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Supplementation with gliadin‐combined plant superoxide dismutase extract promotes antioxidant defences and protects against oxidative stress

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The potential benefits to health of antioxidant enzymes supplied either through dietary intake or supplementation is still a matter of controversy. The development of dietary delivery systems using wheat gliadin biopolymers as a natural carrier represents a new alternative. Combination of antioxidant enzymes with this natural carrier not only delayed their degradation (i.e. the superoxide dismutase, SOD) during the gastrointestinal digestive process, but also promoted, in vivo, the cellular defences by strengthening the antioxidant status. The effects of supplementation for 28 days with a standardized melon SOD extract either combined (Glisodin®) or not with gliadin, were evaluated on various oxidative-stress biomarkers. As already described there was no change either in superoxide dismutase, catalase or glutathione peroxidase activities in blood circulation or in the liver following non-protected SOD supplementation. However, animals supplemented with Glisodin® showed a significant elevation in circulated antioxidant enzymes activities, correlated with an increased resistance of red blood cells to oxidative stress-induced hemolysis. In the presence of Sin-1, a chemical donor of peroxynitrites, mitochondria from hepatocytes regularly underwent membrane depolarization as the primary biological event of the apoptosis cascade. Hepatocytes isolated from animals supplemented with Glisodin® presented a delayed depolarization response and an enhanced resistance to oxidative stress-induced apoptosis. It is concluded that supplementation with gliadin-combined standardized melon SOD extract (Glisodin®) promoted the cellular antioxidant status and protected against oxidative stress-induced cell death. Copyright © 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 957-'-962 (2004)
Published online in Wiley InterScience ( DOl: 10.1002/ptr.1542
Supplementation with Glladin-combined
Plant Superoxide Dismutase Extract Promotes
Antioxidant Defences and Protects Against
Oxidative Stress
Ioannis Veuldeukis-", Marc Contf; Pascal Krauss', Caroline Kamaté'r', Samantha Blazquez',
Maurel Tefif, Dominiqne Mazier", Alphonse Calenda! and Bernard Dngas
'ISOCELL Nutra SAS. 53 bd du Général Martial Valin, 75015, Paris, France
2INSER\1 U511, Irnmunobiologie Cellulaire et Moléculaire des Infections Parasitaires, CHU-Pitié Salptérière Paris VI, 75013,
Paris, France .
'INSER"! U477, Hôpital Cochin, 75006 Paris, France
The potential benefits to health of antioxidant enzymes supplied either through dietary intake or supple-
mentation is still a matter of controversy, The development of dietary delivery systems using wheat gliadin
biopolymers as a natural carrier represents a new alternative. Combination of antioxidant enzymes with this
natural carrier not only delayed their degradation (i.e. the superoxide dismutase, SOD) during the gastrointestinal
digestive process, but also promoted,
in vivo,
the cellular defences by strengthening the antioxidant statns.
The effects of supplementation for 28 days with a standamized melon SOD extract either combined (Gllsodin"
or not with gliadin, were evaluated on various oxidarive-stress biomarkers, As already described there was no
change either in superoxide dismutase, catalase or glutathione peroxidase activities in blood circulation or in
the liver' following non-protected SOD supplementaticn, Bowever, aninIals supplemented with GlisodinllD
showed a significant elevation in circulated antioxidant enzymes activities, correlated with an increased resist-
ance of red blood cells to oxidative stress-induced hemolysis, ln the presence of Sin-I, a chemical donor of
peroxynitrltes, mitochondria from hepatocytes regularly underwent membrane depolarîzation as the primary
biological event of the apoptosis cascade. Hepatocytes isolated from animais supplemented with Glisodinll>
presented a delayed depolarîzation response and an enbanced resistance to oxidative stress-induced apoptosis.
It is concluded that supplementation with gliadin-combined standardized melon son extract (Glisodin"
promoted the cellular antioxidant statns and protected against oxidative stress-induced cell death. Copyright
© 2004 John Wiley
Sons, Ltd.
Keywords: antioxidant; plant superoxide dismutase; gliadin; oxidative stress.
ln all aerobic organisms, the consumption of oxygen is
crucial for life. It also produces reactive oxygen species
involved in the regulation of many different biological
pro cesses (Forman and Torres, 2002) and survival from
invading pathogens. Under physiological conditions the
production of these pro-oxidant molecules is control-
led at different levels by the antioxidant defences that
normally !imit the excess of free radical species (Wei
and Lee, 2002). These natural defences are essentially
composed of specialized enzymes such as superoxide
dismutase (SOD), catalase (Cat) and glutathione-
peroxidase (Gpx) and also by non-enzymatic antioxi-
dant molecules such as vitamins, thiols and f3-carotene.
Inflammatory or aging processes (Wickens, 2001) are
Correspondence to: Professor A. B. Dugas, ISOCELL Nutra SAS, 53
blvd du Général Martial Valin, 75015, Paris, France.
E-mail: bdugasïêtibcrtysurf.ïr
Contract/grant Sponsor: CIFRE fellowship ISOCELL Pharma SAS.
2004 John Wiley
Sons, Ltd.
associated with the disruption of the oxidant/antioxidant
(redox) balance resulting in cellular and tissue oxidative
stress and cell death by apoptosis (Lang et al., 2002;
Chandra et al., 2000). Indeed, the progressive and dis-
crete imbalance of the endogenous redox system can
lead to the development of chronic degenerative dis-
eases (Lavrovsky et al., 2000; Tak et al., 2000). Thus it
seemed evident that nutritional antioxidant supple-
mentation could have health-promoting effects if it could
control the endogenous redox system (Fang et al., 2002;
Kritharides and Stocker, 2002). It is already admitted
that dietary antioxidants are very useful in general
health either by preventing or by supplementing the
usual drug treatments in a variety of diseases (Stephens
1996; Kritchevsky, 1999; Burk, 2002). This sug-
gests that the use of a nutritional antioxidant formula
will provide better prevention of oxidative stress-me di-
ated diseases.
