Hemp seed protein and its hydrolysate compared with casein protein consumption in adults with hypertension: a double-blind crossover study

Abstract

Background

The effects of consuming hemp seed protein (HSP) as well as its hydrolysate-derived bioactive peptide (HSP+) on blood pressure (BP) has not, to our knowledge, been investigated in humans.

Objectives

We aimed to investigate how consumption of HSP and its hydrolysate modulates 24-h systolic (SBP) and diastolic BP (DBP) and plasma biomarkers of BP compared with casein.

Methods

In a double-blind, randomized, crossover design trial, 35 adults who had mild hypertension with SBP between 130 and 160 mmHg and DBP ≤110 mmHg were recruited. Participants were randomly assigned to varying sequences of 3 6-wk treatments, 50 g casein/d, 50 g HSP/d, or 45 g HSP plus 5 g HSP-derived bioactive peptides/d (HSP+), separated by a 2-wk washout period. Treatment effects were assessed with a linear mixed model with repeated measures.

Results

Compared with casein, after HSP+ consumption, 24-h SBP and 24-h DBP decreased from 135.1 and 80.0 mmHg to 128.1 ± 1.6 (P < 0.0001) and 76.0 ± 1.4 mmHg (P < 0.0001), respectively, whereas these values were 133.5 ± 1.6 and 78.9 ± 1.4 mmHg after HSP consumption (P < 0.0001). There were no differences between the HSP and HSP+ consumption in plasma angiotensin-converting enzyme (ACE) activity, renin, or nitric oxide (NO) concentrations. However, these 2 treatments were able to lower both ACE and renin activities and raise NO concentration in plasma compared with casein.

Conclusions

These results suggest that hemp protein consumption, as well as in combination with bioactive peptides, may have a role in the dietary management of hypertension.

This trial was registered at clinicaltrials.gov as NCT03508895.

Introduction

Hypertension is a chronic health issue described as blood pressure (BP) consistently above the normal values (systolic BP [SBP] ≥130 and/or diastolic BP [DBP] ≥80 mmHg) [1,2]. Globally, hypertension affects >30% of adults and causes >8.5 million deaths from vascular and renal diseases [3]. The renin-angiotensin-aldosterone system (RAAS) is the primary hormonal system in body involved in BP regulation. Once renin is secreted from the kidneys into the blood, it converts liver angiotensinogen to angiotensin (Ang) I. Transformation of this physiologically inactive hormone to Ang II is catalyzed by angiotensin-converting enzyme (ACE) while passing through the lungs and kidneys [4,5]. Ang II exerts various biological functions that conclusively result in vasoconstriction [6]. Nitric oxide (NO), known as a vasodilation mediator, is another powerful regulator of BP. Once produced by endothelial NO synthase from L-arginine in the vascular endothelium, NO decreases vascular tone and increases blood flow [7,8].

The pharmaceutical industry has developed BP medications in particular by utilizing ACE inhibition to block the RAAS. ACE inhibitors such as enalapril, captopril, and lisinopril are the most commonly prescribed medications in the treatment of cardiovascular and renal diseases and hypertension [9,10]. Although pharmacologically effective, long-term use of these agents has been associated with adverse effects from cough to more serious complications, such as hyperkalemia and angioedema [11]. The American Heart Association estimated that the national annual direct cost of hypertension in 2012–2013 was $47.3 billion, where about half of this cost was for antihypertensive medications [12]. Alternatives to taking medicine have been identified as desirable by many patients [13]. Thus, innovative yet effective nonpharmacologic approaches to reducing hypertension are of interest, particularly in patients considered to be at high risk of negative issues from combination therapy or those desiring to minimize their medication dose [14].

Nonpharmacologic food-based interventions have gained increasing attention in ACE inhibition and subsequently hypertension prevention and control. The potential benefits of implementing nutrition interventions for hypertension may be cost effective with minor or no adverse reactions [15,16]. Dietary factors including greater consumption of protein have been strongly supported by epidemiologic studies in terms of their ability to lower BP [17]. However, the antihypertensive effects of dietary protein may not solely depend on its quantity but are also influenced by its source. A cohort study of 272 men in 5 y revealed a negative correlation between plant protein intake and BP [18]. The distinction between animal-based and plant-based proteins may not be as pivotal as the composition of amino acids within the protein source in terms of its BP-lowering capabilities. This distinction is significant because the specific amino acid composition is likely a crucial factor in determining the antihypertensive potential of a protein. Furthermore, different protein sources may impact BP through various mechanisms, depending on their amino acid profiles. Certain amino acids, such as arginine (Arg), cysteine, tryptophan, and glutamic acid, have been identified as potentially possessing BP-reducing properties [19].

