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131
CHAPTER – 7
INVITRO AND INVIVO ANTI-OXIDANT ACTIVITY
Chapter No Contents Page No.
Invitro Anti-Oxidant Activity of Combined Extract of CissusQuadrangularis and Aegle Marmelos
7.1. Introduction 132
7.2. Materials and Methods 133
7.3. Results and Discussion 138
7.4. Conclusions 139
Invivo Antioxidant Activity of Combined Extract of Cissus
Quadrangularis and Aegle Marmelos
7.5. Introduction 139
7.6. Materials and Methods 141
7.7. Results and Discussion 147
7.8. Conclusions 151
132
INVITRO ANTI-OXIDANT ACTIVITY OF COMBINED EXTRACT OF
CISSUS QUADRANGULARIS AND AEGLE MARMELOS
7.1. Introduction
The antioxidants are radical scavengers which protect the human body against
free radicals1. The reactive oxygen species, with superoxide, hydroxyl radical and
hydrogen peroxide are produced in definite organelles in the cell under normal
physiological conditions. Excessive production of these ROS, beyond antioxidant
defense capacity of the body can cause oxidase stress2. The reactive oxygen species
(ROS) and free radicals mediated reactions are involved in various pathological
conditions such as anaemia, asthma, inflammation, neurodegeneration against ageing
process and perhaps dementia. Diet has a main role in the growth of chronic diseases,
such as, cancer, coronary heart disease, diabetes, hypertension and cataract3.
Researches have also indicates that utilization of food and beverages high in phenolic
content is correlated with reduced incidence of heart diseases, anaemia, asthma, arthritis,
inflammation, neurodegeneration. Recently, there has been considerable interest in finding
natural antioxidants from plant sources to replace synthetic ones. Natural antioxidant
substances are considered to be safe since they occur in plant foods and are desirable than
their synthetic counter parts. The scientific reports and experimental studies have shown that
plants contain a large variety of phytochemicals that have antioxidant property4. The most
familiar plant phenolic antioxidants consist of flavonoid compounds, cinnamic acid
derivatives, coumarins, tocopherols and poly functional organic acids. Studies on antioxidants
have been explored in various plants and plant products. In this antioxidant survey two
common plants like Sauropus androgynous and Alternanthera pungens were selected based
on their use as edible greens5. There is scanty information on the antioxidant property of these
plants and hence the present work was taken up to fill the gap.
133
Recent study with main bioactive substances in various plants and food substances
are of greater interest6. It has been exposed that free radicals could induce cellular damage
and may be involved in numerous human diseases such as cancer, arteriosclerosis, diabetic
mellitus, hypertension,AIDS and in aging processes of different types of natural antioxidants,
flavonoids and phenolic compounds have achieved more attention7. Polyphenolic compounds,
like flavonoids and phenolic acids, generally originate in plants have been reported to have
many biological effects, including antioxidant activity8.
A. lanata has been studied for flavonoid glycosides, β-sitosterol, α-amyrin,
compesterol, chrysin, four new alkaloids viz. aervine, methylaervine, aervoside and
aervolanine. Similarly, from A. persica alkaloids, leucoanthocyanidins, flavonoids,
triterpenoids, cardiac-glycosides, coumarins and saponins are reported to possess antioxidant
activity9.
7.2. MATERIALS AND METHODS
7.2.1. Materials
Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit
pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of Cissus
quadrangularis and fruit pulp of Aegle marmelos (c-ECA).
