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RESEARCH ARTICLE
Adaptogenic potential of royal jelly in liver of
rats exposed to chronic stress
Douglas Carvalho Caixeta
1☯
, Renata Roland Teixeira
1☯
, Leonardo Gomes Peixoto
1
, Helen
Lara Machado
1
, Nathalia Belele Baptista
1
, Adriele Vieira de Souza
1
, Danielle Diniz Vilela
1
,
Celso Rodrigues Franci
2
, Foued Salmen Espindola
1
*
1Institute of Biotechnology, Federal University of Uberla
ˆndia, Uberla
ˆndia, Minas Gerais, Brazil,
2Department of Physiology, University of São Paulo, Ribeirão Preto, Minas Gerais, Brazil
☯These authors contributed equally to this work.
*foued@ufu.br
Abstract
Restraint and cold stress increase both corticosterone and glycemia, which lead to oxidative
damages in hepatic tissue. This study assessed the effect of royal jelly (RJ) supplementa-
tion on the corticosterone level, glycemia, plasma enzymes and hepatic antioxidant system
in restraint and cold stressed rats. Wistar rats were allocated into no-stress, stress, no-
stress supplemented with RJ and stress supplemented with RJ groups. Initially, RJ (200mg/
Kg) was administered for fourteen days and stressed groups were submitted to chronic
stress from the seventh day. The results showed that RJ supplementation decreases corti-
costerone levels and improves glycemia control after stress induction. RJ supplementation
also decreased the body weight, AST, ALP and GGT. Moreover, RJ improved total antioxi-
dant capacity, SOD activity and reduced GSH, GR and lipoperoxidation in the liver. Thus,
RJ supplementation reestablished the corticosterone levels and the hepatic antioxidant sys-
tem in stressed rats, indicating an adaptogenic and hepatoprotective potential of RJ.
Introduction
The adaptive response to stress is characterized by psychophysiological adaptations of an
organism to restore homeostasis [1]. It is well documented that chronic restraint and cold
stress effectively mimics physical and psychological stress [2], elevate metabolic rate and also
increase production of reactive oxygen species (ROS). Physical and psychological stress acti-
vates the hypothalamic-pituitary-adrenal (HPA) axis and the autonomic nervous system
(ANS), increasing plasma glucocorticoid levels [3]. Increased corticosterone levels were
observed in stress responses using the stress models, such as restraint and cold [2], immobiliza-
tion [4], cold [5], cold water immersion [5], electric foot shock [6] and social isolation stress
[7]. Corticosterone increases gluconeogenesis and hepatic glycogenolysis in rats, resulting in
an increase in the availability of metabolic substrates [8].
A metabolic byproduct of stress-induced increase in energy production is the formation of
ROS [9] e.g. hydrogen peroxide (H
2
O
2
), hydroxyl radicals (HO
) and superoxide anion radi-
cals (O2
–
), which cause lipid peroxidation. In addition, the increased corticosterone levels
PLOS ONE | https://doi.org/10.1371/journal.pone.0191889 January 29, 2018 1 / 13
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OPEN ACCESS
Citation: Caixeta DC, Teixeira RR, Peixoto LG,
Machado HL, Baptista NB, de Souza AV, et al.
(2018) Adaptogenic potential of royal jelly in liver of
rats exposed to chronic stress. PLoS ONE 13(1):
e0191889. https://doi.org/10.1371/journal.
pone.0191889
Editor: M. Faadiel Essop, Stellenbosch University,
SOUTH AFRICA
Received: August 3, 2017
Accepted: January 12, 2018
Published: January 29, 2018
Copyright: ©2018 Caixeta et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors received funding support for
this work by Foundation of Support Research of
the State of Minas Gerais (FAPEMIG), National
Council for Scientific and Technological
Development (CNPq) and Coordination for the
Improvement of Higher Education Personnel
(CAPES). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
trigger ROS production and promote redox imbalance in different tissues of the body [10,11].
Oxidative stress also associated with physical stress induces changes in antioxidant defense sys-
tems [11]. Therefore, to neutralize reactive oxygen species, the body uses mainly enzymatic
and non-enzymatic antioxidant defense system [12].
