The effects of date seed (Phoenix dactylifera) supplementation on exercise-induced
oxidative stress and aerobic and anaerobic performance following high-intensity interval
training sessions: A randomized, double-blind, placebo-controlled trial
Elham Moslemi 1, Parvin Dehghan 2*, Mostafa Khani 3, Parvin Sarbakhsh4, Bahareh Sarmadi 5
1
Student research committee, Faculty of Nutrition and Food Science, Tabriz University of
Medical Sciences, Tabriz, Iran
2
Nutrition Research Center, Department of Biochemistry and Diet Therapy, Faculty of Nutrition
and Food Science, Tabriz University of Medical Sciences, Tabriz, Iran
3
Faculty of Physical Education and Sport Sciences, University of Tabriz, Tabriz, Iran
4
Department of Statistics and Epidemiology, School of Public Health, Tabriz University of
Medical Sciences, Tabriz, Iran
5
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, University
Putra Malaysia, 43400, Serdang, Selangor, Malaysia
*Corresponding authors: Parvin Dehghan, PhD in Nutrition, Tabriz University of Medical
Sciences, Tel.: +98 4133376229; Fax: +984133340634. Postal code: 5166614711.
E-mail address:dehghan.nut@gmail.com.
This peer-reviewed article has been accepted for publication but not yet copyedited or typeset,
and so may be subject to change during the production process. The article is considered
published and may be cited using its DOI
10.1017/S0007114522002124
The British Journal of Nutrition is published by Cambridge University Press on behalf of The
Nutrition Society
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
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Abstract
Although high-intensity interval training (HIIT) is an efficient method to improve vascular
function, maximal oxygen consumption, and muscle mitochondrial capacity, acute HIIT
overstresses the oxidative and immune systems and causes muscle soreness and damage. The
aim of the present study was to investigate the effects of date seeds on exercise-induced
oxidative stress and aerobic and anaerobic performance following HIIT sessions. Thirty-six
physically active men and women aged 18-35 years were assigned to take 26g/d of date seed
powder (DSP, n=18) or wheat bran powder (placebo, n=18) before and after HIIT workouts for
14 days. Total antioxidant capacity (TAC), oxidative stress index (OSI), total oxidant status
(TOS), superoxide dismutase (SOD), glutathione peroxidase (GPx), uric acid, malondialdehyde
(MDA), and 8-iso-prostaglandin F2α (8-iso-PGF2) were determined at baseline, at the end of the
intervention, and 24 hours’ post-intervention. We used the Cooper and running-based anaerobic
sprint test (RAST) to assess aerobic and anaerobic performance at the study's beginning and end.
Independent Student’s t tests, analysis of covariance, and repeated-measures analysis of variance
(ANOVA) were used to compare the quantitative variables. Positive changes were observed in
TAC, TOS, OSI, GPx, MDA, and VAS after intervention and at 24 h post-exercise (p<0.05).
Likewise, peak power and fatigue index were significantly improved in DSP in comparison to
the placebo group. Levels of SOD, uric acid, 8-iso-PGF2, VO2 max, and average power were not
changed after training. Our results showed that date seed supplementation in active participants
performing HIIT bouts ameliorated oxidative stress and improved performance parameters.
Keywords: Date seed, HIIT, Exercise performance, Oxidative stress, Polyphenol, Functional
food, Prebiotic.
Abbreviations: ANCOVA: Analysis of covariance; ANOVA: Analysis of variance; BMI: Body
mass index; DSP: Date seed powder; FI: Fatigue index; GPx: Glutathione peroxidase; HR: Heart
rate; HRR: Heart rate reserve; HIIT: High-intensity interval training; IPAQ: International
Physical Activity Questionnaire; MDA: Malondialdehyde; OSI: Oxidative Stress Index; PAR-Q:
Physical Activity Readiness Questionnaire; 8-iso-PGF2α: 8-iso-Prostaglandin F2α; RAST:
Running-based anaerobic sprint test; RCT: Randomized clinical trial; ROS: Reactive oxygen
species; SOD: Superoxide dismutase; TAC: Total antioxidant capacity; THR: Target heart rate;
TOS: Total oxidant status; VAS: Visual analog scale; W: Watts
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1. Introduction
As a highly nutritious food, dates (Phoenix dactylifera) are grown mainly in North Africa and the
Middle East. Iran is the second-largest producer of date fruits, accounting for 21% of world
production
(1)
. Date seeds are waste products generated in large quantities in the production
process of dates. Recently, it has been the focus of growing interest regarding its application as a
functional food in both animal studies and clinical trials, owing to its high content of dietary
fiber (75–80 g/100 g), antioxidants [phenolic acids (2697–5342 mg gallic acid equivalents/100
g), total flavonoids (1224–1844 mg of rutin equivalent/100g) and carotenoids] and considerable
amounts of minerals, vitamins, protein, and fat
(2, 3)
. Earlier studies highlighted flavan-3-ols
(catechin, epicatechin, and procyanidins) as the most important polyphenolic compounds found
in date seeds (4). Several studies have reported health benefits of date seeds, including antioxidant
activity, anticarcinogenic, antimutagenic, and anti-inflammatory effects, as well as the
amelioration of hyperglycemia, hyperlipidemia, and memory, and learning disorders
(5-9)
. The
safety of date seeds has been reported at a dose of 0.5 g/kg/d (10).
High-intensity interval training (HIIT) is a useful exercise condition that facilitates metabolic
adaptations, and improves muscle mitochondrial capacity, vascular function, maximal oxygen
consumption, and alleviates hyperglycemia, cardiometabolic risk factors, and body fat
(11-14)
.
