Positive effects of an oral supplementation by Glisodin, a gliadin-combined SOD-rich melon extract, in an animal model of dietary-induced oxidative stress

Abstract

We investigated the potential protective effects of two antioxidant molecules: Glisodin, a gliadin combined copper-zinc superoxide dismutase SOD (Cu,Zn SOD)-rich melon extract, SOD is a known enzyme that has been best studied as a regulator of antioxidant defence, and an antioxidant agent N-acetylcysteine (NAC). Glisodin, given orally to rats fed a chow diet, as 180 U/d during 2 weeks in a preconditioning treatment, and then for 8 weeks, combined to a high fat/high fructose diet (HF/HFr), had more positive effects than NAC (100 mg/d), not only on oxidative stress parameters, but also on features of the metabolic-syndrome. DNA oxidative damages, lipid peroxidation, and fasting glycaemia were lower in rats receiving Glisodin than in those supplemented by NAC. In addition, insulin sensitivity was improved and mesenteric fat was significantly lower in rats fed the Fr/Fe diet plus Glisodin than in animals fed NAC supplementation.

These data suggest potential beneficial effects of oral Glisodin supplementation in preventing metabolic alterations related to the metabolic syndrome.

Full text

PHYTOTHÉRAPIE EXPÉRIMENTALE
Positive effects of an oral supplementation by Glisodin,
a gliadin-combined SOD-rich melon extract, in an animal model
of dietary-induced oxidative stress
Effets bénéfiques d’une supplémentation orale par le Glisodin, un extrait de melon combiné
à de la gliadine, dans un modèle animal de stress oxydant induit par l’alimentation
I. Hininger-Favier · M. Osman · A.M. Roussel · L. Intes · B. Montanari
© Lavoisier SAS 2015
Abstract We investigated the potential protective effects of
two antioxidant molecules: Glisodin, a gliadin combined
copper-zinc superoxide dismutase SOD (Cu,Zn SOD)-rich
melon extract, SOD is a known enzyme that has been best
studied as a regulator of antioxidant defence , and an antiox-
idant agent N-acetylcysteine (NAC). Glisodin, given orally
to rats fed a chow diet, as 180 U/d during 2 weeks in a pre-
conditioning treatment, and then for 8 weeks, combined to a
high fat/high fructose diet (HF/HFr), had more positive
effects than NAC (100 mg/d), not only on oxidative stress
parameters, but also on features of the metabolic-syndrome.
DNA oxidative damages, lipid peroxidation, and fasting gly-
caemia were lower in rats receiving Glisodin than in those
supplemented by NAC. In addition, insulin sensitivity was
improved and mesenteric fat was significantly lower in rats
fed the Fr/Fe diet plus Glisodin than in animals fed NAC
supplementation.
These data suggest potential beneficial effects of oral Gli-
sodin supplementation in preventing metabolic alterations
related to the metabolic syndrome.
Keywords Glisodin · SOD melon extract · Oxidative stress ·
Metabolic syndrome
Résumé Nous avons comparé les effets de deux molécules
antioxydantes, le Glisodin, un extrait de melon riche en
superoxide dismutase Cu, Zn (Cu, Zn-SOD) combiné à de
la gliadine, et un agent antioxydant la NAC, N acétyl cysté-
ine. Les deux antioxydants ont été administrés oralement
2 semaines en pré-conditionnement à des rats nourris au
régime d’entretien, puis 8 semaines associées à un régime
riche en fer et en fructose induisant un stress oxydant. Le
Glisodin (180 U/j) a des effets bénéfiques supérieurs à
ceux de la NAC (100 mg/j) non seulement sur le stress oxy-
dant (dommages oxydatifs aux ADN, peroxydation lipidi-
que) mais aussi sur les paramètres du syndrome métabolique
(glycémie a jeun, insulinémie, graisse mésentérique).
Ces résultats suggèrent une utilisation thérapeutique du
Glisodin dans le syndrome métabolique.
Mots clés Glisodin · SOD de melon · Stress oxydant ·
Syndrome métabolique
Introduction
Oxidative stress, as a cause or consequence of insulin resis-
tance and overweight, is involved in the early steps of the
metabolic syndrome [1] and increases the risk of pathologies
and oxidative complications such as cardiovascular diseases,
diabetes retinopathies, glomerulopathies, or early cognitive
decline. Compared to Mediterranean diet, the western-style
diet has a higher content of highly processed and refined
foods (i.e high sugar contents and refined cereals), a high
consumption of red meat and a lower content in fruit and
vegetables, and it is associated to a higher risk of oxidative
stress and insulin-resistance. It is well documented that pro-
longed hyperglycemia and overweight, that leading to an
insulin resistant state, might result in an oxidative burst [2].
Accumulating data suggest that overfeeding, high fat and
high fructose intake, or excessive meat consumption, are
the main causes of dietary-induced oxidative stress in West-
ern countries [3]. Recent evidence suggests that oxidative
I. Hininger-Favier (*) · M. Osman · A.M. Roussel
LBFA/Inserm 1055, Joseph Fourier University,
38000 Grenoble, France
e-mail : isabelle.hininger@ujf-grenoble.fr
L. Intes · B. Montanari
ISOCELL NUTRA, 53 boulevard du général Martial Valin,
75015 Paris, France
Phytothérapie
DOI 10.1007/s10298-015-0928-4

