The Journal of Nutrition

Nutrition and Disease

Dietary Hemp Seeds More Effectively

Attenuate Disorders in Genetically Obese

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Rats than Their Lipid Fraction
Paulina M Opyd, Adam Jurgoński, Bartosz Fotschki, and Jerzy Juśkiewicz

Department of Biological Function of Food, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn,
Poland

ABSTRACT
Background: Hemp seeds are rich in PUFAs and other bioactives that can attenuate the development of obesity-related
disorders; however, the extent to which their lipid fraction is responsible for this effect is unknown.
Objective: We hypothesized that hemp seed or hemp oil supplementation can attenuate genetically determined
disorders and that the former are more effective in doing so.
Methods: Lean and obese male Zucker rats, aged 8 wk, weighing 174 ± 4.2 g and 223 ± 3.8 g, respectively, were
allocated to 4 groups. The lean (LC) and obese controls (OC) were fed a standard diet, whereas the other 2 obese
groups were fed a modified diet in which hemp oil (4% diet; O + HO) or hemp seeds (12% diet; O + HS) were included.
All diets had the same proportions of protein (18%), fat (8%), and fiber (5%) and a similar carbohydrate proportion
(∼52%). Diets fed to O + HO and O + HS had similar fatty acid profiles. After 4 wk, markers of gut and liver function,
antioxidant status, and lipid metabolism were measured.
Results: The total SCFA concentration in the cecal digesta was lower in OC (64.8 ± 4.21 mol/g) compared with LC
(78.1 ± 2.83 mol/g) (P ≤ 0.05), whereas it was greater in O + HS (89 ± 4.41 mol/g) compared with LC, OC, and
O + HO (69.7 ± 2.68 mol/g) (P ≤ 0.05). Plasma total cholesterol was greater in OC (6.20 ± 0.198 mmol/L) and O + HO
(5.60 ± 0.084 mmol/L) compared with LC (2.71 ± 0.094 mmol/L) (P ≤ 0.05); in O + HS, the concentration did not differ
from the other groups (5.16 ± 0.278 mmol/L). The liver cholesterol concentration was greater in OC (1.79 ± 0.379 mg/g)
compared with the other groups (1.28–1.43 mg/g) (P ≤ 0.05). Hepatic expression of peroxisome proliferator-activated
receptor γ was lower in OC (11.9 ± 0.93 units) compared with LC (17.3 ± 1.3 units) (P ≤ 0.05), whereas it was greater
in O + HS (19.2 ± 1.04 units) compared with OC and O + HO (14.0 ± 1.33 units) (P ≤ 0.05).
Conclusions: Dietary hemp seeds more effectively attenuate metabolic disorders in genetically obese rats than the oil
extracted from them, which suggests that the lipid fraction is only partly responsible for these effects. J Nutr 2020;00:1–
9.

Keywords: hemp seeds, hemp seed oil, gut, metabolic disorders, genetic obesity

Introduction including the most studied cannabinoids; however, only traces

of these compounds are present in the seeds of industrial hemp,
Cannabis sativa L., commonly called industrial hemp, can
but they contain significant amounts of lignanamides (7, 8).
play an important role in the human diet, providing both
These phenolic amides are claimed to have cytotoxic, anti-
nutrients and bioactive compounds (1). Hemp seeds contain
inflammatory, antineoplastic, and cardioprotective activity (7).
∼26% protein rich in arginine, aspartic acid, and glutamic acid,
Nevertheless, hemp seeds also contain some antinutritional
whereas lysine is the limiting amino acid (1–3). Antioxidative
components, especially phytic acid (∼63 mg/g), which can
and antihypertensive peptides from hemp seeds have already
reduce the absorption of mineral elements (9).
been isolated (4, 5), and some oligopeptides exhibited α-
Hemp seeds contain a high percentage of oil (∼36%), which
glucosidase inhibitory activity, which suggests their use as
is usually extracted by cold pressing, and have a characteristic
potential antihyperglycemic agents (6). Hemp seeds are also rich
green color and fresh nutty taste and smell (10). Hemp seed
in dietary fiber, the content of which is ∼28%, including its
oil has a high percentage of PUFAs, including linoleic acid
mainly insoluble fraction (22%) that can beneficially affect an
(56%), α-linolenic acid (20%), γ -linolenic acid (4%), and
organism, especially gut function (1).
stearidonic acid (2%); the latter 2 are rare in nature (1, 11).
A distinctive feature of the Cannabis genus, especially the C.
Moreover, hemp seed oil is characterized by the optimal ratio
indica variety, is that it contains over 480 bioactive compounds,

Copyright  C The Author(s) 2020.