However, during a HIIT session, exercise-related physiological responses incline muscles to
produce more reactive oxygen species (ROS), which results in the development of oxidative
stress
(15)
. Oxidative stress is regarded as an imbalance between the generation and degradation
of reactive molecules that results in predomination of pro-oxidants over antioxidants and
disruption of redox signaling and molecular damage
(16, 17)
. In HIIT sessions, oxidative stress
reduces calcium uptake by the sarcoplasmic reticulum, influences muscle contraction, and
consequently, causes an acute decrease in physical performance (namely, muscle soreness and
fatigue) (18).
Antioxidant supplements can reduce exercise-induced oxidative stress. However, the
antioxidants effects on exercise performance can be antagonistic which depends on the dose,
type, and duration of the antioxidant administration. Several studies have shown that isolated
bioactive compounds (e.g., vitamin C, vitamin E, and lipoic acid) may have adverse effects on
signs and adaptive responses to exercise
(19, 20)
. In contrast, no evidence of such relations
following the consumption of antioxidant-rich foods and/or extracts was reported
(21, 22)
.
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Furthermore, studies suggest that consuming polyphenols during exercise, regardless of the
length of the intervention
(23)
, may provide antioxidant protection and thus may minimize the
negative physiological responses that occur during and following exercise, like fatigue and
muscle pain
(24)
. A study reported a significant increase in urinary polyphenol metabolites as
biomarkers of date seed polyphenol intake for up to 24 h in the urine samples
(10)
. Limited
clinical trials (6, 8, 10, 25) and animal studies (26-31) have reported modulating effects of date seed on
metabolic parameters, oxidative stress, and inflammation. To our knowledge, no previous study
has investigated the impact of date seed powder supplementation on oxidative stress and aerobic
and anaerobic performance in humans performing high-intensity interval training. Therefore, this
study aimed to examine the effects of date seeds as a rich source of polyphenol antioxidants on
oxidative stress markers, muscle pain, and aerobic and anaerobic performance following HIIT.
2. Materials and methods
2.1. Ethical approval
This research was performed following the principles of the Declaration of Helsinki. After a
detailed explanation of the study methodology, all volunteer participants signed an informed
consent form at the beginning of the study. The ethics committee at Tabriz University of Medical
Sciences approved the study plan (IR.TBZMED.REC.1399.1011), which was then registered on
the
Iranian
Registry
of
Clinical
Trials
(https://www.irct.ir/)
with
the
number
IRCT20150205020965N9.
2.2. Participants
Between October and November 2021, 38 healthy and physically active men and women
(recreational runners, Image 1) participated in this study. Their eligibility to participate actively
in the study was determined by the Physical Activity Readiness Questionnaire (PAR-Q)
(32)
under the supervision of a physician. The study protocol followed the Consolidated Standards of
Reporting Trials (CONSORT) checklist, and Figure 1 shows the diagram of the study protocol
(33)
. The inclusion criteria were as follows: male and female aged between 18 and 35 years;
physically active subjects based on a) 3 days of vigorous activity (minimum of 20 min/day, or b)
five days of any combination of walking, vigorous-intensity or moderate-intensity activities
(minimum of 600 MET-min/week), or c) 5 days of walking or moderate-intensity exercise (for
the minimum of 30 min/day); stable body mass index (BMI, changes of < 3 kg were acceptable)
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within the last five months; body mass index of 18.5–25 kg/m2 ; no HIIT in the past three
months; and willingness of subjects to participate in this study. The exclusion criteria were:
light-intensity activities; a history of chronic diseases such as cardiovascular, thyroid,
gastrointestinal, kidney, diabetic, cancer, or pancreatic disease; infectious diseases; cognitive
disorders; smoking; pregnancy or lactation; anemia (Hb <13g/dl); musculoskeletal injuries;
antacids; taking antibiotics; antidiarrheals; anti-inflammatory; antihypertensive, or laxative
medications, anabolic steroids, ergogenic agents (arginine, carnitine, creatine, and caffeine), or
other medications during the previous month; subjects with special diets or dietary restrictions;
and recent consumption of antioxidants. Also, during the study, if an individual lost more than
10% of the supplementation packets and did not attend at least 90% of each week's training
sessions, he/she was considered non-compliant and was consequently excluded from the study.
2.3. Sample size
The sample size was estimated based on the changes in the parameter malondialdehyde (MDA)
as the main outcome following Platat et al,
(10)
. Using Stata software (version 16), the sample
size in this study was estimated to be at least 16 subjects for each group, with a power of 90%
and 95% confidence levels. Based on a 25% decrease in the level of expected MDA through
supplementation and a 10% dropout rate for each group, the sample size in each group increased
to 18.
2.4. Randomization and allocation concealment
After a run-in period, we used the PAR-Q, which is a pre-study screening questionnaire to assess
a person's medical history, lifestyle, eating habits, and physical fitness in several areas (32). When
the participant answered "yes" to a question on the questionnaire, he or she was excluded from
the study. According to the PAR-Q analysis, 36 eligible volunteers from Tabriz stadiums were
randomly allocated to the intervention group (date seed powder, n = 18) and the placebo group (n
= 18) for 14 days. The randomization procedure was performed into the two groups (1:1)
following stratified randomization based on sex and VO2 max. We used random allocation
software to perform randomized blocks of sizes 2 and 4. To ensure blinding of the study, a third
person allocated the subjects into groups. The main researcher was blinded to the groups of
subjects until the end of the analysis.