7.2.2. Experiment
7.2.2.1. DPPH Method
The free radical scavenging activity by different plant extracts was done according
to the method reported10. The plant extract of volume fifty micro liters is mixed with
methanol, provides 100 μg/ml, mix 450 μl of 50 mM Tris-HCl buffer (pH 7.4) and 1ml
of 0.1mM DPPH in methanol solution in every reaction. Methanol (50 μl) without any
substances was taken as control. The reduction of the DPPH free radical was measured
reading the absorbance at 517 nm after incubation at room temperature for 30 min. BHT
134
and L-Ascorbic acid were used as controls. The percent inhibition was determinedfrom the
following formulae:
% DPPH radical-
scavenging= 100)(
)(
controlofAbsorbance
sampletestofAbsorbancecontrolofAbsorbance
Table 7.1: Antioxidant activity by DPPH method
S.No. Sample Absorbance % of scavenging DPPH
1. c-EACA(100µg) 0.21 60.38
2. c-ECA(100µg) 0.23 56.60
3. Negative control (Water) 0.53 -
4. Methanol 0.51 -
5. Standard (Vit E) 0.05 90.57
Sample 1 Sample 2Control MethanolStandard0.000.050.100.150.200.250.300.350.400.450.500.55
Groups
Abso
rban
ce
Fig.7.1: Antioxidant activity by DPPH method
135
7.2.2.2. FTC Method
The standard method as described by Palombo et al.,11 was followed. A mixture
was prepared by mixing 4.0 mg plant extract in 4 ml absolute ethanol, 4.1 ml of 2.5%
linolenic acid in absolute ethanol, 8.0 ml of 0.05 M phosphate buffer (pH 7.0) and 3.9
ml of water was filled in a vial by means of a screw cap and then dried in an oven at 40 °C.
To 0.1 ml of this solution was mixed 9.7 ml of 75% ethanol and 0.1 ml of 30%
ammonium thiocyanate. Accurately 3 min after addition of 0.1 ml of 0.02 M ferrous
chloride in 3.5% HCl to the reaction mixture, the absorbance of red colour was calculated
at 500 nm each 24 hr til the day after absorbance of control attained maximum. α-
tocopherol was considered as positive controls, mixture without plant sample was
considered as the negative control.
Table 7.2: Antioxidant activity by FTC method
S.No Sample Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
1. c-EACA (100µg) 0.76 0.92 1.06 0.69 0.68 0.68
2. c-ECA (100µg) 0.77 0.91 1.05 0.69 0.69 0.69
3. -ve control 0.20 0.39 0.40 0.41 0.44 0.44
4. +ve control 0.20 0.23 1.04 1.06 1.06 1.07
136
Sample 1 Sample 2 Control Standard0.00.10.20.30.40.50.60.70.80.91.01.1
GroupsAb
sorb
ance
Figure 7. 2: Antioxidant activity by FTC method
7.2.2.3. Thiobarbituric acid (TBA) method
The method of Koleva et al., 12 was used. Two ml of 20% trichloroacetic acid and 2
ml of 0.67% 2-thiobarbituric acid was mixed with 1 ml of sample solution, as such in
FTC method. Then it is immersed in a boiling water bath and cooled, it was subjected to
centrifugation for 20 min at 3000 rpm. Absorbance of supernatant was calculated at 552
nm. Antioxidant activity depends on the absorbance on the final day.
Table 7.3: Antioxidant activity by TBA method
S.No. Sample Absorbance
1. c-EACA (100µg) 0.15
2. c-ECA (100µg) 0.36
3. -ve control 0.08
4. +ve control 0.12
137
Sample 1 Sample 2 Control Standard0.0
0.1
0.2
0.3
0.4
Groups
Abso
rban
ce
Fig.7.3: Antioxidant activity by TBA method
7.2.2.4. Qualitative analysis
For chromatographic separation, spot the ethanol extract on a TLC plate (100
μg/ml) using the mobile phase methanol: chloroform (95:5, v/v). The chromatogram is
made to develop for 30 minutes. After the chromatogram is completed the entire plate was
sprinkled with DPPH (0.15 % w/v) solution by means of an atomizer. In pinkish
background yellowish colour is developed on the TLC plate were noted which represents
the presence of antioxidant substances.
Fig.7.4.Chromatographic separation of the extract using TLC
138
7. 4. RESULTS AND DISCUSSION
It is familiar that free radicals are the one of the reason of numerous diseases,
Parkinson’s disease, coronary heart disease and cancer13. This study demonstrates that
ethanolic and ethyl acetate extracts of Aegle marmelos and Cissus quadrangularis, have
tremendous antioxidant activity. It is remarkable and valuable to scrutinize the latent
efficiency of combined extracts of Aegle marmelos and Cissus quadrangularis
The importance of natural antioxidants as of combined extracts of Aegle
marmelos and Cissus quadrangularis can be distinguished, For health conscious
consumer, the word “free radicals and antioxidants” has become very important.
Antioxidants assist the organisms to face oxidative stress. It is possible to
diminish the threat of chronic diseases and avoid their development by either rising the
body’s antioxidant defense or by enrichment with established nutritional antioxidants14.