Several studies have investigated whether nutraceutical supplementation results in enhance-
ment of the antioxidant defense system. In relation to stress management, nutraceutical sup-
plements have been used as an adaptogenic agent to support the body’s adaptation in stressful
situations [13]. Thus, royal jelly (RJ) could be used as a nutraceutical product. RJ is secreted by
the mandibular and hypopharyngeal glands of worker bees (Apis mellifera L.). Royal Jelly com-
position includes major royal jelly proteins, free amino acids, sugars, vitamins (B1, B2, B6,
folic acid, pantothenic acid, nicotinic acid, and biotin) and lipids such as 10-hydroxy-2-dece-
noic acid (HDA-10). The RJ has several biological properties including anti-inflammatory
[14], vasodilator and hypotensive [15], antimicrobial [16], immunomodulatory [17], hypocho-
lesterolemic [18] and antioxidant [19] activities.
In a previous study from our research group, we demonstrated a neuroprotective effect of
royal jelly supplementation and a reduction of corticosterone levels in a stress condition [20].
With these interesting results, the interest arose to evaluate the effect of the royal jelly supple-
mentation on the liver, which is a central organ of metabolism and is related to the synthesis of
cholesterol, a precursor of corticosterone. As well as, to investigate the effect of RJ supplemen-
tation on a non-stressful situation. As glucocorticoids have direct and indirect modulatory
roles in oxidative stress [9], our hypothesis was that RJ could decrease corticosterone levels,
even in the absence of stress, and oxidative stress in liver tissue. Thus, the aim of the study was
to evaluate the adaptogenic and antioxidant effect of RJ supplementation in rats submitted to
chronic stress induced by restraint and cold.
Materials and methods
Samples
RJ was imported from China and provided by Apia
´rio Girassol Ltda. (Uberla
ˆndia—MG, Bra-
zil). The centesimal analysis of the royal jelly was carried out by the Laboratory of Bromatology
and Animal Nutrition of the Federal University of Uberla
ˆndia. The royal jelly has 67% of
humidity and 33% of dry matter, of these, 14.19% are crude protein, 2.01% are lipids, 0.87%
are ash and 15.93% are carbohydrates. The RJ was stored at -20˚C until use. Daily, 200 mg/kg
b.w. of RJ samples were prepared for use in supplementation.
Animals
Wistar rats (207–250g) were obtained and kept in the Center for Bioterism and Experimenta-
tion at the Federal University of Uberla
ˆndia, Uberla
ˆndia, Brazil. Animals were kept in con-
trolled conditions (22 ±1 ˚C, humidity 60% ±5 and 12-hour light-dark cycles– 6:00/18:00 h
lights on/off) with a standard diet and water ad libitum. Body weight was measured at the
beginning and end of the study, while water and food intake per animal were measured daily.
All experimental procedures were approved and conducted by the Brazilian Society of Labora-
tory Animal Science and the Ethics Committee for Animal Research of the Federal University
of Uberla
ˆndia, Brazil (CEUA No. 047/14).
Induction of stress by restraint and cold
Animals were randomly allocated into four groups (n = 10/group): no stress (NS); no stress
supplemented with royal jelly (NSRJ); stress (S); and stress supplemented with royal jelly
Royal jelly in chronic stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0191889 January 29, 2018 2 / 13
Competing interests: The authors have declared
that no competing interests exist.
(SRJ). The animals were exposed to stress by restraint and cold, using the method of Paula-
Freire et al. [21]. These method effectively mimics a condition of physical and psychological
stress [2]. The restraint stress was carried out using individual acrylic hemicylindrical plastic
tubes (4.5 cm diameter, 12 cm long) for 2 hours daily in the morning (8:00 h– 10:00 h.). The
cold stress was carried at 10˚C for 2 hours daily in the afternoon (16:00 h– 18:00 h).
Supplementation with RJ began seven days before the stress sessions with subsequent sup-
plementation for seven more days during the period of stress induction [21]. RJ was adminis-
tered by oral gavage 45 minutes before the stress session. The NS and S rats received a placebo
(water) and the NS and NSRJ rats were not exposed to stressors.