Hemp seed (Cannabis sativa L.) is an emerging source of plant protein that is rich in Arg and may have hypotensive properties. Although hemp seed protein (HSP) is known to be abundant in arginine, its impact on BP has not been thoroughly characterized; therefore, the potential exists to utilize HSP for its antihypertensive properties or to produce the hypotensive bioactive peptides [20,21].

Studies have demonstrated some bioactive peptides obtained through enzymatic hydrolysis from food protein sources such as HSP exert ACE and renin inhibitory properties [22]. The hydrolysis of HSP could produce bioactive peptides that perform biological activities such as antihypertensive effects [9]. In an animal study, oral administration of 200 mg HSP hydrolysate (HPH)/kg body weight to spontaneously hypertensive rats (SHRs) lowered SBP by 30 mmHg and demonstrated ACE and renin inhibitory effects [23]. In addition, feeding growing SHRs with HPH prevented the expected increase in SBP compared with casein (∼120 mmHg and 158 mmHg, respectively) [24]. In a meta-analysis that included 17 randomized controlled trials (RCTs) examining how dietary protein affects BP, the interventions involved protein intake ranging from 26 to 74 g/d, sourced from both animal and plant origins [25]. The objective of this study was to investigate the ability of 50 g HSP/d and 45 g HSP plus 5 g of its hydrolysate-derived bioactive peptides/d on 24-h BP in a double-blind randomized crossover trial.

Methods

Study design

A randomized, double-blind, crossover trial was designed with 3 6-wk treatment periods and 2-wk washouts between periods during which the volunteers followed their regular diet. Methodological details of this study have been published [26]. This trial was conducted at the Clinical Nutrition Research Unit of the Richardson Centre for Food Technology and Research (RCFTR), University of Manitoba. Participant randomization was completed in a 1:1:1 ratio by an external research assistant through sealed envelopes. A 3 × 3 Latin-square design followed by a random number generator was used to randomly assign the eligible participants to the following sequences: ABC, ACB, BAC, BCA, CAB, and CBA in which each letter (A, B, and C) had been randomly assigned to 1 of the 3 treatments. The research participants and the researchers were unaware of which letter was assigned to which treatment. The trial was conducted in compliance with the principles of good clinical practice. The research protocol was reviewed and approved by the University of Manitoba’s Biomedical Research Ethics Board in Winnipeg, Manitoba, Canada (protocol no. B2016:125). In addition, the Non-prescription Health Products Directorate of Health Canada approved this study with the protocol number HEMP BP 001. The trial was registered on the Registry Databank at clinicaltrials.gov as NCT03508895.

Study population

Thirty-five volunteers with hypertension aged 18–75 y were screened and recruited from Winnipeg, Manitoba, Canada. News articles in Winnipeg Free Press newspaper and study posters were published and circulated, and University of Manitoba e-mail lists were used to recruit for the study. Volunteers were initially screened over the phone using a brief screening questionnaire where they were asked about their general health and medical history. Then, potentially eligible participants were scheduled for an in-person screening process at the RCFTR to have their BP and blood samples taken. Before the in-person screening started, the participants were provided written informed consent forms by the research coordinator to sign. Then, while they were fasting, 20-mL blood samples were taken by a phlebotomist via venipuncture to test blood biomarkers related to the exclusion criteria.

Individuals with SBP 130-160 mmHg, DBP ≤110 mmHg, and BMI of 18.5–40 kg/m² were recruited. For those participants who were on medication, to be included in the study, they needed to be on a stable type and dosage of medication for ≥3 mo. Participants were excluded if their blood test results reported sodium concentration of <134 mmol/L or >148 mmol/L and fasting glucose >6.1 mmol/L. Individuals with any clinically significant biochemistry abnormalities, secondary hypertension, active cardiovascular disease, diabetes, a history of cancer or malignancy in the last 5 y, and gastrointestinal (GI) disorder were also excluded. Smoking, consuming alcohol >14 drinks/wk, people with child-bearing potential not using birth control, pregnancy, lactation, and having weight change ≥5 kg in the past 3 mo were also exclusion criteria. Finally, if determined as eligible, the study coordinator assigned the participants to interventions by opening a sealed envelope that contained a sequence of blinded treatments.

Both research coordinators and participants were blinded to the study treatments. The participants were instructed to avoid inconsistency in their physical activities and dietary patterns during the study and they were asked to perform their typical daily routines and abstain from prolonged strenuous exercise on measurement days. In addition, they were asked to limit their alcohol and caffeine consumption to ≤2 alcoholic beverages and <3 8-oz caffeinated beverages/d, respectively. We chose not to collect food recalls or records during the study to minimize participant burden and to avoid potential alterations in dietary behavior that might impact our study outcomes. Our primary outcome was to evaluate 24-h SBP, which was less likely to be influenced by minor variations in calorie or protein intake. Under the randomized crossover design, the participants were administered distinct treatments in a random order, effectively mitigating individual variability in diet and protein intake. By comparing each participant’s response to different treatments, we minimized the influence of individual differences in baseline diet on our study outcomes. The study screening and recruitment process was from April 2018 until November 2018, and the intervention periods and follow-up visits took place from May 2018 until May 2019. After trial commencement, we expanded the range of eligible participants to include those with DBP ≤110 mmHg instead of 100 mmHg to improve the recruitment rate.