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2.5. Intervention
The intervention group received date seed powder (DSP) at a dosage of 26 g/day (date seeds,
Flavinea Co., Iran) for 14 days according to a pilot study of date seed powder for two weeks in
active people (data not reported). The placebo group received the same amount of placebo
(wheat bran powder, Nazhvangiah Co., Iran). The powder was divided into two 13-g packages,
and it was given to subjects one hour before HIIT activity and one hour after HIIT with a cup of
water. Both date seed and placebo powder were tasteless, odorless brown powders distributed to
participants in identical opaque packages. The powders were delivered to participants weekly for
two weeks. The main researcher was in daily contact with the subjects via text message to
emphasize physical activity maintenance, clarify supplement use problems, and ensure
compliance. Participants were also given a checklist to tick off after each powder intake to check
for non-compliance.
2.6.Exercise protocol
Based on the American College of Sports Medicine (ACSM) recommendations for physical
activity, a high-intensity interval training (HIIT) exercise protocol was established for each
participant in both groups
(34)
. The first phase involved habituation to the intensity of the HIIT
program. After consuming DSP or a placebo, the HIIT sessions in the following phases followed
the same pattern. The subjects ate a standardized breakfast two hours before the HIIT session.
The intervention was given to subjects one hour prior to HIIT activity and one hour later.
Subjects participated in a two-week HIIT program (5 exercise sessions per week; 10 sessions
during the study period). Each session began with a 15-minute warm-up at 50% heart rate
reserve (HRR) (including various stretches, flexibility, walking, and running). Both groups' main
actives comprised two sessions of three to four repetitions and 30 seconds of running at 90-100%
of the HRR on each repetition. There were 90–180 seconds of active rest after each repetition
and 2.5–4 minutes of active recovery after each phase, respectively. Also, each session finished
with a 5-min cool-down with 45% HRR (35). This strategy has been demonstrated to be effective
in causing oxidative stress in physically active individuals (36).
Also, heart rate (HR) was continuously monitored during the supervised exercise intervention,
and participants’ heart rates were recorded using a Polar heart rate (Polar, RS800CX, Kempele,
Finland) to ensure training at the intended intensity. The Karvonen formula was used to calculate
the target heart rate (THR) zone for each participant, and mean of THR was reported:
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Max HR = 220 - age
Target Heart Rate = [(max HR - resting HR) × % Intensity] + resting HR (37).
Throughout the trial, the research team kept in touch with individuals daily. A blinded researcher
conducted the 17-minute HIIT sessions about the consumption of DSP or a placebo. All
participants were guided to make their cooperative decisions during high-intensity exercise
training. The number of sessions attended is used to assess adherence to the training program.
2.7. Primary and secondary outcomes
Changes in total antioxidant capacity (TAC), total oxidant status (TOS), Oxidative Stress Index
(OSI), superoxide dismutase (SOD), glutathione peroxidase (GPx), uric acid, malondialdehyde
(MDA), 8-iso-prostaglandin F2α (8-iso-PGF2), and muscle fatigue are the primary outcomes of
the current research. VO2 max, peak power, average power, and fatigue index (FI) were
considered the secondary outcomes.
2.8. Assessment of dietary antioxidants intake
A dietary protocol was used to check the intake of macronutrients and antioxidants (vitamin A,
vitamin C, vitamin E, α- tocopherol, β-carotene, lycopene, β-cryptoxanthin, zinc, and selenium)
to ensure that they were having their habitual diet and were not consuming additional
antioxidants via diet. The regular food and beverage intake was assessed using three-day food
records (two weekdays and one weekend) before starting the HIIT protocol and supplements and
at the study's end during the last week (38). At the beginning of the interview, a dietitian informed
the participants about the recording procedure and asked them to record their consumed
beverages and foods. The information recorded by the subjects during home measurements was
standardized and converted to grams and/or milliliters of the foods and/or drinks. The
"Nutritionist 4" software (First Databank Inc., Hearst Corp., San Bruno, CA, USA) was used to
analyze the nutritional data.
2.9. Characterization of study subjects
Anthropometric parameters (height, weight, and BMI) were assessed at the baseline and at the
end of the study. A reliable scale (Seca, Hamburg, Germany) was used to measure weight while
barefoot and wearing light clothing to the nearest 0.5 kg. A centimeter tape with a precision of
0.1 cm was used to measure height with no shoes. The BMI was calculated by the weight (kg)
divided by the height (m) squared. Based on the World Health Organization's classifications,
BMI has been adopted as a measure of nutritional status (39).
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We determined their physical activity level using the International Physical Activity
Questionnaire (IPAQ–short version)
(40)
. The following formula was used to calculate the
physical activity rate: moderate activity = (4.0 × moderate activity minutes’ × days); vigorous
activity = (8.0 × vigorous activity minutes × days). The cut-off levels were divided into three
groups based on the IPAQ scoring protocol's current PA guidelines: 1. Low: some activities were
reported but not enough to meet categories 2 or 3, 2. Moderate: 5 or more days of any
combination of walking, moderate- or vigorous-intensity activities (at least 600 METs min.wk1
accumulatively), 3. High: 7 or more days of any combination of walking, moderate-intensity, or
vigorous-intensity activities (at least 3000 METs min.wk1 accumulatively)
(41)
. The intensity of
pain was quantified using a visual analog scale (VAS). On a 10 cm ruler, "0" represented the
absence of pain and "10" the maximum pain level that active men or women can tolerate
(42)
.
Each participant was instructed to record the VAS at baseline (after the Cooper 12-minute run
test and running-based anaerobic sprint test (RAST)), after the intervention, and 24 hours later.