Cancer chemoprevention is by means of antioxidant advancements has been
recommended to propose a good possibility in providing betterment to health and is
accepted by many researchers for inhibiting, hindering, or reverse the course of
carcinogenesis15,16 .
DPPH is a constant radical which is used to estimate the antioxidant activity of
plant17. FTC test measures the peroxide compound which is produced at the preliminary
stage of oxidation while TBA test measures the minor products such as aldehyde and
ketone18.
139
7. 5. CONCLUSION
Both extracts exhibited significant antioxidant activity in all the four methods.
In DPPH assay method Both Sample 1(c-ECA) and Sample 2 (c-EACA) have
better antioxidant activity. Sample 2 have more antioxidant activity when compared to
Sample 1.
In FTC method, Both Sample 1(c-ECA) and Sample 2 (c-EACA) have better
antioxidant activity. Sample 1 has more antioxidant activity when compared to Sample 2.
In TBA method, Both Sample 1(c-ECA) and Sample 2 (c-EACA) have better
antioxidant activity. Sample 1 has more antioxidant activity when compared to Sample 2.
In the qualitative analysis by TLC plate method. Both Sample 1(c-ECA) and
Sample 2 (c-EACA) have better antioxidant activity.
INVIVO ANTIOXIDANT ACTIVITY OF COMBINED EXTRACT
OF CISSUS QUADRANGULARIS AND AEGLE MARMELOS
7.5. Introduction
Phagocytic cells generate poisonous reactive oxygen intermediates (ROI) along with
injury and swelling of tissues as a method to destroy attack microbes19, 20.
Oxidative stress has been exposed to produe lesser harm through late cellular death
and swelling consequently, reducing oxidative stress can stop cellular death, reduces swelling
and avoid morbidity and mortality21.
Oxidative stress is related with numerous neonatal diseases for instance retinopathy
of prematurity, periventricular leucomalacia, necrotizing enterocolitis and bronchopulmonary
dysplasia22.
140
Heavy metal pollution of soils expected substantial awareness as a value of the
amplified ecological pollution from industrialized and agricultural source23, 24.
Antioxidants play a main role in the body defense system against reactive oxygen
species (ROS), which are dangerous by yields which are produced during ordinary cell
aerobic respiration25. Antioxidants are radical scavengers, which protect the human from
diseases like ischemia, anaemia, asthma, arthritis, inflammation, neuro-degeneration and
ageing process26.
Free radicals attack the unsaturated fatty acids in the cell biomembranes leads to
membrane lipid peroxidation, decline in membrane fluidity, enzyme loss and receptor tend to
activates and membrane proteins damage causing cell inactivation27. If the in vivo activity of
scavengers is very low to slow down these radicals, produce diseases like arteriosclerosis,
liver disease, diabetes, inflammation, renal failure or aging 28.
Lipid peroxidation is linked with aging and carcinogenesis29. Though, living
systems are guarded from active oxygen species by enzymes like superoxide
dismutase, glutathione peroxidase, glutathione reductase and catalase30.
Therefore, to justify the traditional claims the present study was undertaken to
find out if combined ethyl acetate extract of stem bark of Cissus quadrangularis and
fruit pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of
Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA) demonstrates the
antioxidant activity against CCl4 induced rats models of erythrocyte damage by
estimation of anti-oxidant enzymes such as and the antioxidants superoxide dismutase
(SOD), catalase, glutathione peroxidase, glutathione reductase and lipid peroxidation
as biomarkers. Hence, the current research was considered to confirm the state of the
native practitioners.
141
7.6. MATERIALS AND METHODS
7.6.1. Materials
Combined ethyl acetate extract of stem bark of Cissus quadrangularis and
fruit pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of
Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA), carbon
tetrachloride, Vehicle -1% tween 80.
7.6.2. Experimental Animals
Wistar male albino rats bearing the weight of 150-220 gm were used. They
maintained in Santhiram College of Pharmacy, Nandyal, Andhra Pradesh, India. The animals
were kept in a well-ventilated room with at 12:12 hr light, dark cycle in polypropylene cages.
Institutional Animal Ethical Committee (IAEC) clearance was done with reference no
1519/PO/a/11/CPCSEA).