stress is a cornerstone of the metabolic mechanisms by
which overfeeding leads to insulin resistance [4]. Metabolic
syndrome (MetS) is defined as a constellation of clinical
criteria including visceral adiposity, elevated blood pressure,
hypertriglyceridemia, insulin resistance, and elevated fasting
glycemia. Its prevalence is high worldwide, although it var-
ies from one country to another and depends on defining
criteria. Using the NCEP (National Cholesterol Educa-tion
Program) definition, MetS reached 14.1% of the population
in France in 2006–2007 [5] and 22.9% in the US in 2010 [6].
In western countries, in particular in US, high fructose
corn syrup in particular by beverage has increased in the
last hundred years and correlates closely with the rise of obe-
sity, metabolic syndrome and diabetes [1,7]. This concern
was supported mainly by observations in high-fructose-fed
rodents that reported hyperinsulinemia, increase of body fat,
and hepatomegaly associated with accumulation of lipids in
the liver leading to the syndrome of non-alcoholic fatty liver
disease [8]. The feeding of rats with high fructose is there-
fore a suitable and pertinent model for MetS [8].
In addition, several line of evidence suggest that the risk
of type 2 diabetes in the normal population is increased by
iron intake due to high consumption of meat as the major
source of iron [9]. An excessive amount of elemental iron
has been proposed to induce free radical formation and being
the leading causes of dietary-induced oxidative stress [10].
Iron, in the form of free transition metal, is a very potent
catalyst leading to free radical production from hydrogen
peroxide in the Fenton’s reaction [11]. Due to its potential
pro-oxidant effect, high iron store have been reported to
increase the risk of diabetes type 2 [9], cardiovascular dis-
eases [12] or neurodegenerative diseases [13]. To our knowl-
edge there is no experimental study on the effects of the
interaction of these two western diet constituants on hyper-
glycemia and oxidative stress.
For that, we proposed an original model of a diet both rich
in fructose and iron to evaluate the risk of these contents of
western diet type, on oxidative stress and hyperglycemia.
So far the studies on trace elementsand vitaminssupple-
mentation to counteract oxidative metabolic pathologies are
inconclusive [14].
Our aim was to design an experimental study to evaluate
the protective effects of two antioxidants acting at cellular
levels to counteract the oxidative stress induced by a high
fructose/high iron diet (Fr/Fe). The N acetyl cysteine a pre-
cursor of glutathione, an important endogenous antioxidant,
and the Glisodin, a stable and bioavailable gliadin-combined
melon SOD superoxide extract [15], as antioxidant enzyme
inducer, were compared regarding their potential in-vivo
protective effects against oxidative markers (on DNA oxida-
tive damages, lipid peroxidation, antioxidant total power)
and metabolic parameters (fasting glycaemia, fasting insuli-
nemia and body composition).
Material and methods
Animals and diets
The experimental procedure was reviewed and approved by
the Joseph Fourier University Institutional Ethic Committee
for Animal Experimentation (Grenoble, France).
The rats were maintained and handled in accordance
with the Guide for the Care and Use of Laboratory Animals
(NIH, 1985). Forty eight male Wistar rats (Charles River,
l’Arbresle, France) of 200-220 g, were housed in a room with
20-22°C ambient temperature, 50±10% relative humidity, less
than 50dB white noise and 12h/12hcircadian cycle. Food was
purchased from Safe (Augis, France). Glisodin was pro-
vided from (Isocell Nutra, Paris). NAC was purchased from
Sigma-Aldrich (Lyon, Fr). Consideration was taken daily to
avoid potential pain of the animals in agreement with Ethic
Committee Recommendations. None of the animals exhibited
signs of pain or distress during the study.
As reported in Table 1, the Fructose/Iron (Fr/Fe) diet con-
tained 650 g fructose and 320 mg Fe/kg.
Design of the study
After 5 days of habituation, animals were distributed in three
groups of 16 animals (Fig. 1).
Table 1 Composition of the diets (g/100g diet).
Purina chow Fructose/Fe
diet
Starch 62 0
Fructose 0 65
Casein 22.7 20
Vegetal oils 4.5 5
Mineral and vitamin mixture 6.25 6.25
with Fe mg/100g diet 10 32
cellulose 4.50 5
kCal/100g diet 379 385
Fig. 1 Design of the study
2 Phytothérapie

