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Food Research International 76 (2015) 666674

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Antioxidant potential of dietary chia seed and oil (Salvia hispanica L.) in
diet-induced obese rats
Rafaela da Silva Marineli, Sabrina Alves Lenquiste, rica Aguiar Moraes, Mrio Roberto Marstica Jr.
Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas UNICAMP, Campinas, Sao Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to investigate the effects of dietary chia seed and oil on plasma and liver oxidative status in diet-
Received 15 May 2015 induced obese rats. Thirty-six Wistar rats were divided in six groups (6 animals each): control group was fed the
Received in revised form 20 July 2015 American Institute of Nutrition (AIN)-93 M diet; HFF group was fed a high-fat and high-fructose (HFF) diet;
Accepted 24 July 2015
chia seed short (6-weeks) and long (12-weeks) treatments received an HFF diet with chia seed; chia oil short
Available online 28 July 2015
(6-weeks) and long (12-weeks) treatments received an HFF diet with chia oil. Plasma and hepatic biomarkers
of lipid peroxidation, endogenous enzymatic and non-enzymatic antioxidant systems and antioxidant capacity
Chia (Salvia hispanica) were determined. HFF diet induced weight gain, oxidative stress and lipid peroxidation in plasma and liver of an-
Omega-3 fatty acid imals. Compared to HFF group chia seed and chia oil (12 and 6 weeks) intake increased plasma reduced thiol
Oxidative stress (GSH) levels, plasma catalase (CAT) and glutathione peroxidase (GPx) activities. In the liver glutathione reduc-
Lipid peroxidation tase (GRd) activity was enhanced, while CAT and GPx activities did not change. There were no differences in plas-
Antioxidant enzymes ma and liver superoxide dismutase activity among chia diets and HFF group. Chia (seed and oil) intake did not
Rats modify liver lipid peroxidation, but was able to reduce plasma thiobarbituric acid reactive substances (TBARS)
and 8-isoprostane levels increased by HFF group. Plasma and hepatic antioxidant capacity values were increased
in chia seed and oil groups about 35% and 47%, respectively, compared to HFF group. Chia groups presented sim-
ilar antioxidant potential, regardless of treatment time. Dietary chia seed and oil reduced oxidative stress in vivo,
since it improved antioxidant status and reduced lipid peroxidation in diet-induced obese rats.
2015 Elsevier Ltd. All rights reserved.

1. Introduction (GPx) and glutathione reductase (GRd) activities, and also affects the
non-enzymatic antioxidant systems (reduced thiol, minerals, vitamins
Obesity has become a worldwide epidemic and may be a state of and polyphenols) which play essential roles in many antioxidant mech-
chronic oxidative stress, which consists in an imbalance between pro- anisms (Brambilla et al., 2008; Fernndez-Snchez et al., 2011). Many
tector endogenous antioxidant systems and free radicals generation investigations have shown that consumption of natural dietary sources
(Fernndez-Snchez et al., 2011). The reactive oxygen species (ROS) ex- (fruits, nuts, vegetables) with antioxidant bioactive compounds
cess can damage cell proteins, lipids and DNA, which might result in loss (polyphenols, tocopherols, carotenoids, vitamins) may support the pre-
of function and even cellular death. This mechanism has been linked to vention of oxidative stress and could be a natural alternative for
many diseases, such as cardiovascular disease, neurodegeneration, can- preventing and controlling chronic diseases (Avignon, Hokayem,
cer and diabetes (Brambilla et al., 2008; Pandey & Rizv, 2010). Further- Bisbal, & Lambert, 2012; Bullo, Lamuela-Raventos, & Salas-Salvado,
more, obesity impairs the enzymatic antioxidant system, with reduction 2011; Landete, 2012). Several mechanisms have been proposed for
of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase this health protection, such as the improvement of antioxidant defense
system which protects and reduces ROS damage in the organism
Abbreviations: AIN, American Institute of Nutrition; ALA, alpha-linolenic acid; CAT, cat- (Avignon et al., 2012; Brambilla et al., 2008). Therefore, the evaluation
alase; FAME, fatty acids methyl esters; FRAP, ferric reducing antioxidant power; GC, gas of potential functional foods that may improve antioxidant systems ef-
chromatography; GPx, glutathione peroxidase; GRd, glutathione reductase; GSH, reduced
cacy or alleviate reactive oxygen and nitrogen species formation
thiol; HFF, high-fat and high-fructose diet; LDL, low-density lipoprotein; MDA,
malondialdehyde; PB, phosphate buffer; ROS, reactive oxygen species; SOD, superoxide could be a strategy to prevent obesity complications.
dismutase; TBA, 2-thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; In this context, chia (Salvia hispanica L.) is an herbaceous plant that
TCA, trichloroacetic acid. belongs to Lamiaceae family native from southern Mexico and northern
Corresponding author at: Monteiro Lobato St., 80, Zip Code 13083-862, Campinas, Sao Guatemala (Ayerza, 1995). Today it is commercially available for human
Paulo, Brazil.
E-mail addresses: (R.S. Marineli),
consumption as whole seeds, our, and oil in the Americas, Australia, (S.A. Lenquiste), Europe and Southeast Asia (Mohd Ali et al., 2012). Chia seed is the
(.A. Moraes), (M.R. Marstica). richest known botanical source of omega-3 -linolenic acid (C18:3,
0963-9969/ 2015 Elsevier Ltd. All rights reserved.
R.S. Marineli et al. / Food Research International 76 (2015) 666674 667