On the fourteenth day, animals were exposed to the two stressors simultaneously for 2
hours [21] until euthanasia. Glucose levels were measured before and after this last session of
stress (Sb—stress before; SRJb—stress supplemented with RJ before stress session; Sa—stress
after; SRJa—stress supplemented with RJ after stress session) by puncturing the tail vein, using
reactive strips (Accu-Chek Performa, Roche Diagnostic Systems, Basel, Switzerland).
The NS, NSRJ, S, SRJ rats were anaesthetized with ketamine (90 mg/kg) and xylazine (20
mg/kg), in accordance the methods of Arnold and Langhans, 2010 [22]. All groups were sub-
jected to the same manipulation procedure. Plasma samples were used to assess corticosterone
levels and hepatic enzyme activities, whereas liver tissues were used for oxidative stress
analysis.
Determination of corticosterone levels by radioimmunoassay
Blood samples were collected (08:00 h– 11:00 h in heparinized plastic tubes and centrifuged at
1200g at 4˚C for 15 min.) after the last session of stress via cardiac puncture in the right ventri-
cle. Plasma was separated and frozen at -20˚C until the assay. Radioimmunoassay (RIA) used
H3-corticosterone from NEN Life Science Products (Boston, USA) and a standard reference
specific antibody from Sigma (St. Louis, MO, USA). Corticosterone was used to measure triti-
ated recovery [23]. The intra-assay error was 4.5% and the minimum detectable dose was 0.08
ng/ml.
Hepatic enzymes activities in plasma
Aspartate transaminase (AST), alanine transaminase (ALT), γ-glutamyl transferase (GGT) and
alkaline phosphatase (ALP) were measured at the Laboratory of Clinical Analyses, School of
Veterinary Medicine, Federal University of Uberla
ˆndia, using an automatic analyzer (Cobas
Mira, Roche Diagnostic Systems, Basel—Switzerland), by using commercial kits (Labtest Diag-
no
´stica, Lagoa Santa—Brazil).
Sample collection and tissue preparation
The liver tissues were quickly removed, separated in lobes, washed (NaCl 0.9% buffer) and
immersed in liquid nitrogen. Then, the same part of liver tissues were thawed and homoge-
nized in phosphate buffer (1:10 w/v, pH 7.4). The homogenates were centrifuged at 800 x g for
15 min at 4˚C, and the total protein concentration in the supernatant samples was measured,
according to the Bradford assay [24].
Oxidative stress marker analysis
Thiobarbituric acid reactive substances (TBARS). Lipid peroxidation was measured by
the reaction between malondialdehyde in the liver samples (MDA) and thiobarbituric acid
(0.67% TBA). Organic-phase fluorescence was evaluated at 515 nm (excitation) and at 553 nm
Royal jelly in chronic stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0191889 January 29, 2018 3 / 13
(emission). A MDA standard curve allowed the quantification of the compound in the samples
by linear regression [25]. TBARS levels were calculated as nmol TBARS/mg of protein.
Total antioxidant capacity (FRAP). Total antioxidant capacity was evaluated by the
capacity of the samples to reduce Fe
+3
to Fe
+2
, which was then chelated by TPTZ (2,4,6-Tris
(2-pyridyl)-s-triazine) in order to form the deep-blue colored Fe
+2
-TPTZ complex [25]. This
complex was measured in a spectrophotometer at 593 nm.
Superoxide dismutase (SOD) activity. SOD activity was measured by the inhibition
autoxidative capacity of pyrogallol. The SOD activity was evaluated using a spectrophotometer
at 420 nm. A calibration curve was constructed using SOD as standard. A 50% inhibition of
autoxidation of pyrogallol was defined as one SOD unit [25].
Reduced glutathione (GSH). The protein content of the samples was initially precipitated
by metaphosphoric acid (MPA) at the ratio of 1:1 (homogenate/MPA). The samples were cen-
trifuged at 7000xg for 10 minutes. The supernatant was collected and mixed with sodium
phosphate buffer (100 mM, pH 8.0), containing EDTA (5mM) and ortho-phthaldialdehyde (1
mg/mL in methanol). The mixture was incubated in the dark at room temperature for 15 min
and fluorescence was measured at 350 nm (excitation) and 420 nm (emission). A standard
curve of GSH (0.001–0.1 mM) was used for linear regression [25].