Study intervention, treatment preparation, and masking

The protocol has been previously published [26]. In this trial, participants consumed their regular diet plus 50 g casein/d, 50 g HSP/d, or 45 g HSP with an added 5 g of bioactive peptides (HSP+)/d for 6 wk. The method used to prepare HSP hydrolysate (Supplemental Table 1) was developed to simulate GI tract digestion [23]. The treatments were in the form of smoothies, and each smoothie consisted of 25 g of protein from treatment powder mixed with frozen fruit, diet fruit juice, and sorbet, which were consumed twice a day (Supplemental Table 2). Each treatment contributed an average of 205 kcal to the daily calorie intake. The treatments were consumed under supervision twice a week at RCFTR, and the rest of the treatments were taken to be consumed at home. Furthermore, to ensure consistency in treatment preparation and for participants’ convenience, the treatments were prepared and provided to participants in single portions for consumption at home over the subsequent 2–3 d. They were instructed to store the treatments in the refrigerator and to mix them prior to consumption at home. A member of the RCFTR metabolic kitchen staff was responsible for preparing the treatments according to the protocol, and the treatments were served in single portions with identical stainless-steel cups and straws. The HSP powder for the study was provided by Manitoba Harvest (HEMP Pro 70 soluble hemp protein concentrate), whereas the casein protein powder was purchased from Nutrablend Foods. Casein and HSP powder both have a light tan color and look similar when combined with frozen fruit and sorbet in the smoothies. A treatment consumption log was given to the participants to enter the date and time when they consumed the smoothies. They were required to return the treatment containers on the next visit and the consumption checklist at the end of each treatment period to monitor for compliance. Returning the containers or being present for treatment consumption under supervision in ≥80% of the treatments per treatment period was defined as compliant. In addition, a GI tolerability questionnaire was completed by participants weekly during each intervention period to evaluate the possible adverse events on the GI tract or complaints about tolerability.

Blood sample collection

Samples of fasting blood (∼20 mL) were collected by a phlebotomist on the morning of day 1 and day 42 of each treatment period (6 time points). Other than treatment consumption, missing 2 consecutive sessions of blood sampling and measurement were considered noncompliance. Blood samples were drawn from the top of the forearm via venipuncture for the assessment of plasma biochemical markers. The participants were instructed to abstain from alcoholic beverages within 48 h prior to blood draws and food or caffeinated beverages within 12 h prior to blood draws during the trial phases. After blood collection, the samples were centrifuged at 1000 (g for 20 min at 4°C) to separate and aliquot plasma samples, which were then stored at −80°C for further analyses.

Outcome measurements

The following outcomes were measured and there was no change to trial outcomes after the trial commencement.

24-h SBP and DBP

The primary outcome of this study was 24-h SBP, it was measured on day 1 of the first treatment period and the last day of each period using ambulatory BP monitors (ABPMs, OnTrak, Spacelabs Healthcare Inc.) [27]. Participants were fitted with an ABPM to wear for a full day, and the continuous SBP and DBP measured over 24 h were recorded. In addition, the participants were asked to record the time and the activity they were involved in each time the ABPM went off in an ABPM log provided to them and to drop off the log with the ABPM after 24 h. The continuous SBP and DBP were measured every 30 min during the daytime and every 1 h during nighttime according to the participant’s sleep routine. Both written and verbal ABPM instructions were provided to the participants to minimize measurement errors. The report of the results was transferred to a computer and read by sentinel software. Of 36 measures, 27 readings (75% cutoff) were considered a successful wear period; otherwise, the participant was asked to wear the ABPM for another 24 h to obtain the minimum acceptable number of readings [28].

Determination of plasma ACE activity

The spectrophotometric method with furanacryloyl-L-phenylalanylglycylglycine as the substrate was used [23] with minor modifications, and the ACE enzyme (from rabbit lungs) was purchased from Sigma-Aldrich Canada to determine the plasma activity of ACE. Concisely, 1 mL 0.5 mM furanacryloyl-L-phenylalanylglycylglycine (dissolved in 50 mM Tris–HCl buffering solution containing 0.3 M NaCl, pH 7.5) was mixed with 20 μL plasma or ACE (different enzyme concentrations were prepared: 0.0313, 0.0625, 0.125, 0.25, and 0.5 U/mL) and 200 μL 50 mM Tris–HCl buffer. The absorption intensity was determined at 345 nm and was recorded for 2 min at 23°C in a spectrophotometer (BioTek PowerWave XS Microplate Reader). The standard curve was defined using the results expressed as change in absorbance per minute plotted against ACE enzyme concentration. Finally, a linear regression model of the standard curve was used to calculate the plasma ACE activity (units per milliliter).