Preconditioning period
As reported in Figure 1, during two preconditioning weeks
all rats were fed at libitum with the Purina chow containing
100 mg Fe/kg diet and they received by forced feeding 1 ml
water (group I), either 1 ml N acetyl cysteine at 100 mg/ml
(group II), or 1 ml Glisodin 180 U/ml (group III). At the end
of the preconditioning period, six animals of each group
were sacrificed and analysis performed.
Antioxidant molecules effect
After the preconditioning period, the diet was replaced by a
Fructose/Iron rich diet (Fr/Fe), inducing oxidative stress, for
8 weeks at libitum. In addition, the 10 animals remaining/
group kept on to receive each day, by forced feeding, 1 ml
water (group I), either 1 ml N acetyl cysteine at 100 mg/ml
(Group II), or 1 ml Glisodin 180 U/ml (group III).
At the end of the 8 weeks of treatment, the animals were
sacrificed, and analyses were performed.
Analysis
Sampling
After overnight fasting, rats were weighted and intraperito-
neally anesthetized with sodium pentobarbital. Blood was
collected by heart puncture in heparinized tubes protected
from light and centrifuged at room temperature for 10 min
at 3000 g. Plasma was immediately isolated, aliquoted,
stored at –80 °C, and analyzed within 2 months.
After blood collection, the rats were sacrificed. Fat depos-
its were excised and weighted. Mesenteric fat pad was com-
posed of the adipose tissue surrounding the gastrointestinal
tract, from the gastro-oesophageal sphincter to the rectum.
Liver was removed, weighted, frozen in liquid nitrogen and
stored until analysis. For the comet assay and DNA damage
determinations, total blood was stored as described previ-
ously [16].
Plasma fasting Glucose and insulin
Fasting glucose levels were evaluated, as routine laboratory
analyses, by enzymatic and colorimetric methods on Roche/
Hitachi modular P 12.
Fasting insulinemia was measured by ELISA, American
Laboratory Products Co., Windham at the end of the study.
Lipid peroxidation
Plasma and liver TBAR’s concentrations were assessed as
described by Richard et al. (1992) [17].
DNA oxidative damages
The evaluation of DNA damages was achieved by the
comet assay (single-cell gel electrophoresis) on total
blood as described previously [16]. Results were expressed
as tail moment (TEM). Three samples per animals were
assayed. The mean of three determinations was calculated
for each rat.
FRAP (Ferric Reducing Antioxidant Power)
Plasma antioxidant status was estimated using ferric reducing
antioxidant power (FRAP) assay. The FRAP assay uses anti-
oxidants as reductants in a redox-linked colorimetric method.
In this assay, at low pH, a ferric-tripyridyltriazine (FeIII-
TPTZ) complex is reduced to the ferrous form, which is
blue, and monitored by measuring the change in absorption
at 593 nm. The change in absorbance is directly proportional
to the reducing power of the electron-donating antioxidants
present in plasma. The absorbance change is translated into
a FRAP value (in μmol/l) by relating the change of absor-
bance at 593 nm of test sample to that of a standard solution
of known FRAP value [18].
Liver Cu-Zn SOD
Liver homogenates were performed in buffer (10 mmol/L
Tris-NaOH, 1 mmol/L DPTA, 1 mmol LLPMSF, pH = 7.4)
and centrifuged at 3000 g and 4 °C for 10 min. The superna-
tant was then submitted to three cycles of frost and defrost in
liquid nitrogen followed by a bath at 37°C before to be sub-
mitted to a second centrifugation at 10.000g for 20 min.
Afterwards SOD activity was determined on the supernatant
by monitoring the auto-oxidation of pyrogallol [19].
Statistical analyses
Values were expressed as mean ± SEM. Statistical signifi-
cance was set at p<0.05. Data statistical analyses were per-
formed using the statistical software package (Statistica Pro-
gram, Statistical Software, Paris, France). Statistical analyses
of the data were performed by analysis of variance, using a
Student‘s test for comparison of the means.
Results
Effects of the preconditioning supplementation
After two weeks, the glycaemia, lipid peroxidation and
FRAP were significantly improved in the group III (chow
diet plus Glisodin) compared to the group I (chow diet plus
water) (Table 2a).
Phytothérapie 3

