Oxidative initiated by an unbalanced production of reactive oxygen

Oxidative
stress plays an important role in the pathogenesis of various diseases such as
atherosclerosis, alcoholic liver cirrhosis and cancer (1). Oxidative stress can be initiated by an unbalanced production of
reactive oxygen species (ROS), such as super-oxide anion (O-2),
peroxide radical (HOO·) and
hydroxyl radical (HO·). These radicals are formed by one electron
reduction of molecular oxygen (O2). Of pathological significance,
ROS can easily initiate a self-propagating lipid peroxidation (LP) of the
membrane lipids, which can cause permanent damage to the cell membrane and
lipoprotein structure (2). It is now generally accepted that LP and its
products play an important role Exogenous chemicals and radiation produce
peroxidation of lipids leading to structural and functional damage to cellular
membranes(3). Ionizing radiation damages cellular molecules directly by
transferring energy or indirectly by generation of oxygen-derived free
radicals. Excited states and other reactive species are collectively known as
reactive oxygen species (ROS) (4), in liver, kidney and brain toxicity (5).

Exogenous chemicals and radiation produce peroxidation of lipids leading to
structural and functional damage to cellular membranes (6). Polyunsaturated
fatty acids present in cellular membranes are especially prone to damage by ROS
and the resulting LP can have serious consequences. LP plays a major role in
mediating oxidative-damage in biological systems. There are also several toxic
by-products of peroxidation which can damage other biomolecules away from the
site of generation (7,8). Among the subcellular organelles mitochondria are one
of the key components of the cell killed by radiation-induced oxidative stress.

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Endogenous antioxidants constitute important defence systems in cells and
elicit their action by suppressing the formation of ROS, their scavenging or by
repairing the damage caused. Besides this, a number of natural antioxidants are
found in plant materials, such as oil seeds, cereal crops, vegetables, fruits,
leaves, roots, spices and herbs (9, 10, 11, 12, 13, 14, 15). Some of them
exhibit significant antioxidant activity (16, 17, 18, 19 ) and are commonly
utilized for pharmaceutical purposes and in health foods. Recent evidence
indicates that medicinal plants contain a large number of biologically active
components that offer protection against degenerative diseases. A number of
medicinal plant have recently been reported to possess significant antioxidant
activity (20, 21, 22,). Ginger Zingiber Officinale is one of the world’s
best known spices, and it has also been universally used throughout history
for its health benefits. The dried extract of ginger contains mono
terpenes and ses quiterpenes. The main antioxidant active principles
in ginger are the gingerols and shogaols and some related phenolic
ketone derivatives. Ginger extract possesses anti-oxidative characteristics,
since it can scavenge super-oxide anion and hydroxyl radicals (23,
24). In line with this, Gingerol can inhibit ascorbate/ferrous complex induced
lipid peroxidation in rat liver microsomes (25). Ginger was also
suggested to interfere with inflammation processes (26).

Furthermore, ginger acts as a hypo-lipidemic agent in cholesterol-fed
rabbits (27, 28) and can increase the excretion of cholesterol via bile in rats
(29). In folk medicine, dates palm Phoenix
dactylifera, have been used in cases of cold, anemia, asthma, bronchitis,
catarrh, cough and congestion, fatigue, fever, flu, diarrhea, hemorrhoids,
stomachache, gonorrhea, thirst, toothache, tuberculosis, and vaginitis. In line
with this, dates have been reported to exhibit aphrodisiac, contraceptive,
demulcent, diuretic, emollient, estrogenic, expectorant, laxative, pectoral,
and purgative properties. These beneficial effects can be related to its high
content in vitamins and natural fiber, but this fruit can also be an important
source of calcium, sulfur, iron, potassium, phosphorous, manganese, copper, and
magnesium (30, 31). Key lime Citrus
aurantifolia  is popularly used for treating nausea and fainting and locally
the juice is a good astringent and is used as a gargle for sore throats (32). C.