Manuscript received October 8, 2019. Initial review completed January 16, 2020. Revision accepted March 9, 2020.
First published online 0, 2020; doi: https://doi.org/10.1093/jn/nxaa081. 1
of n–6 to n–3 PUFAs (3:1), which is especially important for
cardiovascular health (12). Furthermore, Montserrat-de la Paz
et al. (13) reported some amounts of other bioactive compounds
in the oil, such as sterols and their derivatives (β-sitosterol,
campesterol, cycloartenol) and γ -tocopherol (14). Interestingly,
Al-Khalifa et al. (15) demonstrated that dietary hemp seeds can
provide significant cardioprotective effects during postischemic
reperfusion. The authors suggested that it is due to its high PUFA
content; however, the extent to which the oil is responsible for
such effects remains unknown.
Among various animal models of diseases, Zucker rats
deserve special attention as an example of the best known and
most widely used animal model of genetic obesity (16). Obesity
in Zucker rats is inherited as an autosomal recessive trait caused
by a mutation in the leptin receptor gene (17). The mutation
relies on a single adenine to cytosine change at nucleotide
880, which results in a glutamine to proline substitution at
residue 269 (18). Leptin is produced by adipose tissue and
affects the brain via the leptin receptor, which leads to decreased
food intake and increased energy expenditure (19). As a result
of the mutation, obese Zucker (fa/fa) rats are insensitive to
leptin circulating in their body (20). They are characterized
by food overconsumption, disturbed energy homeostasis, and
drastic weight gain. An additional aspect of their phenotype is
disorders similar to those seen in human metabolic syndrome,
such as fatty liver, dyslipidemia, insulin resistance, chronic
inflammation, and some endocrinological abnormalities (19).
Most of the available studies have focused either on the
antioxidative and antihypertensive activities of peptides isolated
from hemp seeds (4, 5) or on the cardioprotective properties of
hemp seed oil (12). However, to date, the extent to which the
lipid fraction can contribute to the health effects of hemp seed
consumption has not been studied. At the same time, a similar
lack of knowledge is seen when considering the extent to which
dietary factors can affect the development of genetic obesity in
general. Therefore, the aim of this study was to compare the
effect of dietary supplementation with hemp seeds and hemp
seed oil on the development of metabolic disorders in genetically
obese Zucker rats. We hypothesized that hemp seed or hemp
seed oil supplementation can attenuate genetically determined
metabolic disorders and that the former is more effective due to
a wider range of potentially bioactive compounds present in the
seeds.
Methods
Chemical composition of hemp seeds and hemp seed
oil
This study used whole hemp seeds (Cannabis sativa L.), which were a
certified organic food product from the Ekogram company (Zielonki),
whereas unrefined, cold-pressed hemp seed oil was from the Ol’Vita
This research was financially supported by the National Science Centre, Poland
(project number: 2016/23/B/NZ9/01012).
Author disclosures: The authors report no conflicts of interest.
Supplemental Tables 1 and 2 are available from the “Supplementary data” link
in the online posting of the article and from the same link in the online table of
contents at https://academic.oup.com/jn.
Address correspondence to AJ (e-mail: a.jurgonski@pan.olszty.pl).
Abbreviations used: Actb, β-actin; ALP, alkaline phosphatase; ALT, alanine
transaminase; AST, aspartate transaminase; DM, dry matter; GSH, glutathione;
GSSG, glutathione disulfide; LC, lean control; OC, obese control; O + HO, obese
fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp
seeds; Pparα, peroxisome proliferator-activated receptor α; Pparγ , peroxisome
proliferator-activated receptor γ ; Srebp1c, sterol regulatory element-binding
protein 1c; TBARS, thiobarbituric acid-reactive substances; TFA, trans fatty acid.
2 Opyd et al.
company (Panków). The chemical composition of hemp seeds and
hemp seed oil was determined by an accredited research laboratory
(Nuscana, Mrowino, Poland). In the seeds, the dry matter (DM) and
ash content were determined using the gravimetric method after drying
at 105◦ C and ashing at ∼580◦ C. Total dietary fiber was determined
by the enzymatic-gravimetric method, crude protein was determined
by the Kjeldahl method, crude fat was determined by the Soxhlet
extraction method and then the nitrogen-free extract was calculated.
The fatty acid profile of the oil and oil fraction extracted from the
seeds was determined by GC with flame-ionization detection after
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previous conversion of the fatty acids into their respective methyl esters.
Supplementary Table 1 details the basic chemical composition of the
seeds and fatty acid profile of the seeds and oil.
Animals and experimental design
The feeding experiment was conducted on 30 lean (Fa/?) and obese
(fa/fa) Zucker male rats aged 8 wk, which were allocated to 4 groups
(1 lean and 3 obese). Initial body weight (BW) of the rats is shown in
Table 1. The rats were individually housed in plastic cages and
a controlled environment (12-h light-dark cycle, a temperature of
21 ± 1◦ C, relative humidity of 50% to 70%, and 20 air changes
per hour). For 29 d, each group was fed a modified version of the
semipurified casein diet recommended for rodents by Reeves (21). The
lean control (LC) and obese control (OC) groups were fed a diet
containing casein, cellulose, and rapeseed (canola type) and palm oil
(4% of each oil) as the source of protein, fiber, and fat, respectively. The
other 2 obese groups were fed a modification of the standard diet in
which hemp seed oil was added at the expense of palm oil (4% diet; O
+ HO group) or ground hemp seeds were added at the expense of palm
oil, cellulose, and casein (12.05% diet; O + HS group). Hemp seeds were
ground for 1 min at a temperature below 37◦ C prior to their inclusion in
the diet. All diets had the same proportion of protein (18%), fat (8.3%),
and fiber (5%) and a similar carbohydrate proportion (∼52%). Diets
fed to the O + HO and O + HS groups also had similar fatty acid
profiles, including a >2-fold increase in the proportion of PUFAs that
originated either from hemp seeds or hemp seed oil. This experimental
design allowed us to assess the extent to which the lipid fraction of
hemp seeds can contribute to the health effects of their consumption.
The detailed composition of the diets, which were freely available to
rats for the entire experimental period, is shown in Supplementary
Table 2. All diets were in a powdery form and during preparation,
powdered ingredients of commercial origin, such as casein preparation,
corn starch preparation, vitamin mixture, etc. were mixed together in an
ordinary mixer and under standard conditions. After preparation, the
diets were stored at 4◦ C. The experiment was conducted in compliance
with the European guidelines for the care and use of laboratory animals,
and the animal protocol employed in this study was approved by the
local Institutional Animal Care and Use Committee in Olsztyn, Poland
(permission number: 37/2017).
Analysis of body composition in rats
At the beginning and end of the feeding experiment, the body lean and
fat masses of the rats were determined by time-domain NMR using the
Minispec LF 90II analyzer (Bruker). The method relies on transmitting
various radio frequency pulses into soft tissues to reorient the nuclear
magnetic spins of the hydrogen and then detects radio frequency signals
generated by the hydrogen spins from these tissues. The contrast in
relaxation times of the hydrogen spins found between adipose tissue
and water-rich tissues is used to estimate fat and lean body masses.
Collection of biological material and analytical
procedures
After 4 wk of experimental feeding, rats were anesthetized with a
mixture of xylazine and ketamine in physiological salt (10 mg and
100 mg/kg BW, respectively). Each animal was then weighed, and the
abdomen was cut open. Blood was subsequently collected from the vena
cava into heparinized tubes, centrifuged for 10 min at 380 × g and
4◦ C, and the obtained plasma was then frozen until analysis. Next, the
small intestine, cecum, colon, kidneys, liver, and epididymal fat were
TABLE 1 Diet intake, body weight, and body composition of lean and obese Zucker rats fed a diet containing hemp seed oil or hemp
seeds for 4 wk1