ALA, up to 68%) (Ayerza, 1995) compared to axseed (50.6%), rapeseed male Wistar rats aged 21 to 23 days were obtained from Multidisciplin-
(8.1%), soybean (7.6%) and sunower (1.8%) oils (Tuberoso, Kowalczyk, ary Center for Biological Investigation, University of Campinas. Animals
Sarritzu, & Cabras, 2007); and has been also described as an important remained in individual cages for growth with free access to water and
source of protein, dietary ber, minerals and bioactive compounds chow diet for 4 weeks and were maintained under controlled conditions
(Marineli et al., 2014; Reyes-Caudillo, Tecante, & Valdivia-Lpez, (22 1 C, 6070% humidity, 12 h light/dark cycle). After growth, ani-
2008). Literature data indicates that chia seed and oil possess high anti- mals (234.08 g 19.12) were randomly divided in six experimental
oxidant potential conrmed by different in vitro assays (Marineli et al., groups (n = 6/group) for 12 weeks.
2014; Martnez-Cruz & Paredes-Lpez, 2014; Reyes-Caudillo et al., Diets were based on the American Institute of Nutrition (AIN)-93 M
2008). Chia seed and chia oil are considered new sources of natural an- diet (Reeves, Nielsen, & Fahey, 1993) with protein concentration of 12%.
tioxidants, due to the content of tocopherols, phytosterols, carotenoids Control group received the standard diet (AIN-93 M); high-fat and high-
(lvarez-Chvez, Valdivia-Lpez, Aburto-Jurez, & Tecante, 2008; fructose (HFF) group received a diet containing 4% (w/w) soybean oil,
Ixtaina et al., 2011) and phenolic compounds (Martnez-Cruz & 31% (w/w) lard and 20% fructose (w/w) (Shapiro, Tumer, Gao, Cheng,
Paredes-Lpez, 2014; Reyes-Caudillo et al., 2008), which have the po- & Scarpace, 2011); chia seed short and long treatments received an
tential to protect consumers against many diseases and also promotes HFF diet with 13.3% (w/w) of chia seed; chia oil short and long treat-
benecial effects on human health (Avignon et al., 2012; Landete, ments received an HFF diet with 4% (w/w) of chia oil. There were a
2012). Chia lipids could be an omega-3 alternative source to vegetarians long (12 weeks) and a short (6 weeks) treatment with chia seed or
and people with sh allergies, since chia seed and oil have not shown chia oil. Animals from the long treatment were fed only an HFF diet con-
problems associated with other nutritional sources such as axseed taining chia seed or chia oil for 12 weeks. Short groups were initially fed
and marine products, including sh avor, animal weight loss and di- only an HFF diet for the rst 6 weeks, followed by an additional 6 weeks
gestive problems (Mohd Ali et al., 2012). with an HFF diet containing chia seed or chia oil. Soybean oil for both
Our previous study Marineli et al. (2015) reported that chia inges- chia seed and oil diet was replaced by the oil content of chia seed or
tion induced heat shock protein expression and restored SOD and GPx chia oil. Chia diets were formulated to provide equal quantities of oil
expression in skeletal muscle, with a higher potential of oil relative to and ALA from chia (Ayerza & Coates, 2007). The seeds contained 30.2%
seed. However, chia seed and oil has been little explored from a scientif- of oil (Marineli et al., 2014). Protein and dietary ber content in HFF-
ic point of view, especially in relation to its antioxidant potential in vivo. chia seed groups was balanced taking into account the amount of such
To the best of our knowledge, this is the rst report about antioxidant nutrients present in chia seed (Marineli et al., 2014). Diets composition
effects in liver and plasma after chia seed and oil intake. We hypothe- is presented in Table 1. Diets were prepared monthly and packed in dark
sized that dietary chia seed and chia oil could protect against oxidative polyethylene bags and stored at 20 C to minimize the oxidation of
stress by improving antioxidant defenses and reducing lipid peroxida- fatty acids. Weight gain was monitored weekly and food intake every
tion in diet-induced obese rats, due to the content of bioactive com- 2 days.
pounds. Therefore, the present study aimed to evaluate the effects of
dietary chia seed and chia oil on oxidative stress and antioxidant bio- 2.2.1. Diet analyses
markers of plasma and liver in diet-induced obese Wistar rats. Calorie values of diets were determined using isoperibol automatic
calorimeter (PARR 1261) instrument equipped with oxygen bomb
2. Materials and methods

2.1. Chemical, chia seed and oil Table 1

Ingredients and fatty acid composition of experimental diets.