In the NAC supplemented group (group II), surprisingly,
we measured an increased DNA oxidative damages despite a
higher level of FRAP.
Effects of Glisodin and NAC oral supplementation
in Fr/Fe rats
After 8 weeks of (Fr/Fe) rich diet (Table 2b), positive and
significant effects of the supplementation were observed in
the Glisodin group on fasting glycaemia, fasting insuline-
mia, plasma TBAR’s, and oxidative DNA damages assessed
by the comet assay. Hepatic SOD activity and plasma FRAP
were also increased (Tables 2b, 2c).
In the group receiving NAC supplementation, FRAP was
also increased and DNA damages trended to decrease, but at
a lesser extent than in the glisodin supplemented group. Fast-
ing glycemia, insulinemia, plasma TBAR’s and liver SOD
activity remained unchanged.
Total weight and mesenteric fat were significantly
reduced in both Glisodin and NAC supplemented groups
as reported in Table 2c.
Discussion
By using the Fr/Fe diet as an original animal model that
mimics some aspects of deleterious dietary habits of the
Western countries, and as an “in vivo”inducer of oxidative
stress, we aimed to investigate and to compare the potential
impact of two molecules reported to act as antioxidant at
cellular levels, Glisodin and NAC.
Fructose consumption that continues to increase in West-
ern countries due in part to the high consumption of soft
drinks, is one of the major causes of the worldwide epidemic
of the metabolic syndrome, insulin resistant states, and oxi-
dative stress [21]. As reported by numerous studies, high
fructose consumption, in men and animals, induces altera-
tions in glucose oxidative pathway, and results not only in
decreased insulin sensitivity, but also in enhanced produc-
tion of radical oxygen species (ROS) [22]. In animals, fruc-
tose rich [23,24] or high fructose/high fat [7] diets have been
frequently proposed to investigate dietary-induced oxida-
tive stress. In addition to fructose consumption, high dietary
Table 2a Effects of preconditioning NAC and Glisodin supplementation.
Groups (n=6) and diets Glycaemia μmol/L TBAR’s
μmol/L
Cometes (TEM) Liver SOD
U/g prot
FRAP
Purina Chow plus water (group I) 8.97±0.40 5.27±0.19 4.53±0.14 21.62±0.92 259±19
Purina Chow plus NAC (group II) 8.61±0.78 5.18±0.17 5.72±0.15* 21.47±0.88 326±19*
Purina Chow plus Glisodin (group III) 7.66±0.50* 4.79±0.17* 4.96±0.14 21.75±0.92 299±19*
*p<0.05 vs Purina chow plus water.
Table 2b Effects of Glisodin and NAC oral supplementation on metabolic and oxidative stress markers.
Groups and diets
(n=10)
Glycaemia μmol/L insulinemia Cometes
(TEM)
Liver SOD
U/g prot
FRAP TBAR’sμmol/L
Fr/Fe+ water
(Group I)
11,34±0.32 11.07± 5.91 5.30±0.40 22.98±0.39 275+8 5.76±0.14
Fr/Fe+ NAC
(Group II)
10.68±0.59 4.29± 2.14* 4.71±0.06 22.82±0.81 347+21* 5.67±0.22
Fr/Fe + Glisodin
(group III)
10.36±0.39* 4,37 ± 3,58* 4.50±0.09* 24.48±0.85* 302+11* 5.41±0.13*
*p<0.05 vs Fr/Fe plus water.
Table 2c Effects of Glisodin and NAC supplementation on
body weight and fat pad.
Groups (n=10)
and diets
Total weight (g) Mesenteric fat (g)
Fr/Fe+ water
(Group I)
420±5 12.81±1.34
Fr/Fe+ NAC
(Group II)
362±5* 8.84±1.02*
Fr/Fe + Glisodin
(group III)
385±7* 8.61±1.00*
4 Phytothérapie