7.6.3. Acute Toxicity Study
As per the OECD guideline no. 423 (Acute Toxic Method) the acute toxicity of c-
EACA and c-ECA was estimated. The test extract was found to be safe even at 2000 mg/kg
dose. Hence, 1/8th (250 mg/kg) and 1/4th (500 mg/kg) of this dose were preferred for further
study31.
7.6.4. Experimental Design:
Body weight of animals was noted and they were separated into 6 groups each
group is comprised of 6 rats each.
1% Tween 80 was used as a vehicle of extracts in addition to carbon
tetrachloride (1 ml/kg body weight), given intraperitoneally in alternate days for 14
days.
The following groups were used:
142
Group I - Vehicle 1% Tween 80 (5 ml/kg, p.o) [Normal Control]
Group II - CCl4 (1 ml/kg of body weight), i.p[Negative Control]
Group III - Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit
pulp of Aegle marmelos (c-EACA) 250 mg/kg, p.o + CCl4 (1 ml/kg of body weight), i.p
Group IV - Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit
pulp of Aegle marmelos (c-EACA) 500 mg/kg, p.o + CCl4 (1 ml/kg of body weight), i.p
Group V - Combined ethanol extract of stem bark of Cissus quadrangularis and fruit pulp of
Aegle marmelos (c-ECA) 250 mg, p.o + CCl4 (1 ml/kg of body weight), i.p
Group VI - Combined ethanol extract of stem bark of Cissus quadrangularis and fruit pulp
of Aegle marmelos (c-ECA) 500 mg/kg, p.o + CCl4 (1 ml/kg of body weight), i.p
Vehicle and extract treatments were given once daily for 14 days, but CCl4 (1 ml/kg
of body weight, i.p), was given in alternate days for 14 days.
On the 15th day all group of animals were fasted for 16 hours then it was
sacrificed by cervical dislocation. The blood was collected from the jugular vein into
tubes containing heparin (anticoagulant), the buffy coat obtained was removed by
using centrifugation at 3000 rpm for 15 min. The packed cells were washed with
physiological saline (0.9% NaCl) for three times, by suspending them in cold distilled
water It is lysed, and again centrifuged at 7000 rpm for 30 min. The pellet was formed
along with the erythrocyte membrane and the supernatant denotes the haemolysate.
7.6.4.1. Biochemical estimation
As a result of centrifugation, plasma was formed that measures the lipid
peroxidation by the method of Gutteridge and Wilkins32 Superoxide dismutase was
estimated by haemolysate 33 and catalase34 activities. Lipids were extracted from the
143
erythrocyte membrane by means of method of Folch et al35. The concentration of
cholesterol and phospholipids were evaluated by established methods36, 37. The
cholesterol/phospholipid ratio was determined.
7.6.4.2. Estimation of Lipid Peroxidation
It was determined in the plasma sample by quantifying the amount of
malondialdehyde (Gutteridge and Wilkins, 1982)32.
Reagents
1. 20% Acetic acid
2. 8.1% Sodium dodecyl sulphate (SDS)
3. 0.8 % Thiobarbituric acid (TBA)
4. N-Butanol-Pyridine mixture (15:1v/v)
Procedure
0.2 ml of plasma sample was added to 1.5 ml of 20% acetic acid, 0.2 ml of sodium
dodecyl sulphate and 1.5 ml of thiobarbituric acid, then it was made up to the volume to 4.0
ml with distilled water. It was heated at 95oC for 60 min in a water bath and it was incubated
and cooled to room temperature and made up to the final volume of 5.0 ml in all tubes. Then
5.0 ml n-butanol pyridine (15: 1) mixture was added and mixed carefully by vortexing for 2
minutes. Centrifugation was done at 3000 rpm for 10 min, the organic upper layer was taken
and its optical density was read at 532 nm against a suitable blank.
The levels of lipid peroxides were represented by n moles of malondialdehyde
(MDA)/min/mg protein in plasma sample.
7.6.4.3. Estimation of Superoxide Dismutase
Superoxide dismutase was assayed by Marklund and Marklund, 1974. Method33.