Determination of plasma renin concentration

A pre-established fluorometric technique was used with slight changes [29]. A renin inhibitor screening assay kit was purchased from Cayman Chemical to quantify the plasma renin concentration. To prepare various concentrations (4.15, 8.3, 16.5, 33, 66, 132, and 250 μg protein/mL), renin was diluted with assay buffer containing 50 mM of Tris–HCl at pH 8.0 and 100 mM NaCl. A 96-well microplate was filled with 20 μL renin substrate and 160 μL assay buffer. The chemical reaction was activated by adding 10 μL plasma or each diluted renin solution followed by 10 s of shaking and 15 min incubation at 37°C in a fluorometric microplate reader (Spectra MAX Gemini; Molecular Devices). Afterward, fluorescence intensity (FI) was documented using emission spectra in the wavelength of 490 nm upon excitation at 340 nm, and the results were presented as change in FI per minute. The linear regression from the plot of change in FI per minute compared with renin concentration was used to obtain the standard curve. The change in FI per minute obtained from the regression equation for each plasma sample was used to calculate plasma renin concentration (micrograms per milliliter).

Determination of plasma NO concentration

The plasma NO concentration was determined using a nitrate/nitrite colorimetric assay kit (Cayman Chemical) according to the manufacturer’s protocol. Briefly, nitrate standard concentrations were prepared by mixing nitrate standard with assay buffer (0, 5, 10, 15, 20, 25, 30, and 35 μM). A 96-well plate was filled with the solutions in the following order: 80 μL plasma (diluted 1:1 with assay buffer), 10 μL enzyme cofactor mixture (containing 1.2 mL nitrate/nitrite assay buffer), and 10 μL nitrate reductase mixture (containing 1.2 mL nitrate/nitrite assay buffer) then incubated at room temperature for 1 h while covered by the plate cover. Afterward, 50 μL Griess reagent R1 and 50 μL Griess reagent R2 were added to the wells, and the plate was incubated at room temperature for 10 min for color development. The absorbance was then read at 550 nm in a spectrophotometer (BioTek PowerWave XS Microplate Reader). The plot of the absorbance at 550 nm was made, and the nitrate standard curve was used to determine NO products.

[Nitrate + Nitrite] (μM) = (A₅₅₀ – y-intercept) (200 μL) × dilution

Sample size and statistical analysis

A total sample size of 32 participants was determined to allow detection of a clinical 4.0 mmHg change in the mean 24-h SBP (primary outcome) with an estimated within-participant SD of 5.5 mmHg. The power to show the significant difference (type 1 error [α] of 5%) in 24-h SBP between groups was 0.80 [[30], [31], [32]]. A total recruitment goal of 35 participants was set to achieve the calculated sample size, which allowed for potential participant dropouts. Study outcomes were compared between treatments using endpoint values, and statistical analysis was performed using a mixed-effects model with repeated measures in SAS (OnDemand for Academics, version 9.04.01, SAS Institute Inc.) with treatment, period, and sequence as fixed factors and participant as a random effect [33]. Baseline values of the selected outcomes, period, sequence, and treatments were considered in the model as independent variables. There were no binary outcomes and no interim or adjusted analyses performed in this study. Results are presented as estimated least square means ± SEM for all values. Statistical significance was considered at P < 0.05 for all analyses. Pairwise comparisons of hemp treatments with casein were performed using Dunnett’s test, and a Bonferroni correction was used to account for multiple comparisons between the hemp treatments.

Results

Baseline characteristics of the participants

As shown in Figure 1, a total of 35 participants were randomly assigned to consume the treatments according to the assigned sequences. Four of 35 participants withdrew from the trial owing to schedule conflicts, traveling, and personal reasons. These 4 participants dropped out before the end of the first period and were excluded from the analyses. The study was completed with a total of 31 participants included in the outcome analyses. The baseline characteristics of these 31 participants are represented in Table 1. At screening, the mean ± SD age was 61.1 ± 9.3 y, the BMI was 28.5 ± 4.9 kg/m², and the waist circumference was 98.9 ± 12.6 cm. The office SBP was 145.6 ± 14.6 mmHg, and the office DBP was 89.9 ± 9.7 mmHg. Of 31 participants, 11 (35.5%) were on antihypertensive medications. We also assessed any possible adverse effect of protein consumption on GI tract. Twenty-nine participants reported no GI symptoms. Six participants reported mild flatulence and abdominal discomfort during the initial 2 wk of treatment consumption, but these side effects subsided within 2 wk of commencing the study.