aurantifolia juice is also a very effective bactericide and can be used for
treating rheumatic conditions, malaria and other fevers. The skin of the
ripe fruit is carminative (33). Loranthus
europeaus   has been used in
narcotic, antispasmodic, diaphoretic, headache 
tonic, tearing, rending rheumatic or neuralgic pains, coming on in
paroxysms; weak, irregular heart- action, with dyspnoea, cardiac hypertrophy,
and valvular insufficiency All parts of the plant contain viscin; also called bird-glue; curiously
miscalled birdlime
(from the German Vogelleim),
deriving its name from the fact that it has been  used in Germany in catching small birds. It
is very adhesive, soft, and elastic, having a greenish or brownish color;
insoluble in water and fixed oils, slightly soluble in alcohol, very soluble in
ether.  The proof of their antioxidant
activity can also explain their mechanism of action and  hence, it was considered desirable to
evaluate the effect of these plant extracts 
against lipid peroxidation induced by ? -radiation and
1,2-Dimethyhydrazine (DMH), which generates two potent ROS capable of inducing
LP, namely hydroxyl radical (OH.) and peroxyl radical (ROO.).

1, 2-Dimethyl hydrazine (DMH) generates model peroxyl radicals. These radicals
are similar to such peroxyl conditions that are physiologically active (1,2). ?
-Radiation also generates physiologically relevant ROS such as hydroxyl
radical, superoxide, hydrogen peroxide, single oxygen, etc. that are capable of
damaging many crucial cellular molecules, including membrane lipids (6,12,13).

Hence, inhibition of lipid peroxidation induced by these two agents is
physiologically relevant.   

 

Material & Methods

Hydrogen peroxide, ethylene diamine tetra acetic acid (EDTA),
2-thiobarbituric acid, triphenylphosphine (TPP), trichloroacetic acid, xylenol
orange and butylated hydroxyl toluene (BHT) were purchased from Sigma Chemical
Co, USA.  (DMH)  was from Aldrich Chemical Co, USA. Other
chemicals used in our study were of the highest quality commercially available
from local suppliers. The plants which selected in Phoenix dactylifera
,Loranthus europeaus ,Zingiber officinale, Citrus aurantifolia. The
powdered material was defatted with petroleum ether in Soxhlet apparatus for 8
to 10 h and the defatted material was then extracted with 70% methanol.  dissolving 0.1 g of the extract in 10 ml of
distilled water and stirring for 1 h. Above extracts were centrifuged for 15
min and supernatants were stored at –20°C. The supernatants were used to
examine the antioxidant properties (28). 
We had used three different concentrations, i.e. 0.2, 0.4 and 0.8%.  Three-month-old female Wistar rats (weighing
about 250 g) were used for the preparation of mitochondria. In brief, rat liver
and brain tissues were excised, homogenized in 0.25 M sucrose containing 1 mM
EDTA. The homogenate was centrifuged at 3000 g for 10 min to remove cell
debris and nuclear fraction. The resultant supernatant was centrifuged at
10,000 g for 10 min to sediment mitochondria. This pellet was washed
thrice with 50 mM phosphate buffer, pH 7.4 to remove sucrose. The protein was
estimated (29) and pellets were suspended in the same buffer (3). The
mitochondria were suspended in buffer and exposed to ? -radiation from 60Co
source at a dose rate of 15 Gy/min. The effect of extract on the
oxidative-damage caused by radiation was studied at a dose of 450 Gy.

Mitochondria (2.0 mg protein/ml) were suspended in the buffer and exposed to
radiation with or without the extracts. The effect of extracts on the
oxidative- damage caused by  (DMH)  was also studied. The mitochondria (2.0 mg
protein/ml) were exposed to  (DMH) (10
mM) with or without extract for 30 min. The mitochondria after exposed to ?
-radiation and (DMH) were evaluated for LP. Aliquots (90 ml) of brain/liver
mitochondria, after exposure to radiation sample, were transferred to
microcentrifuge tubes together with 10 ml of TPP in methanol/10 ml of methanol
in blank and test samples respectively. The samples were then vortexed and
subsequently incubated for 30 min at room temperature. Next 900 ml of Fox II
reagent (xylenol orange (100 mM), butylated hydroxy toluene (4.4 mM), sulphuric
acid (25 mM), ammonium ferrous sulphate (250 mM)) was added and samples were
incubated for a further 30 min in dark. The samples were centrifuged at 12000 g
for 10 min prior to reading absorbance of supernatant at 560 nm. The level
of peroxide in the sample was then determined using the difference between mean
absorbance of samples with and without TPP treatment and the final volume was
extrapolated to H2O2 concentrations in the standard graph. The effect of plants
extracts under study on hydroperoxide induction by  (DMH) at varying time intervals was also
determined (31). Thiobarbituric acid reactive substances (TBARS) assay was
performed by standard method using malonaldehyde equivalents derived from tetra
methoxypropane. Malonaldehyde and other aldehydes have been identified as
products of LP that react with thiobarbituric acid (TBA) to give a pink
coloured species at 532 nm. The method involved heating of the samples after
exposure to radiation and (DMH) with TBA reagent for 20 min in a boiling water
bath. TBA reagent contains 50 ml TCA (20%), 25 ml TBA (500 mg), 2.5 M HCl, 224
mg EDTA and the final volume is made up to 100 ml. After cooling, the solution
was centrifuged at 2000 g for 10 min and the precipitate obtained was
removed. The absorbance of the supernatant was determined at 532 nm against a
blank that contained all the reagents minus the sample. The malonaldehyde
equivalents of the sample were calculated using an extinction coefficient of
1.56 ´ 105 M–1cm–1. For collection of endogenous TBARS, fresh samples were
boiled without radiation exposure, and values were subtracted (30, 31, 32).