Solvents were all analytical grade from J.T. Baker (Phillipsburg, NJ, AIN-93 M HFF Chia seed Chia oil
USA). Metaphosphoric acid was purchased from Vetec Qumica Fina Ingredients (g/kg diet)
(Sao Paulo, Brazil). L-Glutathione reduced, glutathione reductase, gluta- Caseina 139.53 139.53 106.53 139.53
thione oxidized form disodium salt, -nicotinamide adenine dinucleo- Maltodextrin 155.0 45.40 45.40 45.40
tide 2-phosphate reduced tetrasodium salt hydrate (NADPH), 55- Corn starch 465.69 136.44 123.44 136.44
Sucrose 100.0 29.32 29.32 29.32
dithio-bis-2-nitrobenzoic acid (DTNB), albumin from bovine serum
Soybean oil 40 40
(BSA), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid Chia oil 40
(Trolox), 2,4,6- tris(2-pyridyl)-s-triazine (TPTZ), 2-thiobarbituric acid Chia seed 133
(TBA) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Tri- Lard 310 310 310
Fructose 200 200 200
chloroacetic acid (TCA) was purchased from Synth Laboratorios (Sao
Cellulose 50 50 3.6 50
Paulo, Brazil), Bradford reagent was purchased from BioAgency (Sao Mineral mixture 35 35 35 35
Paulo, Brazil) and malondialdehyde (MDA) was purchased from Vitamin mixture 10 10 10 10
Cayman Chemical Company (Ann Arbor, MI, USA). Fresh chia seed and L-Cystine 1.8 1.8 1.8 1.8
chia oil from FTP S.A. Santiago, Chile were purchased from R&S Blumos Choline bitartrate 2.5 2.5 2.5 2.5
Comercial de Produtos Alimentcios Ltda., Brazil. According to the pro- tert-Butyl hydroquinone 0.008 0.008 0.008 0.008
Energy density (kcal/g diet) 3.74 5.89 5.84 5.88
ducer, chia oil was obtained by cold pressing (b40 C) and stored at
28 C until use in amber glass bottles without head space. Seeds Fatty acidsb
were ground in a laboratory impact mill (Marconi MA 630/1, SFA 7.38 127.86 125.35 125.99
MUFA 10.20 146.94 138.87 140.11
Piracicaba Sao Paulo, Brazil) and passed through a 0.850 mm sieve.
PUFA 22.42 71.10 81.11 79.54
Chemical analysis and antioxidant activity of Chilean chia seed and oil Linoleic (C18:2n6) 19.89 66.74 54.97 53.53
samples used in this study were previously determined by Marineli -Linolenic (C18:3n3) 2.20 4.06 25.88 25.73
et al. (2014) in the same raw material. n6/n3 ratio 9.04 16.44 2.12 2.08

AIN-93 M group: control standard diet; HFF group: high-fat and high-fructose diet; Chia seed
2.2. Animals, diets and interventions groups: HFF diet plus 13.3% (w/w) chia seed; Chia oil groups: HFF diet plus 4% (w/w) chia oil.
SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty
This work was approved by the Ethics Commission on Animal Use a
Amount was calculated based on protein content equal to 86% to provide 12 g of
(CEUA/UNICAMP, protocol no. 29361) and followed the University protein/100 g of diet.
guidelines for the use of animals in experimental studies. Thirty-six b
Fatty acids expressed as g/kg diet.
668 R.S. Marineli et al. / Food Research International 76 (2015) 666674

(PARR 1108) (Instrument Co, Moline, IL, USA). Lipids were extracted ac- Catalase (CAT) activity. CAT activity was analyzed in plasma and
cording to the method of Bligh and Dyer (1959) and samples were sa- liver PB homogenates using commercial assay kits (#707002, Cayman
ponied in 0.5 mol/L methanolic KOH. Fatty acids methyl esters Chemical Company, Ann Arbor, MI, USA). The method is based on
(FAME) were prepared as previously described by Maia and reaction of the enzyme with methanol in the presence of an optimal
Rodriguez-Amaya (1993). The fatty acid composition of the experimen- concentration of H2O2. The formaldehyde produced is measured colori-
tal diets was determined by gas chromatography (GC) (Kramer et al., metrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole
1997) using a capillary GC Agilent 6850 Series GC System (Agilent Tech- (Purpald) as the chromogen. Results were expressed as nmol CAT activ-
nologies, Wilmington, DE, USA), capillary column DB-23 Agilent ity/min/g protein or nmol CAT activity/min/mL plasma.
(50% cyanopropyl-methylpolysiloxane, 60 m 0.25 mm 0.25 mm)
(Wilmington, DE, USA), with ame ionization detector (FID). Oven tem- Superoxide dismutase (SOD) activity. SOD activity was analyzed in
perature was 110 C for 5 min, 110215 C (5 C/min), 215 C for plasma and liver PB homogenates using commercial assay kits
24 min; detector temperature: 280 C; injector temperature: 250 C; (#706002, Cayman Chemical Company, Ann Arbor, MI, USA). The meth-
carrier gas: helium; split ratio 1:50; injection volume: 1.0 L. FAME od utilizes a tetrazolium salt for detection of superoxide radicals gener-
were identied by comparison of retention times with FAME standard ated by xanthine oxidase and hypoxanthine. SOD assay measures all
mixture under the same conditions (FAME Mix GLC-68A; Nu-Chek- three types of SOD (Cu/Zn, Mn, and FeSOD). One unit of SOD is dened
Prep, Inc., Elysian, MN, USA) and quantied using area normalization. as the amount of enzyme needed to exhibit 50% dismutation of the su-
peroxide radical. Results were expressed as U SOD activity/mg protein
2.3. Blood and tissue collection or U SOD activity/mL plasma.