Group2
Index LC OC O + HO O + HS ANOVA P value
Initial body weight, g 174 ± 4.2b 224 ± 5.1a 227 ± 8.8a 219 ± 6.6a ≤0.001
Initial body fat, % 16.3 ± 0.54b 47.1 ± 0.58a 47.7 ± 2.33a 46.4 ± 1.25a ≤0.001
Initial body lean, % 77.6 ± 0.59a 45.3 ± 0.54b 45.5 ± 2.80b 46.3 ± 1.17b ≤0.001
± 0.248b ± 0.854a ± 0.580a ± 0.591a ≤0.001

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Diet intake, g/d 15.2 23.9 23.7 24.0
Final body weight, g 266 ± 4.23b 367 ± 10.7a 373 ± 8.62a 350 ± 8.68a ≤0.001
Final body fat, % 22.3 ± 0.854b 68.0 ± 0.869a 65.6 ± 0.610a 67.6 ± 1.95a ≤0.001
Final body lean, % 67.1 ± 1.11a 24.2 ± 1.07c 27.4 ± 0.6851b,c 29.4 ± 2.05b ≤0.001
Body weight gain, g 91.8 ± 4.07b 143.6 ± 6.69a 145.6 ± 7.692a 132.2 ± 7.10a ≤0.001
Body fat gain, g 31.0 ± 2.01b 145.0 ± 8.28a 137.0 ± 5.123a 133.1 ± 5.77a ≤0.001
Body lean gain, g 43.4 ± 3.69a − 13.2 ± 3.18c − 0.270 ± 5.85b,c 2.97 ± 5.25b ≤0.001
1
Values are means ± SEMs, n = 7–8. Labeled means in a row without a common letter (a, b, c) differ at P ≤0.05 (Duncan’s or Dunn’s post hoc test).
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.

removed, weighed, and frozen using liquid nitrogen or were used for
further treatment.
Disaccharidase activity (lactase, maltase, and sucrase) was assayed in
jejunal mucosal samples collected using a microscope slide according to
the method of Dahlqvist with previously described modifications (22).
The disaccharidase activity was expressed as mol of glucose liberated
from disaccharide per min per gram of protein. The mucosal protein
content was determined using the Bradford method with BSA as the
standard.
Samples of fresh ileal, cecal, and colonic digesta were collected, and
the pH values were measured using a microelectrode and pH/ION meter
(model 301, Hanna Instruments). The ammonia concentration in the
fresh cecal digesta was determined in Conway dishes according to the
method described by Hofirek and Haas (23). The SCFA concentration
was determined in cecal digesta after storage at −20◦ C using GC (Shi-
madzu Co.) and a capillary column (SGE BP21, 30 m × 0.53 mm; SGE
Europe Ltd.) as previously described (24). Microbial glycolytic activity
in the colonic digesta (α- and β-glucosidase, α- and β-galactosidase,
and β-glucuronidase) was measured spectrophotometrically using the
rate of p- or o-nitrophenol release from nitrophenylglucosides (Sigma-
Aldrich) according to the method described by Cholewińska et al. (25).
The enzymatic activity (α- and β-glucosidase, α- and β-galactosidase,
and β-glucuronidase) was expressed as micromoles of product formed
per hour per gram of digesta.
The glutathione (GSH) and glutathione disulfide (GSSG) content
in the liver tissue was determined spectrophotometrically using the
method of Rahman et al. (26). Thiobarbituric acid-reactive substances
(TBARS) were determined in the kidney and liver tissue after storage
at −20◦ C using a procedure developed by Botsoglou et al. (27). The
TBARS content was determined spectrophotometrically at 532 nm and
expressed as micrograms of malondialdehyde per gram of tissue. Liver
lipids were extracted according to the method of Folch et al. (28)
with previously described modifications (29). The liver cholesterol and
triglyceride concentrations were determined spectrophotometrically in
the extracted lipid phase using reagents from Alpha Diagnostics Ltd.
The plasma concentration of cholesterol (total and its HDL and
LDL fractions), triglycerides, urea, uric acid, creatinine and the plasma
activities of aspartate transaminase (AST), alanine transaminase (ALT),
and alkaline phosphatase (ALP) were determined using an automatic
biochemical analyzer (Pentra C200, Horiba Ltd.).
Gene expression analysis in the liver
qRT-PCR was performed according to the previously described method
(29). Briefy, RNA was extracted from the liver using TRI Reagent
solution (Thermo Fisher Scientific) according to the manufacturer’s
instructions. β-actin (Actb) was selected as a reference gene. The levels
of peroxisome proliferator-activated receptor α (Pparα), peroxisome
proliferator-activated receptor γ (Pparγ ), sterol regulatory element-
binding protein 1c (Srebp1c), and Actb mRNA expression were
analyzed using Single Tube TaqManVR Gene Expression Assays (Life
Technologies). Amplification was performed using a 7900HT Fast Real-
Time PCR System. The mRNA expression levels of Pparα, Pparγ , and
Srebp1c were normalized to Actb and multiplied by 10.
Statistical analysis
The results are expressed as the mean ± SEM: however, the chemical
composition of hemp seeds and hemp seed oil is expressed as the
mean ± SD. One-factor ANOVA and Duncan’s multiple range post
hoc test were used to determine significant differences between groups
(P ≤ 0.05). If the variance was not homogenous, the Kruskal–Wallis
1-factor ANOVA by ranks was used followed by Dunn’s post hoc test
(P ≤ 0.05). All calculations were performed using STATISTICA version
12 (StatSoft Corp.).
Results
The chemical composition of hemp seeds and hemp seed oil is
shown in Supplementary Table 1. The main component of the
seeds was fat, the proportion of which equaled 33.2% DM.
Hemp seeds were also a good source of crude protein and
dietary fiber; the proportions of these components were 26.3%
and 27.5% DM, respectively. The fatty acid content of the seeds
and oil was similar and expressed as the percentage of total
fatty acids. Both hemp seeds and hemp seed oil were rich in
PUFAs (76.4% and 76.1%, respectively), especially in linoleic
acid (52.3% and 52.8%, respectively), α-linolenic acid (18.1%
and 17.5%, respectively), and γ -linolenic acid (4.4% and 4.3%,
respectively). The total content of MUFAs equaled 9.37% and
10.0% for the seeds and oil, respectively, including oleate as the
main MUFA (7.9% and 8.6%, respectively). The overall content
of SFAs in the seeds and oil was 9.34% and 9.02%, respectively,
including palmitate as the main SFA (5.80% and 5.46% of total
fatty acids, respectively). Interestingly, the fatty acid fraction of
the seeds and oil also contained a small proportion of a trans
fatty acid (TFA, cis-9, trans-12-octadecadienoic acid, 0.11%
and 0.12%, respectively).
The effect of dietary supplementation with hemp seeds and
hemp seed oil on rat BW and body composition is shown in
Table 1. The initial BW and body fat percentage were similarly
higher, whereas the initial body lean percentage was similarly
lower in all 3 obese groups compared with the LC group
(P ≤ 0.05). The initial body fat percentage was almost 3 times
lower in the LC group compared with the obese groups, which
did not differ from one another. Moreover, the dietary intake
Effects of dietary hemp seeds and hemp seed oil 3
TABLE 2 Physiological indices of the small intestine, cecum, and colon in lean and obese Zucker rats fed a diet containing hemp
seed oil or hemp seeds for 4 wk1