Until now the development of these new functional
foods has been limited by their poor capacity to pro-
mote efficient oral delivery of antioxidant enzymes
and also by the definition of the correct health bio-
markers to follow (Branca et
2001). However, the
May 2003
Accepteâ 4June 2004
development of new drug delivery and food pack-
aging systems (Weber et al., 2002; Takata et al., 2002)
make this new functional antioxidant formula possible
(Mosca et al., 2002; Stella et al., 1995; Regnault et al.,
1996). Among various different delivery systems the
wheat gliadin biopolymers presented a dual interest:
(i) their capacity to -trap and to delay the release
of the active ingredient during the gastrointestinal
digestive process (Arangoa et al., 2001), and (ii) their
bioadhesive properties with the intestinal mucosa to
improve and/or promote the delivery of the active in-
gredient, thus defining an orally bioactive SOD (Dugas,
This study investigated the froperties of an effective
nutritional formula (Glisodin ) made from the combi-
nation of a melon (Cucumis melo Le.) standardized
superoxide dismutase extract as the active ingredient
and wheat (Triticum vulgare) gliadin biopolymers as
the carrier. The antioxidant properties of the melon
SOD contained in Glisodin" were evaluated on anti-
oxidant biomarkers currently used to assess the poten-
tial health benefits of nutrition al products.
Reagents. Dulbecco's modified Eagle's medium
(DMEM), L-glutamine, glucose, streptomycin-penicil
lin, fetal calf serum (FCS) and most of the chemical
reagents were from Sigma Chemical Co (St Louis, MO).
Hepatocytes were cultured in DMEM medium con-
taining 10% FCS, 1% L-glutamine, 2% streptomycin-
penicillin, in 5%
at 37 "C, The chemical donor
nitrogen peroxide (Sin-I) was the kind gift of Dr J.P.
Kolb (INSER.t\1 U311, Paris, France). 2,2'-azobis-(2-
aminopropane )-dihydrochloride (AAPH) was obtained
from Calbiochem (Meudon, France). Wheat gliadin
(Gliamine") was purchased from HITEX (Vannes,
France). The standardized melon superoxide dismutase
extract (Extramel") was obtained from the strain
Cucumis melo L.e., genetically selected for its higher
grade SOD activity (90 ill/mg of dry powder), BIONOV
(Avignon, France).
The gliadin-combined SOD preparation. Briefly,
Glisodin® is a water dispersible form of superoxide
dismutase lyophilized extract from melon (standardized
to 90 ill/mg) combined with a 40% hydro-alcoholic
soft gel of gliadin at 50 "C, It is spray-dried using
maltodextrin as a support and the various ratios were
adjusted to obtain a theoretical activity of 1 lU/mg of
final dry powder.
The superoxide dismutase activity of the Glisodin"
was certified using a specifie enzymatic assay
(Beauchamp and Fridovich, 1971; Oberley and Spitz,
1984) from 5 g of dry product sonicated into 7 mL of
water. The solution was then centrifuged at 10000 g
for 20 min and the first supernatant
made up to
10 mL with ultra pure water. The pellet was suspended
again in 1 mL of ultra pure water, homogenized and
centrifuged at 10 000 x g for 20 min at 6°-8 "C, The sec-
ond supernatant (S2) was then adjusted to 1 mL. The
activity in both fractions
and S2) was determined
on a native polyacrylamide gel electrophoresis against
the SOD melon extract (90 lU/mg).
Copyright © 2004 John Wiley
Sons, Ltd.
Delayed release of loaded SOD from the gliadin com-
bination. The progressive release of the SOD activity
trapped by the gliadin polymers was compared with
the parallel degradation of the non-protected SOD
(melon extract) during a pro cess that mimicked the
digestive transit (0.1
hydrochloric acid at pH
in the
presence of 1 !lM of pepsin at
DC) as already de-
scribed by Stella et al. (1995).
Animal population and treatment. Balb/c mice were
purchased from IFFA-CREDO (Orleans, France), aged
6-8 weeks and weighing 25-30 g. Each group consisting
of 10 animals randomly selecte d, received either a
normal diet, or a supplementation with gliadin, or a
supplementation with non-protected SOD melon ex-
tract (10 ill/day for 28 days) or Glisodin" (0.1, 0.5, 1,
5 mg/day for 28 days) by force-feeding.
Redox status. Blood samples were collected on heparin
at different time-points along the supplementation pe-
riod (0,7,14,21 and 28 days). Plasma and erythrocytes
were immediately separated by centrifugation at 800
for 20 min at 4 "C, Superoxide dismutase (RANSOD
kit, Randox) glutathione peroxidase (RA.t~SEL kit,
Randox) and catalase activities (was assayed by a
method in which the disappearance of peroxide is
followed spectrophotometrically at 240 nm) were then
deterrnined. Red blood cell (RBC) hemolysis, induced by
the free radical generator 2,2'-azobis-(2-amidinopropane)-
dihydrochloride (AAPH), was deterrnined as previously
described (Miki et al., 1987).
Peroxynitrite-induced apoptosis in hepatocytes. Apo-
ptosis was quantified by using the ApoAlert DNA frag-
mentation detection kit (Clontech, Palo Alto, CA). At
days 0, 7 and 28, hepatic cells were isolated and incu-
bated for 48 h (5 x 105cells/mL) in complete DMEM
medium in the presence or in the absence of 100
mL of Sin-1 (3-morpholinosydnonimine hydrochloride),
a potent generator of nitrogen peroxide. Data are
presented as the percentage of apoptotic cells among
various areas of 200 cells.
Measurement of the mitochondrial depolarization,
'Pmof isolated hepatocytes was measured by flow
cytometry using the J-aggregate-forrning lipophilic
cation, 5,5',6,6'
zolocarbocyanine iodide (JC-1) (Beltran et al., 2000).
Briefiy, aliquots of the cell suspension (106cells) were
incubated with JC-1 at a final concentration of
at 37°C in the dark for 30 min before analysis. Pre-
lirninary experiments demonstrated that under these
conditions the dye reached near equilibrium distribu-
tion and gave a maximal fluorescence response to a
fall in
'Pm induced by the mitochondrial uncoupler
carbonyl cyanide m-chlorophenylhydrazone (5
Flow cytometry was performed on a FACScan instru-
ment (Becton Dickinson). Data were acquired and ana-
lysed by using CELLQUEST software. The results are
expressed as the mean aggregate fluorescence (red)
Statistical analysis. Mean comparisons between the vari-
ous groups (with or without supplemented diets) were
conducted using Student's t-test. Differences were
considered significant when
18, 957-962 (2004)
Supplementation SaD (U/g Hb)
Table 1. Effect of a supplementation with non-protected SOD on circulating antioxidants
Catalase (kU/g Hb)Gpx (U/g Hb)
Non proteeted SaD extra et
1125 ±55
±21 30 ±2
33 ± 6
10) were fed every day with control diet supplemented or not with 10 mg/mouse/day
of the non-protected SOD for 28
Blood sam pies were collected and SaD, Gpx and catalase
activities were evaluated in erythrocytes. Data represent the mean.
ten animals/group
one representative experiment.