2.10. Blood samples
Blood samples (10 mL) were collected at three time points: at the beginning, at the end of the
study (day 14), and 24 hours after that. Blood samples were taken at each time point from the
intermediate vein of the forearm into tubes with/without ethylenediamine-tetra-acetic acid
(EDTA). Aliquots of plasma were used to analyze TAC and TOS using the colorimetric method
(TAC: RANDOX kits, RANDOX Laboratory, UK; TOS: ZellBio GmbH, Ulm, Germany), and
the OSI was calculated using TAC and TOS as follows: OSI =100 × (TOS/TAC)
(43)
. The SOD
and GPx activities were determined via a commercially available kit (Randox Laboratories Ltd.,
Crumlin, County Antrim, UK). Uric acid was measured by using the enzymatic method by an
autoanalyzer using kits (Pars-Azmoon Co., Tehran, Iran). Serum levels of 8-iso-PGF2 were
determined using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge,
UK). The spectrofluorimeter was used to determine MDA levels (Kontron, model SFM 25A,
Italy) (44).
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2.11.Aerobic and anaerobic performance parameters
The Cooper 12-minute run test was used to measure aerobic endurance. Participants ran
continuously for 12 minutes in the 400-meter running track before and after the intervention
period, and the next covered distance was utilized to determine VO 2 max using cooper’s
equation (45):
VO2 max (ml/kg/min) ₌ (Distance - 504.9) / 44.73.
We also measured anaerobic endurance prior and subsequent to the intervention using the RAST.
The RAST test consists of six parallel 35-meter sprints separated by a 10-second rest period.
Participants completed the RAST test, and an electronic timing device automatically recorded
the time spent on each attempt. Then, we calculated anaerobic power output using the formula,
as well as FI as a measure of performance degradation.
• Power (watts) = (Weight ×Distance 2) / Time 3
• Peak Power (watts) = the highest power (the fastest sprint)
• Average Power (watts) = the sum of all six values ÷ 6
• FI (watts/s) = (Maximum Power – Minimum Power) / Time spent in six sprints (46).
During the orientation session, all participants were reminded of the importance of performing
tests and encouraged to give their all. In the 24 hours leading up to the aerobic and anaerobic
tests, they were also forbidden from engaging in any strenuous physical activity. Before
undertaking exercise tests, all subjects were given a warm-up session for 5 min.
2.12. Date seed powder chemical characterization and antioxidants
A specialized company produced the commercial date seed powder samples (date seeds,
Flavinea Co., Iran). The recommended procedures by the Association of Official Analytical
Chemicals were used to determine the chemical analysis
(47)
. The Folin-Ciocalteu colorimetric
technique was used to measure the total phenolic content of the samples
aluminum chloride colorimetric method was used to assess flavonoid levels
(48)
(49)
. Likewise, the
. Table 1 shows
the average of the composition and antioxidant properties of the date seed powder (100 gr) that
were determined three times.
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2.13.Statistical analysis
The SPSS program version 24 was used to analyze the data. The Shapiro-Wilks test was used to
determine the data's normality. We presented qualitative data as frequency (percent), whereas
quantitative data as mean ± standard deviation (SD). The independent sample student's T-test
was used to compare the quantitative variables between groups according to time. Also, analysis
of covariance (ANCOVA) was used to compare after intervention between two groups, adjusting
for baseline value.
For oxidative stress biomarkers and VAS parameter, which were assessed three times, a
repeated-measures analysis of variance (ANOVA) test was performed and p-value
supplement*time in the repeated measure analysis reflect the effect of the intervention. Statistical
significance was defined as a p-value of p <0.05.
3.
Results
Of 36 participants enrolled for trial eligibility, 34 subjects completed the study (17 in the placebo
group and 17 in the date seed group). One subject was dropped out for poor compliance in the
date seed group, and another dropped out for personal reasons in the placebo group. The dropouts did not differ between the two groups, and the participation rate was 94.44%. No side
effects were reported after supplementation with date seeds and placebo. The characteristics of
volunteers at baseline were similar in both groups in terms of gender, age, physical activity, heart
rate, BMI and body weight (Table 2), dietary Nutritional intake (Table 3), TAC, OSI, SOD, GPx,
uric acid, MDA, 8-iso-PGF2α, VAS (Table 4), VO2 max, peak power, average power, and FI
(Table 5) (p > 0.05). No significant differences were observed in weight, BMI and the dietary
antioxidant intake, as efficient factors on oxidative stress status between groups at the end of the
trial (ANCOVA, p > 0.05) (Table 2, Table 3). Based on Table 4, there was a significant
supplement*time interaction for TAC, TOS, OSI, GPx, MDA, and VAS (for all p< 0.05) and no
effect for supplement*time in SOD, uric acid, and 8-iso-PGF2α. Furthermore, we observed a
significant main effect for supplementing in TAC, TOS, OSI, GPx, MDA, 8-iso-PGF2α, and
VAS (for all p< 0.05), whereas there were no changes for SOD and uric acid. TAC, TOS, OSI,
SOD, GPx, uric acid, MDA, 8-iso-PGF2α, and VAS all showed a significant main effect for time
(all p≤ 0.001).
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Our findings showed statistically significant differences in TAC, TOS, OSI, SOD (24h), GPx,
MDA, 8-iso-PGF2α (after), and VAS (24h) in the date seed group when compared to the
placebo. However, there were no differences in SOD (after), uric acid, 8-iso-PGF2α (24h), and
VAS (after) in the date seed group in comparison to the placebo group (Table 4). Figure 2 also,
illustrates oxidative stress marker concentrations and VAS from pre- to post-intervention and 24
h after HIIT.
In aerobic and anaerobic performance, just peak power and FI showed significant differences
between the two groups at the end of the study (ANCOVA, p<0.05) (Table 5).
4.
Discussion
The present study investigated the role of date seed powder on exercise-induced oxidative stress
and parameters of performance following HIIT sessions. To our knowledge, this is the first
randomized, double-blind, placebo-controlled clinical trial of its kind. Our findings demonstrated
that supplementation with DSP for the duration of two weeks attenuated oxidative stress and
improved exercise performance in men and women performing HIIT bouts.
It has been postulated that exercise, which enhances oxygen consumption considerably, can
increase free radicals and oxidative stress
(50)
. HIIT provokes oxidative stress and lipid
peroxidation by increasing nicotinamide adenine dinucleotide phosphate(NADPH) oxidase,
xanthine oxidase, phospholipase A2 activity, cytochrome c from the mitochondria, and
catecholamine oxidation
(36, 51)
. Many adaptations, such as redox signaling cascades and
endogenous antioxidant enzyme upregulation, muscle hypertrophy, glucose uptake by the
skeletal muscle, and mitochondrial biogenesis, contribute to the attenuation of oxidative stress
following HIIT
(52)
. Nonetheless, to achieve ideal recovery time and to strengthen an impaired
antioxidant capacity (that accompanies poor performance), supplementation with an antioxidantrich source like DSP that protects the body from oxidative damage is necessary. In our study,
attenuated oxidative stress response following DSP supplementation was indicated by decreased
TOS, OSI, MDA, and increased TAC, GPx in the DSP group that can be due to the antioxidative
effects of its polyphenolic compounds like flavonoids and/or its ability to enhance endogenous
antioxidants. The decreased OSI in the DSP group is indicative of the beneficial effects of date
seed on increasing TAC and decreasing TOS in this group. Serum GPx activity was significantly
increased in both groups at the end of the study and 24h after that; however, the increments were
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more profound (p<0.05) in the DSP group than the placebo group. GPx is considered a key
barrier against reactive oxygen species as it converts H2O2 to H2O. It is plausible that elevated
levels of H2O2 following HIIT sessions stimulated GPx production, which is, in fact, the body’s
response to increased oxidative stress. Moreover, our findings demonstrated a significant
reduction in MDA concentration in the DSP group in comparison to the placebo group. In
summary, GPx defends cellular membranes against peroxidation by eliminating lipid peroxides;
likewise, in our study reduced MDA levels can be linked to enhanced GPx levels
(53)
. What is
more, uric acid, as the component of plasma's antioxidant capacity and a powerful eliminator of
peroxynitrite and peroxyl radicals
several animal studies
(26, 29, 55, 56)
(54)
did no change in this study. In line with our findings,
, human studies (10, 25) and a systematic review (27) have reported
the beneficial effects of date seed supplementation on the antioxidant defense system under
various health conditions. In a study, supplementation of Wistar rats with date seeds (0.75g/kg,
for seven days) was shown to significantly increase SOD and GPx levels (56). Moreover, Hasan et
al. observed significant changes in MDA and SOD levels following 10 ml of aqueous date seed
extract/day supplementation in diabetic Wistar rats at the end of eight weeks
(29)
. Following 13
weeks of treatment with a diet comprising 2, 4, or 8 g/kg date seeds, MDA levels attenuated
dose-dependently in male Wistar rats (26). In a study by Saryono et al., date seed supplementation
(2.5 g/day for two weeks) in postmenopausal women significantly improved MDA, SOD, and
GPx enzyme activities
(25)
. Platat et al. reported that administration of 0.25 g and 0.5 g date
seeds/kg acute dose reduced MDA in healthy participants dose-dependently (10). The differences
in genotype, the dosage and kind of supplementation, basal oxidative stress status,
supplementation duration, and difference in study design explicate the differences in findings.
Date seeds are rich sources of polyphenols
(26, 55, 56)
and their polyphenols, particularly their
flavonoids, can be responsible for attenuation of the exercise-induced oxidative stress observed
in the present study. Although the exact mechanisms through which date seed polyphenols
modulate oxidative stress were mainly unexplored, some of the proposed mechanisms can be as
follows: scavenging free radicals through chelating and/or reducing metal ions by the OH groups
attached to the aromatic ring of polyphenol (57), enhancing the expression of nuclear transcription
factor-erythroid 2-related factor 2 (Nrf2) as a major transcriptional regulator of antioxidant
enzymes such as SOD, CAT, GPx
(58)
, impeding the nuclear factor-kappa B (NF-κβ) cascade
pathway at different steps as inducer of the expression of the target genes including IL-6, IL-2,
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IL-8, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS)
(59)
, activating
sirtuins 1 (SIRT1) to suppresses pro-apoptotic factors and proinflammatory factors by
downregulating p53 and NF-κB
(60)
, and modulating metabolic endotoxemia involved in
oxidative stress and MAPK- NF-κβ cascade pathway
stimulates ROS production
(62)
(61)
. High oxygen uptake during exercise
. While ROS can support the recovery process
demonstrated that in the state of impaired antioxidant capacity
muscles, thereby decreasing exercise performance
(66, 67)
(65)
(63, 64)
, it has been
, excessive ROS can damage
. Theoretically, supplementation with
antioxidants would reinforce the antioxidant system of the body, would decrease oxidative
stress-induced damage, and consequently would improve performance. Consistently, in the
current study, we investigated the effect of DSP supplementation on the markers of performance
following HIIT bouts. Our findings indicate that DSP supplementation can significantly increase
peak power and decrease pain, which implies enhanced performance following DSP
supplementation. Decreased pain and improved power and performance following date seed
supplementation can be related to its role in speeding up the recovery from ROS generation.
We also found that DSP can lessen fatigue, as indicated by decreased FI. The positive effect of
DSP on exercise-induced fatigue can be related to the ability of its antioxidant content (i.e.,
polyphenols) to neutralize the reactive species produced by HIIT bouts. In a systematic review
and meta-analysis by Blake et al., it was reported that foods rich in polyphenols improve
endurance exercise performance in subjects
(68)
. Morgan et al. found that daily consumption of
330 ml of cacao juice (containing high flavan-3-ols levels) in recreationally active males for
eight days, significantly decreased VAS
(69)
. Roberts et al. reported that decaffeinated green tea
extract supplementation as a flavan-3-ols polyphenol source (571 mg/day for four weeks)
alongside cycle exercise in recreationally active males improved average power (70). Jo´wko et al.
showed non-significant changes in peak power, average power, and FI in subjects with cycle
sprint following green tea extract (245 mg/d for four weeks)
(71)
. In another study, da Silva et al.
reported that green tea supplementation (500 mg/d for 15 days) reduced muscle damage, but not
muscle pain (VAS) in non-trained male subjects
(72)
. A double-blind, randomized clinical trial
indicated that epicatechin supplementation (200 mg daily, for four weeks) resulted in significant
VO2 max, average power, peak power, and FI changes in recreationally-active men and women
(73)
. The different findings may be related to various types of polyphenol, the dosage of
supplements, metabolic and psychological characteristics of study participants, the use of
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript
different exercise protocols, and fitness levels. It is noteworthy that our analysis on the intake of
other dietary antioxidants, like vitamins A, C, E, and α- tocopherol, lycopene, β-carotene, βcryptoxanthin, zinc, and selenium, did not show any significant difference between the two
groups either at the base line or at the end of the study, which eliminates any probable
intervening effects of dietary antioxidants and endorses the pivotal role of date seed on the
amelioration of oxidative stress in individuals performing intensive activities.
HIIT sessions may decline aerobic and anaerobic performance via several mechanisms like
changing in stretch fibers, collapsing membrane surrounding the sarcoplasmic reticulum and
muscle fibers, damaging excitation-contraction coupling, stimulation of proteolytic enzymes,
activating inflammatory response, which can result in muscle edema, and pain, and exacerbated
muscle function
(74, 75)
. However, improved oxidative stress and inflammation have been
mentioned to decrease the amount of pain and improve exercise performance following HIIT
sessions. Other non-oxidative mechanisms can be involved in the performance improvement
following date seed supplementation in the present study. For instance, in vitro studies show
polyphenols can act as an adenosine A1-receptor antagonist and present analgesic effects that
result in the reduction of effort perception or muscle aches and pains during exercise
(76)
.
Furthermore, it was previously found that polyphenols can reduce the conversion of nitric oxide
(NO) to peroxynitrite, which probably contributed to increased vasodilation response, improved
muscle perfusion, and increased oxygenation by increasing NO bioavailability
(77)
. While these
can explain the favorable effects of date seeds on aerobic and anaerobic performance, further
research is required to investigate mechanisms that link polyphenol date seeds with increased
exercise performance.
5.
Strengths and limitations
This study's strengths include its double-blind, placebo-controlled, randomized clinical design;
as well as stratification by gender and VO2 max variables, which eliminate inter-individual
variance and the participation of volunteers. Another strength of the present study was its
novelty, as it is the first clinical experiment that exploited date seed (which could be a waste
product) to determine the effects of its supplementation on recreational runners. Application of
date seeds as alternative sources for functional foods can minimize the cost of waste
management as well as the waste of these valuable by-products. The study's limitations include
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript
using a fixed supplement dose, a short duration, and the lack of assessment of the level of
polyphenols or flavonoid content, glycemic indices, other hormones, and inflammatory
biomarkers. This is knowing that longer-term studies testing date seed powder, its polyphenols
extract, or by-products such as bread or chocolate should be conducted to confirm our findings.
In addition, the sample size was calculated based on the decrease in MDA, with 90 percent
power and 95 percent confidence. The power achieved for this particular variable was sufficient
to provide significant results. However, it appears that a larger sample size and higher power are
required to achieve statistical significance for some other variables.
6.
Conclusion
The present double-blind, placebo-controlled clinical trial revealed that date seed
supplementation can improve exercise-induced oxidative stress and performance parameters in
healthy active subjects. Its findings provided new insights into date seed consumption. Further
studies that investigate the effect of DSP in different doses, with longer intervention periods, and
with other exercises are needed.
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript
Declarations
Contributors
Research Project: Dehghan, P; Khani, M; Moslemi, E. Statistical Analysis: Sarbakhsh, P;
Moslemi, E. Manuscript Preparation: Dehghan, P; Moslemi, E. Review and Critique: Dehghan,
P; Khani, M; Sarmadi, B; Moslemi.
Funding
This work is part of a government-funded project supported by the Tabriz University of Medical
Science. Grant number:66809.
Conflict of interest
None of the authors reported a conflict of interest related to the study
Patient and public involvement
Patients and/or the public were not involved in this research's design, conduct, reporting, or
dissemination plans.
Acknowledgments
We would like to thank all the participants who took part in this study. The study was written
based on data obtained from the M.Sc. thesis of Moslemi, E and sponsored by the Tabriz
University of Medical Sciences (Grant number:66809).
ORCID iDS
Elham Moslemi: http://orcid.org/0000-0001-6820-3718
Parvin Dehghan: http://orcid.org/0000-0001-8929-3302
Mostafa Khani: http://orcid.org/0000-0003-3376-9513
Parvin Sarbakhsh: http://orcid.org/0000-0002-4213-5152
Bahareh Sarmadi: http://orcid.org/0000-0002-7083-7127
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript
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Table 1. Chemical composition, total phenolic acid and flavonoid content of date seeds and placebo (100 g) and
each supplement package 1
Name
Date seeds
Placebo (n=3)
(n=3)
Energy (Kcal)
Date seeds package
Placebo package
(13g)
(13g)
270.79 ± 21.65
252.30 ± 19.64
35.20
32.79
Carbohydrate (g)
13.12 ± 2.56
13.15 ± 2.81
1.70
1.70
Protein (g)
6.10 ± 0.82
7.60 ± 0.93
0.79
0.98
Total lipids (g)
5.70 ± 1.03
6.59 ± 1.37
0.74
0.85
Fiber total (g)
66.76 ± 10.43
50.65 ± 11.02
8.67
6.58
Soluble dietary fiber (g)
57.12 ± 11.48
45.67 ± 9.43
7.42
5.93
Insoluble dietary fiber (g)
9.64 ± 2.06
4.98 ± 0.97
1.25
0.64
Ash (g)
1.30 ± 0.11
7.59 ± 1.13
0.16
0.98
Moisture (%)
7.02 ± 1.42
14.42 ± 2.87
0.91
1.87
3456.86 ±
1033.46 ±
449.39
134.34
522.71
231.40
1624.54 ±
28.67 ± 5.22
211.19
3.72
Antioxidants
Total Phenolic acid (mg
GAE/g)
Flavonoid content (mg QE/g
dry weight)
352.12
GAE:Gallic acid equivalent, QE: quercetin equivalent, 1 Data are presented as mean ± SD.
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript
Table 2. Characteristics of the study participants1
Variables
Date seeds (n=17)
Placebo (n=17)
21 ± 2 (19-26)
23 ± 2 (18-27)
Male
9 (52.9)
8 (47.1)
Female
8 (47.1)
9 (52.9)
Moderate (600-3000METs) min.wk−1
11 (64.7)
10 (58.8)
Vigorous (3000 METs) min.wk−1
6 (35.3)
7 (41.2)
Maximum HR (beats/min)
198 ± 2
196 ± 2
Mean THR (beats/min)
191 ± 10
190 ± 10
Weight at baseline (kg)
67.80 ± 10.90
67.60 ± 9.00
Weight at end of trial (kg)
67.40 ± 11.00
67.20 ± 8.80
171 ± 12
172 ± 12
BMI at baseline (kg/m2)
22.80 ± 1.10
22.75 ± 1.05
BMI at end of trial (kg/m2)
22.70 ± 1.00
22.60 ± 1.00
Age(y) *(range)
Gender, n (%)
PAL, n (%)
Anthropometric indices
Height (cm)
BMI: Body mass index, PAL: physical activity level, HR: Heart rate, THR: Target heart rate. 1Data are
presented as frequency (percent) and mean ± SD
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Table 3. Nutritional intakes of subjects at baseline and at the end of the study1
Variables
Baseline
Energy (kcal/d)
Date seeds
2555.50 ± 585.60
Placebo
2599.55 ± 545.60
Protein (g/d)
Date seeds
141.00 ± 47.45
Placebo
139.55 ± 32.90
Carbohydrate (g/d)
Date seeds
321.40 ± 75.00
Placebo
334.45 ± 86.60
Fat (g/d)
Date seeds
84.00 ± 23.20
Placebo
84.10 ± 23.35
Vitamin A(RAE/d)
Date seeds
436.20 ± 159.70
Placebo
487.00 ± 93.20
Vitamin C (mg/d)
Date seeds
163.05 ± 26.50
Placebo
153.80 ± 27.10
Vitamin E (mg/d)
Date seeds
18.05 ± 3.80
Placebo Group
18.50 ± 4.50
α- tocopherol (mg/d)
Date seeds
25.75 ± 3.10
Placebo
25.65 ± 3.05
β-Carotene (mg/d)
Date seeds
533.90 ± 90.20
Placebo
556.55 ± 115.40
Lycopene (μg/d)
Date seeds
1318.55 ± 371.90
Placebo
1300.40 ± 247.90
β-Cryptoxanthin (μg/d)
Date seeds
120.25 ± 18.90
Placebo
114.00 ± 19.30
Zinc (mg/d)
Date seeds
17.10 ± 4.40
Placebo
16.05 ± 2.80
Selenium (mg/d)
Date seeds
64.10 ± 7.85
Placebo
64.70 ± 8.60
1
Data are presented as mean ± SD. a P<0.05,
After 2 weeks
Change
2638.50 ± 590.70
2680.10 ± 468.85
83.00 ± 137.90
80.60 ± 139.40
P-valuea
0.921
0.284
155.60 ± 35.25
147.35 ± 24.30
14.60 ± 31.30
7.80 ± 18.50
336.80 ± 75.40
364.80 ± 88.40
15.35 ± 23.30
30.35 ± 40.25
79.30 ± 29.60
79.10 ± 16.70
-4.70 ± 16.75
-5.10 ± 12.40
0.158
0.959
0.668
472.70 ± 91.80
470.20 ± 83.95
36.50 ± 165.20
-16.80 ± 91.60
180.40 ± 32.60
166.80 ± 25.50
13 ± 30.70
17.30 ± 45.30
0.215
0.561
19.25 ± 5.15
18.30 ± 3.30
1.20 ± 6.70
-0.20 ± 6.50
0.253
26.50 ± 4.60
24.80 ± 3.80
0.80 ± 4.80
-0.90 ± 5.10
532.40 ± 117.90
516.60 ± 132.90
-1.40 ± 157.10
-39.90 ± 171.90
0.733
0.118
1237.20 ± 252.30
1373.00 ± 230.60
-81.35 ± 501.20
72.60 ± 337.40
0.196
127.95 ± 17.20
118.80 ± 23.50
7.70 ± 26.70
4.74 ± 31.10
14.30 ± 3.20
14.60 ± 2.20
-2.80 ± 5.30
-1.40 ± 3.60
0.739
0.477
62.00 ± 14.20
-2.20 ± 15.90
65.20 ± 11.55
0.50 ± 15.00
Analysis of covariance for comparison of data after 2 weeks
between groups after adjusting for baseline values.
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript
Table 4. Markers of oxidative stress and visual analogue scale status of subjects at baseline, the end of the study
and 24 hours after intervention1
Variables
Baseline
After 2 weeks
24h
P supplement *
time b
TAC (mmol/L)
<0.001
Date seeds
1.32 ± 0.04
1.56 ± 0.06
1.65 ± 0.05
Placebo
1.30 ± 0.05
1.41 ± 0.05
1.37 ± 0.05
P-value a
0.222
<0.001
<0.001
TOS (μmol/L)
0.019
Date seeds
11.87 ± 0.64
12.90 ± 0.85
12.01 ± 0.24
Placebo
12.39 ± 0.56
13.95 ± 0.52
13.24 ± 0.05
P-value
0.020
<0.001
<0.001
OSI
<0.001
Date seeds
0.89 ± 0.07
0.82 ± 0.06
0.72 ± 0.03
Placebo
0.95 ± 0.08
0.98 ± 0.06
0.96 ± 0.04
P-value
0.048
<0.001
<0.001
SOD (U/ml)
0.085
Date seeds
0.17 ± 0.02
0.24 ± 0.04
0.23 ± 0.04
Placebo
0.18 ± 0.02
0.22 ± 0.05
0.20 ± 0.04
P-value
0.066
0.411
0.049
GPx (U/ml)
0.001
Date seeds
4.09 ± 0.36
6.16 ± 0.88
5.64 ± 1.25
Placebo Group
4.36 ± 0.57
5.14 ± 0.59
4.82 ± 0.58
0.113
0.001
0.019
P-value
Uric acid (mg/dL)
0.748
Date seeds
4.17 ± 0.41
5.03 ± 0.62
4.31 ± 0.55
Placebo Group
4.21 ± 0.40
5.20 ± 0.91
4.58 ± 0.61
0.785
0.546
0.180
P-value
MDA ( nmol/mL)
0.046
Date seeds
2.05 ± 0.12
2.19 ± 0.26
2.12 ± 0.28
Placebo Group
2.03 ± 0.24
2.42 ± 0.31
2.39 ± 0.32
0.754
0.027
0.015
P-value
8-iso-PGF2α
0.518
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
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(pg/ml)
Date seeds
26.81 ± 2.56
43.92 ± 2.29
35.13 ± 6.12
Placebo Group
27.11 ± 3.41
46.97 ± 5.21
36.82 ± 6.18
0.777
0.034
0.431
P-value
VAS
<0.001
Date seeds
6.35 ± 1.36
5.06 ± 1.14
3.35 ± 1.05
Placebo Group
6.41 ± 1.46
5.82 ± 1.23
5.00 ± 1.11
0.904
0.070
<0.001
P-value
TAC: Total antioxidant capacity, TOS: Total oxidant status, OSI: Oxidative stress Index, SOD: Superoxide
dismutase, GPx: Glutathione peroxidase, MDA: Malondialdehyde, 8-iso-PGF2α: 8-iso-prostaglandin F2α,
VAS: Visual Analogue Scale.
1
Data are presented as mean ± SD.
a
P<0.05, Independent sample student's T-test for comparison of data
between groups in deferent times of baseline, after 2 weeks and 24 h later.
b
P<0.05, Repeated-measures
analysis of variance (ANOVA) and p-value supplement*time reflect the effect of the intervention.
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript
Table 5. Changes in performance markers of subjects at baseline and the end of the study1
Variables
Baseline
After 2 weeks
P-value b
Change
VO2 max (ml/Kg/min)
0.301
Date seeds
46.40 ± 3.49
49.09 ± 2.34
2.69 ±4.87
Placebo
45.06 ± 3.99
47.60 ± 3.55
2.54 ±1.45
P-value a
0.308
0.160
-
Average Power (W)
0.070
Date seeds
456.91 ± 24.59
497.41 ± 17.31
40.50 ± 26.56
Placebo
460.13 ± 30.26
481.49 ± 36.02
21.35 ± 35.16
P-value
0.735
0.110
-
Peak Power (W)
<0.001
Date seeds
654.48 ± 25.24
693.21 ± 27.73
38.72 ± 15.12
Placebo
665.96 ± 41.05
682.58 ± 37.58
16.61 ± 13.58
P-value
0.333
0.355
-
Fatigue Index (W/s)
0.006
Date seeds
45.27 ± 8.72
37.20 ± 5.54
-8.07 ± 11.99
Placebo Group
49.75 ± 9.75
42.93 ± 5.76
-6.81 ± 10.33
0.168
0.006
P-value a
W: watts, W/s: watts/seconds, 1 Data are presented as mean ± SD.
a
P<0.05, Independent sample student's T-
test for comparison of data between groups in deferent times of baseline, after 2 weeks.
b
P<0.05, Analysis of
covariance for comparison of data after 2 weeks between groups and adjusting for baseline values.
https://doi.org/10.1017/S0007114522002124 Published online by Cambridge University Press
Accepted manuscript