Group2
Index LC OC O + HO O + HS ANOVA P value
Small intestine
Mass with digesta, g/100 g BW 3.13 ± 0.037b 8.68 ± 0.391a 7.29 ± 0.249a 7.15 ± 0.361a,b ≤0.001
pH of digesta 7.06 ± 0.064a 7.06 ± 0.127a 7.02 ± 0.110a 6.57 ± 0.058b <0.01
Mucosal disaccharidase activity, mol · min−1 · g protein−1

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Sucrase 8.22 ± 0.627b 13.1 ± 0.993a 12.4 ± 0.984a 11.9 ± 1.05a <0.005
Maltase 62.4 ± 3.75 66.7 ± 4.91 62.2 ± 6.64 59.7 ± 4.48 NS
Lactase 2.42 ± 0.199 2.82 ± 0.200 2.55 ± 0.188 2.67 ± 0.260 NS
Cecum
Tissue mass, g/100 g BW 0.154 ± 0.007a 0.107 ± 0.005c 0.106 ± 0.004c 0.125 ± 0.004b ≤0.001
Digesta mass, g/g tissue 3.73 ± 0.279 3.40 ± 0.299 3.45 ± 0.193 3.08 ± 0.256 NS
pH of digesta 7.31 ± 0.065 7.55 ± 0.105 7.38 ± 0.091 7.29 ± 0.028 NS
Ammonia, mg/g digesta 0.431 ± 0.019 0.391 ± 0.035 0.428 ± 0.027 0.408 ± 0.009 NS
Microbial enzyme activity, mol · h−1 · g digesta−1
α-glucosidase 13.9 ± 1.25 14.2 ± 1.43 16.8 ± 1.68 15.2 ± 1.12 NS
β-glucosidase 1.93 ± 0.470a,b 1.21 ± 0.177b 1.59 ± 0.124a,b 3.17 ± 0.391a <0.01
α-galactosidase 14.9 ± 1.65a 7.93 ± 1.93b 8.86 ± 1.72b 15.1 ± 1.05a <0.005
β-galactosidase 23.9 ± 2.45b 24.1 ± 3.09b 28.2 ± 3.67b 52.8 ± 5.17a ≤0.001
β-glucuronidase 19.8 ± 3.94 17.3 ± 2.49 22.8 ± 4.46 24.8 ± 1.59 NS
Colon
Tissue mass, g/100 g BW 0.299 ± 0.023a 0.248 ± 0.011b 0.227 ± 0.011b 0.235 ± 0.007b <0.01
Digesta mass, g/g tissue 1.12 ± 0.188 1.59 ± 0.062 1.45 ± 0.107 1.24 ± 0.069 NS
pH of digesta 7.42 ± 0.047a 7.31 ± 0.042a 7.37 ± 0.051a 7.14 ± 0.062b <0.005
1
All values are expressed as the mean ± SEM (n = 7–8). Values not sharing the same superscript (a, b, c) within a row are different at P ≤ 0.05. BW, body weight; NS,
nonsignificant, P >0.05.
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.