Glutathione peroxidase (GPx) activity. To measure the glutathione peroxidase activity,
the homogenate was incubated with GPx buffer (100 mM potassium phosphate containing 1
mM EDTA, pH7.7), sodium azide (40 mM), GSH (diluted in 5% metaphosphoric acid), GR
(diluted in GPx buffer), NADPH (diluted with sodium bicarbonate 5%) and tert-butyl (0.5
mM). The reduction in NADPH concentration was evaluated for 10 minutes in a spectropho-
tometer, at 340 nm [25].
Glutathione reductase (GR) activity. GR activity was evaluated using oxidized glutathi-
one (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH) as substrates. The
activity of the enzyme was determined using sodium phosphate buffer (200 mM, pH 7.5),
EDTA (6.3 mM), GSSG (1 mM), NADPH (1 mM) and the samples [25]. The consumption of
NADPH was measured at 340 nm for 10 minutes. A GR unit is defined as one μmol of reduced
GSSG per minute. The specific activity was calculated as U/mg of protein.
Glucose-6-phosphate dehydrogenase (G6PDH) activity. The activity of glucose-6-phos-
phate dehydrogenase was monitored by the production of NADPH with a consequent increase
in absorbance at 340nm. The samples were incubated with Tris-HCl buffer (100mM, pH 7.5),
magnesium chloride (MgCl
2
, 2 M), NADP
+
(0.5 mM) and glucose-6-phosphate (1mM). The
kinetic readings were monitored for ten minutes [25].
Statistical analyses
Data were used as independent variables (stress by restraint and cold) and as the dependent
variables (body weight, water and food intake, biochemical parameters and oxidative stress).
The data were analyzed using the one-way analysis of variance (ANOVA) followed by the
Tukey Multiple Comparison as a post-hoc test. All analyses were performed using the software
GraphPad Prism (GraphPad Prism version 6.00 for Windows; GraphPad Software, San Diego,
CA, USA). Outliers were detected by performing Grubb’s test using an online GraphPad out-
lier calculator (http://graphpad.com/quickcalcs/Grubbs1.cfm). Only values of p <0.05 were
considered significant. Results were expressed as mean ±SEM.
Results
Table 1 shows the effect of RJ supplementation on body weight, water and food intake and
hepatic enzyme activities in the plasma of restraint and cold stressed rats. S, NSRJ and SRJ
Royal jelly in chronic stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0191889 January 29, 2018 4 / 13
decreased the body weight compared to NS (F
3, 33
= 7.757, p <0.05, F
3, 33
= 7.757, p <0.05;
F
3, 33
= 7.757, p <0.001, respectively), and no change was observed between S and SRJ rats.
No significant difference was observed in the water or food intake among the groups. In addi-
tion, NSRJ and SRJ decreased the AST levels compared to NS (F
3, 29
= 17.64, p <0.001; F
3, 29
=
17.64, p <0.01, respectively) and SRJ compared to S rats (F
3, 29
= 17.64, p <0.05), while ALT
was not different among the groups. S rats had increased GGT compared to NS (F
3, 27
= 14.17,
p<0.001) whereas SRJ rats had decreased GGT levels compared to S rats (F
3, 27
= 14.17,
p<0.05). Furthermore, NSRJ and SRJ had decreased ALP levels compared to NS rats (F
3, 31
=
5.203, p <0.01; F
3, 31
= 5.203, p <0.05, respectively), whereas no change was observed in SRJ
compared to S rats or in S compared to NS rats.
Stress biomarkers were evaluated in plasma samples of NS, NSRJ, S and SRJ rats. Plasma
corticosterone levels increased in S compared to NS rats (F
3,28
= 14.56, p <0.05) whereas RJ
supplementation (NSRJ and SRJ) decreased corticosterone levels compared to NS (F
3,28
=
14.56, p <0.05) and S rats (F
3,28
= 14.56, p <0.001) (Fig 1A). Furthermore, blood glucose lev-
els did not differ in NSRJ and Sb compared to NS rats, whereas an increase was verified in Sa,
Table 1. The effect of the stress-induction by restraint and cold and supplementation of royal jelly on body weight, water intake, food intake and hepatic enzymes
activities in plasma.