Reagents
144
1. 0.1 M, Tris – HCl buffer, pH 8.2
2. 0.5 M, Tris – HCl buffer, pH 7.4
3. 2 mM Pyrogallol solution
4. Absolute alcohol
5. Chloroform
Procedure
1 ml of the sample was added with 0.25 ml of absolute ethanol and 0.15 ml of
chloroform. It was shaken for 15 min by means of a mechanical shaker centrifuge the
suspension and the supernatant attained comprises the enzyme extract. The mixture for auto-
oxidation consists of 2 ml of buffer, 0.5 ml of 2 mM pyrogallol and 1.5 ml of water. Auto-
oxidation rate of pyrogallol was recorded at an interval of 1 min for 3 min. The enzyme
mixture have 2 ml of 0.1 M Tris – HCl buffer, 0.5 ml of pyrogallol, aliquots of the enzyme
preparation and made up to 4 ml with water. The inhibition rate of pyrogallol auto-oxidation
after adding the enzyme was recorded. The superoxide dismutase activity was estimated by
the inhibition of pyrogallol auto-oxidation at 420 nm for 10 min. To bring about 50%
inhibition of auto-oxidation by pyrogallol the amount of one unit of superoxide dismutase
enzyme is essential. The enzyme activity was represented in terms of units/min/mg protein.
7.6.4.4. Estimation of Catalase
Catalase of tissue homogenate was estimated according to the method of Beers RF
and Sizer IW (1952)34.
Reagents
1. Phosphate buffer (M/15, pH 7.0)
2. Hydrogen peroxide – phosphate buffer, pH 7
145
Procedure
Plasma sample (haemolysate) was centrifuged with M/15 phosphate buffer at 1 to 4oC
and. The deposit is stirred with cold phosphate buffer and allowed to stand in the cooled
environment with occasional shaking.This was repeated once or twice; the resulting
supernatants were collected together and used for assay. A cuvette was filled with 3 ml of
H2O2 phosphate buffer and 0.01 – 0.04 ml sample and compared against a control cuvette with
enzyme solution without H2O2 phosphate buffer at 240 nm. It was observed for a decrease in
the optical density from 0.450 to 0.400. The calculations were made by using these values.
7.6.4.5. Estimation of Glutathione Reductase
Glutathione reductase activity of sample was measured by the method of Dobler and
Anderson (1981)35.
Reagents
1. 50 mM Sodium Phosphate buffer pH 7.5
2. 10 mM Ethylene Diamine Tetra Acetic Acid (EDTA)
3. 0.9 mM Glutathione oxidized (GSSG)
4. Nicotinamide Adenine Dinucleotide Phosphate reduced (NADPH)
Procedure
The reaction mixture containing 50 mM sodium Phoshpate buffer pH 7.5, 10 mM
EDTA, 0.67 mM glutathione oxidized and 0.1 mM NADPH was made up to 3ml with water.
The Change in the optical density was monitored after adding 0.1 ml of sample at 340 nm for
3 mins at 30 second interval. The enzyme activity is represented in n moles of GSSG
utilized/min/mg protein in plasma sample.
7.6.4.6. Estimation of Glutathione Peroxidase (GSH-Px)
Glutathione peroxidase of sample was evaluated by the method of Lawrence and
Burk (1976)36.
146
Reagents
1. 75 mM Phosphate buffer, pH 7.0
2. 60 mM Glutathione
3. 30 units/ml Glutathione reductase
4. 15 mM EDTA
5. 7.5 mM H2O2
6. 3 mM NADPH
Procedure
The assay mixture consists of 2 ml of 75mM Phosphate buffer (pH 7.0), 60 mM
Glutathione, 0.1 ml of 30 units/ml Glutathione reductase, 0.1 ml of 15 mM EDTA, 0.1 ml of 3
mM NADPH and supernatant of plasma sample and made upto final volume of 3.0 ml. The
reaction starts by adding 0.1ml of 7.5 mM H2O2.
The change in the absorbance rate during the conversion of NADPH to NADP+ was
measured spectrophotometrically at 340 nm for 3 mins. The activity of glutathione peroxidase
was reprented in moles of NADPH oxidized to NADP+ / min/ mg protein in plasma sample.
7.6.4.7. Statistical Analysis
The data were recorded as mean ± standard error mean (S.E.M). The importance of
variations between the groups was evaluated by means of one way and multiple way analysis
of variance (ANOVA). The test followed by Dunnett’s test p values less than 0.05 were noted
as significance.