Animals were euthanized by decapitation preceded by 12-hour- 2.4.2. Lipid peroxidation

fasting after 12 weeks of experimental period. Blood samples were col-
lected in polyethylene tubes with EDTA anticoagulant to obtain plasma. TBARS (thiobarbituric acid reactive substances) assay. TBARS de-
Tubes were centrifuged at 3000 g (15 min, 4 C). Plasma was separated terminations were done in plasma and liver according to Ohkawa,
and stored in polypropylene microtubes at 80 C until analysis. Liver Ohishi, and Yagi (1979), with adaptations. Hepatic freeze-dried tissue
was removed, washed, frozen in liquid nitrogen and stored at 80 C. (10 mg/mL) was sonicated for 30 min in a 300 mmol/L acetate buffer so-
Liver homogenates were prepared in a ratio of about 100 mg wet tissue lution (pH 3.6). Plasma samples (100 L) were used without previous
per 1 mL of 50 mmol/L phosphate buffer (PB) (pH 7.4) or 5% trichloro- preparation. Working solution was set with 2-thiobarbituric acid
acetic acid (TCA) solution using a homogenizer (MA102/Mini, Marconi, (TBA), acetic acid (20%) and sodium hydroxide (5%) at ratio of
Piracicaba Sao Paulo, Brazil). PB and TCA homogenates were used in 1:100:100 (w:v:v). One hundred microliters of 8.1% sodium dodecyl
the enzymatic antioxidant activities and GSH assays, respectively sulfate solution and 4 mL of work solution were homogenized with
(Batista et al., 2014). Liver was also freeze-dried, manually ground samples and standard and heated at 95 C for 60 min. Samples were
with mortar and pistil and kept at 80 C until analysis of lipid cooled in an ice bath for 10 min and then centrifuged at 10,000 g
peroxidation. (10 min, 4 C). The supernatant was read at 532 nm using a 96-well mi-
croplate. Standard curve (0.62585 nmol MDA/mL) was obtained using
2.4. Biochemical analyses malondialdehyde standard (MDA) (#10009202, Cayman Chemical
Company, Ann Arbor, MI, USA). Results were expressed as nmol MDA/
Absorbance readings were done in a microplate reader Synergy HT, mg tissue or nmol MDA/mL plasma.
Biotek (Winooski, VT, USA); with Gen5 2.0 data analysis software
spectrophotometer. 8-Isoprostane assay. 8-Isoprostane was determined in plasma
samples by an enzyme immunoassay (EIA) kit (#516351, Cayman
2.4.1. Endogenous enzymatic and non-enzymatic antioxidant system Chemical Company, Ann Arbor, MI, USA). The method is based on
The protein concentration of tissue homogenates was determined the competition between 8-isoprostane and an 8-isoprostane-
by Bradford method (Bradford, 1976). acetylcholinesterase (AChE) conjugate (marker) for a limited number
of 8-isoprostane-specic rabbit antiserum binding sites. Results were Reduced thiol (GSH) content. GSH levels were determined in plas- expressed as pg 8-Isoprostane/mL plasma.
ma and liver TCA homogenates by Ellman's reaction using DTNB (Faure
& Lafond, 1995). GSH solution (2.5500 nmol GSH/mL) was used as 2.4.3. Antioxidant capacity measured by ferric reducing antioxidant power
standard curve and absorbance was read at 412 nm. Results were (FRAP) assay
expressed as nmol GSH/g protein or nmol GSH/mL plasma. Plasma samples and liver PB homogenates were treated with etha-
nol:ultrapure water (2:1) and 0.75 mol/L metaphosphoric acid Glutathione reductase (GRd) activity. GRd activity was measured (Batista et al., 2014). Samples were centrifuged at 14,000 g for 5 min
in plasma and liver PB homogenates by the method described by at 4 C and the supernatant removed. The ferric reducing ability of plas-
Carlberg and Mannervik (1985). The decrease in absorbance was mon- ma and liver extracts was determined by FRAP method (Benzie & Strain,
itored at 340 nm after induction with 1 mmol/L oxidized glutathione in 1996), with adaptations. Under dark conditions, FRAP reagent was pre-
the presence of 0.1 mmol/L NADPH in PB. Results were expressed as pared with 10 mmol/L TPTZ, 20 mmol/L FeCl3 and 300 mmol/L acetate
nmol NADPH consumed/min/mg protein or nmol NADPH consumed/ buffer (pH 3.6) following the 1:1:10 ratio. Samples or standard solu-
min/mL plasma. tions, ultrapure water and FRAP reagent were mixed in 1:3:30 ratios
and kept in a water bath for 30 min at 37 C. After cooling to room tem- Glutathione peroxidase (GPx) activity. GPx activity was quantied perature, samples and standard were read at 595 nm. Trolox standard
in plasma and liver PB homogenates according to Flohe and Gunzler curve was made (5400 mol/L). Results were expressed as mol Trolox
(1984). This assay is based on the oxidation of 10 mmol/L reduced glu- equivalent (TE)/g protein or mol TE/L plasma.
tathione by GPx coupled to the oxidation of 4 mmol/L NADPH by 1 unit
(U) enzymatic activity of GRd in the presence of 0.25 mmol H2O2. The 2.5. Statistical analysis
rate of NADPH oxidation was monitored by the decrease in absorbance
at 365 nm. Results were expressed as nmol NADPH consumed/min/mg Data are expressed as mean values standard deviation. Differ-
protein or nmol NADPH consumed/min/mL plasma. ences between AIN-93 M and HFF group were tested by T test.
R.S. Marineli et al. / Food Research International 76 (2015) 666674 669