Parameters NS NSRJ S SRJ
ΔBody weight (g) 51.11±2.54 38.97±2.0439.63±4.1932.22±2.14
Water intake (ml) 41.25±4.19 44.07±4.66 37.56±2.52 38.91±6.25
Food intake (g) 23.51±0.35 24.30±0.71 22.98±0.40 22.82±0.27
AST (U/L) 99.78±3.30 67.13±5.4197±1.19 83.44±3.24#
ALT (U/L) 47.5±4.03 46.38±2.19 48.22±1.66 53.44±2.91
GGT (U/L) 8.71±1.42 7.02±0.70 22.49±3.2514.16±1.59#
ALP (U/L) 336.2±38.04 223±16.35279±8.60 242.3±16.89
Note: Values are expressed as mean ±S.E.M (n = 10).
p<0. 05 vs NS rats;
#
p<0. 05 vs S rats.
No stress (NS), No stress supplemented with royal jelly (NSRJ), Stress(S) and Stress supplemented with royal jelly (SRJ); Aspartate transaminase (AST); Alanine
transaminase (ALT); γ-glutamyl transferase (GGT) and Alkaline phosphatase (ALP).
https://doi.org/10.1371/journal.pone.0191889.t001
Fig 1. Biomarkers of chronic stress in liver tissue of rats stressed by restraint andcold. Plasma corticosterone level after seven days of stress-induction (A). No
stress (NS), No Stress supplemented with Royal Jelly (NSRJ), Stress (S) and Stress supplemented with Royal Jelly (SRJ). Blood glucose level before and after the last
stress induction (B). No stress (NS), No Stress supplemented with Royal Jelly (NSRJ), Stress group before the last stress session (Sb), Stress group after the last stress
session (Sa), Stress group supplemented with RJ before the last stress session (SRJb), Stress group supplemented with RJ after the last stress session (SRJa). Pearson
correlation of mean values of corticosterone levels and blood glucose after the last stress-induction (C). Values are expressed as means ±SEM. p<0.05 vs NS,
# p <0.05 vs S, & p <0.05 vs Sb, § p <0.05 vs Sa (One-way ANOVA followed by Tukey test). Outliers were detected by performing Grubb’s test using an online
GraphPad outlier calculator (http://graphpad.com/quickcalcs/Grubbs1.cfm).
https://doi.org/10.1371/journal.pone.0191889.g001
Royal jelly in chronic stress
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SRJb and SRJa compared with both NS (F
5, 47
= 32.55, p <0.001) and between Sa and SRJa
compared to Sb rats (F
5, 47
= 32.55, p <0.001; F
5, 47
= 32.55, p <0.01, respectively). When gly-
cemia was compared among the stressed rats, SRJa (F
5, 47
= 32.55, p <0.05) and SRJb (F
5, 47
=
32.55, p <0.01) had decreased blood glucose levels compared with Sa rats, whereas no differ-
ence was observed between SRJb compared to Sb and SRJa rats (Fig 1B). In addition, Pearson
correlation between corticosterone levels and glycemia after the last stress induction showed a
strong positive correlation (r = 0.769, p = 0.001) (Fig 1C).
Fig 2 shows the oxidative stress status in liver tissue. Stressed rats (S) decreased the total
antioxidative capacity (FRAP) (F
3, 23
= 11.65, p <0.01) and increased lipid peroxidation
(TBARS) (F
3, 21
= 5.027, p <0.05) compared to NS rats. SRJ rats increased FRAP (F
3, 23
=
11.65, p <0.05) and decreased malondialdehyde levels (F
3, 21
= 5.027, p <0.05) compared to S
rats (Fig 2A and 2B). Pearson correlation between corticosterone and FRAP showed a negative
correlation (r = -0.756, p = 0.001), whereas a positive correlation (r = 0.666, p = 0.0019) was
observed between corticosterone and lipid peroxidation (Fig 2C and 2D).