FIGURE 1.

Flow chart of the participants at each stage of the trial. HSP, hemp seed protein; HSP+, HSP plus HSP-derived bioactive peptides.

TABLE 1.

Characteristics of participants at baseline

	All subjects	Male	Female
n (%)	31	19 (61.2)	12 (38.7)
Age, y	61.1 ± 9.3	58.5 ± 8.9	65.1 ± 8.7
BMI, kg/m2	28.5 ± 4.9	29.0 ± 4.7	27.6 ± 5.1
Waist circumference, cm	98.9 ± 12.6	103.2 ± 10.2	92.1 ± 13.6
Office BP, mmHg
SBP	145.6 ± 14.6	145.7 ± 13.8	145.4 ± 16.4
DBP	89.9 ± 9.7	92.5 ± 8.7	85.8 ± 10.2
Antihypertensivemedication use, n (%)	11 (35.4)	7 (22.5)	4 (12.9)

Abbreviations: BMI, body mass index; BP, blood pressure; DBP, diastolic blood pressure; SBP, systolic blood pressure.

Values are mean ± standard deviation.

Comparison between treatment effects on 24-h SBP and DBP

As shown in Table 2, comparisons of after-treatment consumption revealed that HSP+ had the highest, whereas casein had the lowest, hypotensive effects on both ambulatory SBP and DBP compared with the other 2 treatments (P < 0.0001). Table 3 represents the differences between treatment effects and as shown, the highest reduction was related to the HSP+, which was able to decrease 24-h SBP by −8.9 mmHg compared with casein (σ² = 13.91, P < 0.0001). A separate analysis was conducted on the participants who were on any type of antihypertensive medications and those who were not. The results of these subgroup analyses revealed the same pattern obtained from the analyses of total study samples, and the differences between the treatment groups being compared were statistically significant (P < 0.0001). These results even showed greater hypotensive effects of HSP+ and HSP compared with casein in those participants who were not taking antihypertensive medications. Moreover, period and sequence did not influence changes in 24-h SBD and DBP in these patients (P > 0.05). Figure 2 illustrates the mean 24-h SBP after-treatment consumption. Each dot represents the mean of 24-h SBP in each participant. After consumption of HSP+, 24-h SBP and DBP decreased to 128.1 ± 1.6 and 76.0 ± 1.4 mmHg, respectively (FIGURE 2, FIGURE 3). In comparison, these values were 137.0 ± 1.6 and 81.8 ± 1.4 mmHg after casein and 133.5 ± 1.6 and 78.9 ± 1.4 mmHg after HSP consumption (FIGURE 2, FIGURE 3). These results show that 6-wk consumption of HSP+ led to the lowest ambulatory SBP and DBP among the 3 treatment groups (P < 0.0001) (Supplemental Figure 1). Figure 3 illustrates the mean 24-h DBP after-treatment consumption. Each dot represents the mean of 24-h DBP in each participant. Different treatment consumption effects on frequency distribution of 24-h SBP and DBP are shown in Supplemental Figures 2 and 3. These figures show the aggregated data from multiple participants to create a comprehensive representation of BP distribution. The vertical axis represents the SBP or DBP values. Each point on the vertical axis corresponds to a specific BP value, and the axis extends to cover the range of BP values observed in our study. The horizontal axis represents the frequency of each BP value as recorded by ABPM in all participants over the 24-h monitoring period. The horizontal axis is divided into 0 to 40 intervals, and the number of times a specific BP value fell within each interval is represented by the frequency count.

TABLE 2.

Treatment effects on 24-h systolic and diastolic blood pressure

	Casein	P	HSP	P	HSP+	P
Ambulatory BP, mmHg
Systolic	137.0 ± 1.6	<0.0001	133.5 ± 1.6	<0.0001	128.1 ± 1.6	<0.0001
Diastolic	81.8 ± 1.4	<0.0001	78.9 ± 1.4	<0.0001	75.9 ± 1.4	<0.0001

Abbreviations: BP, blood pressure; HSP, hemp seed protein; HSP+, HSP plus HSP-derived bioactive peptides; SEM, standard error of the mean.

All values are differences in estimated least-squares means ± SEM. P values were derived using SAS MIXED Model.

TABLE 3.