Vehicle controls were used for all the extracts. Methanolic extracts, were dissolved
in distilled water, for LP experiments.*

 

Results and Discussion

Data on
the effect of plants extracts on lipid hydroperoxide (LOOH) induced by   (DMH) in rat liver mitochondria are presented
in Figure 1.

 

P.dactylifera
with the highest inhibition, was the most effective in reducing
1,2-dimethyl hydrazine (DMH)-induced LOOH formation, Followed by Zingiber
officinale.Loranthus europeaus & Citrus aurantifolia. LOOH
formation induced by  (DMH) at varying
time intervals in brain mitochondria and its inhibition by Z.

officinale  & Citrus aurantifolia extracts
was more effective than other extracts. LOOH formation was maximum at 60 min
after exposure to 1,2-Dimethylhydrazin (DMH) . After 60 min, LOOH formation was
inhibited in all treated groups (Figure 2).

A

 

B

 

Figure (1): Effect of plant extracts on
LOOH formation by  (DMH) in Rat Liver Phoenix
dactylifera (PD),Loranthus europeaus(LE) Zingiber officinale (ZO), Citrus
aurantifolia (CA).  Fig(A) 0 time,
Fig (B) 30 min.

Figure (2): Effect of plant extract Phoenix
dactylifera (PD), Loranthus europeaus(LE) Zingiber officinale (ZO), Citrus
aurantifolia (CA). On LOOH formation induced by (DMH) at various time
intervals in rat brain mitochondria.

 

 

The
effect of plants extracts on TBARS formation
induced by  (DMH) in rat liver
mitochondria are given in
Figure 3.  P.dactylifera and &
Citrus aurantifolia were more effective
in reducing LP induced by   (DMH)
compared to other plant extracts. Exposure to radiation, as a function of dose,
ranging from a dose of 0 to 750 Gy, resulted in
enhanced LP as evident by the
formation of TBARS. The increase in TBARS formation was significant with the
increasing doses examined. Exposure
to 300–450 Gy showed steep increases, while higher doses were effective only in marginally enhancing peroxidation. Hence the optimum dose of 450 Gy
was selected for the experiments, as this dose caused
optimum damage in terms of LP in rat liver and brain mitochondria.

 

 

 

 

 

 

A

B

 

Figure (3): Effect of plant  extract Phoenix dactylifera (PD),Loranthus
europeaus(LE) Zingiber officinale (ZO), Citrus aurantifolia (CA)on TBARS
formation induced by  (DMH) in rat liver
mitochondria. Fig(A) 0 time, Fig (B) 30 .

 

Mitochondria are crucial targets for radiation and free
radical-mediated damage. Since mitochondria are devoid of cytosolic
antioxidants, as in a whole cell, they are fairly resistant to ?
-radiation, Hence a dose of 450 Gy is needed to achieve optimum concentration of free radicals capable of
inducing significant damage measurable by simple spectrophotometric means. This
dose is much higher than those used in radiotherapy (1–6 Gy) or for
radioprotection pertinent to mammals (LD50 in the range of 5–7 Gy).

Such in vitro studies with easily measurable systems are
usually animal models involving a large number of animals, and being subject to
animal ethics committees. A case in point are such studies on the
radioprotective effect of caffeine using in vitro systems (33, 34) and
animal models. We plan to extend our investigations to animal studies in
vivo in future. Data on radiation-induced LP and its protection by plants
extracts are given in Figure 4.