Fig 2. Biomarkers of oxidative stress in liver tissue of rats stressed by restraint and cold.Total antioxidant capacity by FRAP method (A). Lipid
Peroxidation by TBARS method (B). No stress (NS), No Stress supplemented with Royal Jelly (NSRJ), Stress (S) and Stress supplemented with Royal Jelly
(SRJ). Values are expressed as mean±SEM, p<0.05 vs. NS, # p <0.05 vs. S (One-way ANOVA followed by Tukey test). Pearson correlation of FRAP and
TBARS (panels C and D) and means values of corticosterone levels. Outliers were detected by performing Grubb’s test using an online GraphPad outlier
calculator (http://graphpad.com/quickcalcs/Grubbs1.cfm).
https://doi.org/10.1371/journal.pone.0191889.g002
Royal jelly in chronic stress
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SOD enzyme activity in liver tissues is shown in Fig 3. The S rats displayed a decreased
SOD activity compared to NS rats (F
3, 40
= 18.18, p <0.05), whereas RJ supplementation
increased the SOD activity in SRJ groups compared to S rats (F
3, 40
= 18.18, p <0.001). Fur-
thermore, NSRJ rats increased the SOD compared to NS (F
3, 40
= 18.18, p <0.05) (Fig 3).
The glutathione defense system analysis in liver tissues is displayed in Fig 4. GSH content
was lower in S compared to NS (F
3, 51
= 17.65, p <0.001), whereas GR and G6PDH levels
were higher in S compared to NS (F
3, 21
= 20.42, p <0.05; F
3, 20
= 17.24, p <0.05, respectively).
No significant difference was observed in GPx activity in S rats compared with NS rats. In
addition, the glutathione defense system analysis of the RJ supplemented S rats showed an
increase in GSH content (F
3, 51
= 17.65, p <0.05), a decrease in the GR level (F
3, 21
= 20.42,
p<0.05), whereas no difference was observed in GPx and G6PDH activity compared to S rats.
Discussion
Studies have been carried out to assess agents that could prevent the damage to liver tissues
triggered by acute and chronic stress, which induce the formation of ROS followed by hepatic
injury [26]
,
[27]. In this study, the adaptogenic potential of RJ was assessed and associated with
the antioxidant activity and anti-stress capacity, to improve the organism’s adaptation to stress
induction and in absence of stress. Our results showed a decrease of corticosterone and blood
glucose levels, weight loss as well as an improvement in the antioxidative parameters in the
liver of stressed RJ-supplemented rats.
Stress promotes the activation of the HPA axis, thus stimulating the release of corticoste-
rone by the adrenal gland [3]. This increase in corticosterone mobilizes energy substrates in
liver tissues to maintain homeostasis in a stress situation [28]. In the present study, restraint
and cold stress not only augmented corticosterone levels, blood glucose levels and biomarkers
of oxidative stress, but also diminished the glutathione antioxidant defense system in the liver
tissues. Our results corroborate other studies that reported an increase of corticosterone levels
under several stress models such as restraint and cold, immobilization and cold, besides other
models of stress induction [2,4–6,20,29]. Besides that, we showed a decreased of the cortico-
sterone level associated with RJ supplementation, even in absence of stress. Teixeira et al.
Fig 3. Superoxide dismutase activity in liver tissue of rats stressed by immobilization and cold. No stress (NS), No
Stress supplemented with Royal Jelly (NSRJ), Stress (S) and Stress supplemented with Royal Jelly (SRJ). Values are
expressed as mean±SEM, p<0.05 vs. NS, # p <0.05 vs. S (One-way ANOVA followed by Tukey test). Outliers were
detected by performing Grubb’s test using an online GraphPad outlier calculator (http://graphpad.com/quickcalcs/
Grubbs1.cfm).
https://doi.org/10.1371/journal.pone.0191889.g003
Royal jelly in chronic stress
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(2017) also demonstrated the effect of RJ in reducing corticosterone levels in stressed rats [20].
Corticosterone synthesis is cholesterol-dependent, which indicates the possibility that RJ is
able to inhibit the synthesis of cholesterol. Major royal jelly protein 1 was identified as a hypo-
cholesterolemic protein [30], indicating a possible mechanism through which RJ could inhibit
corticosterone synthesis. Furthermore, the decreased corticosterone levels improved glucose
uptake [31], which could control glycemia even after stress induction.