Comparison of treatment effects on 24-h systolic and diastolic blood pressure

	ΔHSP vs. casein	P	ΔHSP+ vs. casein	P	ΔHSP+ vs. HSP	P
Ambulatory BP, mmHg
Systolic	−3.5 ± 0.6	<0.0001	−8.9 ± 0.7	<0.0001	−5.4 ± 0.6	<0.0001
Diastolic	−2.9 ± 0.5	<0.0001	−5.8 ± 0.5	<0.0001	−2.9 ± 0.5	<0.0001
Ambulatory SBP, mmHg
On medication1	− 3.0 ± 1.1	0.009	−6.5 ± 1.4	<0.0001	−3.6 ± 1.4	0.025
No medication2	−3.9 ± 0.9	<0.0001	−9.6 ± 0.9	<0.0001	−5.8 ± 0.9	<0.0001
Ambulatory DBP, mmHg
On medication1	−2.7 ± 0.8	0.0008	−3.8 ± 1.0	0.0004	−1.1 ± 1.0	0.53
No medication2	−3.5 ± 0.6	<0.0001	−6.5 ± 0.7	<0.0001	−3.0 ± 0.6	<0.0001

Abbreviations: BP, blood pressure; DBP, diastolic blood pressure; HSP, hemp seed protein; HSP+, HSP plus HSP-derived bioactive peptides; SBP, systolic blood pressure; SEM, standard error of the mean.

All values are differences in estimated least-squares means ± SEM. P values were derived by using SAS MIXED Model.

¹n = 11;

²n = 20.

FIGURE 2.

Box plots representing estimated least-squares means ± SEM of SysBP after consumption of casein, HSP, or HSP+ in 31 participants. Each dot represents the mean of 24-h SysBP in each participant after consumption of each treatment. The results were derived by using SAS MIXED Model. HSP, hemp seed protein; HSP+, HSP plus HSP-derived bioactive peptides; SEM, standard error of the mean; SysBP, systolic blood pressure.

FIGURE 3.

Box plots representing estimated least-squares means ± SEM of DiaBP after consumption of casein, HSP, or HSP+ in 31 participants. Each dot represents the mean of 24-hr DiaBP in each participant after consumption of each treatment. The results are derived by using SAS MIXED Model. DiaBP, diastolic blood pressure; HSP, hemp seed protein; HSP+, HSP plus HSP-derived bioactive peptides; SEM, standard error of the mean.

Plasma ACE activity and renin and NO concentrations

Table 4 shows the comparison of treatment effects relative to baseline. As represented, HSP+ decreased the plasma activity of ACE compared with casein (−0.030 ± 0.006 μg/mL, P < 0.0001) but not HSP. Similar to ACE activity, HSP+ led to a reduction in the plasma concentration of renin compared with casein (−0.010 ± 0.002 U/mL, P = 0.0004) but not HSP. HSP+ also resulted in an increase in the plasma concentrations of NO, compared with casein, (6.29 ± 1.93 μM, P = 0.003) but not HSP. The comparison between HSP and casein revealed that HSP was also able to lower plasma ACE activity (−0.026 ± 0.006 μg/mL, P = 0.0002) and renin concentration (−0.013 ± 0.002 unit/mL, P < 0.0001) and raise the plasma NO concentration (4.27 ± 1.87 μM, P = 0.047). Figure 4 represents the endpoint values of ACE activity and renin and NO concentrations after 6 wk of treatment consumption. As shown in Figure 4A, the lowest ACE activity was observed after HSP+ consumption (0.002 ± 0.004 μg/mL) compared with casein (0.032 ± 0.004 μg/mL, P < 0.0001). As shown in Figure 4B, unlike ACE activity, the renin concentration was lower after consumption of HSP compared with casein (0.011 ± 0.001 U/mL, P < 0.0001). In addition, Figure 4C shows NO had the highest and lowest concentration after HSP+ and casein consumptions, respectively (21.89 ± 1.50 μM, P < 0.0001; 19.87 ± 1.45 μM, P < 0.0001; and 15.59 ± 1.50 μM, P < 0.0001 for HSP+, HSP, and casein).

TABLE 4.

Comparison of treatment effects on plasma ACE, renin, and NO concentrations

	ΔHSP vs. casein	P	ΔHSP+ vs. casein	P	ΔHSP+ vs. HSP	P
ACE, μg/mL	-0.026 ± 0.006	0.0002	-0.030 ± 0.006	<.0001	-0.004 ± 0.006	1
Renin, U/mL	-0.013 ± 0.002	<.0001	-0.010 ± 0.002	0.0002	0.002 ± 0.002	0.37
NO (μM)	4.27 ± 1.87	0.047	6.29 ± 1.93	0.003	2.01 ± 1.88	0.57

Abbreviations: ACE, angiotensin-converting enzyme; HSP, hemp seed protein; HSP+, HSP plus HSP-derived bioactive peptides; NO, nitric oxide; SEM, standard error of the mean.

For all analyses, n = 31. All values are differences in estimated least-squares means ± SEM. P values were derived by using SAS MIXED Model.

FIGURE 4.

ACE activity (A), renin (B), and NO (C) concentrations after-treatment consumption. The bars represent estimated least-squares means ± SEM of plasma biomarkers derived by using SAS MIXED Model. ACE, angiotensin-converting enzyme; NO, nitric oxide; HSP, hemp seed protein; HSP+, HSP plus HSP-derived bioactive peptides; SEM, standard error of the mean.