147
7.7. RESULTS AND DISCUSSION
7.7.1. Effects of c-EACA and c-ECAon lipid peroxidation and primary
antioxidant enzymes of the erythrocytes of carbon tetrachloride -intoxicated rats
Table-7.4 shows the effect of c-EACA and c-ECA on oxidative stress induced
by carbon tetrachloride. The extracts considerably (P <0.01) prohibited the
accumulation of lipid peroxidation substances in the plasma. The rats were intoxicated
by carbon tetrachloride led to considerable increase in superoxide dismutase, catalase,
glutathione reductase and glutathione peroxidase activities, at the same time
instantaneous administration of carbon tetrachloride with the c-EACA and c-ECA
significantly (P <0.01) decreased these activities than control group. c-ECA treated
group showed more significant effect than c-EACA treated group (Fig 7.1 to7. 4).
7.7.2. Effects of c-EACA and c-ECAon cholesterol and phospholipids
Carbon tetrachloride intoxication produces a raise in membrane cholesterol, a
reduction in membrane phospholipid and a consequent enhancement in the cholesterol
to phospholipid ratio. Both extract of c-EACA and c-ECA at the doses of 250 & 500
mg/kg considerably (P<0.01) reduces the cholesterol and phospholipids. Both extracts
showed dose dependent activity and significantly decreased the
cholesterol/phospholipid ratio. (Table 7.2; Fig 7.5
148
Table 7.4: Effects of c-EACA and c-ECAon lipid peroxidation and primary antioxidant enzymes of the
erythrocytes of carbon tetrachloride -intoxicated rats
Values arerepresented asmean ± SEMof sixobservations.Statisticalsignificant testfor comparisonwas made byANOVA,followed byDunnet’stest.a -Comparisonbetween GroupI Vs Group II.b- Comparisonbetween GroupII Vs GroupIII, IV, V &VI. *p<0.05**;p<0.01
Group
Design oftreatments
Enzyme activities
Lipidperoxidation
x 10 -6 (units)
SOD
Units/mgprotein
CatalaseUnits/mgprotein
GlutathioneReductase
GSSGutilized/min/mg protein
Glutathione PeroxidaseNADP+ / min/ mg protein
I Vehicle 1%Tween 80 (5ml/kg, p.o)
27.17±0.7491 192.67±0.8028
1.73±0.076 25.33±0.7601 35.5±0.7638
II CCl4 (1 ml/kg ofbody weight), i.p
49.17±0.9458**a
261±3.777**a 4.53±0.0989**a
15.17±0.4773**a
22±0.5774**a
III c-EACA 250mg/kg +CCl4
38.17±0.4014**b
234.50±1.408**b
3.53±0.0494**b
21±0.5164**b 24.33±0.5578*b
IV c-EACA 500mg/kg +CCl4
34.67±0.6667**b
216.33±1.382**b
2.57±0.0421**b
23.83±0.4773**b
26.67±0.4944**b
V c-ECA250 mg +CCl4
35.17±0.7923**b
229±1.155**b 3.22±0.0477**b
23.67±0.8028**b
25.83±0.4773**b
VI c-ECA 500mg/kg, +CCl4
32.67±0.7149**b
208±1.653**b 2.43±0.0558**b
26.67±0.7491**b
28.17±0.3073**b
149
I II III IV V VI05
10152025303540455055
IIIIIIIVVVI
Groups
Lipi
d pe
roxid
atio
n in
Uni
ts
Fig. 7.5: Effects of c-EACA and c-ECAon lipid peroxidation
I II III IV V VI0
100
200
300IIIIIIIVVVI
Groups
SOD
Units
/mg
prot
ein
Fig. 7.6: Effects of c-EACA and c-ECAon SOD
I II III IV V VI0
1
2
3
4
5IIIIIIIVVVI
Groups
Units
/mg
prot
ein
Fig. 7.7: Effects of c-EACA and c-ECAon Catalase
150
Glut Red Glut Per0
10
20
30
40IIIIIIIVVVI
Groups
Fig. 7.8: Effects of c-EACA and c-ECAon Glutathione Reductase&Glutathione Peroxidase
Table 7.5: Effect of c-EACA and c-ECA on erythrocyte membrane lipids and
cholesterol/phospholipid ratio of carbon tetrachloride - intoxicated rats
Group
Design of treatments Cholesterol
(mg/100μl)
Phospholipid
(mg/100μl)
Cholesterol
/Phospholipid
I Vehicle 1% Tween 80 (5ml/kg,
p.o)
0.66±0.0183 1.11±0.008 0.59
II CCl4 (1 ml/kg of body weight), i.p 0.92±0.0095**a 0.85±0.0109**a 1.08
III c-EACA 250mg/kg + CCl4 0.86±0.0213*b 0.90±0.0119*b 0.96
IV c-EACA 500mg/kg + CCl4 0.73±0.0076**b 0.98±0.011**b 0.74
V c-ECA 250mg + CCl4 0.79±0.0083**b 0.95±0.0128**b 0.83
VI c-ECA 500mg/kg, + CCl4 0.69±0.0159**b 1.01±0.0101**b 0.68
Values are represented as mean ± SEM of six observations.Statistical significant test for comparison was made by ANOVA, followed by Dunnet’s test.a - Comparison between Group I Vs Group II.b - Comparison between Group II Vs Group III, IV, V & VI. *p<0.05**; p<0.01.
151
Cholesterol Phospholipid0.00.10.20.30.40.50.60.70.80.91.01.11.2
IIIIIIIVVVI
Groups
Fig. 7.9: Effect of c-EACA and c-ECA on erythrocyte membrane cholesterol &
phospholipid of Carbon tetrachloride –intoxicated rats
7.8. CONCLUSION
The reports of the current study undoubtedly specified that the rigidity of the membranes
after administration of both extract of c-EACA and c-ECA at the doses of 250 & 500 mg/kg.
Administration of c-EACA and c-ECA prohibited alteration in membrane phospholipids and in
membrane fluidity. Many of the researchers proved free radicals are importantly implicated in
different pathological conditions like cancer, arthritis, inflammation and liver diseases37.
Carbon tetrachloride intoxication breaks the erythrocytes was proved by the elevation of
lipid peroxidation, superoxide dismutase and catalase activities and inversely reduces the
glutathione reductase & glutathione peroxidase in erythrocyte membrane fluidity. The enhanced
superoxide dismutase activity leads to the accumulation of hydrogen peroxide, which enhances
the catalase activity.
152
Experimental animals were pre-treated with the c-EACA and c-ECA reveals a better free
radical scavenging leads toreduced actions of superoxide dismutase and catalase, the
concentration of lipid peroxidation products tends to be regular.
Previous studies documented that changes in glutathione peroxidase activity in
erythrocytes were inversely correlated with intensity of lipid peroxidation. It may be supposed
that decrease in glutathione peroxidase and glutathione reductase activity causes failure of H2O2
detoxification.
H2O2 accumulated in erythrocyte cells ions present may undergo Fenton’s reaction in
which hydroxy radicals are produced. These reactive oxygen species participate in lipid
peroxidation processes38-40. Increases in lipid peroxidation in the present study were dependent
on decrease in glutathione peroxidase & glutathione reductase activity, suggested that oxidative
stress and lipid peroxidation rise might occur after CCl4 administration.
In present study results showed that c-EACA and c-ECA significantly decreased lipid
peroxidation and increased glutathione peroxidase & glutathione reductase. Participation of
oxygen free radicals and oxidative stress in carbon tetrachloride (CCl4) induced erythrocyte
damage may indirectly be confirmed by antioxidant activity of c-EACA and c-ECA extracts.
The cumulative effect of carbon tetrachloride intoxication resulted that micro-viscosity of
a membrane enhances with enhancement in cholesterol to phospholipid ratio results in cellular
rigidity41. Experimental animals were intoxicated with carbon tetrachloride changes the
membrane structure and its role as exposed by the enhancement in cholesterol and consequent
reduction in phospholipid concentrations, therefore enhanced cholesterol to phospholipid ratio.
Cooper et al., 42 states that modification of bio-membrane lipid profile agitates its fluidity,
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permeability, activity of associated enzymes and transport system. Thus c-EACA and c-ECA
extracts plays a key role in peroxidation by hindering the free radical attack on bio-membranes.
The reports of phytochemical test of the c-EACA and c-ECA extractsrevealed that presence of
flavonoids. The presence of flavonoids provides information to guard lipids, blood and body fluids
against the attack of reactive oxygen species such as superoxide, peroxide and hydroxyl radicals43-46. The
presence of flavonoids in c-EACA and c-ECA extractsmay be dependable for their antioxidant activity.
Ever since reactive oxygen species and free radicals were involved in oxidative stress.
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