Wheat gliadin carrier delays the SOD release in
conditions mimicking the digestive pro cess
Many investigations (Zidenberg-Cherr et al., 1983; Giri
and Misra, 1984) now including ours, demonstrated
that oral treatment with non-protected SOD did not
induce significant changes in the circulating redox sta-
tus since the levels of erythrocyte SOD, catalase and
Gpx activities remained constant (Table 1). This is con-
sistent with the poor bioavailability or rapid degrada-
tion of proteins during the digestive process. As a matter
of fact, a rapid disappearance of the non-protected
SOD activity was observed in a medium mimicking the
digestive pro cess (Fig. 1) demonstrating that the anti-
oxidant enzyme was destroyed during gastrointestinal
transit. However, when the SOD activity was trapped
by gliadin biopolymers (Glisodin®) a significant and
progressive increase of SOD activity was observed
probably correlating with the concomitant proteoly-
sis of the gliadin biopolymers. This suggested that
gliadin might delay the release and consequently the
degradation of the. SOD activity during gastrointestinal
Free SOD
--- Glisodin
10 30 60
Time (min)
1. Gliadin polymers delay the release of the melon SOD
activity in a medium mimicking the digestive process. An iden-
tical amount (100 units) of melon-SOD extract was submitted
free or combined with gliadin (Glisodin·"') to conditions mimick-
ing the digestive process, for 1 h at 37°C. The medium was
periodically sampled to measure the residual SOD activity ac-
cording to the reduction of ferricytochrome C. The data repre-
sent the mean
SD of quadruplicate samples of one treatment
out of six different experiments.
Copyright © 2004 John Wiley
Sons, Ltd.
Table 2. Effect of a supplementation with SOD-gliadin combina-
tion on circulating antioxidants
Control Glisodin"
Antioxidant status (rnrnol/Ll
SOD (U/g Hb)
Gpx (U/g Hb)
Catalase (kU/g Hb)
Animais were fed every day with control diet or with con-
trol diet supplemented with 1 mg/mouse/day of Glisodin" for
28 days. Blood samples were collected and SOD, Gpx and
catalase activities were evaluated in erythrocytes. Data repre-
sent the mean
SD of ten animals/group
one representa-
Glisodin@ supplementation modulated the circulating
antioxidant status
Supplementation of normal mice with the gliadin-
combined standardized melon SOD extract (Glisodin®)
for 28 days was.found to promote the circulating anti-
oxidant enzymes SOD, catalase and Gpx (Table 2). This
effect was formula specifie (Glisodin"), because the non-
protected SOD extract or the gliadin alone was unable
to promote these antioxidants. This promoting effect
was time dependent (Fig. 2A) since the circulating SOD
activity began to Increase after 7 days of supplementa-
tion to reach a maximum after 28 days (SOD returned
to the baseline after an addition al 28 days, data not
shown), The promoting effect was dose dependent
(Fig. 2B) since significant effects appeared only for doses
equivalent to 0.5 mg/day or higher with a maximal
effect .obtained at 5 mg/day.
As already demonstrated for different antioxidant
dietary supplementation (Peng
et al.,
2000), the supple-
mentation with Glisodin" for 28 days increased the re-
sistance of RBC
0.01) to oxidative stress-induced
hemolysis (Fig. 3) in response to a chemical donor of
free radicals (AAPH). After 3 h of incubation at 37°C
in the presence of 50 mMAAPH, about 48% vs 74% of
hemolysis was observed for RBC isolated, respectively,
from animaIs supplemented or not with Glisodin".
Hepatoprotective effect of Glisodln" supplementation
As previously described
in vitro
et al.,
2000), the SOD-gliadin combination also induced
in vivo,
a time-dependent increase in SOD activity in
Phytother. Res. 18, 957-962 (2004)
-- -- ----------~.
(J) 5000]
..,.. Free gliadin
~ 4000
Free SOD
4000 .
Q) 1
~ 2000
1000 .
14 21 28 0 0.1 0.5 5
Time of treatment (days) Gllsodirr"
Effect of a supplementation with
on circulating SOD activity. A. Mice were fed for
days, with either a control
diet or supplemented with (a) melon SOD extract
lU of non protected SOD), (b) gliadin
mg) or (e) Glisodin"
mg for
Mice were fed with different doses of Glisodin"
(0.1, 0.5, 1,
mg of Glisodin"/mouse/day). Blood was periodically sampled in
the study (A) while only at day
for study (B). SOD activity was measured as deseribed in materials and methods. Data represent
the mean
SEM of the different groups.
80 -+-Control
-0- Glisodin-treated
n .
1,5 2 2,5 3
Time (Hours)
Effect of a supplementation with Glisodin" on ervthro-
eyte resistanee to oxidative stress-induced hemolysis. After a
day peri ad of supplementation with Glisodin"
RBC were eolleeted and exposed ta the free radical generator
mM). Hemolysis was evaluated as deseribed in mate-
rials and methods. Data represent the mean
SEM of the dif-
ferent groups.
hepatocytes (Fig. 4). This inducing effect appeared to be
<0.05) after 14 days of Glisodin" supple-
mentation and reached a maximal effect after 21-28
0.001). Such stimulation was not restricted to
the SOD activity because catalase and Gpx activities
were also increased (Table 3). As shown in Fig. 5 the
improvement of the hepatocyte antioxidant defences
correlated with an increased resistance (p <0.01) to
oxidative stress-induced apoptosis (Estevez and Jordan,
2002). After 8 h in the presence of the peroxynitrites
chemical donor Sin-I, it was observed that 20% of the
hepatocytes isolated from animals supplemented with
Gliscdin" underwent apoptotsis, whereas this rate in-
creased ta 72% in hepatocytes fram
Copyright © 2004 John Wiley
Table 3. Effect of a supplementation with SOD-gliadin combina-
tion on Iiver antioxidants
Activity (unit/mg of protein)
SOD Gpx Catalase
0.6 0.21 ±0.05
0.80 ±0.02 40
Animais received every day either a control diet with or with-
out supplementation with
mg/mouse/day of Glisodin" for
days. Livers were then collected and then the SOD, catalase
and Gpx activities were evaluated from the various tissue ex-
tracts. Data represent the mean
SD of ten animals/group.
Effect of Glisodin" supplementation on animal
hepatocytes mitchondriaI
exposed ex vivo to
As the mitochondrion is a key compartment involved
in the control of oxidative stress-induced cell death
(Akao et al., 2003a) the mitochondrial functions of
hepatocytes isolated from animaIs receiving a Glisodin"
supplementation were evaluated. As aIready described
(Li et al., 2002; Kahlert and Reiser, 2002; Makani et al.,
2002) mitochondria from normal hepatocytes exposed
to Sin-l showed a gradual decrease in
as described
by the mean aggregate fluorescence of the cationic
lipophilic fluorochrome (Je-l) (Fig. 6). Analysis of
of Sin-l-stimulated hepatocytes from
Glisodin" supplemented animals demonstrated that the
mitochondrial depolarization was substantially delayed.
This study investigated the potential effects of a
supplement containing a gliadin-combined plant SOD
Phytother. Res. 18, 957-962 (2004)
SOO extract
14 21
Time of treatment (days)
Figure 4. Effect of a supplementation with Glisodln" on liver
SOO activity. Mice were fed with a control diet supplemented
or not with
mg/mouse/day of
Animais were killed
periodically each 7 days. Liver proteins were extracted and the
SOO activity was evaluated. The results are expressed as units
per mg of protein and data represent the mean
SEM of the
different grou ps.
B Glisodin
20 .
Figure 5. Effect of a supplementation with Glisodin"' on the
resistance of hepatocytes to nitrogen peroxide-induced
10 per group) were fed a control
diet supplemented or not with , mg/mouse/day of Glisodin"'
and killed after
28 davs,
After isolation Iiver ceIls were submit-
ted to Sin-t. The results are expressed as a percent of apoptotic
ceIls and data represent the mean
SEM of four different
extract on several redox biomarkers. The results of this
animal study were dual: the Glisodin" dietary supple-
mentation not only promoted the circulating and
tissue antioxidant defences (increased SOD, Gpx and
catalase activities) but also improved cell resistance to
oxidative stress. ln the circulation, RBC from animals
receiving Glisodin" were less susceptible to oxidative-
stress-induced hemolysis. ln addition hepatocytes from
animaIs receiving Glisodin" dietary supplementation
Sons, Ltd.
1 •••••••
t: '
120 ~
(I)~ I~'- ~
T ~~
801 ~ ~~ 1
~~ i
= }~~
~ 60 T
Time after serum withdrawal (hours)
Figure 6. Effect of a supplementation with Glisodin"' on
sln-t-tnduced mitochondrial membrane depolarization
hepatocytes. Changes in
of isolated hepatocytes from
normal or Glisodin"'
10 per group) supplemented animais
were followed after exposure to the chemical peroxynitrite
donor, Sin-l
ng/mL) over a period of 4 h as described in
materials and methods. Data represent the mean
SO of ail
were resistant to peroxynitrite-induced apoptosis and
mitochondrial depolarization.
The combination of the melon SOD extract with
gliadin biopolymers
mandatory for obtaining this
health promoting effect, confirrning that the wheat
gliadin is a helpful carrier for the oral delivery of active
food ingredients (Arangoa
et al., 2001) .
Many studies have reported that a long-lasting intake
of fruit and vegetable antioxidants reduced the Iikeli-
hood of cardiovascular and proinfiammatory diseases
as weil as certain cancers (Block
al., 1992; Diplock
et al.,
Madar and Stark,
et al., 2002;
et al.,
2003b): Soit appears that the improvement
of antioxidant defences is a biological key event in the
health promoting effects of antioxidant nutrients. The
present work not only confums and extends these
scientific and clinical studies but also provides useful
information for the development of functionally active
food ingredients.
This new formula shows real benefits for health since
functional antioxidant enzyme supplementation (here
melon SaD) is now able to promote cellular resistance
to stress by strengthening the host antioxidant defences.
Nevertheless, the mechanism by which it exerts its bio-
logical effect remains to be clarified.
The present study does not only confirms the effi-
cacy of dietary antioxidant supplementation but also
describes an orally active plant superoxide dismutase
demonstrating that functional enzymes can be used in
dietary supplementation.
Caroline Kamaté is a PhD student in receipt of a CIFRE fellowship
from Isocell Pharma SAS.
Pliytother. Res.
18, 95ï-962 (2004)
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... Glisodin® is Cucumis melo LC extract rich in SOD coated by a wheat matrix polymer layer gliadin. Studies have found that gliadin carries SOD orally and increases adhesion enzymes into the gastrointestinal epithelium, making it easily absorbed in the J o u r n a l P r e -p r o o f small intestine (Vouldoukis, Krauss, et al., 2004). This combination of Cucumis melo LC gliadin has been widely studied in animals and humans, such as diabetes, reperfusion injury, fibrosarcoma, cognitive, atherosclerosis, and antiaging (Goldberg and Crysler, 2014;Romao, 2015). ...
... The results showed that SOD levels increased significantly on days 14 and 28. The time required for an increase in SOD levels was in line with Vouldoukis et al. (Vouldoukis, Krauss, et al., 2004), which administered SOD-gliadin extract for 28 days. SOD levels increased on day 14 and reached a maximum effect after 28 days. ...
... It has high SOD of 100 IU/NBT per mg dry extract on average, catalase (10 IU/mg), natural antioxidants, and glutathione peroxidase. Antioxidants reduce oxidative stress, while heterologous SODs or antigens may have immunoregulatory properties (Vouldoukis, Krauss, et al., 2004). ...
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Sepsis is a major cause of death in intensive care units whose development is supported by an imbalance of oxidative stress and antioxidant. Superoxide dismutase (SOD) is a primer endogen antioxidant that prevents reactive oxygen species (ROS). Extensive studies on animals and humans have examined Cucumis melo L.C, a cantaloupe rich in SOD, and its combination with gliadin. The studies aimed to determine the effect of enteral administration of Cucumis melo L.C. gliadin (CME-gliadin) 28 days before inducing sepsis in rats. This experimental study aimed to compare four groups of male Wistar rats, including negative and positive control rats and those supplemented with SOD CME-gliadin 1 IU/day and SOD CME-gliadin 5 IU/day. All rats were given the same standard, except the supplementation for 28 days. Sepsis was induced by intraperitoneal injection of LPS 10 mg/kg. Enteral administration of SOD – gliadin extract of CME-gliadin for 28 days was used as antioxidant prophylaxis against oxidative stress due to sepsis. The results showed that enteral administration of CME-gliadin of 1 IU/day and 5 IU/day significantly increased SOD levels based on examination after 14 and 28 days. Also, it significantly decreased MDA (p < 0.001), TNF-α (p < 0.001), and lactate levels in rats induced by sepsis. However, the increase in lactate levels was above >1.64 mmol/l, indicating a high mortality rate. There was no significant difference in SOD, MDA, TNF-α, and Lactate levels between SOD 1 IU and SOD 5 IU. This descriptive data show that SOD 5 IU has a better result in MDA, TNF-α, and Lactate levels than SOD 1 IU.
... Following the technique of SOD encapsulation in gliadin proteins [31], SOD from Cucurbis melo was fused with gliadin peptide (QQPYPQPQPF) in order to improve intestinal adsorption [32]. The results demonstrated that fusing the gliadin peptide to CuZn SOD allowed it to physically interact with a potential receptor on the surface of epithelial cells without altering the antioxidant activity of the SOD protein. ...
... A SOD extract (90 IU/mg and about 4.5 g gliadin/mg SOD) was dissolved in a hydro-alcoholic soft gel of gliadin at mild temperatures and then spray-dried, using maltodextrin as a support, to obtain a theoretical activity of 1 IU/mg of final dry powder [31,142,143]. Glisodin® is currently also marketed and manufactured by other companies and used in pet-health promoting formulations. ...
... Only supplementation with Glisodin® (1 mg/day) for 28 days promoted the circulation of antioxidant enzymes, SOD, catalase and glutathione-peroxidase. Furthermore, supplementation improved red blood cell resistance to oxidative-stress-induced hemolysis and improved the resistance of hepatocytes to peroxynitrite-induced apoptosis [31]. ...
The overproduction of free radicals can cause oxidative-stress damage to a range of biomolecules, and thus potentially contribute to several pathologies, from neurodegenerative disorders to cardiovascular diseases and metabolic disorders. Endogenous antioxidant enzymes, such as superoxide dismutase (SOD), play an important role in diminishing oxidative stress. SOD supplementation could therefore be an effective preventive strategy to reduce the risk of free-radical overproduction. However, the efficacy of SOD administration is hampered by its rapid clearance. Several different approaches to improve the bioavailability of SOD have been explored in recent decades. This review intends to describe the rationale that underlie the various approaches and chemical strategies that have led to the most recent advances in SOD delivery. This critical description includes SOD conjugates, SOD loaded into particulate carriers (micelles, liposomes, nanoparticles, microparticles) and the most promising and suitable formulations for oral delivery, with a particular emphasis on reports of preclinical/clinical results. Likely future directions are also considered and reported.
... Increasingly, veterinarians, owners and trainers are using dietary or nutritive antioxidant supplements to mitigate inflammation associated with excess oxidative damage [23] . The dried and powdered pulp of a particular non-GMO strain a of Cucumis melo LC (cantaloupe or muskmelon) is rich in the antioxidant enzymes superoxide dismutase (SOD) and catalase [24][25][26] and has been shown to provide protection against administered pro-inflammatory compounds [25] . This C. melo pulp (CMP) also reduced markers of oxidative stress and improved antioxidant activity in humans [27] , pigs [28] , felines [29] , mice [30] and horses [31] . ...
... Increasingly, veterinarians, owners and trainers are using dietary or nutritive antioxidant supplements to mitigate inflammation associated with excess oxidative damage [23] . The dried and powdered pulp of a particular non-GMO strain a of Cucumis melo LC (cantaloupe or muskmelon) is rich in the antioxidant enzymes superoxide dismutase (SOD) and catalase [24][25][26] and has been shown to provide protection against administered pro-inflammatory compounds [25] . This C. melo pulp (CMP) also reduced markers of oxidative stress and improved antioxidant activity in humans [27] , pigs [28] , felines [29] , mice [30] and horses [31] . ...
... The mechanism is unclear as plasma SOD activity was unchanged in the CMP group, but perhaps indicating that activity was adequate for the demands. It is also possible that upregulated activities of glutathione peroxidase or catalase may have contributed to the increase in plasma TAS [25,47] , and this needs to be investigated in future studies. ...
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We evaluated the antioxidative and anti-inflammatory potential of daily oral supplementation with a proprietary powdered Cucumis melo pulp (CMP) on exercise-induced markers of articular and muscular oxidative stress and inflammation in 12 horses. Horses performed a high-intensity exercise test immediately prior to, and then following, 3 weeks of daily supplementation of 1 g powdered CMP (CMP; n=8). Controls (Co; n=8) underwent the same exercise and sampling regime but were not supplemented. Blood and synovial fluid (SF) samples were taken 24 h prior to exercise (BL), and at 1 and 24 h following exercise. Plasma and SF were analysed for prostaglandin E2 (PGE2), total antioxidant status (TAS), nitrite and superoxide dismutase (SOD) activity. SF was analysed for glycosaminoglycans (GAG), and plasma was analysed for thiobarbituric acid reactive substances (TBARS). Comparisons were made using repeated measures with the initial exercise test as a covariate. There was an increase in SF SOD activity in the CMP group. Compared to Co at 1 h, CMP reduced nitrite and GAG in SF, as well as maintained plasma TAS and lymphocyte levels. At 24 h, plasma PGE2 and creatine kinase were lower in horses receiving CMP. Three weeks of supplementation with CMP reduced markers of articular and skeletal muscle oxidative stress and inflammation in response to high-intensity exercise in horses. Nutritive antioxidants may provide a useful adjunct to the daily nutrition plan of horses undergoing regular exercise training and competition.
... 16 Studies, performed in animal models and in humans, have confirmed the superiority of using gliadinprotected SOD (GP-SOD), when compared to placebo or other antioxidants in neuroprotection, atherosclerosis or UV-induced erythema. [17][18][19][20] The objective of this study was to evaluate the efficacy of a combination treatment comprising narrowband ultraviolet B (NB-UVB) associated with GP-SOD for widespread nonsegmental vitiligo. ...
... [11][12][13] The combination of SOD with a wheat gliadin biopolymer has been shown to protect it from degradation as it passes through the gastrointestinal tract. Furthermore, as the activity of GP-SOD has been demonstrated in several animal models and clinical studies, [16][17][18][19][20] it appears to be promising candidate for vitiligo treatment. Our results show almost 20% repigmentation after 24 weeks of treatment with GP-SOD combined with NB-UVB compared to 9% when NB-UVB was associated with placebo. ...
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Background: Despite a solid rationale, the usefulness of antioxidants in treating vitiligo has not been clearly demonstrated. Combining superoxide dismutase (SOD) with a wheat gliadin biopolymer protects it during the passage through the gastrointestinal tract. Objective: To evaluate the efficacy of gliadin-protected SOD (GP-SOD), associated with narrowband ultraviolet B(NB-UVB), for treating vitiligo. Methods: We conducted a 24-week monocentric interventional prospective randomized placebo-controlled trial in the tertiary center for vitiligo care in the department of Dermatology of Nice University hospital, Nice, France. Subjects with non-segmental vitiligo affecting more than 5% of the total body surface were included. The subjects received gliadin-protected SOD (GP-SOD; 1g/d for 12 weeks followed by 0.5g/d for 12 weeks) or placebo in combination with twice weekly sessions of NB-UVB. The primary endpoint was the total repigmentation rate at 24 weeks, compared to baseline, as assessed by investigator-assessed Vitiligo Extent Score (VES) on standardized pictures. Results: A total of 50 patients were included. After 24 weeks, a greater improvement in VES was observed in the GP-SOD group (19.85%; SE 4.63, p<0.0001) compared to the placebo group (8.83%; SE 4.72, p=0.0676). Tolerance was good in both groups. No related side-effect was reported. Conclusions: The use of GP-SOD appears to be a useful add-on to phototherapy in the treatment of vitiligo patients.
... Many past researchers have described the routes of administration for SOD in pharmacological studies. Animal models involving pathological diseases were mostly treated with intravenous, subcutaneous, intramuscular, and intraperitoneal (i.p.) injections of SOD (Cloarec et al., 2007;Décordé et al., 2010;Jadot & Michelson, 1986;Kick et al., 2007;Kim et al., 2011;Laursen et al., 1997;Lin, Pape, & Friedrich, 1994;Naito et al., 2004;Nakazono et al., 1991;Okada et al., 2006;Regnault et al., 1995;Robbins et al., 2010;Simonson et al., 1997;Stone, Bjorling, Southard, Galbreath, & Lindsay, 1992;Suzuki, Matsumoto, Okamoto, & Hibi, 2008;Tanaka et al., 2011;Tarhini et al., 2011;Trea, Ouali, Baba-Ahmed, & Kadi, 2013;Vaille, Jadot, & Elizagaray, 1990;Vouldoukis et al., 2004;Watterlot et al., 2010;Welsh et al., 2012;Welty-Wolf et al., 1997;Weydert et al., 2006;Zhang, Zhao, Zhang, Domann, & Oberley, 2002). Several researchers have supplied SOD directly at the site of infection, for instance plant Cu/Zn-SOD was applied as ointment against skin fibrosis in breast irradiated women to evaluate the anti-fibrotic properties of SOD (Houghton, Steels, Fassett, & Coombes, 2011;Teoh-Fitzgerald & Domann, 2012). ...
... Oral administration of wheat-gliadin encapsulated melon SOD has remarkably reduced oxidative stress and prevented severe ailments including Type 2 diabetes, ischemia-reperfusion injury, tumor formation, atherosclerosis, Alzheimer's, viral infections, and inflammation in animal and human models (Carillon, Rouanet, Cristol, & Brion, 2013;Kick et al., 2007;Romao, 2015). Effectiveness of oral gliadin biopolymer-SOD administration against several health conditions was credible to the bioadhesive properties of gliadin, which enhances the delivery of SOD at target sites across intestinal barriers, and its ability to entrap and protect the physical structure of SOD during digestion in the stomach (Cloarec et al., 2007;Houghton et al., 2011;Naito et al., 2004;Vouldoukis et al., 2004). The promising effect of gliadin-SOD in multiple diseases led to the formulation of clinically proven commercialized health supplement, GliSODin (Menvielle-Bourg, 2005). ...
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Superoxide dismutase (SOD) is an antioxidant enzyme functional for physiological defense strategies in animals and plants against free radicals and reactive oxygen species (ROS) generated from biotic and abiotic stress. Supplementation of SOD from plants in mammalian diet is a new approach in terms of health improvement against pathological conditions. There is a research gap about the feasibility of including plant-derived SOD in animal diet as health enhancer due to poor bioavailability upon oral administration. Commercially available wheat gliadin encapsulated melon SOD has been proven to enhance mammalian health, but gluten/gliadin intolerance in certain animals and human may limit its marketability. Therefore, this review aims to highlight the sources of SOD from underutilized plants and potential encapsulation of SOD using soluble dietary fibers to be incorporated in animal diet as health enhancing supplements. This review provides a sustainable solution for the development of therapeutic approaches in agricultural industry.
... It is also a very good source of dietary fiber, folate, niacin, pantothenic acid, and thiamine. It is also rich in superoxide dismutase (SOD), and studies had shown that cantaloupe SOD extract promotes cellular antioxidant activity, protects against oxidative stress-induced cell death, reduced the diabetes-induced oxidative stress, kidney cell damage and as an anti-inflammatory [4]. Cantaloupe are rich in -carotenes, which lower the risk of cancers of the larynx, oesophagus and lungs without the risk associated with -carotene supplements and also contains the compound adenosine, which is used in patients with heart disease as a blood-thinning agent, and as a relief from angina [5]. ...
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The cantaloupe or sweet melon Cucumis melo Linn seed oil was extracted from powder sample of the seeds using Soxhlet extraction method with n-hexane as the solvent. The powder seeds sample gave 38.33% oil yield with density of 0.94 g/cm 3. The oil was yellow in colour with a fried smell. The chemical analysis of the oil revealed iodine value of 36.42 ± 0.36 Ig/100g, acid value of 0.60 ± 0.30 mg KOH/g and saponification value of 116.88 ± 0.97 mg KOH/g. The GC-MS analysis of the oil revealed six different compounds among which three are free fatty acids palmitic acid, cis-9-cis-12-linoleic acid (grape seed oil) and methyl ester oleic acid and the rest are, two ketonic compounds 3,3-dimethyl-2-hexanone and 2,3-epoxy-2-hexanone and a nitro-alkane 2-nitro hexane. The oil was used to produce a soft texture soap with a milky colour which is slightly soluble in water. The solution of the soap has a foam height of 6 cm and pH value of 8.5 which falls within the range approved by the National Agency for Food and Drug Administration and Control. This oil from cantaloupe seeds shows that it has a promising future not only in cosmetics and pharmaceutical industries but also in food industries.
... Observations from studies have shown that SOD knockout mice accelerate Aβ plaque deposition [126], increase tau phosphorylation [127] and worsen behavioural deficits [128], all suggesting that SOD plays a pivotal role in human ageing and AD. Unfortunately, it has been found that the SOD molecule is deactivated and does not become bioavailable as it passes through the GI tract once it encounters acids and enzymes [129,130]. As a result, scientists have worked around this problem by having SOD coupled with a protective protein derived from wheat, which can then sustain the gastric acids and be delivered in full form and absorbed into the bloodstream, thus effectively enhancing the body's own primary defence system [131,132]. ...
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Oxidative stress, the imbalance of the antioxidant system, results in an accumulation of neurotoxic proteins in Alzheimer's disease (AD). The antioxidant system is composed of exogenous and endogenous antioxidants to maintain homeostasis. Superoxide dismutase (SOD) is an endogenous enzymatic antioxidant that converts superoxide ions to hydrogen peroxide in cells. SOD supplementation in mice prevented cognitive decline in stress-induced cells by reducing lipid peroxidation and maintaining neurogenesis in the hippocampus. Furthermore, SOD decreased expression of BACE1 while reducing plaque burden in the brain. Additionally, Astaxanthin (AST), a potent exogenous carotenoid, scavenges superoxide anion radicals. Mice treated with AST showed slower memory decline and decreased depositions of amyloid-beta (A β ) and tau protein. Currently, the neuroprotective potential of these supplements has only been examined separately in studies. However, a single antioxidant cannot sufficiently resist oxidative damage to the brain, therefore, a combinatory approach is proposed as a relevant therapy for ameliorating pathological changes in AD.
Ascorbic acid (AA) is one of the foremost antioxidants. Unfortunately, its sensitivity to different external stimuli such as light, heat and oxygen are concrete limitations for its use. Various approaches have been investigated in order to circumvent this problem and enhance the stability of the active compound, besides promoting its use for different applications. In this investigation, AA was encapsulated in a vegetal protein-based matrix made up of gliadin, the prolamin obtained from wheat kernels, with the aim of proposing a novel nutraceutical formulation. The nanosystems were characterized by an average diameter of <200 nm and a negative surface charge of ∼-40 mV. The samples were not destabilized after incubation at different temperatures (up to 70°C) or after the pasteurization procedure. Suitable stability was also observed in NaCl 100 mM, as well as after cryodesiccation when 10% w/v of mannose was used. The gliadin nanoparticles showed the ability to retain high amounts of AA, promoting its prolonged release in PBS and under simulated gastrointestinal conditions. The nanosystems enhanced the antioxidant features of the compound as compared to its free form and preserved its chemical stability following UV exposition. The results demonstrate the potential application of the investigated nanoparticles as a novel nutraceutical formulation or as food fortificants.
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Superoxide dismutases (SODs) are metalloenzymes that play a major role in antioxidant defense against oxidative stress in the body. SOD supplementation may therefore trigger the endogenous antioxidant machinery for the neutralization of free-radical excess and be used in a variety of pathological settings. This paper aimed to provide an extensive review of the possible uses of SODs in a range of pathological settings, as well as describe the current pitfalls and the delivery strategies that are in development to solve bioavailability issues. We carried out a PubMed query, using the keywords “SOD”, “SOD mimetics”, “SOD supplementation”, which included papers published in the English language, between 2012 and 2020, on the potential therapeutic applications of SODs, including detoxification strategies. As highlighted in this paper, it can be argued that the generic antioxidant effects of SODs are beneficial under all tested conditions, from ocular and cardiovascular diseases to neurodegenerative disorders and metabolic diseases, including diabetes and its complications and obesity. However, it must be underlined that clinical evidence for its efficacy is limited and consequently, this efficacy is currently far from being demonstrated.
For many years, natural products have been considered as the backbone of medical therapy. Recently, different extracted active moieties of these botanical plants were employed as innovative drug delivery vehicles. Protein-based nanocarriers have acquired great engrossment as colloidal vehicles for delivering different therapeutic agents, anticancer, anti-inflammatory, vitamins, or even biomedical devices. Gliadin is a natural protein, accounts for 80-85% of the total wheat protein, and forms a vital part in the quality and nutritional value of flour. Gliadin is not just a dietary protein, its properties created a novel and surprising applications in medical fields, pharmaceutical, biomedical devices, and drug delivery. Gliadin is safe, biocompatible, and superior over synthetic proteins. The current review highlights the nature, characteristics, quantitation, and extraction of gliadin moreover sheds the light on its different applications. The high bioadhesive ability endorsed its interaction with gastric mucosa facilitating the delivery of various drug molecules, its structure, and the disulfide bonds expanded the encapsulation of lipophilic molecules, vitamins, and enzymes. Add on this its antimicrobial film capability and its flexibility as fibers. Gliadin is applied extensively in food and its safety to the patient with celiac disease, and cytotoxicity to normal cells has not been fully elaborated.
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Abstract: On expose une méthode pour la préparation de capsules molles (GI.C) et de gomme masticable (GC.G) de la proteine hydrophobe du blé, la gliadine brute. Les films de Gliadine se sont révélés être plus hydrophyles que des films comparables de gélatine. La libération de paracétamol a été significativement durable, indiquant la distribution du médicament. Les profiles de libération de médicaments dans un milieu d’acide chlorhydrique à 0,1 M, ont consisté en trois zones pour GI.C: une prériode de latence initiale, suivie par une zone de libération faible et essentiellement stable: et une zone pour GCG avec une libération trés lente de paracetamol. Un mécanisme de libération de médicament est proposé, impliquant une interaction hydrophobe entre la gliadine et des ligands non-polaires. Ces résultats sont discutés et selon cette cette étude, la gliadine apparaît être une proteine trés prometteuse, de bas coût, bioacceptable, pour la réalisation de formulation de médicaments avec un potentiel trés interressant de libération contrôlée. Mots Clés de l’auteur: Gliadine; Capsule; gomme masticable: paracetamol: libération contrôlée. A method for the preparation of soft capsules (Gl.C) and chewable gums (GCG) of the hydrophobic wheat protein, crude gliadin, is reported. Gliadin films were found to be more hydrophilic than comparable gelatin films. The release of paracetamol was significantly sustained, indicating drug delivery. Drug release profiles in 0.1 M hydrochloric acid media consisted of three regions for Gl.C: these were an initial latency period, followed by a low release region and essentially constant rate; and one region for GCG with very slow release of paracetamol. A mechanism of drug release is proposed involving hydrophobic interaction between gliadin and non-polar ligands. These results are discussed and based on this study, gliadin appears to be a highly promising, low-cost, bioacceptable protein for the manufacture of drug formulations with a very interesting controlled released potency.
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The mitochondrion is a key organelle in the control of cell death. Nitric oxide (NO) inhibits complex IV in the respiratory chain and is reported to possess both proapoptotic and antiapoptotic actions. We investigated the effects of continuous inhibition of respiration by NO on mitochondrial energy status and cell viability. Serum-deprived human T cell leukemia (Jurkat) cells were exposed to NO at a concentration that caused continuous and complete (∼85%) inhibition of respiration. Serum deprivation caused progressive loss of mitochondrial membrane potential (Δψm) and apoptotic cell death. In the presence of NO, Δψm was maintained compared to controls, and cells were protected from apoptosis. Similar results were obtained by using staurosporin as the apoptotic stimulus. As exposure of serum-deprived cells to NO progressed (>5 h), however, Δψm fell, correlating with the appearance of early apoptotic features and a decrease in cell viability. Glucose deprivation or iodoacetate treatment of cells in the presence of NO resulted in a collapse of Δψm, demonstrating involvement of glycolytic ATP in its maintenance. Under these conditions cell viability also was decreased. Treatment with oligomycin and/or bongkrekic acid indicated that the maintenance of Δψm during exposure to NO is caused by reversal of the ATP synthase and other electrogenic pumps. Thus, blockade of complex IV by NO initiates a protective action in the mitochondrion to maintain Δψm; this results in prevention of apoptosis. It is likely that during cellular stress involving increased generation of NO this compound will trigger a similar sequence of events, depending on its concentration and duration of release.
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Approximately 200 studies that examined the relationship between fruit and vegetable intake and cancers of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, bladder, pancreas, and ovary are reviewed. A statistically significant protective effect of fruit and vegetable consumption was found in 128 of 156 dietary studies in which results were expressed in terms of relative risk. For most cancer sites, persons with low fruit and vegetable intake (at least the lower one-fourth of the population) experience about twice the risk of cancer compared with those with high intake, even after control for potentially confounding factors. For lung cancer, significant protection was found in 24 of 25 studies after control for smoking in most instances. Fruits, in particular, were significantly protective in cancers of the esophagus, oral cavity, and larynx, for which 28 of 29 studies were significant. Strong evidence of a protective effect of fruit and vegetable consumption was seen in cancers of the pancreas and stomach (26 of 30 studies), as well as in colorectal and bladder cancers (23 of 38 studies). For cancers of the cervix, ovary, and endometrium, a significant protective effect was shown in 11 of 13 studies, and for breast cancer a protective effect was found to be strong and consistent in a meta analysis. It would appear that major public health benefits could be achieved by substantially increasing consumption of these foods.
Nitro blue tetrazolium has been used to intercept O2⁻ generated enzymically or photochemically. The reduction of NBT by O2⁻ has been utilized as the basis of assays for superoxide dismutase, which exposes its presence by inhibiting the reduction of NBT. Superoxide dismutase could thus be assayed either in crude extracts or in purified protein fractions. The assays described are sensitive to ng/ml levels of super-oxide dismutase and were applicable in free solution or on polyacrylamide gels. The staining procedure for localizing superoxide dismutase on polyacrylamide electrophoretograms has been applied to extracts obtained from a variety of sources. E. coli has been found to contain two superoxide dismutases whereas bovine heart, brain, lung, and erthrocytes contain only one.
Oxidative stress at sites of chronic inflammation can cause permanent genetic changes. The development of mutations in the p53 tumor suppressor gene and other key regulatory genes could help convert inflammation into chronic disease in rheumatoid arthritis and other inflammatory disorders.
The oxidation of rat red blood cells (RBC) by molecular oxygen was performed in an aqueous suspension with an azo compound as a free-radical initiator. The RBC were oxidized at a constant rate by a free-radical chain mechanism, resulting in hemolysis. The extent of hemolysis was proportional to the concentration of free radical. α-Tocopherol in RBC membranes suppressed the oxidation and hemolysis to produce an induction period. Tocopherol was constantly consumed during the induction period, and hemolysis developed when tocopherol concentrations fell below a critically low level. Among the membrane lipids, phosphatidylethanolamine, phosphatidylserine, and arachidonic acids were predominantly oxidized in the absence of tocopherol. In the presence of tocopherol, however, such lipid changes were suppressed during a 120-min incubation even when hemolysis started. Membrane proteins as well as lipids were oxidized. The formation of proteins with high molecular weight and concomitant decrease of the lowmolecular-weight proteins were observed on gel electrophoresis with the onset of hemolysis. This study clearly showed the damage of RBC membranes caused by oxygen radical attack from outside of the membranes, and suggested that membrane tocopherol even below a critically low level could suppress lipid oxidation but that it could not prevent protein oxidation and hemolysis.
Nitro blue tetrazolium has been used to intercept O2− generated enzymically or photochemically. The reduction of NBT by O2− has been utilized as the basis of assays for superoxide dismutase, which exposes its presence by inhibiting the reduction of NBT. Superoxide dismutase could thus be assayed either in crude extracts or in purified protein fractions. The assays described are sensitive to ng/ml levels of super-oxide dismutase and were applicable in free solution or on polyacrylamide gels. The staining procedure for localizing superoxide dismutase on polyacrylamide electrophoretograms has been applied to extracts obtained from a variety of sources. E. coli has been found to contain two superoxide dismutases whereas bovine heart, brain, lung, and erthrocytes contain only one.