Differences among HFF, chia seed 12 weeks, chia seed 6 weeks, chia oil
12 weeks and chia oil 6 week groups were tested by one-way analysis of
variance (ANOVA) followed by Tukey's test using the GraphPad Prism
5.0 (GraphPad Software, Inc., La Jolla, CA, USA) software. P b 0.05 was
considered statistically signicant.

3. Results

3.1. Food intake and weight gain

Total food intake was lower (Fig. 1A) and total energy intake was
higher (Fig. 1B) in HFF-fed animals compared to AIN-93 M group.
These parameters did not differ among the chia (seed or oil) and HFF
groups. Animals fed on the HFF diet showed an increase in weight
gain compared to AIN-93 M group (Fig. 1C). Chia (seed or oil) intake
did not attenuate rat's weight gain, regardless of treatment time,
when compared to HFF group.

3.2. Enzymatic antioxidant activities and GSH levels

Animals fed with HFF diet showed the lowest plasma and liver CAT
activity when compared to AIN-93 M group (Figs. 2A and 3A, respec-
tively). All chia seed and chia oil groups presented higher plasma CAT
activity and no changes in liver CAT activity in relation to HFF group. Al-
though plasma SOD activity did not differ among the experimental
groups (Fig. 2B), HFF group presented lower liver SOD activity com-
pared to AIN-93 M group (Fig. 3B). However, the groups that consumed
chia seed or chia oil diet did not show differences in liver SOD activity
compared to HFF group.
Plasma and liver GSH values decreased in HFF-fed animals compared
to AIN-93 M group (Figs. 2E and 3E, respectively). Chia groups (seed and
oil) showed an increase in plasma GSH level compared to HFF group.
Moreover, the group that received chia seed diet for 12 weeks showed
a higher GSH level in liver (28%) compared to HFF group and also com-
pared to other chia groups.
HFF-fed animals decreased plasma and liver GPx and GRd activities
compared to AIN-93 M group (Figs. 2C, D and 3C, D respectively). Chia
groups (seed and oil) showed an increase in plasma GPx and liver
GRd activities, but not in plasma GRd and liver GPx activities compared
to HFF group.

3.3. Lipid peroxidation

HFF-fed animals increased plasma 8-isoprostane levels and plasma

and liver TBARS compared to AIN-93 M group (Fig. 4A, B and C, respec-
tively). Chia (seed or oil) intake did not modify liver TBARS levels in
comparison to HFF group. On the other hand, chia diets were able to re-
duce plasma TBARS and 8-isoprostane levels compared to HFF group.

3.4. Antioxidant capacity by FRAP assay

HFF diet promoted a decrease in plasma and liver antioxidant values

compared to AIN-93 M group (Fig. 5A, B). The addition of chia (seed or
oil) to the diets reversed the effect caused by the HFF diet. Plasma FRAP
values were increased in chia groups about 35% and liver FRAP values
were higher in chia groups about 47% as compared to HFF group.

4. Discussion
Fig. 1. Total food intake (A), total energy intake (B) and total weight gain (C) of rats fed on
Excessive energy intake from saturated fatty acids leads to an in- different diets: control standard diet (AIN-93 M); high-fat and high-fructose (HFF) diet;
crease in energy storage as fat, accompanied by an elevation in mito- HFF diet plus 13.3% chia seed during 12 weeks (chia seed 12-weeks) or 6 weeks (chia
chondrial macronutrient oxidation, favoring an excessive production seed 6-weeks); HFF plus 4% chia oil during 12 weeks (chia oil 12-weeks) or 6 weeks
of free radicals and oxidative stress (Avignon et al., 2012). Obesity and (chia oil 6-weeks). Different capital letters (AB) mean signicant differences between
AIN-93 M and HFF group according to T test (P b 0.05). Different small letters (ab)
more specically visceral fat are associated with oxidative stress in mean signicant differences between HFF, chia seed and chia oil groups by one-way anal-
humans, which may contribute to metabolic syndrome development ysis of variance followed by Tukey's test (P b 0.05). Data expressed as mean standard
(Fernndez-Snchez et al., 2011). deviation (n = 6/group).
670 R.S. Marineli et al. / Food Research International 76 (2015) 666674