Loranthus europeaus & Citrus aurantifolia showed significant ability to inhibit
radiation-induced LP in rat liver mitochondria, rather than .  P.dactylifera & Z. officinale  at a concentration of 1% reduced TBARS
formation significantly when it was present at the time of irradiation. The
formation of LOOH, an intermediate of peroxidation, showed that LOOH formation
induced by ? -radiation in rat liver mitochondria was inhibited more
effectively by Z. officinale & Citrus aurantifolia than other 
extracts, and the data are represented in Figure 5. Prevention of free-radical
formation and maintenance of cellular structural integrity and of chemical
environment are fundamental requirements of all cells. In biological systems,
radiation-induced free radicals impair antioxidant defence leading to increased
membrane lipid peroxidation. Generation of ROS by ionizing radiation
(especially with low-LET radiation) and (DMH) and its profound impact on
cellular biomolecules are well established (35, 36).

The present investigation demonstrates that (DMH) and radiation
induced significant LP in mitochondria. Increase in peroxidation is observed as
a function of radiation dose. Radiation generated ROS and is also capable of
initiating LP. The initial products of peroxidation are conjugated dienes, to
which is added oxygen to form LOOH that further breaks down to stable aldehydes
and reacts with TBA to form thiobarbituric acid–malonaldehyde adduct (37).

 

 

 

A

     B

Figure (4): Effect of plant extract Phoenix
dactylifera (PD),Loranthus europeaus(LE) Zingiber officinale (ZO), Citrus aurantifolia
(CA)on TBARS formation induced by ?- 
ray in rat liver mitochondria. Fig(A) 0 time, Fig (B) 30 .

 

Radiation therapy is one of the most important and popular tools
for cancer treatment. Because human tissues contain 80% water, the major
radiation damage is due to aqueous free radicals, generated by the action of
radiation on water. The major free radicals resulting from aqueous radiolysis
are ·OH, ·H, eaq –, HO2 ·, H3O+, etc. (33). Among them ·OH is the most potent,
capable of inflicting severe molecular damage. This free radical reacts with
cellular macromolecules such as DNA, proteins, lipids, etc. and causes
dysfunction and mortality. These reactions take place in tumor as well as
normal cells when exposed to radiation. LP causes membrane damage as well as
oxidative modification of critical targets. Agents that can interact with these
secondary radicals formed during peroxidation and scavenging them, would be
effective in inhibiting LP and in turn protect against radiation and
1,2-Dimethylhydrazin (DMH) -induced damage. Removal of excess reactive species,
suppression of their generation or protection against peroxidation by repair of
membrane damage may be an efficient way of preventing cancer and other
diseases.

The effects of plant extracts on LP show significant inhibition of
LOOH and TBARS formation. Our earlier studies have indicated that plant
extracts are effective scavengers of both primary and secondary radicals (32).

Protection of membranes at both primary and secondary levels explains the possible mechanism by which plants
inhibit LP by radiation and 1,2-Dimethylhydrazin (DMH) .

Phenolics are a group of non-essential dietary components that have
been associated with inhibition of atherosclerosis and cancer, by chelating
metals, inhibiting lipo oxygenases and scavenging of free radicals. plants
phenolic compounds are found to be excellent antioxidants and synergists that
are not mutagenic(32). Our earlier results also have shown that plant extracts
possess significant radical scavenging properties of both primary and secondary
radicals, in a concentration-dependent manner. Hence the components present in
plants may inhibit LP by scavenging of radicals that initiate or propagate LP.

Our earlier studies have revealed that all the plant extracts employed in this
study possess antioxidant properties, mainly measured as radical scavenging (19,
20).

 

 

 

A

B

Figure (5):
Effect of plant extract Phoenix dactylifera (PD),Loranthus europeaus(LE)
Zingiber officinale (ZO), Citrus aurantifolia (CA)on LOOH formation induced
by ?  ray in rat liver
mitochondria. Fig(A) 0 time, Fig (B) 30

 

The
present finding strongly suggests that the use of these extracts to prevent LP
leading to membrane damage consequent to exposure to radiation and to certain
chemicals which generate potent ROS in the form of ·OH or ROO·. This also
explains the possible mechanisms behind the observed health benefits of these
plants.