Fontella et al. (2005) demonstrated that the repeated exposure of adult rats to restraint stress
causes a temporary suppression of food intake and reduction of body weight [32]. Herein, we
observed a decrease of body weight in both groups of stressed rats and non-stressed rats sup-
plemented with RJ, even without a diminution in food intake. These results corroborate a
prior study [32] demonstrating that the diminution of body weight in restriction and cold
stressed rats is attributable to an elevation of corticosterone levels. Furthermore, our data indi-
cate that the RJ can also diminish body weight even in the absence of stress induction.
Although we did not evaluate the mechanism by which supplementation with RJ reduces body
weight, other studies have also showed that RJ supplementation can diminish body weight [33,
34]. New studies must be conducted to investigate the mechanism by which this effect occurs.
Fig 4. Glutathione antioxidant defense system in liver tissue of rats stressed by immobilization and cold. Glutathione level (GSH) (A).
Glutathione peroxidase activity (GPx) (B). Glutathione reductase activity (GR) (C). Glucose-6-phosphate dehydrogenase activity (G6PDH) (D).
No stress (NS), No Stress supplemented with Royal Jelly (NSRJ), Stress (S) and Stress supplemented with Royal Jelly (SRJ). Values are expressed
as mean±SEM, p<0.05 vs. NS, # p <0.05 vs. S (One-way ANOVA followed by Tukey teste). Outliers were detected by performing Grubb’s
test using an online GraphPad outlier calculator (http://graphpad.com/quickcalcs/Grubbs1.cfm).
https://doi.org/10.1371/journal.pone.0191889.g004
Royal jelly in chronic stress
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Stress alters the availability of metabolic substrates and can influence blood glucose levels,
leading to an increase in oxidative stress [35]. Our results indicate that the glycemia of non-
stressed rats supplemented with royal jelly did not differ from the non-stressed group.
Although we did not measure the plasmatic insulin, a prior study demonstrated that RJ did
not affect insulin levels [36]. Thus, the augmentation of corticosterone levels is probably
responsible for increased the blood glucose level under this stress situation, due to its role in
hepatic gluconeogenesis [37]. Furthermore, a strong positive correlation between glycemia
and corticosterone levels was observed after the last stress induction session. These results
indicate not only that glycemic control is corticosterone-dependent in the stress response, but
also that after the last stress session (Sa and SRJa) the RJ supplementation diminished the
increase of blood glucose levels compared to Sa rats. Thus, the capacity to inhibit the increase
in blood glucose levels after stress induction indicates a potential anti-stress and adaptogenic
effects of RJ.
Corticosterone acts on the liver, increasing glucose production especially through gluco-
neogenesis [37]. It can also increase oxidative stress and induce damage in the liver tissue [38].
Stressed rats presented increased plasma GGT compared with NS rats, indicating hepatic dam-
age [39]. RJ supplementation reduced both GGT levels and AST compared with S rats, and
also reduced AST and ALP compared with NS rats. Other studies using models of toxicity
induced by lambda-cyhalothrin and azathioprine also found a decrease in these enzymes, sup-
porting the protective effect of RJ towards liver tissue [40,41]. Thus, data observed here sug-
gests a hepatoprotective effect of RJ, not only by improving the liver enzymes, but also the
antioxidative systems.
High levels of glucocorticoids and exposure to stress increase ROS [10,11]. Herein, stressed
rats had increased lipid peroxidation associated with the corticosterone level, corroborating
other studies [38,42]. However, RJ supplementation decreased the lipid peroxidation in the
liver of stressed rats. RJ peptides can diminish the peroxidation of linoleic acid and eliminate
hydroxyl radicals, inhibiting lipid peroxidation [43]. In addition, in the present study, stressed
rats decreased total antioxidant capacity associated with the increase of corticosterone levels,
whereas RJ supplementation restored FRAP to levels comparable to those of NS rats. RJ con-
tains vitamins B and E, zinc, copper [44], phenolic compounds [45] and peptides with antioxi-
dant actions [43]. These RJ compounds can act as antioxidant agents preventing the oxidative
damage in the liver, suggesting that RJ supplementation may improve the antioxidant defense
of stressed rats.
Furthermore, we analyzed the enzymatic and glutathione antioxidant defense system in the
liver tissues of the stressed rats. SOD activity decreased in stressed rats compared with NS and
increased in rats supplemented with RJ compared with S rats. Other studies have also shown a
decrease in this enzyme activity in the liver of rats undergoing stress, thereby corroborating
our results [46,47]. Oishi and Mashida (2009) reported a decrease in hepatic SOD mRNA six
hours after stress by immobilization and cold [48]. Furthermore, our findings of decreased
GSH and increased GR and G6PDH activities in stressed rats, indicate a compensatory mecha-
nism to maintain the redox cycle of GSH, as shown in other studies [11,42].
RJ supplementation also increased the GSH level, corroborating Karadeniz et al. (2011),
who showed that RJ-supplemented rats present increased GSH in the liver and kidney in an
oxidative stress model induced by cisplatin [49]. Furthermore, RJ supplementation decreased
GR activity compared to S rats, and increased GPx and G6PDH activities similar to NS rats.
Studies employing models of toxicity for cisplatin [49] and paracetamol [50] also showed
increased GPx activity in rat livers. The unchanged GPx activity between stressed rats shown
in the present study was not found in previous studies. Our finding may be attributable to the
stress model used in this study. To the best of our knowledge, this is the first study that has
Royal jelly in chronic stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0191889 January 29, 2018 9 / 13
observed the effect of RJ supplementation on the activity of GR and G6PDH in the liver during
a stress situation. The RJ antioxidant property may be derived from the short-chain peptides
[43], phenolic compounds (flavonoids and cinnamic acid derivatives) [45], some antioxidant
type vitamins (A and E) and fatty acids (trans-10-Hydroxy-2-decenoic) [19]. Therefore, these
results support the antioxidant activity of RJ and indicate a prevention of hepatic oxidative
damage caused by stress.
Conclusion
RJ decreases corticosterone and improves glycemia control after stress induction. Moreover,
RJ showed a hepatoprotective effect against oxidative damage, reducing lipoperoxidation and
increasing the total antioxidant capacity in liver tissues of restraint and cold stressed rats.
Taken together, these results highlight an adaptogenic role of RJ in situations of stress and oxi-
dative damage.
Acknowledgments
The authors gratefully acknowledge the Apia
´rio Girassol Ltda. (Uberla
ˆndia, Brazil) for donat-
ing the royal jelly for the development of this study; Center of Animal Experimentation
(CBEA-UFU) of the Federal University of Uberla
ˆndia for supplying the animals and infra-
structural support; Foundation of Support Research of the State of Minas Gerais (FAPEMIG)
for financial support; Laboratory of Clinical Analyses, School of Veterinary Medicine, Federal
University of Uberla
ˆndia for help with the plasma biochemical analysis, Laboratory of Broma-
tology and Animal Nutrition of the Federal University of Uberla
ˆndia for centesimal analysis of
the royal jelly and the support from the National Institute of Science and Technology in Thera-
nostics and Nanobiotechnology (INCT). DCC, HLM, AVS and DDV received graduate fellow-
ships from the National Council for Scientific and Technological Development (CNPq) and
Coordination for the Improvement of Higher Education Personnel (CAPES). LGP received
postdoctoral fellowships from the National Postdoctoral Program PNPD/CAPES and FSE is
recipient of grant from CNPq (308965/2015-9).
Author Contributions
Conceptualization: Renata Roland Teixeira, Helen Lara Machado, Foued Salmen Espindola.
Formal analysis: Douglas Carvalho Caixeta, Renata Roland Teixeira, Leonardo Gomes
Peixoto.
Funding acquisition: Foued Salmen Espindola.
Investigation: Douglas Carvalho Caixeta, Renata Roland Teixeira, Nathalia Belele Baptista,
Adriele Vieira de Souza, Danielle Diniz Vilela, Celso Rodrigues Franci.
Methodology: Renata Roland Teixeira, Helen Lara Machado, Foued Salmen Espindola.
Project administration: Renata Roland Teixeira, Helen Lara Machado.
Writing – original draft: Douglas Carvalho Caixeta, Renata Roland Teixeira, Leonardo
Gomes Peixoto.
Writing – review & editing: Douglas Carvalho Caixeta, Renata Roland Teixeira, Leonardo
Gomes Peixoto, Foued Salmen Espindola.
Royal jelly in chronic stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0191889 January 29, 2018 10 / 13
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