Fig. 2. Plasma antioxidant enzymes: catalase (CAT) activity (A), superoxide dismutase (SOD) activity (B), glutathione peroxidase (GPx) (C), glutathione reductase (GRd) (D), and thiol
groups (GSH) (E) in plasma of rats fed on different diets: control standard diet (AIN-93 M); high-fat and high-fructose (HFF) diet; HFF diet plus 13.3% chia seed during 12 weeks (chia
seed 12-weeks) or 6 weeks (chia seed 6-weeks); HFF plus 4% chia oil during 12 weeks (chia oil 12-weeks) or 6 weeks (chia oil 6-weeks). Different capital letters (AB) mean signicant
differences between AIN-93 M and HFF group according to T test (P b 0.05). Different small letters (ab) mean signicant differences between HFF, chia seed and chia oil groups by one-
way analysis of variance followed by Tukey's test (P b 0.05). Data expressed as mean standard deviation (n = 6/group).

Our data show that a HFF diet intake induced lipid peroxidation, re- (Reddy, Ramatholisamma, Karuna, & Saralakumari, 2009). In the pres-
duced antioxidant capacity and enzyme activities involved in the anti- ent study, a daily consumption of chia seed and oil diets enhanced plas-
oxidant defense system in plasma and liver of rats. These ndings are ma antioxidant status through CAT and GPx activity and GSH levels
in agreement with those reported in earlier animals (Feillet-Coudray reduction. However, no effect was observed in SOD and GRd activities,
et al., 2009; Laissouf, Mokhtari-Soulimane, Merzouk, & Benhabib, regardless of treatment time. It is important to consider that different
2013) and humans studies (Patel et al., 2007), which demonstrated nutrients such as natural antioxidants and other substances should
that high-fat and high-carbohydrate diets are able to induce oxidative not necessarily affect all enzymes of redox system in the same fashion
stress. Furthermore, previous studies showed that high-fructose diet in- and to the same extent. Antioxidant enzymes respond independently
take negatively affects the balance between antioxidant defense and to different inducing radicals and therefore we should not expect
free radical production in rats, leading to increased lipid susceptibility these enzymes respond identically (Kohen & Nyska, 2002).
to peroxidation (Busserolles et al., 2002). The increasing of fructose ca- Although plasma and liver SOD activity did not differ statistically
tabolism causes a reduction in total glutathione levels and the suppres- after chia ingestion, our previous study showed a restored SOD expres-
sion of hepatic antioxidant enzyme activities, such as CAT, SOD and GPx sion in skeletal muscle after chia seed and oil intake. These results
R.S. Marineli et al. / Food Research International 76 (2015) 666674 671

Fig. 3. Liver antioxidant enzymes: catalase (CAT) activity (A), superoxide dismutase (SOD) activity (B), glutathione peroxidase (GPx) (C), glutathione reductase (GRd) (D) and thiol groups
(GSH) (E) in liver of rats fed on different diets: control standard diet (AIN-93 M); high-fat and high-fructose (HFF) diet; HFF diet plus 13.3% chia seed during 12 weeks (chia seed 12-weeks)
or 6 weeks (chia seed 6-weeks); HFF plus 4% chia oil during 12 weeks (chia oil 12-weeks) or 6 weeks (chia oil 6-weeks). Different capital letters (AB) mean signicant differences be-
tween AIN-93 M and HFF group according to T test (P b 0.05). Different small letters (ab) mean signicant differences between HFF, chia seed and chia oil groups by one-way analysis of
variance followed by Tukey's test (P b 0.05). Data expressed as mean standard deviation (n = 6/group).

suggest that tissues respond differently to the same diet intervention. non-enzymatic mechanism which involves free radical-catalyzed per-
Furthermore, we should highlight that different methodologies were oxidation of arachidonic acid and is the most specic biological indicator
used in both experiments (Marineli et al., 2015). A previous study for the assessment of oxidative stress in vivo (Musiek, Yin, Milne, &
(Laissouf et al., 2013) found that axseed oil diet (2.5 and 5%) intake at- Morrow, 2005). In this way, the dietary intake of chia prevents plasma
tenuated lipid and protein oxidation in different tissues and improved 8-isoprostane increase in rats fed with HFF diet which endorse the ben-
antioxidant status reducing CAT and GPx in obese aged rats. ecial effect of chia seed and oil in reducing this non-enzymatic product
With regard to effects of chia seed and oil intake on lipid peroxida- of lipid oxidation in vivo.
tion, we found a signicant reduction in plasma 8-isoprostane and Dietary contribution of antioxidant compounds affects the overall
TBARS levels, suggesting that both seed and oil could act as antioxidant resistance of lipoproteins to lipid peroxidation (Shinitzky, 1984). In
agents and counteract the pro-oxidative effect caused by HFF consump- this way, the reduction in plasma lipid peroxidation may be involved
tion. 8-Isoprostane is a specic lipid peroxidation product formed via a to hypolipidemic effect of chia seed and chia oil previous shown by
672 R.S. Marineli et al. / Food Research International 76 (2015) 666674

Ayerza and Coates (2007) and Poudyal, Panchal, Waanders, Ward, and
Brown (2012). This effect is associated with reduced susceptibility of
low-density lipoprotein (LDL) to oxidation, apparently due to the re-
duction in plasma LDL-cholesterol levels.
Although lipid peroxidation reduction demonstrated in plasma
through TBARS levels, it was not reversed in hepatic tissue, since liver
TBARS levels remained unchanged in chia seed and oil groups in both
6 and 12 weeks of treatment compared to HFF group. Lipid peroxidation
is a basic cellular deteriorating process induced by oxidative stress and
occurs readily in the tissues rich in highly oxidizable polyunsaturated
fatty acids (PUFA), as in the liver (Halliwell, 1994). Furthermore, inves-
tigations have shown that different types of fatty acids intake affect fatty
acid composition in a tissue-specic manner (Valenzuela, Barrera,
Gonzlez-Astorga, Sanhueza, & Valenzuela, 2014). It indicates that met-
abolic rate may be slower in the liver than in the plasma, which the plas-
ma may respond more readily to metabolizing fatty acids (Kang & Choi,
Interestingly, despite the non-reversal of lipid peroxidation in rat
liver, chia seed and oil groups showed an increase in liver GRd activity
and no change in liver GPx activity. In addition, only the group that re-
ceived chia seed diet for 12 weeks increased liver GSH levels. An in-
crease in liver GRd activity may occur to recover reduced glutathione
involved in glutathione cycle. However, there are several interactions
among enzymes and substrates variations in glutathione cycle which
could explain the change or not of the enzymes of this cycle (Kohen &
Nyska, 2002). Moreover, the potential effect of daily chia consumption
on these biomarkers is still recent and not enough studied.
Plasma and liver antioxidant capacity expressed by FRAP assay was
markedly higher in chia seed and oil-fed animals, which seemed to con-
tribute to an improvement in the endogenous antioxidant defenses. In
addition to the assay validity itself, special attention has to be paid to
confounding factors from sample matrix when biological samples are
measured. Many other issues such as metabolism, absorption and
other physiological activities should also be considered (Huang, Ou, &
Prior, 2005). These limitations represent difculties to be overcome in
further studies about bioavailability of bioactive compounds and their
antioxidant capacity in vivo.
Furthermore, higher plasma and liver antioxidant status found in
chia groups could explain previous studies that showed increased car-
dio and hepatoprotective parameters in obese rats fed with chia seed
diet (Poudyal et al., 2012).
Literature data show that different types of fatty acids have variable
effects on free radical production and each fatty acid acts direct on ROS
scavenging and indirect on antioxidant enzymes induction (Bullo et al.,
2011). Chia seed oil is a rich source of ALA (Ayerza, 1995) and previous
studies with the same fatty acid from other source (Pal & Ghosh, 2012a,
2012b) corroborates to our present results on the antioxidant status in
vivo. These authors found that dietary intake of 0.51.0% ALA, extracted
from linseed oil, attenuated organic mercury-induced oxidative stress in
liver, kidney and blood of rats. ALA presented antioxidant effects by re-
ducing lipid peroxidation and restoring antioxidant enzymes such as
CAT, SOD and GPx (Pal & Ghosh, 2012a, 2012b). Thus, it may be possible
that in in vivo conditions ALA might reduce the hydroperoxides forma-
tion by scavenging free radicals and PUFA peroxidation that occurs in
Our results seem to indicate that the protective effects against oxida-
tive stress caused by HFF diet might be due to either natural antioxi-
dants present in chia (Marineli et al., 2014; Martnez-Cruz &
Paredes-Lpez, 2014) or to PUFA action, especially ALA, that give a
Fig. 4. Lipid peroxidation by 8-isoprostane assay in plasma (A) and TBARS assay in
plasma (B) and liver (C) of rats fed on different diets: control standard diet (AIN-93 M); greater mobility in cell membranes and therefore greater membrane ac-
high-fat and high-fructose (HFF) diet; HFF diet plus 13.3% chia seed during 12 weeks cess to the antioxidants (Shinitzky, 1984). A recent study Valenzuela
(chia seed 12-weeks) or 6 weeks (chia seed 6-weeks); HFF plus 4% chia oil during et al. (2014) demonstrated that dietary ALA intake from different vege-
12 weeks (chia oil 12-weeks) or 6 weeks (chia oil 6-weeks). Different capital letters table oils, including chia oil, caused important modications in fatty acid
(AB) mean signicant differences between AIN-93 M and HFF group according to T
test (P b 0.05). Different small letters (ab) mean signicant differences between
composition of phospholipids extracted from the various tissues of rats.
HFF, chia seed and chia oil groups by one-way analysis of variance followed by Tukey's However, the metabolites absorption extension in plasma or tissues was
test (P b 0.05). Data expressed as mean standard deviation (n = 6/group). not assessed in this present study.
R.S. Marineli et al. / Food Research International 76 (2015) 666674 673

Fig. 5. Ferric reducing antioxidant power (FRAP) assay in plasma (A) and liver (B) of rats fed on different diets: control standard diet (AIN-93 M); high-fat and high-fructose (HFF) diet; HFF
diet plus 13.3% chia seed during 12 weeks (chia seed 12-weeks) or 6 weeks (chia seed 6-weeks); HFF plus 4% chia oil during 12 weeks (chia oil 12-weeks) or 6 weeks (chia oil 6-weeks).
Different capital letters (AB) mean signicant differences between AIN-93 M and HFF group according to T test (P b 0.05). Different small letters (ab) mean signicant differences
between HFF, chia seed and chia oil groups by one-way analysis of variance followed by Tukey's test (P b 0.05). Data expressed as mean standard deviation (n = 6/group).

The endogenous antioxidant system is supplemented by exogenous antioxidant enzymes expression in skeletal muscle, depending on the
nutrients, i.e., vitamins, minerals, polyphenols and others. Additionally, treatment time and the ingredient (seed or oil) added to the diet
previous study demonstrated that chia seed and oil could be a rich (Marineli et al., 2015). These ndings lead to the discussion that chia
source of bioactive compounds, due to an elevated content of polyphe- may inuence the oxidative stress process in different ways, and this in-
nolic compounds, including chlorogenic acid, caffeic acid, myricetin, uence is dependent on the biomarker used, including its tissue and de-
quercetin and kaempferol, and lipophilic compounds, such as tocoph- termination method. Moreover, considering the lack of studies
erols, phytosterols, carotenoids and phospholipids (Ixtaina et al., 2011; assessing the antioxidant potential of dietary chia seed and oil in vivo,
Martnez-Cruz & Paredes-Lpez, 2014; Reyes-Caudillo et al., 2008). determination of bioactive compounds and their main in vivo metabo-
Therefore, these compounds may be responsible for the high antioxi- lites in plasma, urine and tissues by specic analytical techniques are
dant potential in vitro as we previously found in Chilean chia seed and absolutely crucial. The results presented in this study are a good starting
oil (Marineli et al., 2014), which could also explain our present ndings. point for further investigations that should conduct further analysis to
The antioxidant activity of a bioactive compound has been attributed to clarify the bioactivity and mechanisms of action related to the benecial
several mechanisms, among which are chain initiation prevention, effects of chia intake.
binding of transition metal ion catalysts, peroxides decomposition, pre- In summary, the present study show that a daily consumption of
vention of continued hydrogen abstraction and radical scavenging chia seed and oil diets enhanced plasma and liver antioxidant status, re-
(Halliwell, 1994). Dietary antioxidants, mainly polyphenolic com- duced plasma lipid peroxidation and promote a protective effect against
pounds, are exposed to an intensive in vivo metabolism which affects oxidative stress arising from obesity. Chia seed and oil presented similar
their chemical structure, bioavailability, tissue retention and subse- in vivo antioxidant properties, independent of treatment time, probably
quently bioactivity (Landete, 2012). A previous study Gladine et al. due to the presence of polyphenols, ALA and other active compounds.
(2007) investigated the effect of plant extracts rich in polyphenols on Thus, chia seed and oil intake could possibly improve antioxidant de-
different tissues of rats fed omega-3 rich diets. These authors demon- fense system in diet-induced obese rats, protecting against cellular oxi-
strated that tissues are differently affected by polyphenols and sug- dative damage and obesity related diseases.
gested that PUFA supplements did not sensibilize tissues in a similar Caution is warranted before extrapolating these results to humans,
extension and that hepatic metabolism of polyphenols could have re- since the amount of chia tested in the present study should be further
duced their activity. Nevertheless, it is necessary to evaluate the antiox- evaluated in clinical studies for chia oil diet replacement or introduction
idant potential of chia seed and oil against oxidative injury including of chia seed in usual human diet.
phenolic compounds absorption mechanism and bioactivity in vitro
and in vivo assays.
Conict of interest
Another important nding was to observe the same antioxidant sta-
tus improvement and oxidative stress reversion after ingestion of both,
This work does not present conicts of interest.
chia seed and chia oil, as well as the treatment time which the animals
were submitted. The absence of signicant differences between both
treatments time could suggest that 6 weeks is the enough time to ob- Acknowledgments
serve signicant changes in plasma and/or tissue biomarkers. Further-
more, a possible explanation for the similar effects of oil and seed This work was supported by Fundao de Amparo Pesquisa do
intake may be related to different food matrices (seed vs. oil), since nu- Estado de So Paulo (FAPESP, 2012/23813-4) and Conselho Nacional
trients interaction between compounds present in chia seed and the de Desenvolvimento Cientco e Tecnolgico (CNPq 140386/2012-2
complexity of food matrix synergism should be considered. The nutrient and 300533/2013-6).
releasing from food matrix, interactions with other food components,
presence of cofactors and stable compounds formation inuence the
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