Discussion

Our results demonstrated that consumption of 50 g HSP or HSP+/d for 6 wk significantly reduced 24-h SBP and DBP compared with casein. HSP+ exhibited an even greater lowering effect on 24-h BP compared with HSP. To date, several studies have investigated the effects of plant protein on BP. Although He et al. [34] did not measure 24-h BP, our results are consistent with their findings relating to the efficacy of plant protein to lower BP; they used soy protein in comparison with milk protein and carbohydrate control. Moreover, their findings did not show a difference between soy and milk protein in terms of the BP-lowering effect. In another RCT, He et al. [35] reported that soybean protein isolate decreased both SBP and DBP compared with complex carbohydrate control. This trial used a parallel design and BP was measured in the office setting. Both HSP and soybean protein have high Arg content, which may contribute to some of their hypotensive properties.

Li et al. [36] investigated bioactive peptides derived from pea protein hydrolysate in patients with hypertension in a randomized crossover trial. Consumption of 3 g pea protein hydrolysate/d resulted in SBP reductions of 5 and 6 mmHg after 2 and 3 wk, respectively. Unlike our study, they did not measure 24-h BP, and the study duration and the hydrolysate dosage were less than that used in our trial. In addition, they did not observe a hypotensive effect from pea protein isolate. In another RCT by Ogawa et al. [37], 12-wk consumption of peptides from rice bran protein showed SBP-lowering effects in patients with grade 1 hypertension. The tripeptide Leu-Arg-Ala was identified as the possible functional substance exerting the hypotensive effects. Kwak et al. [38] compared black soy peptide supplementation with casein as the control in an RCT. Unlike casein and consistent with our results, they showed that black soy peptides reduced both SBP and DBP after 8 wk.

These findings are consistent with the study of Girgih et al. [21], who investigated the BP-lowering effects of HSP or HSP+ compared with casein in an animal experiment. They explored both the preventive and treatment effects of HSP and HSP+ in rats. Although each treatment period was 4 wk, their findings showed a greater hypotensive effect than our results; however, we were not anticipating similar results because animal studies usually do not translate into replications in human studies [39]. Human trials investigating the effects of protein on BP have mostly used animal-based sources of protein such as whey and casein. Data presented by Fekete et al. [40] showed that in an RCT, 8 wk of whey protein (2 × 28 g) supplementation reduced 24-h BP (−3.9 and −2.5 mmHg in SBP and DBP, respectively) compared with maltodextrin as control. In addition, whey protein was able to decrease daytime SBP and DBP compared with casein. It has been suggested that the inhibition of ACE could be the potential mechanism by which whey protein may affect BP [41].

Mollard et al. [42] conducted an acute trial using hemp protein (40 and 20 g) compared with soybean protein and carbohydrate control, but hemp protein did not show any impact at 15, 30, 45, and 60 min on office BP. Other than the acute nature of this trial, inconsistent with our study, they did not recruit from the population with hypertension. Generally healthy and normotensive participants may not have the potential for possible BP-lowering effects of hemp protein with a high Arg content.

Exploratory analysis in this study revealed that the hypotensive effects of HSP and its hydrolysate are even higher in those participants who were not on any type of hypotensive medication. The greater antihypertensive effects in response to hemp protein consumption in these patients could be explained by the potential capacity of the pathways involved in BP regulation because these pathways may become saturated by antihypertensive medications.

The findings of our study add to the collective accumulation of current literature on the hypotensive effects of HSP and its hydrolysate, which, prior to this trial, was based on in vitro and animal studies [23,24,43]. The current study revealed the hypotensive impact of HSP+ compared with HSP and casein; however, we did not observe a significant difference in terms of plasma biomarkers of BP between HSP+ and HSP when the 2 were compared together. Even though RCTs investigating the effects of HSP consumption on BP are lacking, and so far, no clinical trial has evaluated the impacts of HSP-derived bioactive peptide consumption alone in patients with hypertension, our findings are predominantly consistent with other studies that reported a potential relationship between HSP and the bioactive peptide consumption and BP and its plasma biomarkers [24,44,45].

Our results showed that both HSP and HSP+ increased plasma NO concentration, but NO concentration was higher after the consumption of HSP+ compared with HSP. In recent years, increased attention has been generated toward amino acids with hypotensive potentials. Specific amino acids including cysteine, glutamic acid, tryptophan, and Arg have been particularly studied for their hypotensive effects [19,46]. HSP has a high content of Arg, whereas casein is relatively low in Arg [47]. In addition, compared with HSP hydrolysate, HSP contains an even greater amount of Arg [24]. By including a treatment group consuming both HSP and HSP hydrolysate in our study, we aimed to explore whether the antihypertensive effects of HSP and its hydrolysate are additive or synergistic. The NO results suggest that the increasing effect of HSP and its hydrolysate on plasma NO concentration could be modulated through the same underlying mechanism: the conversion of Arg to NO.