intakes of iron as reported by epidemiologic studies which
has been associated for a part to meat overfeeding could also
participate to the pro oxidant effect of the Western diets [25].
It is well documented that the pro-oxidant effect of iron over-
load is related to its capacity to promote the Haber-Weiss
cycle that enhances the production of highly reactive
hydroxyl ions (°OH). In relation with this potential pro-
oxidant effect, high iron stores have been reported to
increase the risk of diabetes type 2 [11] cardiovascular dis-
eases [12] or neurodegenerative diseases [13].
In the present work, we postulated that the addition of
high levels of iron corresponding to two fold the recom-
mended intakes to a fructose rich diet (Fr) could result in
an increased oxidative stress. Our data clearly validated
this hypothesis, since in animals receiving the Fr/Fe diet,
the levels of markers of oxidative stress, especially DNA
oxidative damages, vulnerable to °OH production, were sig-
nificantly higher than in the rats fed the chow diet.
We compared the effects of N acetyl cysteine intakes,
inducer of glutathione synthesis [26], and those of Glisodin,
a melon SOD combined with gliadin, inducer of antioxidant
enzyme activities. Glisodin is a bioavailable and stable form
of SOD. The combination of SOD-rich melon with the wheat
gliadin biopolymer has been reported to allow SOD stability
and delivery during the passage through the gastrointestinal
tract [27].
In the present study, Glisodin supplementation acted pos-
itively not only on oxidative stress parameters, but also on
some important features of the metabolic syndrome. Indeed,
as expected, liver SOD activity was up-regulated, and
protective effects against DNA and lipid oxidation were
observed. In addition, glycaemia, insulinemia and body
composition were improved, suggesting an increased insulin
sensitivity after Glisodin supplementation.
The antioxidant effects of Glisodin, observed in the pres-
ent work, are in agreement with others previous studies
in men and animals. In pigs, with high oxidative stress
induced by ischemia-reperfusion process, Glisodin supple-
mentation resulted in significantly lower levels of induced
DNA damages [28]. In men, submitted to hyperbaric oxy-
gen (HBO)-induced oxidative stress, Glisodin supplementa-
tion protected DNA against oxidative damages and lowered
lipid peroxidation [29]. In patients at risk of cardiovascular
diseases, significant improvements in antioxidant status as
result of Glisodin supplementation were also observed, with
34% reduction in plasma MDA levels [30].
The metabolic syndrome and diabetes are in rise on
worldwide and a tight relationship between hyperglycaemia,
overweight and oxidative stress is largely described. In the
present study, we showed that, beside its antioxidant effect,
Glisodin supplementation positively impacted also fasting
blood glucose levels, fasting insulinemia and fat distribution.
Those changes are strongly involved in the risk of metabolic
syndrome, diabetes, cardiovascular diseases and related oxi-
dative complications.
Few studies, so far, have focused on the interest of Gliso-
din supplementation in the metabolic syndrome and diabetes
type 2. In diabetic mice supplemented with the melon extract-
gliadin combination, significant reductions in the levels of
8 hydroxydeoxyguanosine (8-OH-dG), marker of oxidative
stress, were observed, but in contrast with our data, no signif-
icant decrease in blood glucose levels or body weight were
observed [31]. More recently, Carillon et al. (2013) [32]
reported that after 1 month of supplementation with a melon
superoxide dismutase extract, body weight and insulin resis-
tance induced by a cafeteria diet, composed of high fat, high
sugar and high salt products, were reduced, while hepatic oxi-
dative stress was corrected. These data are in agreement with
our findings. The authors suggested that these beneficial
effects could be due to an increased expression of the liver
antioxidant enzymes. This mechanism, involving the up-
regulation of genes coding for antioxidant enzyme activity
rather than a ROS scavenging process, could explain the bet-
ter antioxidant efficiency of the Glisodin supplementation
compared to those of the NAC supplementation.
Conclusion
In conclusion, this study clearly showed that feeding rats with
a high-fructose high iron diet has detrimental consequence on
oxidative stress and glucose tolerance and strongly suggests
that Glisodin, might be a novel approach for preventing met-
abolic syndrome and related oxidative stress. In this work,
using an animal model of dietary-induced oxidative stress
by high levels of iron and fructose intakes, we showed that
an oral supplementation with Glisodin is effective, not only in
controlling oxidative stress, but also in improving important
features of the metabolic syndrome. Moreover, based on the
results of our study, it can be assumed that an oral supplemen-
tation with Glisodin advantageously copes to elevated blood
glucose, decreased insulin sensitivity, and mesenteric fat, that
are major risk factors of cardiovascular diseases and diabetes
by increasing SOD activity. At the same time, it protects pro-
teins and lipids against peroxidation. Thus, the above results
indicate that Glisodin may be a natural source of enzymatic
antioxidant protection.
Our data obtained on an experimental model of diet of
metabolic syndrome, supports the view that it is necessary
to continue this research with clinical studies on human met-
abolic syndrome population.
Conflict of interest : les auteurs déclarent ne pas avoir de
liens d’intérêts.
Phytothérapie 5

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