calculated on a daily basis was also similarly higher in the obese
groups compared with the LC group (P ≤ 0.05). As a result, after
4 wk of experimental feeding, obese rats, irrespective of the diet
type, gained similarly more body weight and >4 times the body
fat mass compared with the LC group (P ≤ 0.05). Interestingly,
obese rats from the OC and O + HO groups had negative lean
body gain, whereas dietary hemp seeds significantly increased
body lean gain (compared with the OC group; P ≤ 0.05) but not
to the level of the LC group where it was still >14 times higher.
The final body fat percentage was almost 3 times higher whereas
the final body lean percentage was >2 times lower in the obese
groups compared with those in the LC group. Nevertheless, the
final body lean percentage was significantly increased in the
O + HS group compared with the percentage in the OC group
(P ≤ 0.05).
The effects of hemp seed and hemp seed oil supplementation
on physiological indices of the small intestine, cecum, and colon
in rats are shown in Table 2. In the OC and O + HO groups,
an increase in the mass of the small intestine with digesta
calculated per 100 g of BW was observed (compared with
the LC group; P ≤ 0.05), whereas hemp seed supplementation
prevented a significant increase (P ≤ 0.05). The cecum and
colon relative tissue masses were decreased in all 3 obese
groups (compared with the LC group; P ≤ 0.05); dietary
hemp seeds significantly increased the cecum tissue mass, but
not to the level of the LC group (P ≤ 0.05). Neither the rat
phenotype nor the experimental factors influenced the relative
cecal and colon digesta masses (P = 0.925 and P = 0.095,
respectively). However, dietary hemp seed supplementation
decreased the small intestinal and colonic digesta pH compared
with the pH values of the other groups (P ≤ 0.05), which
did not differ from one another, but the cecal digesta pH
was comparable among all groups (P > 0.05). Moreover, both
the endogenous and exogenous intestinal enzymatic activities
were affected by the rat phenotype and/or dietary factors
(Table 2). As for the endogenous enzymes of the small intestinal
mucosa, the sucrase activity was similarly increased in all obese
groups (compared with the LC group; P ≤ 0.05), whereas
the maltase and lactase activity did not differ among all
groups (P > 0.05). In the cecum, the microbial α-galactosidase
activity was decreased in the OC and O + HO groups
(compared with the LC group; P ≤ 0.05), whereas dietary
hemp seeds increased the activity to the level of the LC group.
When compared with the OC group, supplementation with
hemp seeds increased β-glucosidase and β-galactosidase activity
(P ≤ 0.05).
The concentration, profile, and pool of SCFAs, being the
main microbial fermentation products in the cecum, are shown
in Table 3. The total SCFA concentration was decreased in
the OC group compared with the LC group (P ≤ 0.05).
Dietary supplementation with hemp seeds caused a significant
(compared with the LC and OC groups; P ≤ 0.05) increase in
the total SCFA concentration. This was mainly a result of the
increased acetate concentration in the O + HS group compared
with that in the other groups, which did not differ from one
another, as well as the increased propionate concentration in
the O + HS group compared with the concentration in the
OC and O + HO groups (all at P ≤ 0.05). The acetate,
propionate, and butyrate percentage was comparable among
all groups (P > 0.05). When compared with both control
groups, dietary hemp seeds decreased the SCFA proportion of
putrefactive origin (PSCFA; P ≤ 0.05), which was calculated as
the sum of isobutyrate, isovalerate, and valerate. The SCFA pool
was decreased in the OC group compared with the pool in the

4 Opyd et al.
TABLE 3 SCFA production in the cecal digesta of lean and obese Zucker rats fed a diet containing hemp seed oil or hemp seeds for
4 wk1

Group2
Index LC OC O + HO O + HS ANOVA P value
SCFA concentration, mol/g digesta
Acetate 48.9 ± 2.40b 41.2 ± 3.14b 44.9 ± 2.49b 59.6 ± 4.10a <0.005
Propionate 15.3 ± 0.554a,b 13.3 ± 1.35b 13.7 ± 0.705b 17.6 ± 0.750a <0.05
± ± ± ±

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Butyrate 8.56 0.674 5.72 1.097 6.98 0.827 7.29 0.509 NS
PSCFA3 5.24 ± 0.226 4.59 ± 0.364 4.17 ± 0.232 4.76 ± 0.243 NS
Total 78.1 ± 2.83b 64.8 ± 4.21c 69.7 ± 2.68b,c 89.3 ± 4.41a ≤0.001
SCFA profile, %
Acetate 62.5 ± 1.19 63.3 ± 1.61 64.2 ± 1.64 66.4 ± 1.32 NS
Propionate 19.7 ± 0.319 20.6 ± 1.86 19.7 ± 1.03 19.8 ± 0.492 NS
Butyrate 11.1 ± 0.885 8.91 ± 1.57 10.1 ± 1.26 8.37 ± 0.803 NS
PSCFA 6.74 ± 0.255a 7.20 ± 0.538a 6.01 ± 0.372a,b 5.45 ± 0.436b <0.05
SCFA pool, mol/total digesta mass 119 ± 9.38a 85.2 ± 8.4b 94.6 ± 6.21a,b 117 ± 7.46a <0.05
1
All values are expressed as the mean ± SEM (n = 7–8). Values not sharing the same superscript (a, b, c) within a row are different at P ≤ 0.05. NS, nonsignificant, P >0.05;
PSCFA, SCFA of putrefactive origin.
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.
3
The sum of isobutyrate, isovalerate, and valerate.

LC group (P ≤ 0.05), and dietary hemp seeds prevented this
decrease (P ≤ 0.05).
Markers of liver function, the antioxidant status of the liver,
and the plasma and liver markers of lipid metabolism are shown
in Table 4. The relative liver mass was almost 4 times higher
in the OC and O + HO groups than the LC group, whereas
dietary hemp seeds decreased it to a level comparable with that
of the LC group. The liver fat and triglyceride contents were
>3 times higher in all obese groups compared with content
in the LC group, whereas the liver cholesterol content was
significantly increased only in the OC group (compared with
the other groups; P ≤ 0.05). The antioxidant status of the
liver was comparable between the LC, OC, and O + HO
groups (P > 0.05). In contrast, the liver TBARS content,
as a marker of lipid peroxidation, was significantly reduced
by dietary hemp seeds compared with that of the LC group
(P ≤ 0.05). Moreover, dietary supplementation with hemp seeds
increased the liver GSH and the sum of reduced and oxidized
glutathione (GSH + GSSG) compared with those of the LC
group (P ≤ 0.05). The plasma ALT, AST, and ALP activities
were increased in the OC and O + HO groups compared
with activities in the LC group (P ≤ 0.05), and dietary hemp

TABLE 4 Liver and blood plasma markers of lipid metabolism, antioxidant status, and liver function in lean and obese Zucker rats fed
a diet containing hemp seed oil or hemp seeds for 4 wk1

Group2
Marker LC OC O + HO O + HS ANOVA P value
Liver
Mass, g/100 BW 6.88 ± 0.446b 26.7 ± 2.05a 25.9 ± 2.65a 21.8 ± 1.57a,b ≤0.001
Fat, % 6.89 ± 0.629b 24.7 ± 1.63a 26.0 ± 2.02a 22.8 ± 1.53a ≤0.001
Triglycerides, mg/g 5.92 ± 0.565b 27.5 ± 1.51a 28.7 ± 1.53a 26.1 ± 1.90a ≤0.001
Cholesterol, mg/g 1.43 ± 0.093b 1.79 ± 0.379a 1.28 ± 0.142b 1.28 ± 0.118b <0.05
Antioxidant status
TBARS, g/g 539 ± 22.8a 486 ± 25.6a,b 491 ± 15.7a,b 454 ± 10.8b <0.05
GSH, mol/g 5.84 ± 0.226b 6.00 ± 0.180b 6.34 ± 0.280a,b 6.99 ± 0.262a <0.05
GSSG, mol/g 0.363 ± 0.021 0.363 ± 0.015 0.388 ± 0.025 0.403 ± 0.018 NS
GSH + GSSG, mol/g 6.20 ± 0.237b 6.36 ± 0.189b 6.72 ± 0.297a,b 7.39 ± 0.264a <0.05
GSH/GSSG, mol/g 1.64 ± 0.091 1.66 ± 0.059 1.66 ± 0.080 1.76 ± 0.095 NS
Plasma
Enzyme activity, U/L
AST 72.3 ± 5.12b 255 ± 61.4a 216 ± 34.7a 113 ± 15.1a,b ≤0.001
ALT 41.0 ± 1.96b 263 ± 56.2a 149 ± 12.6a 130 ± 18.5a,b ≤0.001
ALP 235.6 ± 10.9b 593 ± 116.0a 718 ± 80.9a 364 ± 23.2a,b ≤0.001
Lipid profile, mmol/L
Total cholesterol 2.71 ± 0.094b 6.20 ± 0.198a 5.60 ± 0.084a 5.16 ± 0.278a,b ≤0.001
HDL cholesterol 0.984 ± 0.028c 2.19 ± 0.099a 1.71 ± 0.053b 1.62 ± 0.065b ≤0.001
Non-HDL cholesterol 1.73 ± 0.076b 4.01 ± 0.120a 3.89 ± 0.086a 3.54 ± 0.250a,b ≤0.001
Triglycerides 2.42 ± 0.322b 5.14 ± 0.692a 3.42 ± 0.679b 4.01 ± 0.372a,b <0.05
1
All values are expressed as the mean ± SEM (n = 7–8). Values not sharing the same superscript (a, b, c) within a row are different at P ≤ 0.05. BW, body weight; GSH,
glutathione; GSH/GSSG, glutathione to glutathione disulfide ratio; GSSG, glutathione disulfide; NS, nonsignificant, P >0.05; TBARS, thiobarbituric acid-reacting substances.
2
LC, lean control; OC, obese control; O + HO, obese fed a diet containing hemp seed oil; O + HS, obese fed a diet containing hemp seeds.

Effects of dietary hemp seeds and hemp seed oil 5


FIGURE 1 qRT-PCR analysis of Pparα, Pparγ , and Srebp1c mRNA
expression in the liver of lean and obese Zucker rats fed a diet
containing hemp seed oil or hemp seeds for 4 wk. Values are
means ± SEMs, n = 7–8. Labeled means without a common letter
(a, b, c) differ at P ≤0.05 (Duncan’s or Dunn’s post hoc test). Actb,
β-actin; LC, lean control; OC, obese control; O + HO, obese fed a
diet containing hemp seed oil; O + HS, obese fed a diet containing
hemp seeds; Pparα, peroxisome proliferator-activated receptor α;
Pparγ , peroxisome proliferator-activated receptor γ ; Srebp1c, sterol
regulatory element-binding protein 1c.
seeds slightly decreased the activity; however, the activity was
comparable to that of the LC group (Table 4). The plasma
cholesterol concentration, including the HDL and non-HDL
fractions, and the plasma triglyceride concentrations were at
least twice as high in the OC group compared with the LC
group. Both dietary oil and seeds decreased the HDL cholesterol
concentration (compared with the OC group; P ≤ 0.05), but not
to the level of the LC group (P >0.05). The total cholesterol
and non-HDL cholesterol concentrations were decreased by
dietary hemp seeds to the level of the LC group. The triglyceride
concentrations were significantly decreased only by dietary
hemp seed oil (compared with the OC group; P ≤ 0.05), but
their concentrations both in the O + HO and O + HS groups
corresponded with those of the LC group.
The effects of dietary hemp seed and hemp seed oil
supplementation on the mRNA expression of Pparα, Pparγ ,
and Srebp1c in rat livers are shown in Figure 1. Pparα and
Srebp1c expression was similarly decreased in all 3 obese groups
compared with expression in the LC group (P ≤ 0.05). Pparγ
expression was decreased in the OC group compared with the
LC group (P ≤ 0.05), whereas dietary supplementation with
hemp seeds significantly increased Pparγ expression (P ≤ 0.05)
to a level comparable with that of the LC group.
Discussion
The aim of this study was to compare the effect of dietary
supplementation with hemp seed oil and hemp seeds on
metabolic disorders in genetically obese rats. The study required
a precise design of semipurified diets to assess the extent to
which the lipid fraction could contribute to the health effects of
hemp seed supplementation. Thus, the first stage of the study
was to analyze, in detail, the chemical composition of hemp
seeds and hemp seed oil, so that the experimental diets would
be adequately formulated (results in Supplementary Table 1).
The chemical composition of hemp seeds can be affected by
6 Opyd et al.
the cultivar, the environment, including soil conditions, and
the grade of postharvest processing, which causes variability
in the available study results (2, 30). Nevertheless, our findings
confirm that hemp seeds are a good source of protein, fat, and
fiber. The protein content was slightly higher in the present
study (26.3% DM) than in the literature, where it ranged from
24.0 to 25.6% (1, 2, 31). The fiber content was similar to that
reported by Callaway et al. (1) and was ∼6% lower than in
the study by Mattila et al. (3). The fat content in hemp seeds
Downloaded from https://academic.oup.com/jn/advance-article-abstract/doi/10.1093/jn/nxaa081/5818727 by Macquarie University user on 12 April 2020
reportedly ranged from 29.2 to 35.5% DM (1–3, 31), which
was in accordance with this study, where it was 33.3% DM. Of
importance, from the study design perspective, was that the fatty
acid profile of hemp seed oil and hemp seeds was very similar,
which may indicate sufficient stability of fatty acids during oil
extraction. Moreover, da Porto et al. (11) showed that PUFAs
can comprise over 80% of the total fatty acids in hemp seed
oil, which was higher than the proportions found for hemp
seed oil (76.1%) and hemp seeds (76.4%) in the present study.
The main PUFAs determined in similar quantities in the seeds
and oil were linoleic acid, α-linolenic acid, and γ -linolenic acid
(∼52%, 18%, and 4%, respectively), which was generally in
accordance with the literature (1, 11, 32), although the linoleic
acid percentage was 4–7% lower in the present study. Linoleic
acid and α-linolenic acid are 2 essential fatty acids belonging to
the n–6 and n–3 fatty acid families, respectively. Consumption
of n–6 and n–3 fatty acids is associated with an improvement in
blood lipid profile and the regulation of inflammatory processes
in the body through the production of eicosanoids (33). γ -
Linolenic acid is rarely found in nature, but it occurs in human
milk and has anti-inflammatory and anticancer activities (34).
Hemp seeds also contained a small amount of TFA (cis-9, trans-
12-octadecadienoic acid), which can potentially be detrimental
to the body because TFAs are known to raise the risk of
atherosclerosis and coronary heart disease by inhibiting the
synthesis of PUFAs in the phospholipids of arterial cells (35).
Obesity in Zucker (fa/fa) rats begins manifesting in the
first month of their life (19). At the beginning of the present
experiment, obese rats aged 8 wk weighed ∼30% more than
their lean counterparts, and their body fat percentage was
almost 3 times higher (Table 1). As a result of leptin resistance,
obese Zucker rats develop hyperphagia 17 d after birth(19). In
the present study, an increased diet intake in obese rats was
also observed, which in turn led to the increase in BW and
fat percentage and to the decrease in lean body percentage by
the end of the experiment. Dietary supplementation with hemp
seeds slightly, but statistically significantly, increased the lean
body content, which might be due to some favorable changes in
the amino acid profile of the diet. It is known that the quality,
digestibility, and amino acid profile of consumed protein can
trigger a rise in muscle protein synthesis (36). Hemp seed protein
is highly digestible and the overall pattern of amino acid supply
positions it as a good source of vegetable-based protein (2, 3).
It is thought that a single genetic mutation in a mammalian
gene responsible for regulating food intake can affect the
composition of the gut microbiota within the body (37). An in
vivo experiment on Zucker rats has shown that the obesogenic
phenotype is characterized by significantly lower bacterial
counts in the hindgut than the lean phenotype (38). This finding
seems to be in accordance with the present study because both
the cecal α-galactosidase activity and SCFA production were
decreased in the OC group, indicating that the metabolic activity
of the microbiota was reduced (Table 2 and 3). Interestingly,
dietary hemp seed supplementation increased the microbial
glycolytic activity (β-glucosidase and α- and β-galactosidase),
which was apparently due to the fiber fraction of hemp seeds.
This fraction constituted 66% of the dietary fiber in the O + HS
group, and although mostly insoluble, it was apparently a good
substrate for the gut microbiota as the cecal SCFA production
was also considerably increased in this group. Fermentation of
dietary fiber within the hindgut affects the microbiota and its
activity. The main product of fiber fermentation is SCFAs, which
play an important role in the regulation of energy metabolism
and fulfill a number of important metabolic functions within
the body (39). Moreover, the present study shows that the
endogenic enzyme activity is also changed in obese Zucker rats,
namely, the mucosal sucrase activity in the small intestine was
considerably increased. One of the explanations for this change
is overconsumption of the diet, including sucrose, which is an
important dietary source of carbohydrates.
GSH is a tripeptide involved in the elimination of reactive
oxygen species from the organism, especially from the liver.
In our study, dietary hemp seeds significantly elevated the
content of total glutathione (GSH + GSSG) and its reduced
form (GSH) in the liver (Table 4). This might be partly due
to a relatively high content of cysteine in hemp seed protein
compared with its 5 times lower content in casein, which was
used as the main source of protein in all experimental diets.
Cysteine is 1 of the primary amino acids necessary for GSH
synthesis, and the intracellular pool of cysteine is relatively
small compared with the metabolically active pool of GSH,
so it is generally considered the limiting amino acid for GSH
synthesis (40). Moreover, in the present study, dietary hemp
seeds also decreased the TBARS content in the liver as a marker
of lipid peroxidation (Table 4), which is in accordance with the
study by Girgih et al. (5), who showed that hemp seed peptides
were effective inhibitors of oxidative stress in spontaneously
hypertensive rats. Nevertheless, the amino acid profile of protein
is not the only factor that may be responsible for the antioxidant
activity of hemp seeds. For example, Chen et al. (41) reported a
high antioxidant activity of hemp seed hull extract and amides
and lignanamides responsible for it (N-trans-caffeoyltyramine
and cannabisin B). Moreover, an in vitro study by Yan et al.
(8) showed that lignanamides, which are 1 of the primary
phenolic compounds in hemp seeds, exhibited powerful radical
scavenging activity.
In addition to obesity, Zucker (fa/fa) rats are also character-
ized by liver disorders, including liver enlargement and steatosis
(17). In our study, these disorders are exhibited by the several-
fold increased liver fat percentage and plasma aminotransferase
activities (ALP and AST) as well as the >2-fold increased plasma
ALP activity (Table 4). This indicates liver damage and problems
with bile outflow to the gut. Interestingly, 4-wk feeding with
dietary hemp seeds reduced ALT, AST, and ALP activity to levels
comparable with those of the LC group.
In the present study, obese rats were characterized by >2-fold
increased plasma concentrations of triglycerides and choles-
terol, including the HDL and non-HDL fractions (Table 4).
These results are in agreement with the study by Liao et al. (42),
who observed elevated plasma concentrations of cholesterol
and triglycerides in obese Zucker rats. De Artinano and Castro
(19) showed that an increase in plasma lipid and lipoprotein
concentrations is 1 of the first abnormalities that can be
observed in obese Zucker rats. In this study, obese Zucker
rats were also characterized by liver enlargement, which was
mainly due to the >3-fold higher fat content (Table 4). The liver
triglyceride and cholesterol contents were also considerably
increased in the OC group, which is in accordance with previous
studies on this animal model (43). In the present study, all of
the aforementioned disorders were accompanied by reduced
hepatic expression of Pparα, Pparγ , and Srebp1c (Figure 1),
which are transcription factors involved in the regulation of
lipid metabolism. Genes activated by Pparα or Pparγ stimulate
β-oxidation of fatty acids or adipogenesis, respectively, whereas
genes activated by Srebp1c stimulate de novo lipogenesis (44,
45). This suggests that the lipid metabolism in Zucker rats is
heavily disturbed at the molecular level as well. Interestingly, it
has been shown that Pparγ -deficient mice develop severe fatty
Downloaded from https://academic.oup.com/jn/advance-article-abstract/doi/10.1093/jn/nxaa081/5818727 by Macquarie University user on 12 April 2020
liver associated with a failure to store and process triglycerides
(46, 47), which seems to be in accordance with our results.
Nevertheless, both dietary hemp seed oil and hemp seeds were
able to affect lipid metabolism, although the effectiveness of
the seeds was better. To the best of our knowledge, this is the
first study examining the effect of hemp seed supplementation
on lipid metabolism, whereas the effect of hemp seed oil
has already been studied to some extent. Sargolzaie et al.
(48) showed that an 8-d long hemp seed oil intraperitoneal
injection decreased total and HDL cholesterol and triglyceride
concentrations in the serum of rats. Schwab et al. (49) observed
the lipid-lowering effects of hemp seed oil consumed by healthy
humans. In the present study, both dietary supplements reduced
the hepatic cholesterol content and plasma HDL cholesterol
concentration, whereas seed supplementation also decreased
the plasma concentration of total cholesterol and its non-HDL
fraction (Table 4). On the other hand, the plasma triglyceride
concentration was not significantly decreased by the seeds,
as it was by the oil. All the aforementioned effects can be
considered favorable for the organism, except for the reduction
in plasma HDL cholesterol observed after hemp seed and hemp
seed oil supplementation. Since rats in the OC group had
>2 times higher plasma HDL cholesterol concentrations, this
detrimental effect could have been strain related. Indeed, our
latest study comparing the effects of hemp seed oil in lean
and obese Zucker rats indicated that a decrease in the plasma
HDL cholesterol concentration was observed only in obese
animals (data not shown). Nevertheless, the modest presence of
cis-9, trans-12-octadecadienoic acid in the lipid fraction might
also have been a factor, since it has been shown that TFAs
can considerably decrease HDL cholesterol in rat blood (50).
Moreover, our study suggests that the lipid-lowering effects of
hemp seeds were not strictly associated with PUFAs and that
other components present in the seeds also played a role. One
of the factors could be the fiber fraction and, thus, the increased
cecal formation of SCFAs, especially propionate, which after
absorption, can decrease hepatic lipogenesis and improve the
blood lipid profile (51). Interestingly, Al-Lahham et al. (52)
suggested that propionic acid can activate the expression of
Pparγ , and in the present study, both the hepatic expression of
Pparγ and the cecal production of propionate were increased
after dietary hemp seed supplementation. However, further
studies on the PPARG protein concentration and expression
of genes activated by this transcription factor are necessary to
confirm this finding.
Obese Zucker rats are characterized by a number of
metabolic disorders, including those found within the gut and
mainly associated with reduced metabolic activity of the micro-
biota, as well as many others, such as hyperlipidemia, fatty liver,
signs of liver injury, and decreased hepatic mRNA expression
of transcription factors that regulate lipid metabolism. Dietary
supplementation with hemp seeds can improve the fermentation
efficiency of the gut microbiota and liver functions, including
its antioxidant status, and has lipid-lowering effects; however,
it is not able to attenuate the development of obesity
Effects of dietary hemp seeds and hemp seed oil 7
itself. Moreover, dietary supplementation with hemp seeds is
more effective at attenuating genetically determined disorders
than is the oil extracted from them, which suggests that
the lipid fraction, including mainly PUFAs, is only partly
responsible for these effects. Moreover, this study suggests
that dietary supplementation with seeds rich in nutrients and
containing other bioactive compounds can attenuate genetically
determined metabolic disorders, although it is only possible to
a limited extent.
Acknowledgments
We thank the technicians from the Department of Biological
Function of Food and Animal Quarters, Institute of Animal
Reproduction and Food Research of PAS, for their assistance in
conducting the experiment. The authors’ responsibilities were
as follows—AJ and PMO: designed the research; AJ, PMO, BF,
and JJ: performed the experiments; AJ and PMO: analyzed the
data; PMO and AJ: wrote the draft and final manuscript; AJ:
had primary responsibility for the final content; and all authors:
read and approved the final manuscript.
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