This trial has several strengths including the crossover design with the advantage of within-participant comparisons of response to treatment, which reduces much of the potential for an interindividual variation in the study outcomes [33]. This study has addressed a few contextual knowledge gaps in the association between individuals’ protein intake and their BP. The most unique aspect of this study is that, to our knowledge, this is the first clinical trial investigating the effects of HSP and its bioactive peptides in patients with hypertension. The ABPM devices used in the present study are considered the gold standard for measuring BP compared with in-office and home measurements. This trial also has some limitations that must be considered. First, we were not able to observe the daily consumption of treatments, did not include a run-in period, and only had a 2-wk washout period between treatments. Moreover, the extent to which disparities in dietary intake and physical activity patterns between treatments may have modified the BP or plasma biomarker responses to the study treatments in our trial remains speculative as we did not tightly control diet or exercise in participants in this free-living trial. Further studies will be needed to confirm the potential of HSP and its hydrolysate-derived bioactive peptides in BP reduction. Dose–response research is strongly encouraged in the future to investigate the highest dose of hemp seed bioactive peptides with the optimum hypotensive response.

For this study, we enrolled both males and females whose ages ranged from 18 to 75 y with SBP between 130 and 160 mmHg and DBP <110 mmHg. Importantly, participants were selected regardless of their BP medication status, including those who were not taking any BP medications and those on stable dosages of such medications. This inclusive approach allowed us to examine the effects of our intervention in a real-world context. As such, the findings of this study hold relevance not only for this specific study population but also offer insights into the potential benefits of our intervention for a broader demographic of adults with mild hypertension. However, it is essential to note that the generalizability of our results to individuals with more severe hypertension or specific medical conditions may warrant further investigation.

Although the intervention of 50 g protein/d was efficacious in our study, it is important to acknowledge a daily commitment to 50 g of protein powder might pose challenges for some patients, whether due to taste preferences or dietary restrictions, such as those of individuals with specific medical conditions, such as chronic kidney disease. Additionally, the measurement of 24-h BP, although robust in assessing treatment effects, may not be universally convenient for all individuals. Therefore, although our findings offer promising insights into the potential benefits of this intervention, it is essential to consider individual circumstances and preferences when projecting these results to a broader population. Further research is warranted to explore the feasibility and acceptability of this protein regimen in diverse patient populations.

In conclusion, we found evidence that consumption of both HSP and HSP+ decreased 24-h SBP and DBP compared with casein. Also, HSP+ demonstrated a greater lowering effect on 24-h SBP and DBP compared with HSP. Similar to the hypotensive effects, HSP and HSP+ reduced plasma ACE activity and increased renin and NO concentrations when compared with casein. However, we did not observe a difference between HSP and HSP+ in terms of their effects on plasma BP biomarkers. These findings imply that the inclusion of hemp protein in the diet may potentially play a role in managing mild hypertension among adults. It is important to note that our study primarily focused on assessing the impact of hemp protein on BP within the parameters of our trial. Although our findings indicate modest changes, we recognize that extrapolating to broader efficacy and dose–response relationships lies beyond the scope of this study. These results mostly align with the prior animal data that suggested a beneficial association between intake of HSP and its hydrolysate and hypertension management. Further research, including larger, longer trials, may be required to confirm these fundings and discover potential mechanisms.

Acknowledgments

We appreciate Dennis Joseph’s technical assistance on this project.

Author contributions

The authors’ responsibilities were as follows − REA, DSM, RCM: conceptualized the research and designed the research program; REA, MS: submitted Ethics and Health Canada documents for approval; AMA, MS: were involved in the development of HSP hydrolysate; MS: was involved in the preparation of the HSP hydrolysate in the laboratory; MS: was responsible for the screening, data collection, and clinical trial coordination; MS, AMA: analyzed all the samples; MS: performed the statistical analyses and wrote the first draft of the manuscript; REA: was the Principal Investigator and administrative lead for the study; all authors: critically reviewed the manuscript; and all authors: read and approved the final manuscript.

Funding

Financial support for this study is provided by Heart and Stroke Foundation of Canada (Grant-in-Aid [GIA] program) and Richardson Centre for Food Technology and Research. Manitoba Harvest provided hemp seed protein powder for this research. REA and DSM obtained the funding. The supporting source had no such involvement or restrictions regarding publication.

Data availability

Deidentified data described in the manuscript, code book, and analytic code will be made available upon request pending application to and approval of the corresponding author.

Conflict of interest

The authors report no conflicts of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: