Spinacia oleracea L. protects against gamma radiations : a study on glutathione and lipid peroxidation in mouse liver

ARTICLE IN PRESS
Phytomedicine 11 (2004) 607–615
www.elsevier.de/phymed
Spinacia oleracea L. protects against gamma radiations:
a study on glutathione and lipid peroxidation in mouse liver
A.L. Bhatia*, M. Jain
Department of Zoology, Radiation Biology Laboratory, University of Rajasthan, Jaipur, India
Received 17 June 2003; accepted 16 July 2003
Abstract
The present study deals with the protective effect of Spinacia oleracea L. against radiation-induced oxidative stress,
which is evaluated in terms of lipid peroxidation (LPO) product and tissue levels of glutathione. Swiss albino male mice
aged 6–8 weeks, weighing 2273 g, each were selected from an inbred colony and divided into four groups. One group
served as normal and a second group (extract of S. oleracea L. (SE) treated un-irradiated) were administered
methanolic (50%) SE at a dose of 1100 mg/kg body wt./day dissolved in distilled water. A third group (untreated-
irradiated) was administered distilled water orally, which served as control. A fourth group (SE pre-treated irradiated)
was administered methanolic (50%) SE at a dose of 1100 mg/kg body wt./day dissolved in distilled water. Two groups,
one untreated-irradiated and another S. oleracea pre-treated irradiated were exposed to 5 Gy of gamma radiation at a
rate of 1.07 Gy/min with a source-to-surface distance of 77.5 cm. The animals were autopsied at 1, 3, 7, 15, and 30 days
post-exposure. LPO increased after irradiation up to day 15 in the untreated-irradiated group and up to day 7 in SE
pre-treated irradiated mice. LPO values were signi?cantly lower in the SE pre-treated irradiated group as compared to
their respective untreated-irradiated group at all intervals, which reached normal values from day 7 onward. The
percentage of protection observed in the SE pre-treated irradiated group was, 22.22%, 24.8%, 33.25%, 42.84% and
26.36% at 1, 3, 7, 15, 30 days post-exposure, respectively. Radiation-induced glutathione depletion was checked after 7
days’ exposure in SE pre-treated irradiated as compared to untreated-irradiated in which recovery started after day 15.
Values were signi?cantly higher in the SE pre-treated irradiated group from their respective untreated-irradiated group
at all intervals. The percentage of protection observed in the SE pre-treated irradiated group was, 29.41%, 42.68%,
43.55%, 53.81%, 39.28% at 1, 3, 7, 15, 30 days post-exposure, respectively. It is found that radiation-induced
augmentation in malondialdehyde contents and depletion in glutathione changes in liver can be altered by S. oleracea
L. The protection may be attributed to the combined effects of its constituents rather than to any single factor as the
leaves are rich in carotenoid content (b-carotene, lutein, Zeaxanthine), ascorbic acid, ?avonoids and p-coumaric acid.
Thus Spinacia, showing protection in liver, may prove promising as a rich source of antioxidants because its use is cost-
effective, especially for peoples in adverse and hazardous circumstances who are living in poverty.
r 2004 Elsevier GmbH. All rights reserved.
Keywords: Spinacia oleracea L.; Radioprotection; Liver; Lipid peroxidation; Glutathione
Introduction
*Corresponding author. 301, C-89, Mangalam Apartments, Jagraj
With extensive use of nuclear power plants, it has
Marg, BapuNagar, Jaipur 302004, India. Tel.: +91-141-2711304; fax:
become
imperative
to
safeguard
human
popula-
+91-141-2701137.
E-mail address: armbha@sancharnet.in (A.L. Bhatia).
tions inhabiting the surrounding areas against high
0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2003.07.004

---

ARTICLE IN PRESS
608
A.L. Bhatia, M. Jain / Phytomedicine 11 (2004) 607–615
background radiation. A need exists for non-toxic and
a molecular mechanism of cell injury leading to the
inexpensive drugs for clinical radiation protection.
generation of peroxides and lipid hydroperoxides, which
Recent studies have indicated that some commonly
can decompose to yield a wide range of cytotoxic
used medicinal plants may be good sources of potent but
products, most of which are aldehydes such as
non-toxic radioprotectors. Antioxidants of plant origin
malondialdehyde (MDA) and 4 hydroxynonenal. The
include vitamin E, C, selenium, phenolic compounds,
stimulation of LPO as a consequence of tissue injury can
carotenoids and ?avonoids (Chandha, 1996). Spinacia
sometimes contribute signi?cantly to a worsening of
oleracea L. (Eng.-Spinach; Hindi, Punjabi-Palak) be-
injury. LPO is a highly destructive process that affects
longs to the family Chenopodiaceae, and is commonly
cellular organelles and causes them to lose biochemical
reported to be a good source of minerals, vitamin B
function and/or structural integrity, which may lead to
complex, ascorbic acid, carotenoids (b-carotene, lutein,
irreparable damage or cell death.
zeaxanthine), ?avonoids, apocyanin and p-coumaric
The present study has therefore been undertaken to
acid (Burton, 1976; Zennie et al., 1977; Gerster, 1993;
determine the possible protective role of S. oleracea L.
Bergan et al., 2001). The entire plant is used as a remedy
against radiation-induced OS, which is evaluated in
for urinary calculi and the leaves are used for bowel and
terms of LPO product and tissue levels of glutathione.
lung in?ammation, febrile af?iction, and cooling (Jain
The present study was also aimed at unraveling the
and De Fillipps, 1991).
mechanism of radiation protection by S. oleracea L.,
Oxidative stress (OS) is a state of imbalance between
with special reference to aforementioned endpoints.
generation of ROS and the level of antioxidant defence
system. OS and free-radical-mediated processes have
been implicated in the pathogenesis of a variety of
Materials and methods
diseases such as atherosclerosis, cancer, liver damage,
rheumatoid arthritis, immunological competence, and
Animals
neurodegenerative disorders. Radiation-induced free
radicals in turn impair the antioxidative defence
Male Swiss albino mice (Mus musculus), 6–8 weeks
mechanism, leading to an increased membrane lipid
old, weighing 2273 g each, from an inbred colony, were
peroxidation (LPO) which results in damage of the
selected and maintained under controlled conditions of
membrane-bound enzymes (Halliwell et al., 1989).
temperature (2572C) and light (light:dark=14:10 h).
Antioxidant enzymes are part of the endogenous system
They were provided standard mice feed (procured from
available for the removal or detoxi?cation of free
Hindustan Lever Ltd., Mumbai) and water ad libitum.
radicals and their products formed by ionizing radiation
Antibiotic (Tetracycline) water was given once a fort-
(Marklund et al., 1989). Nutritional antioxidant de?-
night as prevention against infection. The animals were
ciency also may lead to OS (Gutteridge and Halliwell,
drawn at random for the study. The experimental
1994).
protocols were approved by the Institutional Animal
The liver of mammals has been reported as a highly
Ethics Committee and were conducted according to the
radiosensitive organ. Hepatic injury can be life threaten-
Indian National Science Academy Guidelines for the use
ing when the entirety or most of the liver is exposed to
and care of experimental animals.
ionizing radiation. The liver is the primary organ
responsible for drug metabolism and mainly detoxi?es
damaging electrophiles generated during OS. This
Irradiation
tissue is rich in endogenous antioxidants and related
enzymes (Sipes and Gandolji, 1983). Endogenous
The Cobalt Teletherapy Unit (ATC-C9) at Cancer
cellular thiol-dependent enzymes play an important role
Treatment Centre, Radiotherapy Department, S.M.S.
in radiation response. Many of the thiol-dependent
Medical College and Hospital, Jaipur, was used for
enzymes rely on reduced Glutathione (GSH). Hence,
irradiation. Unanaesthetized animals were restrained in
post-irradiation damage to normal tissue might be
well-ventilated boxes and exposed whole-body to
prevented by the administration of GSH and endogen-
gamma radiation (5 Gy), at a dose of 1.071 Gy/min
ous thiols, which may involve induction or activation of
and a source-to-surface distance (SSD) of 77.5 cm.
antioxidant enzymes (Bump and Brown, 1990). Biolo-
gical compounds containing thiol groups are essential
Chemicals
for the maintenance of cellular structure and function.
GSH is the most studied and abundant endocellular
Thiobarbituric acid, trichloroacetic acid, and acetic
aminothiol, and plays a key role in physiological
acid were purchased from Central Drug House (Pvt.)
detoxi?cation.
Ltd. Glutathione (reduced form), 1, 1, 3, 3-tetra
LPO is oxidative deterioration of polyunsaturated
methoxy propane, and DTNB (5,5 Dithio-bis 2-Nitro-
lipids and it involves ROS and transition metal ions. It is
benzoic acid) were of analytical grade and were

---

ARTICLE IN PRESS
A.L. Bhatia, M. Jain / Phytomedicine 11 (2004) 607–615
609
procured from Siga Chemicals Co. (St.Louis, MO,
tathione (reduced) was determined according to the
USA).
method described for Ellman’s method modi?ed by
Moron et al. (1979).
Extract preparation
The spinach plant species was identi?ed and placed in
Statistical analysis
Department of Botany Herbarium, University of
Rajasthan, Jaipur, for future reference (Voucher no.
The results obtained in the present study were
RUBL-19867). Fresh S. oleracea L. leaves (Hindi:
expressed as the mean7SD. One-way ANOVA was
Palak; English: Spinach; Family: Chenopodiaceae) were
used for statistical comparison between the studied
collected locally. These were air-dried, powdered, and
groups and the signi?cance was observed at Po0:1;
extracted with 50% methanol by re?uxing for 48 h
Po0:05; Po0:01 and Po0:001 levels, calculated using a
(16 h  3). About 1 kg Spinach (wet) yielded 200 g
statistical software program. Linear trend regression
powdered form when dried and 100 g extracted with
analysis was also conducted.
50% methanol yielded 25 g crude extract. The extract
obtained was vacuum evaporated to produce a pow-
dered form and was dissolved in double-distilled water
Results
(DDW) with the help of a Cyclomixer just before oral
administration. Fixed weight of the extract to volume of
Determination of optimum dose
the solvent after complete dissolution of the extract was
used for administration.
1100 mg/kg body wt./day was determined as the
optimum dose. All irradiated animals without SE
Determination of optimum dose of S: oleracea
treatment showed 100% mortality within 11 days.
extract (SE) against radiation
Maximum survival of 80% (after 30 days) was achieved
by administration of 1100 mg/kg body wt./day. Treated
Mice were divided into 5 groups of 15 animals each
mice showed an improvement body weight gain as
and were administered SE orally (200, 500, 800, 1100,
compared to the irradiated animals.
1400 mg/kg body wt./day) for 15 days. One hour after
the last administration, animals were exposed to whole
Glutathione
body 9.0 Gy gamma radiation. All treated animals were
observed for 30 days for any signs of radiation sickness,
Because there was an insigni?cant variation in
mortality, behavioral toxicity, or morbidity. The opti-
glutathione values from 1 to 30 days post-treatment in
mum dose (1100 mg) obtained was used for the
S. oleracea-treated un-irradiated and normal mice, a
experimental design.
single value was taken for statistical comparison. We
found a signi?cant difference between normal mice and
Experimental design
Spinacia-treated
un-irradiated
[F ð1; 10Þ ¼ 7:112041;
Po0:05]. Glutathione (GSH) decreased after irradiation
Mice selected from an inbred colony were divided into
up to day 15 in untreated-irradiated and it remained
four groups of 30 animals each. The ?rst group did not
lowered (39.46%) as compared to normal at the last
receive any treatment and served as a normal group. The
autopsy interval (Table 1 and Fig. 1). There was a
second group was fed with Spinacia extract (1100-mg/kg
signi?cant difference between normal and untreated-
body wt./day) for 15 days. This group served for the
irradiated
group
at
day
1
[F ð1; 10Þ ¼ 5440:299;
determination of drug tolerance. The third group of
Po0:001], day 3 [Fð1; 10Þ ¼ 16956:75; Po0:001], day 7
animals was fed orally 1100-mg/kg body wt./day SE in
[F ð1; 10Þ ¼ 9722:555; Po0:001], day 15 [F ð1; 10Þ ¼
DDW for 15 days to serve as an experimental group.
15712:87;
Po0:001], day 30 [F ð1; 10Þ ¼ 10203:76;
Mice in the fourth group received an equal volume of
Po0:001]. In S. oleracea pre-treated irradiated groups,
DDW and served as the control group. One hour after
it decreased up to day 7 and afterwards it tended to
the ?nal administration, animals of both groups III and
normalize from day 7 onwards until it reached normal
IV were whole-body exposed to 5 Gy of gamma
values at the last autopsy interval. Statistical difference
radiation at a dose rate of 1.071 Gy/min. The animals
(ANOVA) between normal and Spinacia treated irra-
were autopsied at 1, 3, 7, 15 and 30 days post-
diated at day 1 [F ð1; 10Þ ¼ 346:6902; Po0:001], day 3
irradiation. At least six animals were autopsied at each
[F ð1; 10Þ ¼ 365:8228;
Po0:001], day 7 [F ð1; 10Þ ¼
interval. The liver was removed for biochemical estima-
1557:95;
Po0:001],
day
15
[F ð1; 10Þ ¼ 394:4643;
tion of LPO by the method of Okhawa et al. (1979)
Po0:001], day 30 [Fð1; 10Þ ¼ 0:269454; non-signi?cant]
using tetramethoxy propane as the standard. Glu-
was
recorded.
The
statistical
difference
between

---

610
Table 1. Variations in biochemical parameters in the livers of Swiss albino mice after 5 Gy gamma irradiation in the presence or absence of Spinacia oleracea L. extract.
Biochemical
Group
Days post irradiation
Normal
Spinacia treated
parameter’s
un-irradiated
1
3
7
15
30
Lipid-
Untreated
395.2178.53
420.3178.45
455.2178.82
470.1977.86
400.1879.33
315.25713.3
292.9579.04
A.L.
peroxidation
irradiated
(125.36%), #d
(133.32%), #d
(144.40%), #d
(149.15%), #d
(126.94%), #d
(100%)
(92.93%)c
(TBARS) (n mol/
Bhatia,
gm tissue)
Spinacia pre-
325.1476.88
342.1577.40
350.4177.13
335.1675.95
317.14 77.64
AR
M.
treated irradiated
(103.14%) Ãd,
(108.53%), Ãd,
(111.15%), Ãd,
(106.31%), Ãd,
(100.58%)ÃNS,
Jain
TI
@d
@d
@d
@d
@d
CL
% Protection
22.22%
24.8%
33.25%
42.84%
26.36%
/
Phytom
E
Glutathione
Untreated
4.9770.07
3.8370.05
3.1970.098
3.0770.074
4.6570.05
7.6770.05
7.7470.05
I
N
(n mol/gm tissue)
irradiated
(64.80%), #d
(49.84%), #d
(41.54%), #d
(40.01%), #d
(60.54%), #d
(100%)
(100.86%)b
edicine
P
Spinacia pre-
7.2370.024
7.1070.05
6.5370.05
7.2070.024
7.6670.024
RE
treated irradiated
(94.21%), Ãd,
(92.52%), Ãd,
(85.09%), Ãd
(93.82%), Ãd
(99.817) ÃNS,
11
@d
@d
@d
@d
@d
S
(2004)
S
% Protection
29.41%
42.68%
43.55%
53.81%
39.28%
607
Statistical comparison
Significance level
–
ANOVA
Po0:1 Po0:05 Po0:01 Po0:001
615
Normal vs: Spinacia treated un-irradiated
a
b
c
d
Normal vs: untreated irradiated
a
b
c
d
Normal vs: Spinacia treated irradiated
Ãa
Ãb
Ãc
Ãd
Untreated irradiated vs: Spinacia treated irradiated
@a
@b
@c
@d
NS ¼ non-significant
Each value represent Mean7SD of 6 animals
Values in parentheses indicates %; calculated by treating normal as 100%
Value of normal and Spinacia treated un-irradiated are mean values of all autopsy intervals due to insigni?cant variation at various autopsy interval.

---

ARTICLE IN PRESS
A.L. Bhatia, M. Jain / Phytomedicine 11 (2004) 607–615
611
Variation in Glutathione content in mice liver in various groups
Variation in MDA Content in various groups
9
490
470
y = 0.0215x + 6.8601
8
Normal
Untreated
R2 = 0.3928
450
Irradiated
430
7
Spinacia-treated
y = -0.1427x + 430.1
410
irradiated
R2 = 0.0026
Spinacia treated
390
6
un-irradiated
370
5
350
y = 0.0064x + 3.8569
R2 = 0.008
330
4
y = -0.6429x + 342.49
310
Normal
Spinacia-treated
R2 = 0.3288
290
irradiated
Spinacia treated
3
Untreated
Irradiated
270
un-irradiated
lue (mean ± SD)of LPO in n mol/gm tissue
a 250
lue (mean±SD) of glutathione n mol/gm tissue 2
V
a
0
1
3
7
15
30
V
0
1
3
7
15
30
Days
Days
Fig. 2. Variations in total MDA contents (n mol/g) in liver of
Fig. 1. Variations in Glutathione content (n mol/g) in liver of
Swiss albino mice at various post-irradiation days, in the
Swiss albino mice at various post-irradiation days, in the
presence (experimental) or absence (control) of SE.
presence (experimental) or absence (control) of SE.
was recorded. Statistical difference between untreated-
untreated-irradiated and Spinacia-treated irradiated at
irradiated and Spinacia-treated irradiated at day 1
day 1 [F ð1; 10Þ ¼ 5155:969; Po0:01], day 3 [Fð1; 10Þ ¼
[F ð1; 10Þ ¼ 245:1013;
Po0:001], day 3 [F ð1; 10Þ ¼
1274:88; Po0:01], day 7 [F ð1; 10Þ ¼ 5649:438; Po0:01],
294:4357;
Po0:001],
day
7
[F ð1; 10Þ ¼ 508:6319;
day 15 [F ð1; 10Þ ¼ 17056:9; Po0:01], day 30 [Fð1; 10Þ ¼
Po0:001], day 15 [F ð1; 10Þ ¼ 1129:165; Po0:001], day
16813:46; Po0:01] was observed. Percentage protection
30 [F ð1; 10Þ ¼ 289:4399; Po0:001] was observed.
observed in S. oleracea pre-treated irradiated groups
Percentage protection observed in S. oleracea pre-
was, 29.41%, 42.68%, 43.55%, 53.81% and 39.28% at
treated irradiated groups was 22.22%, 24.8%, 33.25%,
1, 3, 7, 15 and 30 days post exposure, respectively, as
42.84% and 26.36% at 1, 3, 7, 15 and 30 days post-
compared to untreated-irradiated.
exposure, respectively, as compared with untreated-
irradiated.
LPO
LPO increases after irradiation up to day 15 in
Discussion
irradiated and up to day 7 in S. oleracea pre-treated
irradiated groups (Table 1 and Fig. 2). Thereafter,
Statistically, a signi?cant difference between irra-
decrease in LPO values was observed from day 15
diated mice pre-treated with S. oleracea and those
onwards in untreated-irradiated and from day 7 in S.
without such pre-treatment, at the entire interval, shows
oleracea pre-treated irradiated mice. LPO values were
clearly that SE supplementation prevented radiation-
signi?cantly lower in S. oleracea pre-treated irradiated
induced LPO. A statistically signi?cant difference was
groups from their respective untreated-irradiated at all
also noted between S. oleracea-treated un-irradiated and
intervals, which compensated to day 30. In untreated-
normal mice. The basic effect of radiation on the cellular
irradiated, the values were higher (26.94%) than
membrane is believed to be peroxidation of membrane
normal.
lipids. The depletion of the total amount of glutathione
There was a signi?cant difference between normal
at early intervals in treated animals may be due to their
mice and Spinacia-treated un-irradiated [F ð1; 10Þ ¼
utilization in large amount to combat the acute
11:6394; Po0:001]. There was a signi?cant difference
radiation-induced, free-radical damage, as glutathione
between normal and untreated-irradiated at day 1
is major non-enzymatic antioxidant. Depletion of GSH
[F ð1; 10Þ ¼ 154:1571;
Po0:001], day 3 [Fð1; 10Þ ¼
was higher in irradiated animals as compared to spinach
268:8299; Po0:001], day 7 [F ð1; 10Þ ¼ 461:8746; Po
pre-treated irradiated animals as the animals had a high
0:001], day 15 [F ð1; 10Þ ¼ 607:3502; Po0:001], day 30
level of phytoantioxidants after 15 days of chronic
[F ð1; 10Þ ¼ 165:0518;
Po0:001]. Statistical analysis
feeding, hence less utilization of endogenous glu-
(ANOVA) between normal and Spinacia treated irra-
tathione. Afterwards, it tended to be utilized less due
diated at day 1 [F ð1; 10Þ ¼ 2:590996; non-signi?cant],
to the declining impact of radiation and endogenous
day 3 [F ð1; 10Þ ¼ 18:77692; Po0:001], day 7 [Fð1; 10Þ ¼
reparative homeostatic activity. The measurement of
32:75404;
Po0:001], day 15 [Fð1; 10Þ ¼ 11:36509;
LPO is a convenient method to monitor oxidative cell
Po0:001], day 30 [F ð1; 10Þ ¼ 0:091272; non-signi?cant]
damage. Reactive oxygen species (ROS) causes LPO,

---

ARTICLE IN PRESS
612
A.L. Bhatia, M. Jain / Phytomedicine 11 (2004) 607–615
which within the membrane has a devastating effect on
between dietary carotenoids and a reduced incidence of
the functional state. The preservation of cellular
certain disease, including some cancers (van Poppel and
membrane integrity depends on protection or repair
Goldbohm, 1995).
mechanisms capable of neutralizing oxidative reactions.
Zeaxanthine is the carotenoid that protects the
Inhibition of LPO in biomembranes has been caused by
photosynthetic apparatus when photon ?ux density is
antioxidants present in S. oleracea. The above results
high. Similarly Zeaxanthin is only as effective as b-
showed that the SE renders protection against radiation-
carotene in inhibiting autooxidation of lipids in solu-
induced OS.
tion, but is about 50% more effective in retarding
Under normal conditions, the inherent defense
hydroperoxide formation in phosphatidylcholine lipo-
system, including glutathione and the antioxidant
somes (Lim et al., 1992). Some of the greater antioxidant
enzymes, protects against oxidative damage. GSH offers
effectiveness of Zeaxanthin may be due to its relative
protection against oxygen-derived free radicals and
resistance to destruction by oxidative process (Seely and
cellular lethality following exposure to ionizing radia-
Meyer, 1971), but its ability to integrate into membranes
tion (Biaglow et al., 1987). The present study denotes a
appears to be another key factor. Zeaxanthin and Lutein
signi?cant reduction in liver GSH due to radiation. This
are structural isomers, differing only in the placement of
could be due to an enhanced utilization of the
one double bond. People who ate Lutein-rich foods had
antioxidant system during detoxi?cation of the free
been claimed to have a lower risk of developing colon
radicals generated by radiation. Depletion of glu-
cancer. Diets high in Zeaxanthin have also been linked
tathione results in enhanced LPO (Anderstam et al.,
to a lower risk of esophageal cancer. Lutein is effective
1992). Excessive LPO can also cause increased glu-
at inhibiting autooxidation of cellular lipids (Zhang
tathione consumption (Comporti, 1987). Oral adminis-
et al., 1991). Vitamin C or l-ascorbic acid is considered
tration of SE to Swiss albino mice did not signi?cantly
to be the most important antioxidant in extracellular
in?uence endogenous GSH levels in the liver. Further, it
?uids (Stocker and Frei, 1991) and has many cellular
also protected against radiation exposure GSH deple-
activities of an antioxidant nature as well (Moser and
tion. Lower depletion of liver GSH levels in S. oleracea-
Bendich, 1991). Ascorbic acid can protect biomem-
treated irradiated animals might have been due to higher
branes against peroxidation damage. Ascorbic acid also
availability of GSH, which enhanced the capability of
acts to protect membranes against peroxidation by
cells to cope with the free radicals generated by
enhancing the activity of a-tocopherol, the chief lipid-
radiation. GSH is a versatile protector and executes its
soluble and chain-breaking antioxidant. Therefore,
radioprotective function through free-radical scaven-
protection by SE may be attributed to the combined
ging, restoration of the damaged molecules by hydrogen
synergistic effects of its constituents rather than to any
donation, reduction of peroxides, and maintenance of
single factor.
protein thiols in the reduced state (Bump and Brown,
1990).
Natural antioxidant system (NAO) of spinach
Mechanism of action by constituents of spinach
Spinach contains a very effective NAO capable of
preventing LPO in both plant and animal systems
The protection of endogenous antioxidant defense
(Grossman et al., 1994). The potential physiological role
systems of GSH after the supplementation of S. oleracea
of NAO as an antioxidant was shown in several in vitro
appears to be offered by its constituents, including b-
and in vivo systems (Grossman et al., 1994; Zurovsky
carotene, lutein, Zeaxanthine, ?avonoids, vitamin C and
et al., 1994; Lomnitski et al., 2000). Its ef?cacy in
p-coumaric acid. Antioxidant, free-radical scavenging
preventing lipid-peroxidation in the skin of mice, rats
compounds such as b-carotene and vitamin C can
and human has been reported (Grossman et al., 1994).
protect DNA from oxidizing radical reactions. b-
Recently, the ef?cacy of NAO in preventing LPS-
carotene is a potent free-radical quencher, singlet
induced hepatic injury in both rats and rabbits was
oxygen scavenger, and lipid antioxidant (Blot et al.,
demonstrated (Ben-Shaul et al., 1999; Lomnitski et al.,
1995). The level of these constituents has increased in
2000). Aritomi and co-workers (Aritomi and Kawasaki,
blood as reported for b-carotene supplemented meals
1984; Aritomi et al., 1986) reported the presence of
which increased plasma concentration of b-carotene
seven ?avonol glycosides in the methanolic extract of
effectively (van het Hof et al., 1999). b-carotene has
Spinach leaves. Recently, Ferreres et al. (1997) isolated
already been reported to quench not only singlet
and identi?ed ?ve novel, naturally occurring ?avonoids
oxygen, but also to scavenge a variety of free-radical
(Acylated ?avonol glycosides) from alcoholic extracts of
species. b-carotene renders protection against radiation-
Spinach leaves.
induced lipid-peroxidation (Bhatia and Manda, 2000).
Flavonoids are known to display a broad array of
Epidemiological studies have also demonstrated a link
pharmacological and biochemical actions (Middleton

---

ARTICLE IN PRESS
A.L. Bhatia, M. Jain / Phytomedicine 11 (2004) 607–615
613
and Kandaswami, 1992). The ?avonoids are typical
frying oil. In the same manner, the effect of spinach
phenolic compounds and powerful chain-breaking anti-
products on lipid oxidation is affected by processing
oxidants (Torel et al., 1986). It was reported that LPO
(Castenmiller et al., 2002). The consumption of carote-
can be inhibited by ?avonoids, possibly through their
noid-rich foods like spinach, even for a short period of
activity as strong OÀ scavengers (Baumann et al., 1980)
time, gives protection against OS. The results obtained
2
and singlet oxygen quenchers (Sorata et al., 1984). p-
seem to suggest that this protective role is not related
Coumaric acid derivatives are very strong antioxidants.
speci?cally to carotenoids. They may, however, con-
The ability of some phenolic compounds to act as
tribute with other substances present in vegetables to
antioxidants has been shown (Velioglu et al., 1998).
lymphocyte resistance to oxidative damage.
Flavonoids are reported to possess antitumor promoter
(Fuziki et al., 1986), antimetastatic (Bracke et al., 1988)
and antiproliferative (Kandaswami et al., 1991) proper-
Conclusion
ties. Shimoi et al. (1994) demonstrated that some plant
?avonoids protect against radiation-induced micronu-
The lower level of lipid peroxidation products and
cleus formation. Several naturally occurring ?avonoids
lower depletion of Glutathione in irradiated mice after
have been reported to have antioxidant properties which
supplementation of S. oleracea L. for 15 days evidently
scavenge free radicals (Husain et al., 1987; Robak and
showed the check of radiation-induced damage in mice
Gryglewski, 1988; Hu et al., 1995). Shimoi et al. (1996)
liver. The ?ndings with S. oleracea L. as weed and
attributed the radioprotective effect of antioxidant plant
inexpensive herb are signi?cant as the preparation is
?avonoids to their ability to scavenge free radicals.
highly cost-effective and could be given as adjuvant
during radiation treatment as well as in conditions such
Role of micronutrients
as accidental exposure to radiation.
As Spinacia leaves are used as a dietary vegetable and
Copper, manganese and zinc contents are present in
are freely available all over the world, it is worthwhile to
maximum amounts in Spinach (Singh et al., 2001).
conduct detailed studies in order to explore the full
Adequate zinc supplementation inhibits LPO and has
potential of this plant in human radiation protection,
been described as an antioxidant. Increased availability
from the point of view of cost and availability for people
of Zn, Cu, and Mn results in increased/proper activity of
at all socioeconomic levels.
Cu Zn SOD and Mn/Fe SOD.
Miscellaneous factors associated with spinach
Acknowledgements
constituents
A Junior Research Fellowship (JRF) to Manish Jain
from Special Assistance Programme (SAP) of University
Chu et al. (2002) reported that broccoli and spinach
Grant Commission, Department of Zoology, University
possessed the highest total phenolic content among
of Rajasthan, Jaipur is gratefully acknowledged. The
vegetables tested. Antiproliferative activities were also
authors are also thankful to Prof. D.P. Agarwal, Dr.
studied in vitro using HepG(2) human liver cancer cells.
K.S. Jheeta (RSO) and Dr. A.A. Chougle, Department
Spinach showed the highest inhibitory effect, followed
of Radiotherapy, SMS Medical College and Hospital,
by cabbage, red pepper, onion, and broccoli. Kayashima
Jaipur for providing the radiation facility and dosime-
and Katayama (2002) reported that oxalic acid present
try, respectively. The authors also acknowledge the keen
in spinach suppressed in vitro LPO in a concentration-
interest shown by their colleague Dr. M.R. Saini.
dependent manner. Howard et al. (2002) reported that
growing conditions, as well as biotic and abiotic stresses,
in?uenced phenolic metabolism because over-winter
spinach, which was planted in late fall and harvested
References
in the spring, had much higher levels of total phenolics
and antioxidant capacity than spinach planted in early
Anderstam, B., Vacca, C., Ringdahl, M.H., 1992. Lipid
fall and harvested in late fall. Lee et al. (2002) reported
peroxide level in a murine adenocarcinoma exposed to
hyperthermia: the role of glutathione depletion. Radiat.
that spinach in dough decreased accumulation of the
Res. 132, 296–300.
polar compounds in soybean oil during frying, but had
Aritomi, M., Kawasaki, T., 1984. Three highly oxygenated
little effect on fried dough. It also reduced conjugated
?avone glucuronoides in leaves of Spinacia oleracea.
diene and aldehyde formation in the lipid of fried dough
Phytochemistry 23, 2043–2047.
during storage. Improvement in lipid oxidative stability,
Aritomi, M., Kokori, T., Kawaski, T., 1986. Flavonol glyco-
presumably due to pigments in spinach, was more
sides in leaves of Spinacia oleracea. Phytochemistry 25,
noticeable in fried products during storage than in
231–234.

---

ARTICLE IN PRESS
614
A.L. Bhatia, M. Jain / Phytomedicine 11 (2004) 607–615
Baumann, J., Wurm, J., von Bruchhausen, F., 1980. Prosta-
Asada, K., Toshikawa, T. (Eds.), Frontiers of Reactive
glandin synthetase inhibition by ?avonoids and phenolic
Oxygen Species in Biology and Medicine. Elsevier Science,
compounds in relation to their OÀ
2
scavenging properties.
Amsterdam, pp. 343–344.
Arch. Pharm. (Weinheim) 313, 330–337.
Gutteridge, J.M.C., Halliwell, B., 1994. Antioxidants in
Ben-Shaul, V., Sofer, Y., Bergan, M., Zurovsky, Y., Gross-
Nutrition Health and Disease. Oxford University Press,
man, S., 1999. Lipopolysaccharide-induced oxidative stress
Oxford.
in the liver: comparison between rat and rabbit. Shock 12,
Halliwell, B., Gutteridge, J.M.C., 1989. Production of hydro-
288–293.
xyl radicals in living systems In: Free Radicals Biology and
Bergan, M., Varshavsky, L., Gottlieb, H.E., Grossman, S.,
Medicine. Clarendon Press, Oxford, p. 31.
2001. The antioxidant activity of aqueous spinach extract:
Howard, L.R., Pandjaitan, N., Morelock, T., Gil, M.I., 2002.
chemical identi?cation of active fractions. Phytochemistry
Antioxidant capacity and phenolic content of spinach as
58, 143–152.
affected by genetics and growing season. J. Agric. Food
Bhatia, A.L., Manda, K., 2000. Role of b-carotene against
Chem. 50 (21), 5891–5896.
radiation induced lipid-peroxidation in mice testes. Res. J.
Hu, J.D., Calomine, M., Lasure, A., Debruyne, T., Pieters, A.,
Chem. Environ. 4 (1), 59–61.
Vlientinck, A., Vanden Berghe, D.A., 1995. Structure–
Biaglow, J.E., Varnes, M.E., Epp, E.R., Clark, E.P., 1987. In:
activity relationship of ?avonoids with superoxide scaven-
Cerrutti, P.A., Nygaard, O.F., Simic, M.G. (Eds.), Anti-
ging activity. Biol. Trace Element Res. 47, 327–331.
carcinogenesis and Radiation Protection. Plenum Press,
Husain, S.R., Cillarat, J., Cillard, P., 1987. Hydroxyl radical
New York, pp. 387.
scavenging activity of ?avonoids. Phytochemistry 26,
Blot, W.J., Li, J.Y., Taylor, P.R., Guo, W., Dawsey, S.M., Li,
2489–2492.
B., 1995. The initial trials: mortality rates by vitamin-mineral
Jain, S.K., De Fillipps, R.A., 1991. Medicinal Plants of India.
intervention group. Am. J. Clin. Nutr. 62 (142), 45–65.
Reference Publication, Algoriac, MI.
Bracke, M.L., Depestel, G., Castronovo, V., Vijneke, B.,
Kandaswami, C., Perkins, E., Solonink, D.S., Drzewiecki,
Foidart, J.M., Vakaet, L.C.A., Marcel, M.M., 1988.
Middleton Jr., E., 1991. Antiproliferative effects of citrus
Flavonoids inhibit malignant tumors invasion in vitro. In:
?avonoids on a human squamous cell carcinoma in vitro.
Middleton, C.E., Harborne, J.B., Beretz, A. (Eds.),
Cancer Lett. 56, 147–152.
Plant ?avonoids in Biology and Medicine II Biochemical
Kayashima, T., Katayama, T., 2002. Oxalic acid is available as
cellular and Medicinal properties. Alan R Liss, New York,
a natural antioxidant in some systems. Biochim. Biophys.
pp. 219–233.
Acta 1573 (1), 1–3.
Bump, E.A., Brown, J.M., 1990. Role of glutathione in the
Lee, J., Lee, S., Lee, H., Park, K., Choe, E., 2002. Spinach
radiation response of mammalian cells in vitro and in vivo.
(Spinacia oleracea) powder as a natural food-grade anti-
Pharmac. Ther. 47, 117.
oxidant in deep-fat-fried products. J. Agric. Food Chem. 50
Burton, T.B., 1976. Human nutrition. In: Lapedes, D.N. (Ed.),
(20), 5664–5669.
Encyclopaedia of Food Agriculture and Nutrition, 4th
Lim, B.P., Nagaro, A., Terao, J., Tanaka, K., Suzuki, T.,
Edition. H J Heinz/McGraw-Hill company, New York.
Takama, K., 1992. Antioxidant activity of xanthophylls in
Castenmiller, J.J., Linssen, J.P., Heinonen, I.M., HHHopia,
peroxyl radical-mediated phospholipid peroxidation. Bio-
A.I., Schwarz, K., Hollmann, P.C., West, C.E., 2002.
chim. Biophys. Acta 1126, 178–184.
Antioxidant properties of differently processed spinach
Lomnitski, L., Nyaska, A., Ben-Shaul, V., Maronport, R.R.,
products. Nahrung 46 (4), 290–293.
Haseman, J.K., Harrus, T.L., Bergan, M., Grossman, S.,
Chandha, S.L., 1996. Natural sources of antioxidants and their
2000. Effects of antioxidants apocyanin and the natural
adequacy in diet to prevent atherosclerosis. Mediquest 143,
water-soluble antioxidant from spinach on cellular damage
337–351.
induced by lipopolysaccharide in the rat. Toxicol. Pathol.
Chu, Y.F., Sun, J., Wu, X., Liu, R.H., 2002. Antioxidant and
287, 580–587.
antiproliferative activities of common vegetables. J. Agric.
Marklund, S.L., Westman, N.G., Roos, G., Carlesson, J.,
Food. Chem. 50 (23), 6910–6916.
1989. Radiation resistance and the Cu Zn Superoxide
Comporti, M., 1987. Glutathione depleting agents and lipid
dismutases, Mn superoxide dismutase Catalase and Glu-
peroxidation. Chem. Phys. Lipids 45, 143–169.
tathione peroxidase activities of seven human cell lines.
Ferreres, F., Castaner, M., Tomas–Barbern, A., 1997.
Radiat. Res. 100 (1), 115.
Acylated ?avonol glycosides from spinach leaves (Spinacia
Middleton Jr, E., Kandaswami, C., 1992. Effects of ?avonoids
oleracea). Phytochemistry 45, 1701–1705.
on immune and in?ammatory cell functions. Biochem.
Fuziki, H., Horiuchi, T., Yamashita, K., Hakii, H., Iwashima,
Pharmacol. 43, 1167–1179.
A., Hirata, Y., Sugimura, T., 1986. Inhibition of tumour
Moron, M.S., Depierse, J.W., Manrerirk, B., 1979. Levels of
promotion by ?avonoids. In: Middleton, C.E., Harborne,
GSH GR and GST activities in rat lung and liver. Bio.
J.B., Beretz, A. (Eds.), Plant Flavonoids in Biology and
Chem. Biophys. Acta 582, 67.
Medicine II Biochemical Cellular and Medicinal Properties.
Moser, U., Bendich, A., 1991. Vitamin C. In: Machlin, L.J.
Alan R Liss, New York, pp. 429–440.
(Ed.), Handbook of Vitamins, 2nd Edition. Marcel Dekker,
Gerster, H., 1993. Anticarcinogenic effect of common car-
New York, pp. 195–232.
otenoids. Int. J. VII Nutr. Res. 63, 93–121.
Okhawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid
Grossman, S., Reznik, R., Tamari, T., Albeck, M., 1994. New
peroxides in animal tissue by thiobarbituric acid reaction.
plant water-soluble antioxidant (NAO) from Spinach. In:
Anal. Biochem. 95, 351–358.

---

ARTICLE IN PRESS
A.L. Bhatia, M. Jain / Phytomedicine 11 (2004) 607–615
615
Robak, J., Gryglewski, R.J., 1988. Flavonoids are scavengers
Torel, J., Cillard, J., Cillard, P., 1986. Antioxidant activity of
of superoxide anion. Biochem. Pharmacol. 37, 837–841.
?avonoids and reactivity with peroxl radical. Phytochem-
Seely, G.R., Meyer, T.H., 1971. The photosensitised oxidation
istry 25, 383–386.
of b-carotene. Photochem. Photobiol. 13, 27–32.
Van het Hof, K.H., Tijburg, L.B., Pietrzek, K., Weststrate, J.A.,
Shimoi, K., Mishida, S., Furogori, M., Osaki, S., Kinae, N.,
1999. In?uence of feeding different vegetables on plasma
1994. Radioprotective effects of antioxidative ?avonoids in
level of carotenoids folate and vitamin C. Effect of disruption
g-ray irradiated mice. Carcinogenesis 15, 2669–2672.
of the vegetable matrix. Br. J. Nutr. 82 (3), 203–212.
Shimoi, K., Masuda, S., Shen, B., Furugori, M., Kinae, N.,
Van Poppel, G., Goldbohm, R.A., 1995. Epidemiologic
1996. Radioprotective effect of antioxidant plant ?avonoids
evidence for b-carotene and cancer prevention. Am. J.
in mice. Mutat. Res. 35, 153–161.
Clin. Nutr. 62, 1393–1402.
Singh, G., Kawatra, A., Sehgal, S., 2001. Nutritional
Velioglu, Y.S., Mazza, G., Gao, Y.L., Oomah, B.D., 1998.
composition of selected green leafy vegetables herbs and
Antioxidant activity and total phenolics in selected fruits
carrots. Plant Foods Hum. Nutr. 56 (4), 359–364.
vegetables and grain products. J. Agric. Food Chem. 46,
Sipes, G.I., Gandolji, A.J., 1983. Biotransformation of
4113–4117.
toxicants. In: Klaassen, C.D., Amdur, M.O., Doull, J.
Zennie, M., Thomas, D., Ogzewalk, C., 1977. Ascorbic acid
(Eds.), Toxicology: The Science of Poisons. Macmillian,
and vitamin A content of edible wild plants of Ohio and
New York.
Kentucky. J. Econ. Bot. 31, 76–79.
Sorata, Y., Takahama, U., Kimura, M., 1984. Protective effect
Zhang, L.-X., Cooney, R.V., Bertram, J.S., 1991. Carotenoids
of quercetin and rutin on photosensitised lysis of human
entrance gap junctional communication and inhibit lipid-
erythocytes in the presence of hematoporphyrin. Biochim.
peroxidation
in
C3H/10
T1/2
cells:
relationship
to
Biophys. Acta 799, 313–317.
their cancer chemopreventive action. Carcinogenesis 2,
Stocker, R., Frei, B., 1991. Endogenous antioxidant defence in
2109–2114.
human blood plasma. In: Sies, H. (Ed.), Oxidative stress:
Zurovsky, Y., Eligal, Z., Grossman, S., Bergan, M., Gafter,
Oxidants and Antioxidants. Academic Press, London,
U., 1994. Glycerol-induced augmentation of sensitivity to
pp. 213–243.
endotoxin in rats. Toxicon 32, 17–26.

---

Document Outline

• Spinacia oleracea L. protects against gamma radiations: a study on glutathione and lipid peroxidation in mouse liver
  • Introduction
  • Materials and methods
    • Animals
    • Irradiation
    • Chemicals
    • Extract preparation
    • Determination of optimum dose of S.oleracea extract (SE) against radiation
    • Experimental design
  • Statistical analysis
  • Results
    • Determination of optimum dose
    • Glutathione
    • LPO
  • Discussion
    • Mechanism of action by constituents of spinach
    • Natural antioxidant system (NAO) of spinach
    • Role of micronutrients
    • Miscellaneous factors associated with spinach constituents
  • Conclusion
  • Acknowledgements
  • References

---