ADMA is an endogenous inhibitor of NO synthase and has been shown to reduce the sensitivity of NO synthase to L-arginine. However, ADMA inhibition of NO synthase may be overcome with increased dietary arginine consumption. Additionally, arginine rapidly undergoes catabolism by arginase, a metalloenzyme, which hydrolyzes L-arginine to L-ornithine and urea [38]. Arginase is, therefore, a key regulator of NO availability, and arginase inhibitors are being investigated for their therapeutic potential in the treatment of endothelial function and hypertension [39, 40]. As with ADMA, additional dietary arginine consumption may be able to overcome arginase activity, which limits arginine availability for eNOS and NO production.

Typical sources of dietary arginine are meat, fish and poultry; however, soy protein isolate and HSP also possess higher levels of this amino acid [30, 41], while milk protein is relatively low in arginine [42]. Food-based arginine is well-tolerated with few side effects, making it a better source of arginine than free L-arginine which has been associated with nausea, gastrointestinal discomfort and diarrhea [37]. HSP, due to its high arginine content, is, therefore, a protein source, which should be investigated for its anti-hypertensive effects.

Girgih et al. [25] compared the effects of HSP isolate on the prevention and treatment of hypertension in spontaneously hypertensive rats (SHRs). The replacement of 1% casein on a dry-weight basis in the rat diet with HSP isolate was able to prevent the increase in SBP seen in young growing SHRs over 8 weeks and decreased SBP in adult SHRs with established hypertension. Decreased plasma renin concentrations and angiotensin-converting enzyme (ACE) activity were observed with the HSP feeding. Additionally, Girgih et al. [25] produced an HSP hydrolysate from the enzymatic hydrolysis of HSP isolate. Protein hydrolysates contain bioactive peptides, which are typically produced by enzymatic hydrolysis of food proteins to release short peptide sequences [43]. However, bioactive peptides can also be produced endogenously through the digestion of dietary proteins [19]. Numerous bioactive peptides have been shown to modulate RAAS via inhibition of ACE and renin activity [44]. The HSP-derived bioactive peptides made by Girgih et al. [25] were able to lower SBP in adult SHRs with established hypertension and decreased SBP in young growing SHRs to a greater extent than the HSP isolate when substituted for casein at 1%. The HSP-derived bioactive peptides also reduced renin concentrations and ACE activity more than did the HSP isolate in the SHRs, and even reduced plasma ACE activity in the normotensive control rats.

Since there was no difference in the degree of reduction in SBP in the adult SHRs between HSP isolate and bioactive peptide, but the reduction in ACE activity was much greater with the bioactive peptide [45], it was hypothesized that the intake of HSP isolate was likely lowering BP via a different mechanism than the peptide [25]. Indeed, the marked difference in the arginine content between the HSP isolate and the hemp seed protein hydrolysate (HPH) could be responsible for the differing hypotensive mechanisms. The HSP isolate contained more than six times the arginine in the HSH-derived bioactive peptides [25]. However, none of the rats were fed a combination of the HSP isolate with additional HSP bioactive peptides to investigate whether the BP-reducing effects would be additive or synergistic.

Bioactive peptides are specific, short amino-acid sequences that remain inactive when bonded to other amino acids within the primary structure of a food protein, but after release via hydrolysis, the free forms of these peptides can affect biological processes. Bioactive peptides have been shown to act as anti-hypertensive agents by inhibiting in vitro and in vivo renin, ACE and angiotensin-II receptor activities in addition to enhancing blood NO levels [44]. Therefore, there is an interest in characterizing these natural anti-hypertensive compounds so that they can serve as alternatives to anti-hypertensive drugs. Bioactive peptides have the advantage of specificity, with limited side effects, potency and low toxicity such that they can be consumed at high doses [44, 46, 47]. Different types of bioactive peptides, derived from numerous sources including dairy, meat, fish, poultry, canola, buckwheat, algae and hemp, have shown hypotensive effects in animal models [43, 44].

However, only a limited number of the bioactive peptides that have been characterized in animals and in vivo have subsequently been tested in human trials [19]. Most human trials investigating dietary bioactive peptides have been derived from dairy products, with the results from casein- and whey-derived bioactive-peptide feeding trials being mixed [19, 48]. A meta-analysis of human trials investigating dairy-derived bioactive peptides found that the consumption of 2–5 g of bioactive peptides a day could yield approximately 5-mmHg and 2-mmHg reductions in SBP and DBP, respectively. It has been suggested that the amino-acid composition of the bioactive peptides may be critical to the ability of the peptide to inhibit ACE activity. Bioactive peptides with a positive charge due to arginine or lysine residues are thought to be being beneficial for ACE inhibition [49]. A 3-week crossover human intervention trial conducted by our team fed 3 g per day of pea-protein hydrolysate and led to a 5–6 mmHg reduction in SBP in the second and third weeks compared to placebo [18]. To our knowledge the addition of bioactive peptides on top of increased dietary protein has not been previously investigated in humans.

While there is good evidence that replacement of dietary carbohydrate with protein has anti- hypertensive effects [11–13, 22], numerous gaps in the research still remain. First, comparisons of plant versus animal sources of dietary protein, or high-arginine versus low-arginine sources of dietary protein are limited [11, 22]. Second, the effects of additional dietary protein, where it does not explicitly replace carbohydrate, is not known [22]. Third, human trials investigating HSP consumption are limited [32]. Fourth, human trials involving non-dairy-derived bioactive peptides are limited [19]. Lastly, no trials investigating the potential benefit of increased dietary protein plus bioactive peptides have been completed to our knowledge. To address these knowledge gaps, we propose a clinical trial that would evaluate the anti-hypertensive potential of consuming whole HSP, and HSP+, compared to casein protein using a randomized, double-blinded and crossover design. This design should be able to address the knowledge gaps identified above. Although little work has been done in humans in this specific area, for the following reasons we were not able to perform a pilot trial before the main study; the long-term nature of this study was the main factor that prevented us considering a pilot study as the first phase. The recruitment of 35 participants and obtaining their consent to enter a long-term trial was a challenge so, recruitment of more people as a pilot group would increase the duration of study. So, we had to recruit more people to start the main study or have the pilot group on an extra washout period to be able to start the trial over. These participants might lose their interest in continuing with the study and the drop-out rate would increase. In terms of testing the procedure, we mainly followed similar clinical trials with other treatments to evaluate the acceptability of the procedure in the following criteria; treatment consumption frequency, treatment consumption under supervision, free-living trial with basic recommendations for physical activity and alcohol consumption, method and frequency of the outcome measurements, method of randomization and follow-up with participants. Acceptability of the treatments were assessed using a taste and texture testing with random participants. Sample size was calculated based on the expected change in the main outcome using similar studies considered 24-h SBP as their main outcome.